Cysteine String Proteins

Journal Pre-proof Cysteine String Proteins Cameron B. Gundersen

PII:

S0301-0082(20)30013-7

DOI:

https://doi.org/10.1016/j.pneurobio.2020.101758

Reference:

PRONEU 101758

To appear in:

Progress in Neurobiology

Received Date:

26 June 2019

Revised Date:

6 January 2020

Accepted Date:

13 January 2020

Please cite this article as: Gundersen CB, Cysteine String Proteins, Progress in Neurobiology (2020), doi: https://doi.org/10.1016/j.pneurobio.2020.101758

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier.

Cysteine String Proteins by

Cameron B. Gundersen

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Department of Molecular and Medical Pharmacology, UCLA David Geffen School of Medicine, Los Angeles, CA 90095, USA.

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Corresponding author: Tel. 310-825-3423; fax: 310-825-6267

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E-mail address: [email protected] (C.B. Gundersen)

Highlights

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Cysteine string protein- (CSP-) is prominently expressed in regulated secretory cells of multi-cellular organisms. While many organisms have one csp gene, often with multiple splice variants, vertebrate genomes harbor three csp genes (dnajc5) that encode proteins with a J domain, which is a motif that interacts with 70 kDa heat shock proteins, and a cysteine-rich, “string” motif. Abundant data support a chaperone/co-chaperone role for CSP-, but the function of the cysteine string motif remains ambiguous. CSP- is associated with synaptic vesicles and other regulated secretory organelles, and recent data also point to an endosomal/lysosomal localization. The majority, and possibly all, of the cysteine residues of the cysteine string of CSP- are fatty acylated. The tissue distribution and subcellular localization of CSPs- and - have not been clarified. Mutagenesis studies in Drosophila implicated CSP in stimulus-dependent exocytosis at nerve terminals and revealed a role in neuroprotection. Investigations of csp- knockout mice argued that CSP- functions as a molecular chaperone to protect nerve terminals from neurodegeneration, but recent data support additional roles for CSP- in protein quality control. Two distinct mutations of the human csp- gene lead to an autosomal-dominant, neuronal ceroid lipofuscinosis, and ongoing research has identified interesting links between CSPs and other human disorders.

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Abstract:

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Cysteine string protein (CSP) was discovered by use of a synapse-specific, monoclonal antibody to screen a cDNA expression library in Drosophila. A vertebrate CSP homolog was later identified and shown to co-purify with synaptic vesicles. CSP- is now recognized as a membrane constituent of many regulated secretory organelles. Knockout of the csp gene in Drosophila produced temperature-sensitive paralysis reflecting a loss of evoked (but not spontaneous) transmitter release. However, CSP’s role in regulated exocytosis remains ambiguous. Fruit flies lacking the csp gene also exhibited nerve terminal degeneration as did mice deficient in the csp- gene. This phenotype has been ascribed to the depletion of a functional pool of the t-SNARE, SNAP-25. However, recent studies showing that an endosomal pool of CSP- contributes to a novel, protein-export pathway argues that CSP’s role in neurodegeneration is more complex. Clients of this later pathway include tau and -synuclein, proteins linked to neurodegeneration. Additionally, mutations in the csp- gene cause an adultonset, neuronal ceroid lipofuscinosis and diminished CSP- expression is an early event in Alzheimer’s disease. Collectively, these findings indicate that much remains to be learned about the role of CSPs in cellular secretory pathways and human disease.

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Keywords: synaptic vesicles, J domain, neuromuscular transmission, protein palmitoylation, palmitoyltransferase, molecular chaperones, heat-shock proteins, exocytosis, SNARE proteins neurodegeneration

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Abbreviations:

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ANCL: adult onset, autosomal-dominant neuronal ceroid lipofuscinosis; CSP: cysteine string protein; CFTR: cystic fibrosis transmembrane conductance regulator; CHL1: Close Homolog Of L1 cell-adhesion molecule; eGFP: enhanced Green Fluorescent Protein; Hsp70: heat shock protein of 70 kDa; Hsc70: constitutively expressed Hsp70; KO: knockout; pcr: polymerase chain reaction; RT-pcr: reverse transcriptase-pcr; PKA: protein kinase A; SGT: small, glutamine-rich tetratricopeptide repeat protein; SNAP-25: synaptosome-associated protein of 25 kDa; SNARE: soluble, N-ethylmaleimide-sensitive attachment receptor; TDP-43: trans-activation response element DNA-binding protein; UPS: unconventional protein secretion; VAMP: vesicle-associated membrane protein; WT: wild-type

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1. Introduction Exocytosis is the process by which cells turn over constituents of their plasma membrane or release materials into the interstitial space. Eukaryotic cells maintain an elaborate repertoire of proteins that mediate and regulate exocytosis. This is particularly true for the subset of these events referred to as regulated exocytosis. In contrast to constitutive exocytotic pathways, regulated exocytosis occurs in response to a signal. Most commonly, this signal is a transient elevation of cytosolic ionized calcium. However, some forms of regulated exocytosis are initiated by other mechanisms, including protein phosphorylation. A major impetus for the study of regulated exocytosis was the recognition that most neurotransmitters, many hormones, and other substances are secreted from cells via this pathway. Over the last several decades, specific steps in the regulated secretory pathway were identified and functions assigned to specific proteins. Thus, it is now known that most neurotransmitters are released via the following steps: when an action potential propagates into a nerve terminal, it gates the opening of presynaptic, voltage-regulated Ca2+ channels; the attendant Ca2+ influx leads to a transient

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increase of cytosolic ionized calcium and the binding of Ca2+ to the C2 domains of the synaptic vesicle protein, synaptotagmin; the Ca2+-mediated electrostatic screening of synaptotagmin’s C2 domains leads to their insertion into the plasma (or vesicular) membrane; this Ca2+-triggered relocation of the C2 domains directly or indirectly contributes to the fusion of the synaptic vesicle membrane with the presynaptic plasma membrane, thereby enabling the exocytotic discharge of the contents of the synaptic vesicle. At least six other proteins are vital for this process. Foremost among these “other” proteins are the SNAREs (soluble, N-ethylmaleimide-sensitive attachment receptors). At nerve endings, SNAREs typically are represented by the synaptic vesicle protein, synaptobrevin-2, and the plasma membrane proteins, syntaxin-1a/b and SNAP25 (synaptosome-associated protein of 25 kDa). Importantly, SNAREs form a stable, helical complex that almost certainly is necessary to bring synaptic vesicles into close proximity or direct contact with the plasma membrane. In addition to Ca2+-dependent membrane binding, it is widely inferred that synaptotagmin interfaces with SNAREs to catalyze the fusion of the vesicular and plasma membranes. At the same time, a protein that interacts with the SNARE complex, complexin, appears to regulate critical, albeit incompletely defined aspects of this exocytotic sequence. Finally, two other SNARE-interacting proteins, unc/munc-13 and unc/munc-18 appear to be essential components of the molecular machinery that prepares (“primes”) synaptic vesicles for Ca2+-triggered exocytosis. Nevertheless, the exact function of the munc proteins also remains unclear, and some models envision these proteins contributing directly to the exocytotic cascade (for further discussion of the exocytotic proteins, see Rizo, 2018; Brunger et al., 2018). For the purposes of this review, the aforementioned proteins constitute the core molecular machinery for fast, synchronous exocytosis at nerve endings. Beyond this core are auxiliary proteins which make vital contributions to regulated exocytosis. Cysteine string proteins (CSPs) are prominent members of this “auxiliary” category. CSPs are tethered to the surface of synaptic vesicles and other regulated secretory organelles via their fatty acylated cysteine string. Mutagenesis studies in fruit flies indicate that CSPs are important, but not indispensable for fast, synchronous exocytosis at nerve terminals. A major goal of this review is to highlight the disparate observations regarding the molecular role of CSPs in regulated exocytosis at nerve terminals and elsewhere.

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Unexpectedly, deletion of the csp gene in fruit flies and knockout of the csp- gene in mice uncovered a neurodegenerative pathway that has attracted considerable attention. Although it was proposed that neurodegeneration was due to the depletion of a functional pool of the tSNARE, SNAP-25, at nerve endings, recent observations argue that this picture is incomplete. Specifically, in 2016, two groups suggested that an endosomal pool of CSP- was an essential component of an unconventional pathway for exporting misfolded proteins from cells. Clients for this pathway included several proteins (tau, -synuclein and TDP-43) implicated in neurodegenerative diseases. Thus, an additional goal of this review is to integrate biochemical, cell biological, genetic and physiological investigations of CSPs with a goal of identifying areas where targeted investigations could advance the understanding of CSP structure and function in relation to disease etiology. Although an effort has been made to be comprehensive, the reader is referred to earlier reviews of the CSP literature: Buchner and Gundersen, 1997; Chamberlain and Burgoyne, 2000; Zinsmaier and Bronk, 2001; Evans et al., 2003. An update (Zinsmaier, 2010) was followed by commentaries focusing on the CSP link to neurodegeneration (Donnelier and Braun, 2014; Burgoyne & Morgan, 2015; Gorenberg & Sharma, 2017). 2. Discovery and initial characterization of cysteine string proteins (CSPs). 2.1 Drosophila CSP

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The cDNAs for Drosophila CSPs were originally selected by interrogating an expression library with a monoclonal antibody that bound prominently to synaptic boutons in skeletal muscle and neuropil regions in the adult nervous system (Zinsmaier et al., 1990). A pair of alternatively spliced CSP cDNAs emerged from this screen, and the immunohistochemical data suggested that the encoded proteins might contribute to synaptic function. However, although the deduced amino acid sequences offered little insight into the function of these proteins, the fact that both sequences specified a string of 11 consecutive cysteine residues flanked on either side by another pair of cysteines led to the designation of these proteins as CSPs (Fig.1A and Zinsmaier et al., 1990). 2.2. Torpedo CSP

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A vertebrate homolog of fruit fly CSPs emerged during an effort to identify subunits of presynaptic Ca2+ channels (Gundersen and Umbach, 1992). The Torpedo CSP cDNA clone was selected, because its antisense RNA inhibited the expression of N-type Ca channels in frog oocytes injected with mRNA from Torpedo electric lobe (Gundersen and Umbach, 1992). The fish protein was ~70 % identical to the fly protein. However, ongoing investigations quickly dispelled the idea that CSPs might be intrinsic subunits of presynaptic Ca channels: briefly, CSP was found to co-purify with synaptic vesicles from Torpedo electric organ (Mastrogiacomo et al., 1994a) and rat brain (Mastrogiacomo et al., 1995; Braun and Scheller, 1995). In flies, CSP coimmunoprecipitated with synaptotagmin (Zinsmaier et al., 1994) and migrated with synaptic vesicles on glycerol gradients (van de Goor & Kelly, 1996) supporting a vesicular localization. Additionally, there was no evidence for CSP affiliation with the presynaptic plasma membrane in unstimulated electric organ (Mastrogiacomo et al., 1994a). Consequently, it was proposed that CSP might play a role in regulating the function of presynaptic Ca2+ channels once a synaptic vesicle docked at the plasma membrane (Mastrogiacomo et al., 1994a; Umbach et al., 1995). The evolution of this hypothesis is addressed in section 4. 2.3. Genomic insights into CSPs: J domains and cysteine strings

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A widely conserved protein motif, the J domain, was described roughly concurrently with the publication of the Torpedo CSP sequence (Caplan et al., 1993; Cyr et al., 1994; Fig.1B). J domains comprise a stretch of ~70 amino acid residues at the amino (N) terminus of both Drosophila and Torpedo CSPs. This observation turned into a mixed blessing. On one hand, it allowed CSPs to be lumped with a diverse group of other eukaryotic J domain proteins. The distinguishing feature of J domains is that they bind to the nucleotide-binding region and serve as “co-chaperones” for members of the 70 kDa heat shock protein (Hsp70) family (Cyr et al., 1994; Craig & Marszalek; 2017; constitutively expressed versions of Hsp70 are referred to as Hsc70). On the other hand, Hsc70 proteins interface with such a diverse range of targets that it makes it very challenging to illuminate the beneficiary (-ies) of the CSP-Hsp70 chaperone activity. Progress in this area is addressed in sections 3.1 and 4.1. An important upshot of the observation linking cysteine strings with J domains is that the pairing of these motifs could be used to identify CSPs in other species. Early on, it became apparent that CSPs were present in organisms with a nervous system (including, fruit flies and nematode worms), but they were not found in unicellular eukaryotes, like yeast (Buchner and Gundersen, 1997). As more genomes have been sequenced, this distinction appears to persist. For instance, using the original Drosophila J domain or cysteine string sequence, Blast searches of predicted proteins in such organisms as Paramecium, Dictystelium and Arabidopis failed to identify any proteins that contain both of these motifs (CG, unpublished observations; note that proteins such as Ddj1 in Dictystelium have a J domain, but this is not paired with a cysteine string). Hence, while it could be argued that CSPs evolved to play a role (or, roles) in regulated

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secretory events, particularly in animals with a nervous system, the observations linking CSP- to exosomes (Deng et al., 2017) and an unconventional protein secretion pathway (Fontaine et al., 2016; Lee et al., 2016) imply a more complicated evolutionary history for these proteins. Early investigations also identified cDNAs encoding carboxyl (C)-terminally truncated CSP- (Coppola and Gundersen, 1996; Chamberlain and Burgoyne, 1996). However, there is meager evidence that this form of CSP is normally expressed in cells. Thus, this CSP isoform will not be considered further. In addition, as noted (in 2.1), alternative splicing has been observed for the csp gene in Drosophila and other organisms. However, the functional relevance of this splicing has not been clarified.

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2.4. Cellular and subcellular localization of CSP-

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Genome sequencing also led to the recognition that vertebrate genomes harbor three csp genes (csps seeFig.2Awhile invertebrates, like Drosophila (fruit fly) and Caenorhabditis elegans (nematode worm) have one csp gene (but, bear in mind that the Drosophila gene is alternatively spliced; Zinsmaier et al., 1990). A CLUSTAL alignment of csps () from several species is in Fig. 2B. Unexpectedly, an early study suggested that CSP- and CSP- (which are encoded by autosomal genes) were expressed only in testes of mice (Fernandez-Chacon et al., 2004). This conclusion is no longer tenable, and the evidence that CSPs-and -are expressed in other tissues is presented in section 4.

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The original paper (Zinsmaier et al., 1990) documented the robust and apparently ubiquitous expression of CSP immunoreactivity in the synaptic neuropil and motor nerve terminals of adult fruit flies. Concurrently, in situ hybridization revealed the widespread distribution of CSP mRNA in the retina and in neuronal perikarya. These results suggested that CSP was expressed in most, and possibly all fly neurons. A systematic investigation several years later confirmed and extended these conclusions (Eberle et al., 1998). Briefly, by using csp null mutants as negative controls, these investigators showed that CSP was present at a low level in virtually all cells of the fruit fly and that nerve terminals, tall cells of the cardia, ovarian follicular cells and parts of the male reproductive tract had appreciably higher levels of CSP expression. Although csp- knockout (KO) mice are available for use as controls, a comparable study of the tissue distribution of mouse CSP- has yet to be performed. At the same time, although control, KO tissues are not available, data from the Human Protein Atlas (www.proteinatlas.org) indicate a widespread distribution of CSP- immunoreactivity in human tissues which extends the evidence from northern and western data in Coppola and Gundersen (1996).

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By using affinity purified antibody targeting the conserved, C-terminal peptide of Torpedo CSP, Kohan and colleagues (1995) assessed the distribution of CSP- immunoreactivity in rat brain (these antibodies do not bind to CSP- or CSP-). Although CSP- immunoreactivity was prominent in many areas of the synaptic neuropil and in loci with well-characterized axosomatic synapses, there were some unexpected observations. For instance, although strong CSP- immunostaining was detected for mossy fiber terminals in the CA2 and CA3 regions of hippocampus, appreciably weaker staining was seen in the neuropil of the CA1 region. Although several factors could contribute to this difference in staining intensity, an explanation that was not addressed at that time was the possibility of other csp genes being expressed in brain. To date, systematic information concerning the distribution of CSP-or - in vertebrate brain is lacking, and such data could help to explain the relatively lower level of CSP- immunostaining in some regions of the nervous system. Nevertheless, there is clear evidence for the expression of CSP isoforms in certain types of sensory cells. Thus, CSP- is strongly expressed in the inner and outer plexiform layers of the rat retina (Kohan et al., 1995) and in Drosophila

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photoreceptor terminals (Hamanaka and Meinertzhagen, 2010). CSP- is also robustly expressed in inner ear hair cells of rats and guinea pigs, but was absent from outer hair cells (Eybalin et al., 2002). However, a later study reported that while CSP- mRNA was detected only in a subset of cochlear inner hair cells, CSP- mRNA appeared to be expressed more uniformly in these cells (Schmitz et al., 2006). Thus, additional work will be needed to determine whether these two CSPs are expressed together in inner hair cells or at other anatomical loci. Further discussion of the expression of CSP- is in section 4.

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At about the same time that it was reported that CSP- was associated with synaptic vesicles (Mastrogiacomo et al., 1994a; Zinsmaier et al., 1994; van de Goor & Kelly, 1996), numerous groups found that CSP- was also affiliated with other types of regulated secretory organelles. These organelles included pancreatic zymogen granules (Braun and Scheller, 1995; Weng et al., 2009), secretory granules of the anterior pituitary (Jacobsson and Meister, 1996; Pupier et al., 1997), adrenal chromaffin granules (Kohan et al., 1995; Chamberlain et al., 1996), insulincontaining granules of pancreatic  cells (Brown et al., 1998; Zhang et al., 1998), frog oocyte cortical granules (Gundersen et al., 2001; Smith et al., 2005) and mucin granules of bronchial epithelial cells (Park et al., 2009; Raiford et al., 2011). Additionally, Eybalin and co-workers (2002) reported that CSP- was affiliated with synaptic vesicles of ribbon synapses of cochlear inner hair cells. Collectively, these results led several of these groups to predict that CSPs were involved in regulated exocytosis in the parent cells, and evidence for such a role will be discussed in sections 4.2 and 4.3. A prominent exception to the trend of CSP associating with regulated secretory organelles was adipocytes, where CSP- was principally a plasma membrane protein that tended to be excluded from lipid rafts (Chamberlain et al., 2001). CSP- also was detected in a proteomic analysis of lysosomal membranes from the placenta (Schroder et al., 2007; see their Table 1), but it remains to be clarified whether this localization reflects a role for CSP- in lysosomal function, or whether it is testimony to a step in CSP turnover. Ongoing investigations tend to favor lysosomes as the primary locus for degradation of CSP, but this does not exclude a role in lysosomal exocytosis (Chapel, et al., 2013; Sambri et al., 2017; Tharkeshwar et al., 2017; Benitez and Sands, 2017). CSP- also is associated with small vesicles in a mammary epithelial cell line(Gleave et al., 2001), but its function has not been assessed.

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Other cellular loci also harbor pools of CSP-. Work on the trafficking of CFTR, the cystic fibrosis transmembrane conductance regulator, demonstrated a function for CSP- in the endoplasmic reticulum (see section 4.3). Csp is also expressed in enterochromaffin-like cells in the rat stomach (Zhao et al., 1997) and pinealocytes of the gerbil pineal (Redecker et al., 1998).Djengel and colleagues (2012) found CSP- associated with autophagosomes. And, as discussed further in section 5.3, endosomal and exosomal pools of CSP- contribute to protein export from cells. 3. Biochemical investigations of CSP- 3.1. The J domain and its interaction with Hsp/Hsc 70 chaperones The concept that cells employ specific proteins for “chaperone” functions (Ellis et al., 1989; Ellis and Hemmingsen, 1989;) evolved from studies of organism’s responses to thermal stress. The classical observation was that well-defined regions of Drosophila chromosomes became transcriptionally active (showed a new “puffing” pattern) after exposure to elevated temperature (Ritossa, 1962). A series of experiments during the 1970s revealed that the mRNAs transcribed at these “puffs” encoded specific “heat shock” proteins (Tissieres et al., 1974; McKenzie et al., 1975; Spradling et al., 1975; McKenzie and Meselson, 1977; Spradling et al., 1977; Ashburner

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and Bonner, 1979). Typically, the most robustly up-regulated of these proteins was a 70 kDa species designated Hsp70 for heat shock protein of 70 kDa. It was subsequently recognized that E. coli have a single Hsp70 gene (Bardwell and Craig, 1984), while yeast, fruit flies and vertebrates have several genes encoding closely related Hsp70s (Kelley and Schlesinger,1982; Ingolia et al., 1982; Craig et al., 1983). Importantly, a subset of these Hsp70s was found to be expressed in normal (unstressed) cells which suggested that they played some role in ongoing cellular function. These “uninduced” Hsp70s are referred to as cognate or constitutively expressed Hsp70s (ie., Hsc70s). An influential hypothesis (Pelham, 1986) helped to explain why cells would express Hsc70s (and Hsp70s): namely, that these proteins have a general affinity for unfolded, abnormal or denatured proteins and contribute to protein re-folding, disaggregation and/or assembly of protein complexes. Direct support for this idea came from the finding that cells injected with denatured proteins up-regulated the expression of Hsp70 and other heat shock proteins (Ananthan et al., 1986). Hsc70s have also been shown to aid in protein folding during ribosomal translation (Hartl, 1996), and several other lines of work have since supported Pelham’s proposal (reviewed in: Georgopoulos and Welch, 1993; Hartl, 1996). Here, it is important to keep in mind that Hsc70 is a very abundant protein in brain. It was estimated to be ~3% of total spinal cord protein (Aquino et al., 1993). In another study (Wilhelm et al., 2014), individual rat synaptic boutons were reported to contain ~8,000 copies of Hsc70 (~1% of total protein) which is similar to the tubulin content and about one third the level of actin. Because of this abundance and its presence in specific and non-specific binding conditions, the observation that Hsc70 co-purified with SNARE proteins was originally dismissed (Sollner et al., 1993). However, as ensuing discussions will highlight, there are numerous examples of in vitro interactions involving CSPs, SNAREs and Hsc70. Although the purpose of these interactions remains a topic of debate as does the broader question of how, molecularly, Hsp/Hsc70s accomplish their disparate tasks in the cell (a discussion of this later issue is in: Saibil, 2013), the remainder of this section will address results germane to the chaperone activity of CSP-.

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Although mechanistic details of Hsp/Hsc70 action are lacking, numerous functional insights have emerged. The first clues came from biochemical investigations which revealed that E. coli Hsp70 (DnaK) had weak ATPase activity (Zylicz et al., 1983); that ATP hydrolysis was needed for release of substrates from Hsp/Hsc70 (Lewis and Pelham, 1985; Flynn et al., 1989) and that denatured RNA polymerase could be renatured in an ATP-dependent fashion by DnaK (Skowyra et al., 1990). Molecularly, the crystal structure of the ATPase domain of bovine Hsc70 was solved (Flaherty et al., 1990). Shortly thereafter, two other bacterial proteins, the nucleotide exchange factor, GrpE and the DnaJ protein (itself a heat shock protein, Hsp40; Ohki et al., 1986) were found individually to elevate DnaK’s ATPase activity by twofold, but together, ATPase activity went up by as much as 50-fold (Liberek et al., 1991; Jordan and McMacken, 1995; Minami et al., 1996). By monitoring the renaturation of firefly luciferase in vitro (Schroder et al, 1993; Szabo, et al., 1994), it was reported that DnaJ initially bound the substrate and presented it to DnaK. DnaK then hydrolyzed bound ATP while GrpE facilitated the dissociation of ADP from DnaK (reviewed in Bukau and Horwich, 1998; Mayer, 2010). Interestingly, bacterial DnaJ, by itself, binds to denatured proteins and protects them from aggregation (Langer et al., 1992; Hoffmann et al., 1992; Schroder et al., 1993). Concurrently, it was recognized that relatives of bacterial DnaJ were expressed in other species, where the defining feature was a conserved stretch of ~70 amino acid residues at the N terminus of the protein, the J domain (Sadler et al., 1989; Blumberg and Silver, 1991; Caplan and Douglas, 1991, Cheetham et al., 1992; Cyr et al., 1992). J domain proteins in other species also increase the Vmax of the ATPase activity of Hsp70. For instance, the yeast DnaJ protein Ydj1 induces a tenfold increase in the ATPase activity of the yeast Hsc70, Ssa1 (Cyr et al., 1992), while human Hsp40 causes about a 7-fold increase in the ATPase activity of bovine brain Hsc70 (Minami et al., 1996).

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The concept that Hsp/Hsc70 proteins interface with J domain co-chaperones to shield hydrophobic regions of clients and also facilitate protein folding was expanded by observations that these proteins contribute to other events involving the reorganization of macromolecular complexes. For instance, the bacterial DnaK (Hsp70) and DnaJ (Hsp40) proteins were identified because they drive the dissociation of a protein complex at the origin of replication of phage  DNA (reviewed in Georgopoulos and Welch, 1993). Similarly, disassembly of the coat of clathrin-coated vesicles requires the joint action of Hsc70 and the J domain protein, auxilin, plus ATP (Ungewickell et al., 1995). These actions of Hsc70 and J domain proteins have been likened to a molecular “crowbar” (Georgopoulos and Welch, 1993). However, the range of activities of Hsp70s and J domain proteins extends into several other areas as reviewed by Qiu et al. (2006); Kampinga & Craig (2010) and Craig & Marszalek (2017). Collectively, these precedents set the stage for characterizing the J domain of CSP-.

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The first evidence that the J domain of CSP- functioned like other J domain proteins was that recombinant CSP- enhanced the ATPase activity of Hsc70 and Hsp70 by approximately an order of magnitude, in vitro (Braun et al., 1996; Chamberlain and Burgoyne, 1997). While the J domain fragment of CSP- also enhanced Hsc70 ATPase activity, it did not work as well as the full-length protein (Braun et al., 1996). By itself, the ATPase domain of Hsc70 (residues 1-386) was not activated by CSP suggesting that CSP binding to Hsc70 involved sequences in the carboxyl terminus (Braun et al., 1996). These observations were followed by experiments showing that mutation of the highly conserved HPD tri-peptide in the J domain of CSP- abrogated its ability to bind to and stimulate Hsc70 ATPase (Chamberlain and Burgoyne, 1997; Zhang et al., 1999a). Concomitantly, CSP-, by itself, was shown to diminish the aggregation of firefly luciferase (Chamberlain and Burgoyne, 1997). Taken together, these data confirmed that CSP- functioned like a typical J domain protein, in vitro.

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A related study revealed that in certain circumstances, glutamate decarboxylase (GAD), the enzyme responsible for the production of the neurotransmitter, -amino-butyric acid (GABA), copurified with Hsc70 and CSP- (Hsu et al., 2000). These results were interpreted as indicating that CSP- and Hsc70 might play a role in targeting GAD to synaptic vesicles. However, these observations have not been extended, so their functional relevance remains unclear.

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An extension of the idea that CSP subserves a chaperone function came with the finding that CSP- interacted with a pair of Hsc70-interacting proteins, SGT  and  (for small, glutaminerich tetratricopeptide repeat) (Tobaben et al., 2001, 2003). While CSP- binds to Hsc70 via its J domain, both CSP- and Hsc70 apparently interact with SGT via their carboxyl termini and together produce a strong (~20-fold) enhancement of Hsc70 ATPase activity (Tobaben et al., 2001). Later work also implicated the cysteine string region in binding of SGT to CSP (Tobaben et al., 2003). These authors further reported that the combination of CSP-, Hsc70, SGT and ATP produced better re-folding of luciferase than when SGT was omitted. Moreover, although SGT associated with a rat brain synaptic vesicle fraction (here, it is important to note that the labeling of SGT and Hsc70 was reversed in the immunoblot in Fig.4 of Tobaben et al., 2001), it was not detected in several subsequent proteomic inventories of synaptic vesicles (Coughenour et al., 2004; Morciano et al., 2005; Takamori et al., 2006). Nevertheless, a more-recent study found SGTs affiliated with mouse brain synaptic vesicles (Andreyeva et al., 2010). Thus, there appears to be variability in the extent to which the cytosolic, SGT proteins bind to synaptic vesicles. At the same time, overexpression of SGT in cultured neurons blunted transmitter release which was interpreted as suggesting that SGT may cooperate with CSP- and Hsc70 to maintain normal secretory function (Tobaben et al., 2001). However, the molecular mechanism of this result remains obscure.

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A novel addition to efforts to clarify SGT function was work of Andreyeva and colleagues (2010) who made the unexpected observation that the intracellular domain of a cell surface adhesion molecule (CHL1) modulates the function of several presynaptic chaperones. Briefly, they reported that CHL1 (whose intracellular domain has the highly conserved HPD tripeptide of J domains and binds Hsc70) competes with CSP- for binding to Hsc70. This competition was proposed to create two separate chaperone complexes: one composed of CSP and CHL1 and the other composed of CHL1, Hsc70 and SGT. Moreover, CHL1, Hsc70 and SGT showed preference for chaperoning SNAP-25 while CSP-CHL1 favored synaptobrevin-2. Because CHL1 KO mice exhibited reduced presynaptic chaperone activity and SNAREs were found to aggregate abnormally and accumulate in lysosomes of the KO mice, these authors postulated that CHL1 was an important regulator of chaperone function at nerve terminals. The iconoclastic feature of these results is that both CHL1 and CSP- are J domain proteins, so it remains to be determined how Hsc70 uses these two co-chaperones to select its clients.

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An interesting survey of membrane-deforming actions of fruit fly proteins (Uytterhoeven et al., 2015) found that Hsc70-4 could shift its function based on access to CSP, SGT and ATP. Oligomerization and concurrent membrane-deformation by Hsc70-4 were hindered in the presence of ATP or SGT. In contrast, luciferase re-folding by Hsc70-4 was promoted by ATP and further augmented by SGT. However, in contrast to Tobaben et al., (2001), no further enhancement of re-folding was seen when CSP- was added to the mix of Hsc70-4 and SGT. These authors concluded that the membrane-deformation was associated with a role for Hsc704 in microautophagy, while the re-folding function reflects Hsc-70’s conventional role in protein quality control and chaperone activity.

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Independently, Sakisaka and co-workers (2002) presented evidence that CSP- was part of a tripartite chaperone complex (along with Hsc70 and Hsc90) on synaptic membranes that regulated GDP dissociation from rab proteins. Because rab proteins are important in membrane-trafficking events, this result provided an alternative pathway by which CSP might influence secretory events. However, a functional correlate of this interaction is still lacking, and additional studies will be needed to clarify the diverse chaperone pathways at nerve terminals.

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A comprehensive survey (Wilhelm et al., 2014) quantified the copy number per rat brain nerve terminal (synaptosome) of a large number of proteins that are important for exocytosis and endocytosis. Individual rat nerve endings contain roughly 8,000 copies of Hsc70 and about 1,000 copies of CSP-. These numbers dwarf the ~98 copies per synaptosome of SGT(see line 62 of their supplemental Table 2). Thus, additional work will be needed to determine how and when this relatively small number of SGTs functions biologically. Moreover, although the empirical data were not published, it was noted that deletion of the gene encoding SGT in Drosophila did not affect synaptic function, nor did it lead to neurodegeneration (Zinsmaier, 2010). However, a later study (Uytterhoeven et al., 2015) reported that synaptic transmission was enhanced in Drosophila sgt mutants, implying a complex hierarchy of chaperone functions at nerve endings. Clearly, appreciably more work needs to be done to understand the rules governing the myriad roles of CSP, SGT and Hsc70 at nerve terminals. The preceding discussion is the proverbial tip of the iceberg as far as the chaperone role of CSP- is concerned. Considerably more work has been done to assess functional consequences of manipulations involving CSP- and its J domain. These investigations will be considered in section 4. At the same time, it is important to note that little attention has been paid to the J domains of CSPs- and -, because of their presumed testis-specific expression (Fernandez-Chacon et al., 2004; Schmitz and Fernandez-Chacon, 2009). While section 4 considers the evidence that these CSPs are more-widely expressed, it is also noteworthy that

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the J domain of mouse CSP- is truncated, and this led Zinsmaier (2010) to question whether it is functional, in vivo. Although data addressing this point have not appeared, the canonical HPD sequence that is important for Hsc70 binding is retained in mouse CSP-, so it will be interesting to test whether co-chaperone function persists in vitro and in vivo.

3.2. Fatty acylation of the cysteine residues of the cysteine string

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A systematic investigation of brain protein turnover (Fornasiero et al., 2018) included evidence that the half-life of CSP- is ~20 days. Interestingly, this half-life is roughly twice that observed for other key synaptic vesicle proteins, like synaptotagmin-1 and synaptobrevin-2, but appreciably slower than synaptophysin (24-34 d). These data strongly suggest that CSP- turnover involves mechanisms that are different from those targeting its vesicular partners. At the same time, SGT and Hsc70 show turnover rates of 4-5 d and 6-7 d respectively, which indicates that their turnover is also unlikely to be coupled to that of CSP-.

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The 11 consecutive cysteine residues in the deduced amino acid sequence of Drosophila CSPs inspired the naming of these proteins (Zinsmaier et al., 1990). Remarkably, in the ensuing decades, outside of a role in membrane targeting, very little has been learned about the specific biological function of this unusual sequence motif. Nevertheless, crucial biochemical and molecular insights have been gleaned, and a recent proposal concerning the membrane association of this region has led to preliminary results that are addressed: 3.2.1. Biochemical and molecular studies of the “string” region of CSP-

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When Torpedo CSP was synthesized by in vitro translation, the protein product had a mass of ~27 kDa on SDS-polyacrylamide gels (Gundersen et al., 1994). However, CSP-immunoreactive protein in Torpedo electric organ ran more slowly on gels (~34 kDa). This higher mass form of CSP could be reverted to the basal, ~27 kDa mass by treatment with agents that remove fatty acyl moieties from protein thiols. In fact, a stepladder of CSP-immunoreactive bands between 27 and 34 kDa could be observed by interrupting the deacylation reaction before it reached completion. The number of immunoreactive bands gave a provisional estimate that at least 12 of the 13 cysteine residues of Torpedo CSP were fatty acylated (and these results were independently supported by quantification of the incorporation of radioactive iodoacetamide into native versus deacylated CSP). However, when in vitro translated CSP was injected into frog oocytes, only the 27 and 34 kDa species were observed which implied that once the posttranslational modification of CSP started, it rapidly proceeded to completion. Further, it was shown that when oocytes were incubated with 3H-palmitic acid, radioactivity was incorporated into CSP and this radioactivity could be displaced by deacylating reagent. Also, when oocytes containing the 27 kDa and 34 kDa CSP species were fractionated, the 34 kDa form associated with membranes, whereas the un-palmitoylated 27 kDa species remained soluble. This result argued that fatty acylation was necessary for membrane affiliation. In related work (Coppola and Gundersen, 1996; Mastrogiacomo et al., 1994b; 1998a), the 34 kDa form of CSP partitioned into the detergent phase in TritonX-114 partitioning experiments, indicating that it had significant hydrophobic character in contrast to the 27 kDa form, which as noted above, behaved as a soluble protein. Finally, analysis of the fatty acyl moieties displaced from Torpedo CSP indicated that palmitate was the major species, but stearate was also detected. This remains the only analysis of the fatty acids bound to CSPs. Collectively, these data were interpreted as indicating that CSP- was post-translationally modified by the fatty acylation of a large majority of the cysteine residues of the cysteine string. However, to date, it still has not been established whether all of the cysteine residues of the cysteine string of CSP- are fatty acylated. For reasons addressed later, it will be important to clarify the extent to which CSPs are fatty

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acylated. At the same time, two groups presented evidence that the fatty acyl moieties of CSP do not turn over during secretory activity (van de Goor and Kelly, 1996; Gundersen et al., 1996). Thus, from the available data, it appears likely that CSP- is efficiently fatty acylated during or shortly after translation on ribosomes, and this post-translational modification is relatively stable.

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The fact that an electrophoretic mass shift of ~7 kDa occurs upon acylation/deacylation of CSP (Gundersen et al., 1994) has provided a relatively simple means of determining whether CSPs in other species are fatty acylated. By this criterion, CSP- in humans, rats, frogs and fruit flies are fatty acylated (Mastrogiacomo and Gundersen, 1995; van de Goor and Kelly, 1996; Coppola and Gundersen, 1996; Mastrogiacomo et al., 1998a; Chamberlain and Burgoyne, 1998; Gundersen et al., 2001; Chamberlain et al., 2001; Greaves and Chamberlain, 2006; Edmonds et al., 2017). This modification also appears to occur for CSP- (Gorleku and Chamberlain, 2010). Interestingly, this mass-shift strategy was used to identify enzymes that fatty acylated CSP- (Greaves, et al., 2008). Thus, until evidence emerges to the contrary, it appears reasonable to conclude that the cysteine residues of CSP- are fatty acylated in most organisms. Further work will be needed to clarify whether CSPs- and - are fatty acylated, in vivo.

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A minor contretemps emerged when it was reported that deacylation of CSP in Drosophila membranes failed to displace CSP from the membrane environment. This led to the suggestion that some other motif of CSP, besides its fatty acylated cysteine string, was responsible for its affiliation with membranes (van de Goor and Kelly, 1996). Further experiments revealed that the persistent membrane association of deacylated CSP owed to its cysteine string (Mastrogiacomo et al., 1998a). The first result supporting this conclusion is that native or de-acylated CSP could only be displaced from membranes by detergent solubilization. Moreover, the cysteine string was inaccessible to membrane-impermeant alkylating agent after hydroxylamine-mediated deacylation. From these results and prior work (Gundersen et al., 1994), it was clear that CSP- can be produced as a soluble protein that becomes stably membrane associated as a consequence of fatty acylation. However, if the fatty acyl moieties are removed, CSP remains membrane associated via its cysteine string. This interpretation was confirmed and extended by mutagenesis studies (Chamberlain and Burgoyne, 1998), and will be considered further in sections 3.2.2 and 3.2.3. Finally, it is noteworthy that Chamberlain and colleagues (2001) also observed that vertebrate CSP- remained membrane-associated after deacylation.

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The preceding observations raise the interesting question of how a nominally soluble protein, like CSP- (and, specifically, the in vitro translated CSP- that was injected into oocytes in the work of Gundersen et al., 1994) reaches the site where fatty acylation and membrane integration occur. Again, mutagenesis experiments provided important insights into this issue (Greaves and Chamberlain, 2006). Briefly, these investigators observed that several, predominantly hydrophobic residues upstream of the cysteine string were needed for the initial membrane attachment of CSP- constructs that had been fused to eGFP (enhanced Green Fluorescent Protein). Additionally, residues downstream of the cysteine string were required for efficient palmitoylation and correct membrane sorting of the CSP constructs. There are two caveats for interpreting these results. The first is that full-length Torpedo CSP injected into frog oocytes obviously had intact residues upstream of the cysteine string, yet failed detectably to associate with oocyte membranes (Gundersen et al., 1994).Secondly, van de Goor and Kelly (1996) observed that when Drosophila csp was expressed in PC12 cells, it was not fatty acylated and remained exclusively associated with soluble fractions of glycerol gradients. Similar results were obtained (Chamberlain and Burgoyne, 1998) when they expressed myctagged csp in HeLa cells; namely, that un-palmitoylated csp remained in soluble fractions, while palmitoylated csp associated with membranes. Four explanations may contribute to this apparent discrepancy. First, native or myc-tagged CSP may behave differently than the eGFP

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fusion proteins used in the Greaves and Chamberlain (2006) study. Second, processing of mammalian CSPs may vary according to the cell type. Third, the expression level of csp constructs in the Greaves and Chamberlain (2006) study may have produced a pool of unpalmitoylated, membrane-affiliated csp that does not typically accumulate in vivo. Fourth, the membrane affiliation of CSP- seen by Greaves and Chamberlain (2006) may occur cotranslationally which obviously would be an effective, but not an essential mechanism for the cell to adopt (note that this argument does not explain the van de Goor and Kelly (1996) results mentioned earlier). Regardless, the extensive fatty acylation of CSP- presents interesting topological demands that will be addressed further in section 3.2.3.

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A major advance in understanding CSP fatty acylation was the identification of a family of enzymes that transfer fatty acyl groups to cysteine residues of proteins (Lobo et al., 2002; Roth et al., 2002; 2006). Mammalian genomes harbor 23 genes encoding these palmitoyltransferases (Fukata et al., 2004; Huang et al., 2004; Keller et al., 2004; Swarthout et al., 2005; for review see Planey and Zacharias, 2009). A systematic analysis indicated that four of these enzymes effectively palmitoylated CSP- when it was co-expressed in HEK 293 cells, in vitro (Greaves et al., 2008). The HEK 293 cells were chosen as hosts, because the basal palmitoylation of CSP in these cells was relatively low. Although it has not been established whether the four identified palmitoyltransferases (3,7,15 and 17) also mediate CSP- acylation in the brain and other sites where CSP- is expressed, this study did identify three residues (KPK) immediately downstream of the cysteine string as being important for the fatty acylation of CSP-. More recently, Lemonidis and colleagues (2015) identified a sequence motif that is recognized by a sub-set of ankyrin-domain-containing, protein palmitoyltransferases that are abundantly expressed in brain, and this motif is present in several palmitoylated proteins at nerve terminals, including CSP-. Independently, it was shown that CSP palmitoylation and trafficking were impaired in Drosophila deficient in a gene, hip14, which encodes a palmitoyltransferase, suggesting that hip14 is normally involved in the fatty acylation of CSP in Drosophila (Stowers and Isacoff, 2007; Ohyama et al., 2007). 3.2.2. Structural implications of the fatty acylation of CSP-

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The original analysis of the deduced CSP sequences of Drosophila noted the presence of a stretch of 22 residues (106-127 which included much of the cysteine string) that were sufficiently hydrophobic to constitute a candidate membrane-spanning segment (Zinsmaier et al., 1990). However, three observations contributed to dispelling the possibility that this region of CSP formed a typical, membrane-spanning -helix. First, the synaptic vesicle localization of CSP and the presence of a J domain at its amino terminus rendered it very likely that the amino-terminal region of CSP that precedes the cysteine string was oriented toward the cytoplasm (rather than the lumen of the vesicle) to enable CSP’s J domain to interact with cytosolic Hsc70. Second, synaptic vesicles were immunoprecipitated by antibody targeting the C-terminus of CSP- (Mastrogiacomo et al., 1994a). This result strongly implied that the C-end of CSP was also exposed on the vesicle surface. If the region that includes the cysteine string were forming a membrane-spanning domain, either the N- or C-end of CSP would have to extend into the lumen of synaptic vesicles. However, the preceding results argued against this topology. Finally, the recognition that most of the cysteine residues of the cysteine string were modified by fatty acylation (Gundersen et al., 1994; van de Goor and Kelly, 1996) rendered it very unlikely that this motif formed a typical, membrane-spanning -helix. Instead, most models have depicted CSP- tethered to the external surface of secretory vesicle membranes via the fatty acyl moieties of the cysteine string (Gundersen et al., 1995; Umbach et al., 1995; Evans et al., 2003; Takamori et al., 2006; Greaves et al., 2008; Burgoyne and Morgan, 2015). The definitive representation of this mode of membrane association of CSP is in the paper of Burgoyne and

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Morgan (2015). They depicted a synaptic vesicle with 3 copies of CSP attached to the cytosolic surface of the vesicle via multiple (5 or 12) fatty acylated cysteine residues. Concurrently, the Nand C-terminal domains of CSP faced the cytosol. This rendering represents the “traditional” model for the membrane affiliation of CSP. However, as discussed next, we proposed a subtle, but important revision of this model of the membrane association of CSP-.

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The revised model (Fig. 3) of how CSP- affiliates with membranes grew out of four considerations. First, cysteine residues of soluble peptides and proteins prefer to form structure, rather than -helix (a tendency originally quantified by Chou and Fasman, 1974). Second, since the available data indicate that a majority (or, all) of the cysteine residues of the cysteine string are fatty acylated (Gundersen et al., 1994; van de Goor and Kelly, 1996), the mode of membrane association of CSP has to be compatible with shielding of these hydrophobic entities from the cytosolic (aqueous) milieu. Clearly, the notion that CSP- is tethered to a membrane surface via its cysteine string is incompatible with -structure, because this secondary structure would lead to acyl chains projecting into the cytosol. The third consideration is based on the observations that CSPs were not displaced from membranes after de-acylation, and the exposed thiol groups were resistant to alkylation (discussed in 3.2.1). These data also implied that CSPs were not tethered via acyl moieties to the surface of membranes (as is SNAP-25); instead, these findings implied that the cysteine string is “buried” within the membrane. Finally, if one computes the Ramachandran (angles needed to project the R groups of consecutive cysteine residues in the same spatial orientation (which would be necessary to “bury” the affiliated acyl chains in the membrane), at least one of these angles falls in a forbidden zone owing to steric interference involving the planar peptide bonds. Consequently, a model was developed (Gundersen and Umbach, 2013) for the membrane attachment of CSP- that takes into account these considerations. It was proposed that the polypeptide backbone of CSP- penetrates the membrane upstream of the cysteine string and is situated at the interface of the opposed membrane hemi-bilayers as illustrated in Fig.3. The region that is embedded within the membrane adopts structure. This arrangement is propitious, because it ensures that the acyl groups linked to the cysteine residues remain confined within the hydrophobic interior of the membrane (Fig.3). In contrast, if the polypeptide backbone of CSP- were to attach solely to the outer hemi-bilayer of the target membrane, the cysteine string region could NOT adopt  structure, because this would force the fatty acyl moieties of alternating cysteine residues to project into the cytosol. (The reason for this is that the R groups of successive amino acid residues in  conformation project at ~180o relative to one another). Thus, the membrane association proposed in Fig.3 retains the preferred secondary structure of this cysteine-rich region, and shields the acyl moieties from the aqueous cytosol. Clearly, the possibility cannot be excluded that the acyl moieties of the cysteine string are oriented orthogonally to the fatty acyl side chains of the membrane phospholipids, or at some intermediate angle. However, an argument against this is: CSP- is prone to dimerize (Mastrogiacomo and Gundersen,1995; Braun and Scheller, 1996; Chamberlain and Burgoyne, 1998). And, as dimers, hydrogen bonding between pairs of parallel (or, anti-parallel) cysteine strings in a membrane milieu would be effective in further reducing the polarity of this region and facilitating its intra-membrane disposition. While empirical studies will be necessary to distinguish whether the topology depicted in Fig.3 holds true for CSPs, experiments using a palmitated, cysteine-rich peptide with a sequence from synaptotagmin-1 support the model in Fig.3 (Ruchala et al., 2019). Clearly, it will be important to extend this structural work by using full-length, fatty acylated CSP, and to compare and contrast this model with the traditional model of CSP membrane affiliation as exemplified in Figure 2 of the Burgoyne and Morgan (2015) review.

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3.2.3. Challenges for the cellular fatty acylation machinery

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In most cells and tissues in which CSP- has been inferred to play a role in regulated exocytosis, biochemical data indicate that CSP- is fatty acylated, while unacylated CSP- typically is not detected (Gundersen et al., 1994; Mastrogiacomo and Gundersen, 1995; van de Goor and Kelly, 1996; Chamberlain and Burgoyne, 1998; Mastrogiacomo et al., 1998a,b; Poage et al., 1999; Gundersen et al., 2001; Park et al., 2008; note that this conclusion does not extend to cells in which CSP- was artificially over-expressed, such as the HEK 293 cells in the work of Greaves, et al., 2006). These observations suggest that fatty acylation of CSP- normally is very efficient and that little CSP- persists in cells with unmodified cysteine residues. A teleological explanation for this finding is that it may be undesirable for cells to accumulate a protein with as many reactive thiols as CSP. Fatty acylation of CSP’s thiols and the consignment of the fatty acylated string domain to the membrane interior should diminish the potential for many of the other covalent and non-covalent modifications of protein thiols that have been documented (Chalker et al., 2009). Collectively, if one takes into account the fact that many (perhaps, all) of the cysteines in the string region of CSP- are fatty acylated and couples this with the proposal that the “string” of CSP- affiliates with membranes (as in Fig.3), one creates an interesting enzymological challenge for the fatty acylation machinery of the cell:

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The reaction catalyzed by protein palmitoyltransferases involves the transfer of the palmitoyl group of palmitoyl CoA to the thiol group of cysteine residues of target proteins. Because palmitoyltransferases are integral membrane proteins (Politis et al., 2005), they clearly have access to palmitoyl CoA, which is oriented in membranes with the hydrophilic CoA group facing the cytosol and the fatty acyl moiety embedded in the hydrophobic interior of the membrane. Given this topology, if the model in Fig.3 is correct, then the palmitoyltransferase that fatty acylates CSP- must accomplish this task by using a bit of ingenuity. This “ingenuity” is necessary, because the membrane affiliation of CSP shown in Fig.3 demands that the fatty acyl CoA be rotated from its usual orientation in the membrane to a position that will allow the thioltransferase reaction to proceed. Two plausible reaction sequences can be envisioned (Fig.4). In both cases, the cysteine string is assumed to adopt -structure. This means that the -SH groups of successive cysteines project ~180o with respect to one another. This conformation also means that all of the cysteine residues that point toward the membrane interior can be fatty acylated without having to “flip” the palmitoyl CoA. But, once these cysteines are fatty acylated, the challenge is to acylate the remaining thiols. Two likely scenarios are: 1) The partially acylated cysteine string of CSP is threaded into the membrane interior so that the remaining unmodified thiol residues are located between the membrane hemi-bilayers (similar to what is shown for the modified thiols in Fig.3). In this situation, the palmitoyltransferase must rotate the palmitoyl CoA substrate by 90o-180o within the bilayer milieu to enable the thioltransferase reaction to proceed. In other words, the CoA moiety must be inverted relative to its original orientation (Fig.4A); 2) The second possibility is that the cysteine string region binds to the cytosolic face of the palmitoyltransferase such that the cysteine thiols can be palmitoylated at the membrane-cytosol interface. However, because the thiol groups of these cysteines face toward the cytosol (see Fig.4B), the palmitoyltransferase must flip the palmitoyl CoA by 90-180o (into the aqueous cytosol, Fig.4B) to allow the transferase reaction to occur. Subsequently, the fatty acylated portion of the cysteine string must be fed into the membrane to become deployed as shown in Fig.3 (this is necessary to shield the newly attached palmitoyl moiety from the cytosol). These proposals envision the palmitoyltransferase serving either to shield the hydrophilic CoA moiety from the membrane interior (Fig.4A), or buffering the hydrophobic fatty acyl group from the cytosol (Fig.4B). To date, there is no evidence to distinguish between these alternatives. However, observations that are germane to the general issue of protein folding may also be relevant to this problem. Specifically, a well-established function of Hsp70-J

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domain proteins is to bind to nascent polypeptide chains as they emerge from the ribosome (Hartl, 1996). This interaction serves to “protect” hydrophobic domains of nascent polypeptides from the cytosolic milieu and to facilitate effective protein folding once synthesis of the polypeptide backbone is complete. To extend this general theme to CSPs, it is plausible that Hsc70 (recruited by binding to CSP’s J domain or by using an affiliated J domain protein) can aid in the delivery of CSP to the palmitoyltransferase. Alternatively, Hsc70s may help to facilitate the complicated acylation scenarios outlined above (say, by helping to “flip” palmitoyl CoA). Clearly, more work will be needed to illuminate the pathway by which CSP- becomes fatty acylated and to clarify whether Hsc70 contributes to this process. 3.2.4. Does the fatty acylated cysteine string of CSP- have a specific function?

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This semi-rhetorical question will segue into the discussion of the functional investigations of CSP in section 4. The important point here is that in spite of three decades of study, very little insight has emerged concerning the biological role of the cysteine string. From the deduced amino acid sequences, we know that the cysteine string of CSP is conserved from nematode worms through humans (Buchner and Gundersen, 1997). The available evidence also indicates that most, if not all, of the cysteine residues of the cysteine string region of CSP- are fatty acylated (see 3.2.1). When in vitro translated CSP was injected into frog oocytes, only the fatty acylated form of the protein associated with membranes (Gundersen et al., 1994). This implied a link between fatty acylation and membrane affiliation. Additionally, as will be addressed in section 4, mutagenesis studies in Drosophila revealed that deletion of the 11 cysteines of the cysteine string region compromised the targeting of CSP to membranes and prevented it from reaching nerve terminals (Arnold et al., 2004). However, in a strain that lacked 5 of the 11 cysteines of the cysteine string, the mutant protein was targeted to nerve terminals, albeit at lower levels than controls. Interestingly, in PC12 or HeLa cells, replacing seven cysteine residues with serines in vertebrate CSP- prevented membrane targeting of the mutant construct (Chamberlain and Burgoyne, 1998). Thus, in spite of the somewhat different membrane targeting of mutated forms of CSP in invertebrates and vertebrates, it is reasonable to conclude that the cysteine string plays a role in membrane targeting. However, a broader question persists: why does one need the large number of cysteine residues found in the cysteine string of CSPs? After all, a single membrane-spanning domain, or one or two, covalently attached prenyl or fatty acyl (palmitoyl or myristoyl) moieties suffice to target a protein of CSP’s mass to biological membranes (as is observed for the  subunits of G proteins). We consider below two conjectural answers to this question.

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Soon after it was established that the cysteine string of CSP- was fatty acylated, it was proposed that CSP might aid in catalyzing the fusion of biological membranes (Gundersen et al., 1995). A key component of this proposal was the inference that the fatty acylated cysteine string domain of CSP- was sufficiently long to traverse the interface between a docked synaptic vesicle and the plasma membrane. However, there remains no direct evidence that CSP- adopts the kind of interfacial organization that was proposed. And, as discussed further in section 4, while there is considerable support for the idea that CSP- contributes to exocytotic membrane fusion, the molecular details remain sketchy. More recently, it was suggested that CSP might help to shepherd synaptotagmins into a fusogenic complex at the synaptic vesicle-plasma membrane interface (Gundersen and Umbach, 2013). Again, this suggestion is speculative, but it does provide a rationale for the highly conserved structure of the cysteine string: namely, that it enables CSPs to corral other proteins and/or to access and stabilize the interface between a docked secretory organelle and the plasma membrane. Obviously, this is a crucial interface for regulated secretory events, but

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as noted above, there is no direct evidence that CSPs reach this location. Thus, it will require further empirical studies to clarify the specific function(s) of the cysteine string domain. 3.3 Other post-translational modifications of CSP

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Although there is evidence for acetylation of a lysyl residue of CSP- (Choudhary et al., 2009) and ubiquitination of Lys58 (Wagner et al., 2011), the only other established post-translational modification of CSP- is phosphorylation. By subjecting a panel of recombinant proteins to in vitro phosphorylation assays, Evans and colleagues (2001) reported that CSP- was one of a small number of proteins in the exocytotic pathway that is a target for protein kinase A (PKA). Although CSP- was also phosphorylated in vitro by protein kinase C, this group focused on the PKA-dependent modification of CSP-. Immunoprecipitation of CSP- revealed that it was phosphorylated in chromaffin cells and synaptosomes and that 8-bromo-cAMP enhanced this phosphorylation. The site of CSP phosphorylation was identified as Ser10 (which is an atypical PKA site), and this phosphorylation blunted CSP’s interaction with the t-SNARE, syntaxin, but not with Hsc70 or G protein subunits. Functionally, CSP phosphorylation was associated with effects on the kinetics of catecholamine secretion which implied that CSP functions late in the exocytotic sequence in chromaffin cells. A proteomic analysis of synaptosomes confirmed the phosphorylation of Ser10 (Witzmann et al., 2005). Further details of this effect and a recent extension of this work (Chiang et al., 2014) are covered in section 4.3.

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In a follow-up study, Evans and Morgan (2002) reported that CSP- binding to synaptotagmin I was also negatively regulated by phosphorylation of Ser10. It was surmised that this CSPsynaptotagmin interaction might be relevant to independent reports that CSP influences the Ca2+ sensitivity of exocytosis. These results will be addressed further in section 4.

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Additional progress in understanding the role of phosphorylation at Ser10 of CSP- relied on the use of a phospho-specific antibody (Evans and Morgan, 2005; Evans et al., 2006). These authors reported that the level of CSP phosphorylation was variable during development, and a striking difference was seen in the cerebellar granule cell layer where phospho-CSP was abundant in a subset of glutamatergic nerve terminals but undetectable in nearby GABAergic nerve terminals. In addition, it was found that protein kinase B can phosphorylate CSP- at Ser10, and this modification influences catecholamine secretion similarly to PKA (Evans et al., 2006). Interestingly, CSP phosphorylated at Ser10 binds the 14-3-3 protein (Prescott et al., 2008), but the impact of this interaction in vivo has not been clarified. Finally, Patel et al. (2016) reported a major conformational change that takes place in Ser10-phosphorylated CSP- that may explain the functional transitions that have been reported. This investigation used NMRbased analysis to obtain structural insight into a construct representing the first 100 residues of CSP-. This region had seven -helices the longest of which included 17 residues (52-68) and the shortest just 4 residues (7-10). Importantly, the Ser10-phosphorylated form disrupted the short N-terminal helix with phospho-Ser10 shifting into a salt bridge with a lysyl residue (K58) which is highly conserved among J domain proteins. Taken together, these data indicate that CSP function is regulated both developmentally and in a cell and synapse-specific manner. More recently, Shirafuji and colleagues (2018) reported that CSP- was a prominent target for protein kinase C phosphorylation (at Ser10 and Ser34). A phosphomimetic mutant of CSP- enhanced interaction with Hsc70 whereas converting the two serine residues to alanine suppressed this interaction. PC-12 cells expressing the double alanine mutant had more ubiquitinated SNAP-25 and lower overall SNAP-25 content. Finally, in the striatum of protein kinase C KO mice the level of phosphorylated CSP- and the level of SNAP-25 were both reduced implying that these molecular changes might contribute to Parkinson’s disease.

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4. Functional investigations of CSP-

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CSP- function has been probed in a wide assortment of systems, and in spite of the extensive literature, there still is no consensus about the molecular role(s) of CSP- in regulated exocytosis. A partial explanation for this state-of-affairs is the relatively promiscuous interaction profile of CSP-. For instance, a recent study of CSP- interaction partners identified at least 42 proteins prominently expressed at synapses along with several other binding candidates (Trepte et al., 2018). In addition, as noted earlier, the J domain of CSPs mediates interaction with Hsc70 chaperones, which themselves are known to interact with a broad array of proteins and peptides. And, Chamberlain and Burgoyne (1996) showed that CSP-, by itself, exhibited chaperone activity, in vitro. These observations led the authors of earlier reviews to conclude that CSPs should be viewed primarily through the lens of their chaperone functions (Chamberlain and Burgoyne, 2000; Zinsmaier et al., 2001). While the foregoing approaches delineated the myriad interacting partners of CSP-, the following discussion will seek to distill trends that help to constrain the range of functions currently proposed for CSP-.

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4.1. Genetic manipulation 4.1.1. Using Drosophila

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The obvious benefit of the fact that CSPs were discovered in fruit flies was the opportunity to exploit these organisms for genetic manipulations. Thus, 4 years after the discovery of CSPs, Zinsmaier and colleagues (1994) characterized the phenotype of two mutant alleles, cspR1 and cspX1. The former was a null mutation while the latter lacked the promoter and first exon of the csp gene. These mutants exhibited a semi-lethal phenotype in which only a small number of flies survived to adulthood (4% in the case of cspR1). Of those that reached adulthood, none lived beyond 5 days at 22o C, and at 27o C survival was less than one day. This mortality contrasted sharply with the normal lifespan of fruit flies of >40 days. The lethality and early death of both mutant strains were reversed by re-introduction of the csp gene thereby demonstrating the specificity of these effects. In addition to the impact on survival, the most striking physiological characteristic of these mutants was temperature-dependent paralysis. Adult cspR1 flies paralyzed quickly at 29o C. To test for a functional correlate of this paralytic phenotype, electroretinograms documented a loss of “on” and “off” transients suggestive of a blockade of synaptic transmission between photoreceptor terminals and lamina neurons. Finally, electron microscopy revealed a dearth of synaptic vesicles and the presence of electron dense debris at photoreceptor terminals of adult cspX1 mutants. Collectively, these data indicated that although the csp gene was not essential for survival in Drosophila, the block of synaptic transmission at elevated temperature suggested a possible role for CSP in stabilizing components of the synaptic machinery. In addition, the degenerative changes observed at photoreceptor terminals presaged the neurodegeneration subsequently reported in csp- knockout (KO) mice. To obtain a more-definitive understanding of the cellular defect underlying the temperaturesensitive paralysis of the csp mutant alleles, intracellular recordings were made of spontaneous and evoked transmitter release in WT and cspX1 and cspR1 mutant larvae (Umbach et al., 1994). This investigation revealed that the frequency of spontaneous, miniature end plate potentials was modestly diminished (~30%) in the mutants relative to WT controls at permissive temperature. However, the frequency of the spontaneous secretory events did not dramatically change in WT or mutant larvae at elevated temperature (30o C). Nevertheless, even at permissive temperature (22o C), the amplitude of stimulus-evoked end plate potentials was reduced (~50%) at mutant neuromuscular junctions relative to WT controls. Nevertheless,

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synaptic delay and the general kinetics of these evoked responses were similar to WT controls. Then, within minutes of transfer to 30o C, both mutant strains showed a further reduction in the amplitude of the end plate potential. After 15-20 minutes at 30o C, evoked responses were no longer detectable. This thermally induced block of synaptic transmission was reversible, because within ~5 minutes of returning to 22o C, end plate potential amplitude was restored nearly to its original level. Control experiments revealed that this temperature-dependent interruption of neuromuscular transmission was not due to the failure of action potentials to propagate into nerve terminals, nor was it due to a reduction in post-synaptic sensitivity to transmitter. Transgenic expression of csp in the null background restored synaptic function to WT levels. Overall, these results indicated that the absence of CSP unmasked a temperaturedependent feature of synaptic transmission that was not evident in controls. Because nerve impulse propagation and post-synaptic sensitivity to glutamate were not affected, these findings pointed to a presynaptic defect in the regulated secretory pathway at nerve terminals. Moreover, although these data did not identify the precise step in the secretory cascade that was compromised in the mutants at 30o C, the persistence of spontaneous secretion when stimulusevoked events were blocked certainly argued that these two secretory pathways were regulated differently, and that CSP contributed preferentially to the regulated pathway. This conclusion was confirmed and extended in a later investigation that examined the function of various proteins that influence the rate of spontaneous transmitter release (Saitoe et al., 2001). However, the molecular identity of the thermally sensitive step(s) in the secretory cascade at Drosophila nerve endings remains unclear.

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In principle, the reversible blockade of evoked transmitter release in csp mutant larvae could be attributed to a thermally induced defect in one or more of the following: i) presynaptic Ca2+ entry; ii) the efficacy of Ca2+ in triggering the exocytotic cascade; iii) the machinery that triggers the fusion of the synaptic vesicle membrane and the plasma membrane (with the proviso that this machinery must be different for evoked versus spontaneous transmitter release). In addition, there was the remote possibility that synaptic vesicles temporarily vanished, or “lost” their content of neurotransmitter during a thermal challenge, but this seemed unlikely given the persistence of spontaneous secretory events at 30o C and the rapid recovery of evoked secretion at permissive temperature. Nevertheless, as an initial effort to distinguish among the three primary scenarios (i-iii; above), a series of pharmacological investigations was conducted. Importantly, it was observed that two agents, -latrotoxin and Ca2+ ionophore, which circumvent presynaptic Ca channels, triggered quantal discharges at non-permissive temperature in CSP mutant larvae at a time when nerve impulses failed to evoke transmitter release (Umbach and Gundersen, 1997). At the same time, depolarizing (high KCl) solution, or 4-aminopyridine (a K+ channel blocker, both of which demand functional Ca2+ channels, did not elicit quantal secretion in the mutants at elevated temperature. These results were interpreted as favoring scenario i or ii. Similar results were reported by Ranjan et al., (1998), who monitored pre-synaptic secretory dynamics by using FM1-43. They found that both black widow spider venom and Ca2+ ionophore triggered presynaptic de-staining at non-permissive temperature in the csp null mutants, but high K+ solution did not. Their results also suggested that scenarios i and/or ii could explain the secretory defect in csp mutants. Finally, their data provided evidence that the recycling of synaptic vesicles was not significantly impaired in the csp mutants. Further efforts to distinguish cellular/molecular correlates of the disturbance of stimulusdependent transmitter release in csp null mutants led to conflicting results. First, Umbach and colleagues (1998) reported that presynaptic Ca2+ entry was attenuated in csp null mutants at elevated temperature and recovered with cooling. These data were consistent with the idea that CSP modulates presynaptic Ca2+ entry, and that the compensatory mechanisms that operate in the absence of CSP are thermally labile. However, a subsequent study failed to confirm this

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observation (Dawson-Scully et al., 2000), and led instead to the conclusion that CSP regulates the Ca2+-sensitivity of the regulated secretory apparatus. An important element of this latter inference was the observation that intracellular Ca2+ buffering was impaired in the CSP mutants at elevated temperature. Although the mechanism by which CSP influences cytosolic Ca2+ homeostasis remains unclear, experiments in mice (section 4.1.2) independently implicated CSP- in regulating Ca2+ sensing at nerve endings. Hence, the possibility persists that CSPs modulate Ca2+ entry, Ca2+ homeostasis and exocytotic Ca2+-sensing.

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A targeted approach to assess CSP function involved studies of its interaction with other proteins implicated in the regulated secretory pathway. Thus, Nie and coworkers (1999) reported that over-expression of CSP in Drosophila led to modified wing structure and aberrant development of the eye. These effects were suppressed by concomitant over-expression of the t-SNARE, syntaxin, but not by the t-SNARE, SNAP-25. At the same time, syntaxin overexpression interfered with evoked transmitter release at larval neuromuscular junctions, and this effect was reversed by CSP over-expression. Biochemically, syntaxin co-immunoprecipitated with CSP and the recombinant proteins directly interacted, in vitro. Together, these results suggested that syntaxin, which is part of the SNARE complex, and also participates in Ca2+ channel modulation (Bezprozvanny et al., 1995; Wiser et al., 1996) might represent a pathway by which CSP function at nerve terminals is realized. A related study also supported the CSPsyntaxin link. Wu and co-workers (1999) observed that CSP co-immunoprecipitated with syntaxin and synaptotagmin and that CSP competed with syntaxin for binding to the “synprint” site of N-type Ca2+ channels. These later observations further supported a possible modulatory link between CSP and presynaptic Ca2+ channels. However, molecular details of this putative link remain to be clarified.

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An important piece of the CSP puzzle was contributed by Morales and colleagues (1999), who recorded Ca2+ currents at peptidergic terminals in WT and cspR1 mutants at restrictive temperature. There was no decline of Ca2+ current at higher temperatures in the csp nulls. These data paralleled observations in other regulated exocytotic systems (see section 4.2) that CSP influenced regulated exocytosis downstream of Ca2+ entry. However, there are two caveats concerning this study: first, there was no evidence that regulated exocytosis was compromised at the time that Ca2+ currents were recorded, so it is possible that a slowly developing effect was missed; second, the time course of peptide secretion typically is appreciably slower than the secretion of small-molecule neurotransmitters (reviewed in Martin, 2003), so it may be that CSP contributes exclusively to the modulation of Ca2+ channel function at “fast” synapses.

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To test the idea that CSP function is mediated at least in part via its interaction with Hsc70, Bronk and co-workers (2001) investigated the impact of mutant alleles of the Drosophila Hsc4 gene which encodes a protein that most closely resembles mammalian Hsc70. Like csp null mutants, evoked transmitter release was depressed ~50% in Hsc4 hypomorphs at permissive temperature. In addition, evoked responses were further depressed above 30o C and typically were abolished after ~20 minutes at elevated temperature. Spontaneous transmitter release was unaffected in these mutants, but as they had reported for csp null mutants, resting cytosolic Ca2+ was elevated relative to wild type controls at 34oC. At the same time, stimulus-dependent Ca2+ entry was unaffected at motor nerve terminals. Collectively, these data mirrored results for csp null mutants and implied that CSP and Hsc4 functioned together to modulate the Ca2+ sensitivity of the secretory apparatus at nerve endings. By expressing mutated versions of CSP in the null background, Arnold and co-workers (2004) performed a structure-function analysis of specific regions of CSP. These investigators reported a number of interesting phenotypic outcomes of which only the most prominent will be summarized: first, CSP lacking all cysteines failed to bind to membranes and was not targeted

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to nerve terminals (extending the observations of Gundersen et al., 1994 and Chamberlain and Burgoyne, 1998). Second, the construct in which 6 of the 11 cysteines of the cysteine string were deleted was relatively poorly expressed, but was appropriately targeted to the synaptic neuropil. However, transgenic flies expressing this construct still paralyzed at 37o C. Third, when the 11 cysteines of the string were replaced by serines, the mutant protein distributed relatively uniformly throughout the nervous system without CSP’s normal preference for synaptic neuropil in WT flies. At the same time, transgenic larvae with this construct showed even smaller evoked responses at the neuromuscular junction than csp null mutants. Fourth, the mutant construct in which all 11 cysteines of the string were deleted still was weakly expressed in the synaptic neuropil. However, these mutants were not assessed for paralytic behavior or synaptic physiology. Taken together, these data were interpreted as indicating that the number of cysteines in the cysteine string is functionally important. However, the precise role of the string remained uncertain. Finally, this investigation considered the impact of truncating the carboxyl terminus of CSP or the linker region between the cysteine string and the J domain. Here, interpretation of the results may have been complicated by differential expression of the transgenes in larval and adult flies, because confusing discrepancies were reported for paralysis data in the adults and synaptic physiology in the larvae. Thus, it is difficult to draw firm conclusions from these later findings.

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The most-striking physiological outcomes of expressing altered versions of Drosophila CSP in the null background came in the work of Bronk and colleagues (2005). They reported that transgenic larvae expressing a construct lacking the J domain (residues 19-82) were resistant to the temperature-dependent block of evoked transmitter release seen in csp null mutants. These results argue that the J domain of CSP is dispensable for protecting synaptic vesicle exocytosis from failure at elevated temperature. In addition, a point mutation in the J domain (H45Q) that abrogates binding to Hsc70 led to a nearly complete reversal of the ~50% decline of evoked transmitter release seen in csp null mutants at permissive temperature. Together, these findings suggested that the J domain of CSP was not a crucial contributor to CSP’s role in evoked transmitter release. However, as discussed in section 4.2, results in other systems make a strong case for an important function of CSP’s J domain, so future work will be needed to reconcile these disparate findings. Certainly, one possibility is that some other J domaincontaining protein contributes to the restoration of normal thermal sensitivity in fruit fly mutants expressing the J domain-deficient CSP (and, possibly the H45Q mutant). A second finding by this group was that larvae expressing CSP in which the linker region between the J domain and the cysteine string was deleted showed WT thermal sensitivity of transmitter release, and a restoration of WT levels of transmitter release at permissive temperature. These data indicated that CSP’s linker was not essential for CSP’s secretory function at larval nerve terminals. The issue of the role of the linker region will be considered further in section 4.2.

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The work of Bronk and colleagues (2005) also revealed that the number of synaptic boutons at neuromuscular junctions was reduced in cspR1 null mutants to ~60% of WT controls. This loss of boutons could be completely reversed in strains expressing WT CSP; in fact, the overexpression of CSP that occurs with the elav promoter-driven expression of csp led to an increase in bouton number relative to WT controls. Separate experiments indicated that this effect of CSP on bouton number relied on a functional J domain, but the mechanism of this developmental role for CSP has not been elucidated. Certainly, an interesting hypothesis to consider is that CSP may contribute to secretory pathways that insert into the plasma membrane, or release growth factors or other regulatory molecules that are important for synapse formation/stabilization. This investigation also examined further the impact of J domain or linker mutations on the resting level of cytosolic Ca2+ at permissive and non-permissive temperatures. While transgenics expressing the J domain deletion showed a ~5-fold increase in

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cytosolic Ca2+ at 30o C (comparable to mutant alleles lacking CSP altogether), strains expressing the linker deletion showed a partial reduction in the level of cytosolic Ca 2+. Quantitatively, these increases in cytosolic Ca2+ were greater in magnitude than the increase in cytosolic Ca2+ observed in WT larvae after 15 seconds of 30 Hz nerve stimulation. Interestingly, the large increases in cytosolic Ca2+ in these different mutant strains at 30o C had absolutely no effect on the frequency of spontaneous, quantal transmitter release. Although the authors did not comment on the implications of these results, such findings certainly are consistent with their original idea (Dawson-Scully et al., 2000) that the Ca2+ sensitivity of exocytosis is greatly diminished in the csp mutants. Nevertheless, additional work will be needed to clarify how the absence of CSP produces changes both in presynaptic Ca2+ homeostasis and the Ca2+ sensitivity of the secretory apparatus.

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Studies by Brusich and colleagues (2015) revealed a prominent role for CSP in the long-term maintenance of synaptic homeostasis in Drosophila. Interestingly, this paper noted that prior work had argued against the involvement of CSP in short-term homeostatic plasticity, whereas the ongoing maintenance of quantal content did involve CSP. Although the molecular mechanism of this effect remains to be elucidated, this outcome is not surprising given the myriad synaptic changes documented for Drosophila csp mutants, including the reduction in bouton number (Bronk et al., 2005) and the decline in the amplitude of evoked synaptic potentials in the cspR1 and cspX1 alleles relative to WT (Umbach et al., 1994).

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In a study that did not directly involve mutations in the fruit fly csp gene, it was reported that Mind Bomb-1, a post-synaptic density protein and member of the ubiquitin E3 ligase family, profoundly influenced the expression of CSP and glutamate receptors at larval neuromuscular junctions (Sturgeon et al., 2016). These expression results were accompanied by significant morphological and functional changes at these synapses. Clearly, these results are suggestive of developmentally important trans-synaptic interactions whose molecular pathways will require further scrutiny. 4.1.1.1. Drosophila CSP mutants: conclusions and future directions

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Several observations concerning the phenotype of Drosophila csp mutants have been confirmed independently. First, adults and larvae show reversible, temperature-sensitive paralysis (Zinsmaier et al., 1994; Umbach et al., 1994; Arnold et al., 2004). Second, csp null mutant larvae exhibit a decline relative to WT in the amplitude of the evoked synaptic potentials (or, currents) at permissive temperature (Umbach et al., 1994, Umbach and Gundersen, 1997; Heckmann et al., 1997; Nie et al., 1999; Dawson-Scully et al., 2000, 2007; Bronk et al., 2005). Third, at elevated temperature (>25o C), csp null mutants show a gradually developing reduction in the amplitude of evoked synaptic potentials (or currents) that can culminate in a complete blockade of quantal transmitter release (Umbach et al. 1994; Umbach and Gundersen, 1997; Dawson-Scully et al., 2000; Saitoe et al., 2001; Bronk et al., 2005). Fourth, even when evoked transmitter release is completely eliminated at elevated temperature, spontaneous secretory events persist in csp nulls (Umbach et al., 1994; Saitoe et al., 2001; Bronk et al., 2005). These data can reasonably be regarded as representing the core phenotype of the fly, csp null mutants. As will be stressed later (section 4.1.2), the fact that at least two csp genes are expressed in mouse brain limits the interpretation of synaptic data from csp- KO mice, because there is no information concerning the extent to which the expression of other csp genes contributes to the phenotype of these KO animals. Instead, because fruit flies have a single csp gene, the preceding data provide a useful index of CSP’s cellular role(s) in the nervous system (similarities and differences in the phenotype of csp KO Drosophila and C. elegans is covered in section 4.1.3. Conservatively, given the fact that CSP is a synaptic vesicle protein (see section 2), the mutant data reviewed above strongly support the hypothesis that

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CSP plays an important role in regulated exocytosis at “fast” synapses, but not in the pathway responsible for spontaneous secretory events. Unfortunately, this distinction still does not help to identify the most plausible molecular mechanism(s) by which CSP contributes to stimulusdependent, transmitter release. Thus, at this time, the Drosophila data remain consistent with one or more of the hypotheses noted earlier: namely, that CSP participates in the regulation of presynaptic Ca2+ channels; that it influences the Ca2+ sensitivity of fast, synchronous exocytosis; or that it interfaces functionally with one or more of the other key exocytotic proteins mentioned in the Introduction (section 1). The other important point to re-iterate is that the blockade of evoked transmitter release observed in the fruit fly larvae occurs at a developmental stage prior to when neurodegeneration was observed. And, this blockade is rapidly reversible. These observations strongly imply that the temperature-sensitive defect in exocytosis at larval nerve terminals is not directly related to neurodegeneration. This finding contrasts with data from the csp- KO mice in which detectable changes in synaptic function were seen only after neurodegeneration was underway (Fernandez-Chacon et al., 2004 and see 4.1.2).

4.1.2. Using mice

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Obviously, an important future direction is to distinguish among the possible molecular roles for CSP at nerve terminals. As noted before, Drosophila mutants are advantageous in that one does not need to exclude contributions from other csp gene products. However, it has not been determined whether other J domain proteins are also up-regulated at nerve terminals of csp null mutants, and such information could help to clarify the mechanism by which these organisms compensate for the loss of CSP. Obviously, further work could also be done to illuminate the basis of the apparent reduction in the Ca2+ sensitivity of the secretory apparatus in the csp (and, Hsc4) mutants. Since synaptotagmin is widely accepted as the Ca2+ sensor for evoked transmitter release at “fast” synapses (Sudhof and Rizo, 2012; Jahn and Fasshauer, 2012; Meriney et al., 2014), efforts to understand how and why synaptotagmin’s Ca2+-sensing function is modified could be particularly revealing. Finally, more remains to be learned about the proposed CSP-Ca2+ channel link at “fast” synapses, and fruit flies remain a potentially useful system for evaluating molecular mechanisms of this interaction.

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The mouse csp- gene was deleted via homologous recombination (Fernandez-Chacon, et al., 2004). Homozygous KO pups resembled heterozygous or WT controls until about 2 weeks after birth at which time they began to lose weight. By ~3 weeks, there was a rapid increase in mortality and none of the KOs survived past 80 days. A standardized neurological assessment of the KO mice revealed sensorimotor defects which progressively deteriorated with age. Electromyography indicated that neuromuscular function became increasingly impaired after 2 weeks, and this was accompanied by electron microscopic signs of degenerative changes at motor nerve terminals. However, analyses of synaptic function revealed no detectable deficits in the KO mice. Ca2+ currents measured at the calyx of Held of P9-P12 KO mice were indistinguishable from WT controls. The G-protein-mediated regulation of these Ca2+ channels also was unaffected in the csp- KO mice. Synaptic transmission at the calyx synapse was quantified using several different approaches and was found to be virtually unchanged between KOs and WT controls. Collectively, these data were interpreted as indicating that CSP- is not essential for the normal operation of presynaptic Ca2+ channels or for evoked transmitter release. Instead, CSP- was inferred to play a role in protecting nerve terminals from the neurodegenerative changes discussed next. The dominant cellular change observed in csp- KO mice was neurodegeneration (FernandezChacon et al., 2004). As the KO pups aged, their neuromuscular junctions showed increased vacuolization. A high percentage had Schwann cell protrusions and multilamellar bodies.

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Similarly, by P25, calyx nerve terminals occupied much less of the surface of the post-synaptic cell in the KO mice (consistent with terminals having degenerated). Remnants of calyx terminals were electron lucent with aberrant mitochondria and membranous debris. At the same time, some calyx terminals remained relatively normal, implying that there was some other factor involved in triggering the degenerative process. Indeed, the authors commented on the fact that apparently normal nerve terminals co-existed with neighbors that were degenerating. It was proposed that this differential outcome was likely to be due to activity-dependent differences between the terminals. Subsequent publications have expanded on this theme that activity may be an important factor that influences the course of neurodegeneration in the csp- KO mice (Schmitz et al., 2006; Garcia-Junco-Clemente, et al., 2010). At the same time, it was not determined whether other cells in which CSP- is prominently expressed [including, pancreatic  cells (Brown et al., 1998, Zhang et al., 1998a,b); adrenal chromaffin cells (Kohan et al., 1995; Chamberlain et al., 1996) and cells of the anterior pituitary (Jacobsson and Meister, 1996; Pupier et al., 1997)] exhibit degenerative changes in the csp- KO mice. If these cells do not exhibit necrotic or apoptotic changes, then it will be important to evaluate CSP- function in these non-neuronal, regulated secretory cells.

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Because mice (like other vertebrates) have three csp genes, an important consideration in the mouse csp- KO study was the expression status of the other csp gene products. Based on Northern and RT-pcr data, CSPs- and - were reported to be testis specific, and mRNAs encoding these proteins were not detectably up-regulated in the csp- KO animals (FernandezChacon et al., 2004). Immunoblot data were also touted as showing that these CSPs were not expressed in brain. Here, it is important to point out that no evidence was presented that the antibody used specifically detected CSP- (or, CSP-). This distinction is important, because subsequent work (summarized below) demonstrates that CSPs-and - are not testis-specific.

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The first indication that CSP- was not testis-specific was work of Schmitz and colleagues (2006). They reported that the expression of CSP- in auditory epithelium protected inner hair cells from the degeneration observed in retina of csp- KO mice. Their data also showed that CSP- mRNA was detectable in mouse brain (see Fig.5G of Schmitz et al., 2006). Second, gene-expression analysis has revealed 41 tissues expressing CSP- and 68 expressing CSP- (see the Bgee link at Uniprot for DNAJC5B or DNAJC5G: https://www.uniprot.org). Third, the Human Protein Atlas (https://www.proteinatlas.org/) reports human CSP- mRNA in blood, muscle, lung and immune-system tissues and in renal, myeloid and lymphoid cell lines. Immunocytochemical data from this atlas shows a punctate distribution of CSP- in a kidney epithelial cell line (Fig.5). This same resource also reports that CSP- mRNA is widely distributed in pig brain. Finally, the Human Protein Atlas reports low levels of CSP- mRNA in human brain (particularly, the cerebral cortex, hippocampus and amygdala), muscle and blood. Fourth, Human Proteome Map data confirm that CSP- is expressed in human frontal cortex (http://www.humanproteomemap.org/). Fifth, early data documented CSP- expression in cochlear inner hair cells (Eybalin et al., 2002). And, a comparison of mouse inner and outer hair cells revealed that CSP- mRNA was among the transcripts preferentially expressed in inner hair cells (Liu et al., 2014; and (http://www.ebi.ac.uk/arrayexpress/experiments/E-GEOD56866/). Sixth, using antibody that distinguished among CSP isoforms, it was reported that CSP- was expressed as part of a high-mass complex at mouse nerve terminals (Gundersen et al., 2010). Although these results were not confirmed in another study (Gorlecku et al., 2010), as noted above Schmitz et al. (2006) reported CSP- mRNA in mouse brain. Thus, although the issue of CSP-expression in human and mouse brain remains unclear, future research should clarify this situation. Seventh, data (at: https://www.mousephenotype.org/) for mice deficient in

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the gene for csp- or csp- revealed altered blood glucose levels, an abnormal Q/T interval in the electrocardiogram, skeletal modifications, behavioral changes, and neurological and immunological deficits that are incompatible with testis-specific expression of these genes. Data at this link also indicate that CSP- is widely expressed in mouse brain, particularly in the cerebellum. These CSP- expression data are available at: http://www.informatics.jax.org/gxd; see Koscielny et al.(2014). Eighth, a single nucleotide polymorphism in the human DNAJC5B gene is associated with a variable response to statins (Shiffman et al., 2012) implying a role for CSP- in cardiovascular function. Ninth, CSP- is present in and may contribute to the development of esophageal squamous cell carcinoma (Sun et al., 2018), and mRNA for all three csp genes was detected in several cancer cell lines (Mirzaei et al., 2016; note that csp mRNA expression in various types of cancer is archived at the Human Protein Atlas website noted above). Finally, work of Braga et al. (2017) indicates that CSP- mRNA is up-regulated in response to hepatitis C virus infection in various liver cell lines. Collectively, these results argue strongly that the expression of CSPs- and - is not testis-specific. Consequently, it is reasonable to conclude that a re-assessment of the csp- KO data will be needed to determine whether basal or up-regulated expression of CSP- or - influences the phenotypic data reported for the csp- KO mice. For instance, expression of these other CSPs might influence the synaptic phenotype or alter the pattern of neurodegeneration. For the same reason, it is clear that a triple KO of the mouse csp genes will be necessary before definitive conclusions can be drawn concerning CSP function in mouse neurons. After all, there is considerable divergence in the phenotype of the mouse csp- KOs and their Drosophila counterparts, particularly with respect to synaptic function. It will be important to determine whether CSPs- or - (or, other J domain proteins) contribute to these differences. At the same time, several studies (Zhang et al., 2012; Sharma et al., 2011, 2012 and see below) have used the csp- KO mice based on the premise that they represent a clean excision of all CSPs from the nervous system. Although these papers shed light on the phenotype of the csp- KOs, the functional conclusions are constrained by uncertainty regarding the status of the other csp gene products in the KOs.

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The neurodegenerative phenotype of csp- KO mice implied that these animals could be useful to help understand the cellular and molecular processes that lead to death of neurons. Thus, Chandra and colleagues (2005) reported that transgenic expression of -synuclein prevented the neurodegeneration and lethality previously observed in csp- KO mice. It was further inferred that the likely basis of this effect of -synuclein was that it reversed a deficiency in SNARE complex assembly in the csp- KO mice. As attractive as this hypothesis may be, the fact remains that nothing is known about the status of CSP- and CSP- (or, other J domain proteins) and their possible contribution (favorable or unfavorable) to SNARE assembly at nerve endings in the csp- KO mice. Thus, further testing of this hypothesis is warranted.

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Similarly, Schmitz and co-workers (2006) reported prominent photoreceptor degeneration in csp- KO mice. These results were reminiscent of the degenerative changes in photoreceptor terminals originally reported by Zinsmaier and colleagues (1994) in csp null Drosophila. Interestingly, although photoreceptor synapses were affected, auditory hair cell synapses were spared. A striking molecular difference between these two systems is that CSP- mRNA was detected in auditory hair cells, but not in the retina. Thus, it was inferred that the presence of CSP- protected auditory hair cells from the degenerative changes seen in the retina. Note that this inference was made without supporting evidence that bona fide CSP- was expressed in the auditory hair cells (the authors used an antibody that was not independently validated for its ability to bind to CSP-). At the same time, this paper also included the observation that CSP- mRNA was readily detectable via RT-pcr in mouse brain. In fact, Fernandez-Chacon et al.

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(2004) had reported low levels of CSP- mRNA in brain (<1% of CSP- mRNA), but the data of Schmitz et al. (2006) indicated that the mRNA levels for these two CSPs were appreciably closer in magnitude. The rhetorical question that was not addressed was: if one postulates that CSP- can protect auditory hair cell synapses, is it not possible that CSP- (or, CSP-) in neurons also contributes to the variability in nerve terminal degeneration that was originally described by Fernandez-Chacon and colleagues (2004)? This is an important question that will need to be addressed by future research.

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A detailed investigation of neuromuscular transmission in the csp- KO mice focused on the functional deficits that were observed after P14 (Ruiz et al., 2008). Because this period also coincides with the onset of significant neurodegenerative changes at motor nerve terminals (Fernandez-Chacon et al., 2004), it is possible that many of the phenotypic changes (such as, the decrease in the amplitude of evoked response, unusual bursts of spontaneous secretory events, a higher percentage of failures of evoked responses) were due to the ongoing neurodegeneration. Nevertheless, these authors concluded that their data supported a role for CSP- in regulating the Ca2+ sensitivity of the secretory apparatus at motor nerve terminals. As noted earlier (4.1.1.2.), although this remains a plausible role for CSP-, the molecular mechanism of this effect has not been clarified. More recently, studies of neuromuscular function in the csp- KO mice were again the subject of investigation (Rozas et al., 2012). As with the work conducted by Ruiz and colleagues (2008), this group looked at neuromuscular function well beyond the time (P16-20) when overt degenerative changes were originally documented by Fernandez-Chacon and colleagues (2004). In this situation, it is very difficult to conclude whether the functional changes described by Rozas and co-workers (2012) were the cause or a consequence of neurodegeneration. And, of course, the interpretation of these data is complicated owing to the unresolved expression status of CSPs- and - at motor nerve terminals in the KO animals.

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A recent investigation revealed that radial-glia-like, neural stem cells from csp- KO mice exhibited a period of enhanced proliferation beginning about 2 weeks after birth (Nieto-Gonzalez et al., 2019). This hyper-proliferative state was shown to involve direct or indirect signaling between CSP- and the mTOR pathway which leads to a depletion of the hippocampal pool of neural stem cells. These observations clearly mark a new frontier in understanding CSP function, and it will be important to extend these observations in the context of some of the criticisms enumerated above for the csp- KO mice. 4.1.3 Using C. elegans

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Kashyap and colleagues (2014) characterized the phenotype of worm mutants containing a >2 kB deletion that includes the promoter region and most of the coding sequence of the worm CSP homologue. As in Drosophila csp nulls, fewer progeny were obtained and development from egg to adult was slower. Worm mutants also showed roughly a 30% reduction in lifespan which is appreciably less dramatic than observed in fly csp KOs. Although electrophysiological analysis of synaptic transmission was not performed, locomotion and aldicarb assays revealed an age-dependent reduction in motor activity. As discussed further in section 5, worm mutants also exhibited delayed-onset neurodegeneration which was most prominently seen in anterior head neurons. Because these head neurons are predominantly sensory, a further finding was that the mutant worms were severely impaired in a food-race assay even at developmental stages (2 days) when overt anatomical signs of degeneration were lacking. Collectively, these results indicate considerable congruence between fly and worm mutants, but with obvious phenotypical differences that will require further investigation.

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4.2. CSP function that involves interactions with target proteins The presence in CSPs of a J domain raises the possibility that CSP alone or in conjunction with Hsc70 engages in general chaperone functions, as well as reactions that target specific clients. Thus, it is not surprising that CSP has been implicated in a broad spectrum of macromolecular interactions that are potentially important for regulated exocytosis at nerve terminals and elsewhere (Trepte et al., 2018). Whereas the preceding sections have taken a chronological view of progress in illuminating CSP structure and function, this section will consider the data supporting CSP’s interactions with specific target proteins and focus on the possible functional implications of these results. 4.2.1. CSPs and presynaptic Ca2+ channels

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The voltage-gated Ca2+ channels that regulate transmitter release at “fast” synapses are minimally composed of a pore-forming 1 subunit which associates with 2 and  subunits (Dolphin, 2009; Catterall, 2011; Weiss and Zamponi, 2012; Meriney et al., 2014). The aggregate mass of each Ca2+ channel is well in excess of 300 kDa, and this channel complex is regulated by other proteins and by post-translational modifications (Dolphin, 2009; Catterall, 2011; Weiss and Zamponi, 2012; Mochida, 2019). Two recent observations at frog neuromuscular junctions have helped to clarify the function of presynaptic Ca2+ channels: first, individual presynaptic Ca2+ channels show a low probability of opening, and second, Ca2+ entry via a single open channel can be sufficient to trigger exocytosis of a vesicle located within its “nanodomain” (Tarr et al., 2012; Dittrich et al., 2013; Meriney and Dittrich, 2013). Although the probability of Ca2+ channel opening is variable at other nerve terminals, there are many instances where individual Ca2+ channel nanodomains suffice to trigger vesicular exocytosis (Eggermann et al., 2011; Tarr et al., 2012). In addition to G protein / subunits and the proteins that bind to the “synprint” (synaptic protein interaction) site of presynaptic Ca channels, several other nerve terminal proteins have been shown functionally to modulate presynaptic Ca2+ channels (Eggermann et al., 2011; Weiss and Zamponi, 2012; Meriney et al., 2014; Mochida, 2019). These interactions are likely to play a major role in regulating the probability of channel opening in response to depolarization. CSPs were originally proposed to be among the modulators of presynaptic Ca2+ channels (Mastrogiacomo et al., 1994; Seagar et al., 1999; Weiss and Zamponi, 2012). However, as summarized in the following discussion, the evidence for this role remains equivocal.

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A vertebrate CSP was identified, because its antisense RNA inhibited the heterologous expression of N-type Ca channels in Xenopus oocytes (Gundersen and Umbach, 1992). Although the mechanism of this effect was not clarified, the subsequent finding that CSPs were synaptic vesicle proteins (Mastrogiacomo et al., 1994a) eliminated the possibility that CSPs were integral subunits of N-type channels. Instead, the hypothesis was advanced that CSPs might be part of the machinery by which a synaptic vesicle reports its presence to a nearby Ca2+ channel. Teleologically, this signaling was proposed to help limit Ca2+ entry to presynaptic sites where synaptic vesicles were suitably prepared to respond to a Ca2+ signal (Umbach et al., 1995) Evidence has accumulated both in favor of and against a link between CSPs and presynaptic Ca2+channels. First, it is important to acknowledge that various groups showed clearly that CSPs have a role in regulated exocytosis that is independent of Ca2+ channels in a number of non-neuronal secretory cells (Brown et al., 1998; Chamberlain and Burgoyne, 1998b; Zhang et al., 1999b; Smith et al., 2005). In addition, it was noted earlier that Ca2+ currents in peptidergic nerve terminals of Drosophila CSP mutants were unaffected at non-permissive temperature (Morales et al., 1999). From these results, it is clear that if CSPs have a role in Ca2+ channel

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regulation, this function is restricted to “fast” synapses, where transmitter is released within a fraction of a millisecond after nerve terminal depolarization.

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The first sign of a molecular mechanism that could underlie the putative CSP-Ca2+channel link was that recombinant CSP bound directly to the cytoplasmic loop connecting homologous domains II and III of P/Q type Ca channels, but not to any other region of the P/Q type channel (Leveque et al., 1998). However, although CSP co-immunoprecipitated with synaptobrevin (a result that appeared to be indirect), co-immunoprecipitation was not seen for P/Q type Ca2+ channels or other members (SNAP-25 or syntaxin) of the SNARE complex. As noted by the authors, a possible explanation for this discrepancy is that the detergents used to solubilize the P/Q channels may have hampered the co-immunoprecipitation. At the same time, recombinant CSP was found not to bind directly to recombinant synaptobrevin, syntaxin or SNAP-25. Because the II-III loop includes the synaptic-protein interaction site (“synprint” site, which also binds syntaxin 1a and SNAP-25; Rettig et al., 1996), these data suggested that CSP might play a role in the assembly or disassembly of regulatory complexes for exocytosis.

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As discussed earlier (section 4.1.1), Drosophila CSP mutants have also been used to examine the underlying basis of their temperature-dependent paralysis. Although one set of observations (Umbach et al., 1998) was consistent with the conclusion that presynaptic Ca2+ entry was impaired at elevated temperature in CSP null mutants, this conclusion was not supported by later work (Dawson-Scully et al., 2000). To date, this discrepancy remains unresolved.

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In a provocative study, Chen and co-workers (2002) observed that infusion of Drosophila CSP into presynaptic terminals of the chick calyx synapse led to an increase in Ca2+ current amplitude. This increase was not due to changes in channel kinetics, voltage dependence, prepulse inactivation or G protein modulation. Instead, CSP appeared to recruit dormant Ca2+ channels. Because this system theoretically could be used to undertake a structure-function analysis of the domain(s) of CSP that mediate this effect, it would be extremely helpful to extend this work. A similar infusion strategy evaluated the impact of CSP antibody delivered presynaptically at neuromuscular synapses formed between embryonic frog motor neurons and muscle cells, in vitro (Poage et al., 1999). These experiments revealed a block of stimulusevoked transmitter release, but no significant change in the frequency of spontaneous release events. Again, although these data were consistent with the idea that binding of antibody to CSP occluded its interaction with presynaptic Ca2+ channels, other explanations (such as, altered Ca2+ sensing, or a direct effect on the release apparatus) could not be excluded.

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Independently, CSPs have been implicated in regulating the interaction of G protein subunits with N-type Ca channels (Magga et al., 2000; Miller et al., 2003a,b; Swayne et al., 2005). In particular, these authors found that the N-terminus of CSP bound to G protein  subunits, while the C-terminus of CSP bound to G or to the heterotrimeric complex. These CSP-G protein interactions contributed to a substantial reduction in the amplitude of N-type Ca2+ channel current in heterologous cells in which these proteins were expressed. Interestingly, this inhibitory effect of CSP/G proteins was blocked by fragments of the huntingtin protein with a polyglutamine expansion. Additionally, recombinant CSP, like syntaxin 1a and mutant huntingtin, bound to the synprint site of N-type Ca2+ channels. Collectively, these macromolecular interactions imply that N-type Ca channels are subject to complex regulatory interactions. However, the preceding results are difficult to reconcile with several observations, including the csp KO data in fruit flies and mice. In particular, if one of CSP’s chief roles were to inhibit presynaptic Ca2+ channels, one would expect Ca2+ currents and evoked transmitter release to increase at nerve terminals of csp KO organisms. This clearly is not the case (Zinsmaier et al., 1994; Umbach et al., 1994; Umbach et al., 1998; Dawson-Scully et al., 2000;

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Fernandez-Chacon et al., 2004). Similarly, CSP infusion into the chick calyx synapse should have blunted Ca2+ currents instead of the augmentation that was reported (Chen et al., 2002), and changed the direction of the CSP antibody-mediated reduction in evoked synaptic responses at frog motor nerve terminals (Poage et al., 2009). Given these discordant observations, it remains a challenge to integrate the inhibition of N-type Ca2+ channel function into the picture that has emerged from work of several other groups.

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Beyond the preceding studies, there have been few other direct investigations of the CSP-Ca2+ channel link at nerve endings. An earlier section (4.1.1.) reviewed the controversy concerning CSP regulation of presynaptic Ca2+ entry in larval motor nerve terminals of Drosophila. One point that bears mention in this later context is that the Ca2+ channels that regulate fast neurotransmitter release in Drosophila lack the synprint site (Littleton and Ganetzky, 2000). Thus, the interactions between P/Q, R or N type Ca2+ channels and such proteins as syntaxin, SNAP-25, synaptotagmin and CSP that have been reported in vertebrates either do not take place in Drosophila, or involve different regulatory regions of the fruit fly channel. Nevertheless, this system retains attractive features that should aid in future efforts to clarify CSP function. 4.2.2. CSPs and syntaxin

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In spite of the initial report that recombinant CSP did not bind to recombinant syntaxin 1 (Leveque et al., 1998), subsequent investigations provided evidence for a direct association of these proteins (Nie et al., 1999; Wu et al., 1999; Evans et al., 2001; Swayne et al., 2006; but, see Boal et al., 2004 for a negative result). Thus, as noted earlier (4.1.1), Nie and colleagues (1999) found that CSP interacted with syntaxin during fruit fly development and CSP overexpression suppressed the inhibitory effect of syntaxin over-expression on neuromuscular transmission. Biochemically, CSP and syntaxin co-immunoprecipitated, and CSP bound directly to syntaxin, as judged by using recombinant proteins. Cumulatively, these data were interpreted as indicating that CSP and syntaxin may interact to modulate Ca2+ channel function or a later step in the secretory cascade (Nie et al., 1999). Independently, Wu and co-workers (1999) reported that CSP co-immunoprecipitated with syntaxin and that CSP effectively competed with syntaxin for binding to the synprint site of N-type Ca channels. These data led to the suggestion that CSP might relieve the syntaxin-mediated inhibition of N-type Ca2+ channels. Then, in an investigation of the impact of phosphorylating CSP on Ser10, it was observed that syntaxin bound much less effectively to phospho-CSP (Evans et al., 2001). Subsequently, Swayne and colleagues (2006; also see 4.2.1.) also detected a competition of CSP and syntaxin for binding to the synprint region of N-type Ca2+ channels. Clearly, the reproducibility of this CSP-syntaxin interaction in vitro suggests that it is likely to have functional relevance, in vivo. However, the role of this interaction in the context both of the other regulatory influences that impinge on presynaptic Ca2+ channels (reviewed in Eggermann et al., 2011; Weiss and Zamponi, 2012; Meriney et al., 2014; Mochida, 2019) and the general secretory apparatus remain to be elucidated. In experiments focusing on glucose transported trafficking, it was observed that CSP- was predominantly associated with the plasma membrane of 3T3-L1 adipocytes where it interacted with syntaxin 4 (Chamberlain et al., 2001). However, the precise role of this interaction has not been determined. Finally, Shimomura et al., (2013) indicated that CSP- in parotid acinar cells bound to syntaxin 3, but not to syntaxin 4 in a reaction that may influence SNARE complex assembly in these cells. However, the extent to which these observations can be generalized (especially, in light of the preceding work of Chamberlain et al.) is unclear. 4.2.3. CSPs and SNAP-25

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An early biochemical investigation (Leveque et al., 1998) found no co-immunoprecipitation of CSP and SNAP-25. Also, although CSP over-expression caused developmental defects in Drosophila that were relieved by syntaxin over-expression, similar relief was not obtained with SNAP-25 over-expression (Nie et al., 1999). Nevertheless, while it was originally noted that several synaptic proteins (including, complexin and synaptobrevin-2) were unchanged in csp- KO mice (Fernandez-Chacon et al., 2004), a later report indicated that SNAP-25 levels decreased by ~50% and SNARE complex assembly was also impaired (Chandra et al., 2005). In a follow-up study (Sharma et al., 2011), it was reported that a trimeric complex (CSP- Hsc70 and SGT) bound directly to SNAP-25, and this binding was important for subsequent SNARE complex formation. Moreover, deletion of csp- led to an abnormal SNAP-25 conformer that inhibited SNARE complex assembly and was a target for ubiquitination and proteasomal degradation. Evidence was also presented that the influence of CSP- on SNAP-25 folding was important during normal cycles of synaptic activity. This conclusion was extended in an investigation of the contribution of defects in SNAP-25 and SNARE complex assembly to neurodegeneration in the csp- KO mice (Sharma et al., 2012). Key observations were that SNAP-25 over-expression blunted the neurodegeneration seen in csp- KOs while reduced SNAP-25 expression exacerbated the phenotype of the csp- KOs. The upshot of this work was the proposal that the primary impact of the chaperone function of CSP- is to preserve the function of SNAP-25 (Sharma et al., 2012). In a similar vein, a large decline of SNAP-25 was also reported at the neuromuscular junction of csp- KO mice (Rozas et al., 2012). However, as noted previously (4.1.2), this later study used mice well beyond the age when neurodegenerative hallmarks are present at neuromuscular junctions. Thus, the decline of SNAP-25 could be a consequence rather than a cause of the degeneration.

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Although the preceding studies provide a plausible explanation for the neuroprotective role of CSP-, several concerns need to be addressed with respect to the CSP-SNAP-25 interaction and its role in neurodegeneration. The first issue was mentioned earlier (4.1.2) which is that csp- KO mice do not represent a complete excision of all CSPs from mouse brain. Thus, it needs to be clarified whether CSP- or - (or other J domain proteins) influence SNAP-25 levels or function in these animals. Second, it is noteworthy that a ~50% decline of SNAP-25 in the brain of mice lacking one SNAP-25 allele produced no discernible phenotype (Washbourne et al., 2002). This result raises the question of the extent to which the SNAP-25 pool at nerve terminals needs to be compromised for secretory and degenerative phenotypes to emerge. In vitro knockdown evidence in Sharma et al. (2012) revealed negligible changes in synaptic function with ~90% reduction of SNAP-25 immunoreactivity. Here, it is useful to note that individual rat brain synaptosomes have about 27,000 SNAP-25 molecules (Wilhelm et al., 2014). Thus, a ~90% reduction of SNAP-25, argues that nerve-terminal function can be maintained with fewer than 3,000 SNAP-25s per terminal. At the same time the half-life of SNAP-25 is ~3 days (Fornasiero et al., 2018). This result means that ~4,500 “new” SNAP-25 molecules are delivered per terminal per day. Since <3,000 SNAP-25 molecules per terminal are adequate to sustain transmitter release, and 4,500 replacement SNAP-25 molecules are delivered daily, these data raise quantitative questions about why the delivery of “fresh” SNAP25 is inadequate to maintain secretory function and presynaptic integrity in the csp-KOs. Third, it would be helpful to determine whether SNAP-25 levels and function are altered in csp KO Drosophila. Since there is only one csp gene in fruit flies, the outcome of these experiments would be easier to interpret. A fourth issue is that acute down-regulation of CSP- in pancreatic cells does not lead to a decline of SNAP-25 (Zhang et al., 1999b). Minimally, this result implies that there are differences between neurons and endocrine cells with respect to CSP function (a similar argument was advanced regarding CSP’s impact on Ca2+ channel function in endocrine cells; see 4.2.1). Fifth, studies of the cortical reaction in frog oocytes have shown that this

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massive exocytotic response relies on CSP’s interaction with Hsc70 (Smith et al., 2005). Because the cortical reaction is a one-time exocytotic event, it does not involve repetitive cycles of exocytosis and endocytosis that could compromise SNAP-25 function. At the same time, efforts to detect SNAP-25 or SNAP-23 in these oocytes have been unsuccessful (C. Gundersen and C. Schietroma, unpublished data), which means that CSP function in this system almost certainly is independent of SNAP-25. By analogy, it is possible that the neurodegenerative phenotype in the csp- KO mice involves targets besides SNAP-25. Sixth, the idea that CSP- exists to re-fold SNAP-25 is inconsistent with its synaptic vesicle localization. As a vesicular protein, CSP- will have greatly restricted access to SNAP-25: binding should be possible when vesicles are docked at the plasma membrane or prior to endocytic retrieval of CSP- after fullcollapse, vesicular exocytosis. It would make more sense for CSP- to be a soluble or plasma membrane protein, if its primary role is to maintain a functional pool of SNAP-25. After all, of the hundreds of synaptic vesicles in a synaptosome, only a tiny percentage are close enough to the plasma membrane (“docked”) to have access to SNAP-25 (Wilhelm et al., 2014). Extending this argument, quantitative analyses indicate that 2-3 SNARE complexes (and therefore, 2-3 SNAP25 molecules) suffice for exocytosis from a single synaptic vesicle (Mohrmann et al., 2010; Sinha et al., 2011). Interestingly, this small number of SNARE complexes is very close to the estimates for the number (3) of CSP- molecules per synaptic vesicle (Takamori et al., 2006; Wilhelm et al., 2014). These considerations further stress the importance of determining quantitatively whether the pool of functional SNAP-25 is depleted to a precarious level in the absence of CSP-. Seventh, Boal and coworkers (2010) reported that CSP- did not bind directly to SNAP-25 (judged by using recombinant proteins), but it did associate indirectly via synaptotagmin-9 in pancreatic  cells. While this result does not support the idea that CSP is a general SNAP-25 chaperone (as proposed by Sharma et al., 2011), it again is possible that CSP- function varies among cell types. Eighth, type A botulinum toxin cleaves and inactivates SNAP-25 which profoundly impairs evoked transmitter release (Schiavo et al., 2000). Botulinum type A intoxication also has no discernible effect on nerve terminal ultrastructure and leads to nerve-terminal sprouting, not neurodegeneration (Thesleff, 1960; Duchen and Strich, 1968; Duchen, 1971; Angaut-Petit et al., 1990). Thus, because type A botulinum toxin depletes the functional pool of SNAP-25 without causing neurodegeneration, it needs to be clarified why the residual SNAP-25 pool in csp- KOs is a trigger for neurodegeneration. Finally, as noted earlier, the work of Andreyeva and colleagues (2010) revealed considerable specificity in chaperone functions at nerve terminals: the CHL1-Hsc70-SGT complex was effective at chaperoning SNAP-25, but not synaptobrevin-2, whereas the opposite was true for the CHL1-CSP- complex. From the perspective that CSP- and synaptobrevin-2 are both synaptic vesicle proteins and will be delivered simultaneously to the plasma membrane (where CHL1 resides) during full-collapse exocytosis, this distinction makes sense. Similarly, because SNAP-25, like CHL1, is a plasma membrane protein, it is readily accessible for chaperone actions in conjunction with soluble Hsc70 and SGT. These results raise clear questions about why CHL1 chaperone function cannot supplant CSP- in maintaining SNAP-25 in the csp- KO mice. In conclusion, the proposed neuroprotective link between CSP- and its chaperoning of SNAP-25 will require further scrutiny. 4.2.4. CSPs and synaptotagmin Direct evidence for a CSP-synaptotagmin link first emerged in studies of the implications of the PKA-dependent phosphorylation of CSP at Ser10 (Evans and Morgan, 2002, and see section 3.3). These investigators identified synaptotagmin-1 as among the proteins that bound to recombinant CSP, in vitro. In addition, CSP and synaptotagmin-1 co-immunoprecipitated (similar co-immunoprecipitation data were reported by Wu et al., 1999). PKA-dependent

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phosphorylation of CSP reduced synaptotagmin’s binding affinity by nearly an order of magnitude. Similar results (Evans et al., 2006) were obtained for the Akt-dependent phosphorylation of CSP on Ser10. Collectively, these findings were advanced as a possible molecular mechanism to explain how CSP could influence the Ca2+ sensitivity of exocytosis. However, it remains unclear how these results relate to the hypothesized changes in Ca2+sensing at nerve terminals of organisms (like, fruit fly larvae) that are deficient in CSP.

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In a separate investigation (Boal et al., 2010), the C2A domain of synaptotagmin 9 was found to bind to CSP via charged residues in the linker region between the J domain and the cysteine string. This interaction was Ca2+ dependent and was studied in cells by using fluorescence resonance energy transfer (FRET) which indicated that these proteins achieve close proximity. These authors speculated that the Ca2+-dependent interaction of CSP and synaptotagmin 9 might aid in positioning CSP at the site of membrane fusion for subsequent interaction with Hsc70 and a target protein. It remains unclear whether these results apply to “fast” synapses which typically use synaptotagmin-1 or -2.

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4.2.5 CSPs and synaptobrevin (VAMP)

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In a speculative scenario, it was hypothesized that CSP might aid in the positioning of synaptotagmin at the interface between a synaptic vesicle and the plasma membrane (Gundersen and Umbach, 2013). This proposal drew on the idea that the disposition of CSP’s fatty acylated cysteine string domain (shown in Fig.3) creates a membrane motif that would enable CSP to “shepherd” synaptotagmins to the site of vesicle-plasma membrane contact. Once the synaptotagmins reach this interface, it was suggested that they play a direct role in catalyzing the membrane fusion step of regulated exocytosis (Gundersen and Umbach, 2013). Obviously, this hypothesis has not been explicitly tested, but it does provide a rationale for the evolutionary conservation of the cysteine string of CSPs, and it offers an indirect mechanism by which CSP could influence the Ca2+ sensing machinery for exocytosis.

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Synaptobrevin-2 (also known as VAMP-2 for vesicle-associated membrane protein) is the major v-SNARE of synaptic vesicles, and synaptobrevins are important for regulated exocytosis in many cell types (Jahn and Scheller, 2006). CSP was originally found to co-immunoprecipitate with synaptobrevin-2 (Leveque et al., 1998), but this interaction apparently was indirect, because recombinant CSP did not bind to synaptobrevin-2, in vitro. Subsequently, a Ca2+dependent, CSP-synaptobrevin-2 interaction was described for pancreatic  cells (Boal et al., 2004), and mutagenesis of the regions of CSP involved in this interaction (linker and Cterminus) largely blunted the impact of CSP overexpression on secretion by these cells. The Ca2+-dependence of the CSP-synaptobrevin interaction reported by Boal and colleagues (2004) may account for the difference between their observations and the results of Leveque et al., (1998). Independently, Weng and co-workers (2009) reported an interaction of CSP- with synaptobrevin-8 on the surface of zymogen granules which they postulated was involved in secretory activity in these cells. At the same time, they could not detect significant interaction of CSP- with synaptobrevin-2. However, there has been no further analysis of the functional relevance of the CSP-synaptobrevin-8 interaction. 4.2.6. CSPs and heterotrimeric G proteins In a series of investigations, CSP- was reported to harbor two discrete sites for interaction with heterotrimeric G proteins or individual subunits of this complex (Magga et al., 2000; Miller et al., 2003a,b; Natochin et al., 2005; Swayne et al., 2005). The region of CSP encoded by amino acid residues 83-112 associated with G and/or G, while a segment of CSP’s N-terminus bound the  subunit, but not the heterotrimeric complex. Occupancy of either of these binding sites

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inhibited N-type Ca2+ channels, as assessed in a transient expression system. Further insight into CSP’s role in this G protein dependent modulation of N type Ca2+ channels was the finding that CSP apparently serves as a catalyst of GDP/GTP exchange for the Gs subunit (but, not Gi). However, as noted in section 4.2.2, the relevance of these results to CSP function in vivo and in other systems remains ambiguous and will require further examination. 4.2.7 CSPs and other prospective “clients”

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In an approach that relies on discerning relative differences in individual protein levels between two empirical situations, Zhang and colleagues (2012) contrasted the constituents of synaptic vesicles, cytosol and synaptic plasma membranes of WT and csp- KO mice. This study identified more than three dozen candidate CSP- clients. A client that the authors regarded with particular interest was dynamin-1, a protein involved in endocytosis. Several additional experiments supported CSP binding directly to dynamin-1 and regulating its polymerization. However, there are concerns regarding the conclusions drawn from this investigation. The most serious issue is that the authors based their work on data from the brains of P28 csp- KO mice. Although they implied that this developmental stage precedes overt neurodegeneration in these mice, this contention is not supported by the published record. Here are specific examples which show convincingly that P28 csp- KO mice exhibit profound neurodegeneration. First, to quote Fernandez-Chacon et al., (2004), “…ultrastructural examination of the Calyx by electron microscopy revealed severe degeneration of nerve terminals in csp- KO mice at P25”. If “severe degeneration” was evident at P25, one can assume that the situation was worse by P28. Second, Fernandez-Chacon et al., (2004) performed an ultrastructural analysis of motor nerve terminals at P23 and reported that 44% had Schwann cell protrusions, 24% had vacuoles and 22% had multilamellar bodies. These results were all significantly different from controls and supported the conclusion that neurodegeneration was a hallmark of the P23 csp- KO mice. Third, in Schmitz et al.(2006), electron microscopic analysis of photoreceptor synapses in P28 KO mice revealed, “…most of the ribbon synapses in the OPL of csp- KO mice displayed a strongly pathological morphology…”. Again, this work showed that by P28, presynaptic degenerative changes were prominent in the csp- KO mice. Finally, Fernandez-Chacon, et al., (2004) reported that by P28, csp- KO mice were less than half the mass of littermate controls and showed profound sensorimotor deficits consistent with a degenerative phenotype. Taken together, the preceding results uniformly support the conclusion that empirical results obtained using P28 mice will be strongly influenced by progressive neurodegeneration. Thus, while the findings of Zhang et al. (2012) may be germane to neurodegeneration, their relevance to CSP function is questionable.

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There are four additional reasons to be skeptical of the proposed role for CSPs in endocytosis. First, FM1-43 dye labeling experiments in csp KO Drosophila revealed no bulk impairment of vesicle recycling (Ranjan et al., 1998). As noted earlier, Drosophila KO data are more reliable in terms of functional insights than the mouse data, because there is a single csp gene in these organisms. Second, the number of quanta discharged in response to black widow spider toxin was indistinguishable between WT and csp mutant Drosophila (Umbach and Gundersen, 1997; Ranjan et al., 1998). These later results indicate that the pool of releasable synaptic vesicles is comparable for control and KO terminals which argues against a large-scale, vesicle-recycling deficit in the absence of CSP. The third argument relies on data from the csp- KO mice. All of the electrophysiological data in Fernandez-Chacon et al., (2004) emphasized the functional equivalence of synaptic parameters in the WT and KO alleles up to two weeks of age. If synaptic vesicle recycling had been significantly impaired in the mutants, transmitter release should have shown decrements. The fact that KOs were indistinguishable from controls two weeks after birth blunts the argument for a recycling defect. This conclusion is also supported

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by electron microscopic data. As these authors noted, at P25 “normal-looking” Calyx nerve terminals were observed next to degenerating terminals. If CSP- were important for synaptic vesicle recycling, Calyx terminals in P25 mice should have had demonstrably fewer vesicles. Thus, the preponderance of evidence currently weighs against a prominent role for CSPs in synaptic vesicle recycling. Nevertheless, observations indicating that dynamin modulates secretory dynamics (Kawasaki et al., 2000; Graham et al., 2002; Shin et al., 2017) offer an alternative pathway that could involve CSP and dynamin, and more research will be needed to evaluate this possibility. 4.3. Functional investigations using antibodies, siRNA, antisense RNA, over-expression or mutagenesis, in vitro

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CSP- has been functionally implicated in the regulated secretory pathway in a wide variety of cells. As the following commentary emphasizes, diverse strategies have been used to investigate the secretory function of CSP-. However, in spite of the wealth of data, we still lack a coherent picture of the molecular role(s) of CSP-in this process. Specific suggestions will be made for areas where future investigations might be particularly informative.

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With the finding that CSPs were affiliated with secretory organelles in non-neuronal cells (see section 2.4), several groups embarked on efforts to discern possible roles for CSP in membrane trafficking. The first clear indication that CSP- had a role in regulated exocytosis independent of Ca2+ channel modulation was the work of Chamberlain and Burgoyne (1998b). They generated PC12 cells exhibiting stable, ~15-fold over-expression of CSP- relative to controls. Cells over-expressing CSP- displayed no detectable change in depolarization-dependent Ca2+ fluxes. However, using digitonin-permeabilized cells, Ca2+-dependent dopamine release was significantly augmented by ~40% relative to controls. Enhanced secretion was also obtained using GTPS as the secretogogue. Overall, these data indicated that CSP did not detectably influence Ca2+ channel function in these cells and that CSP positively modulated exocytosis independently of transmembrane Ca2+ flux. However, the cellular/molecular mechanism of this effect was not further clarified.

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As a counterpoint to the preceding results, a study of transient over-expression of CSP- in a pancreatic -cell line revealed a significant blunting of stimulus-dependent exocytosis (Brown et al., 1998). This effect was observed separately from any impact of CSP- on voltage-dependent Ca2+ channel current, and led the authors to conclude that CSP had a role in exocytosis that could be dissociated from an influence on Ca2+ channel function. A similar conclusion was drawn in a study that used CSP antisense RNA to reduce CSP expression in an insulinsecreting, hamster  cell line (Zhang et al., 1998b). In this study, the authors found that diminished CSP- expression led to a reduction of exocytosis triggered either by KCl depolarization, or by Ca2+ after permeabilization using streptolysin. Extending this theme of a secretory function for CSP in pancreatic  cells, it was reported that CSP- was among several exocytotic proteins that were down-regulated in pancreatic islets of diabetic rats (Zhang et al., 2002). However, the precise function of CSP in the exocytotic cascade was not established. Here, it is worth addressing the somewhat confusing findings in the preceding paragraphs. On one hand, stable over-expression of CSP- augmented exocytosis in PC12 cells, while transient over-expression of CSP- blunted exocytosis in insulin-secreting cells. At the same time, a reduction of CSP- expression also impaired exocytosis in insulin-secreting cells. How might these apparently discordant observations be reconciled? One possibility is that the transient over-expression scenario creates a pool of CSP that is not affiliated with secretory organelles. Then, rather than participating in the normal exocytotic cascade, this pool of CSP may either

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sequester other proteins that are necessary for exocytosis, or interfere with the normal function of vesicle-bound CSP. If one of these scenarios is correct, then the transient over-expression results would be concordant with the other observations. A second consideration is that chromaffin cells typically have many more secretory granules per cell (compared with PC12 cells, which tonically secrete catecholamines; Greene and Rein, 1977a,b), and the vast majority of these granules are not docked at the plasma membrane. Thus, if CSP contributes to a predocking or priming process, empirical interventions should be more apparent in chromaffin cells. Nevertheless, additional work still is needed to clarify the molecular basis of these nominally divergent results.

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As a further step toward clarifying CSP’s secretory role in non-neuronal cells, mutagenesis experiments were undertaken (Zhang et al., 1999a). The model system used in this work relied on the finding that transient over-expression of CSP- in hamster insulinoma cells inhibited exocytosis triggered either by KCl depolarization, or by Ca2+ in permeabilized cells (these results were reminiscent of the data of Brown et al., 1998). Mutations that diminished the extent of secretory inhibition were interpreted as giving insight into functional domains of CSP-. A somewhat unexpected result was that mutation of the HPD triplet in CSP’s J domain (which interferes with activation of the ATPase activity of Hsc70; see section 3.1) did not affect regulated exocytosis in these cells. Instead, the HPD mutation had to be combined with a truncation of the C-terminus of CSP to have an effect. In addition, mutation of the linker region between the J domain and the cysteine string influenced exocytosis. Although these data identified several regions of CSP that were keys to its role in regulating insulin exocytosis, they still did not identify a particular step in the exocytotic sequence that relied on CSP.

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Just as transient CSP- over-expression inhibited insulin secretion (Brown et al., 1998), it also inhibited catecholamine release in bovine chromaffin cells (Graham and Burgoyne, 2000). An important feature of this investigation is that it used amperometry to characterize the effect of CSP- on the total number of secretory events as well as the amplitude and kinetics of individual secretory events. Prominently, the number of amperometric spikes declined by more than 80% in cells over-expressing CSP-. Interestingly, the residual spikes in CSP overexpressing cells showed altered kinetics: a delayed rise time and increased half-width that led to a net increase in the total charge transfer associated with each exocytotic event. These results were interpreted as indicating that CSP- contributes to protein interactions at a late stage in the exocytotic cascade that can influence the characteristics of the fusion pore opening (note that this may be germane to recent findings that dynamin regulates fusion pore opening and that dynamin appears to be an interaction partner for CSP, as noted in 4.2.7). Further insight into the effect of CSP over-expression on individual secretory events in chromaffin cells assessed the impact of a non-phosphorylatable mutant (S10A) of CSP- (Evans et al., 2002). Notably, the S10A mutant partially relieved the ~80% decline in spike number provoked by WT CSP-, and also completely reversed the changes in spike shape. Although the molecular mechanism of these effects on secretory dynamics remains unclear, a recent investigation (Chiang et al., 2014) extended these observations. This group analyzed individual secretory events in PC12 cells over-expressing WT CSP-, the S10A mutant (which cannot be phosphorylated), and two phosphomimetics, S10D and S10E. The data from PC12 cells was somewhat different from chromaffin cells. First, relative to cells with control vector, over-expression of WT CSP- did not significantly reduce the rate of amperometric spikes elicited by KCl depolarization. Second, cells expressing the S10A mutant showed approximately a 30% reduction in secretion rate compared to cells transfected with control vector. Third, the phosphomimetic constructs had the opposite effect with the secretion rate increasing ~25% relative to control cells or those over-expressing WT CSP. Finally, expression of the phosphomimetics prolonged fusion pore lifetime relative to

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WT CSP or S10A, and kinetic analysis suggested that this was due to a delay of fusion pore closing. These results were interpreted as indicating that CSP- participates in regulating fusion pore dynamics and that this role can be modulated by phosphorylation. At this stage in the evolution of our understanding of CSP function, these studies have provided some of the best insights into specific stages of the secretory cascade that are modulated by CSPs. An important step for the future will be to develop and test specific models for how CSP- contributes to the regulation of fusion pore dynamics.

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It is worth digressing again to consider possible explanations for the divergence in results in the amperometric studies using chromaffin cells versus PC12 cells. Of course, beyond the species difference (cow versus rat), a major factor is likely to be the difference in secretory granule dynamics between these cell types. Whereas chromaffin cells maintain a large and relatively stable pool of granules (see discussion in Graham and Burgoyne, 2000), PC12 cells tonically secrete catecholamines (Greene and Rein, 1977a,b). This is important, because it means that newly synthesized CSP constructs have a higher probability of being incorporated into newly formed secretory organelles in the PC12 cells than in the chromaffin cells. Thus, while most of the over-expressed CSP (and CSP variants) in chromaffin cells is probably mis-localized (accumulating in such compartments as the endoplasmic reticulum, Golgi and plasma membrane), a higher proportion of the CSP variants expressed in PC12 cells is likely to be affiliated with secretory granules. This would help to explain why WT CSP did not interfere with secretion in PC12 cells, but had a dominant-negative effect in chromaffin cells. Although additional work will be needed to determine whether this explanation is tenable, it highlights the possibility that the subcellular distribution of CSP is likely to be an important determinant of the functional outcome that is obtained in such experiments. Ideally, future investigations will include data that address the subcellular distribution of the CSP constructs that are expressed in the model system.

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To extend the theme of the preceding paragraph, it is interesting to note that pharmacological up-regulation of CSP- expression in PC12 cells was correlated with augmented secretory function (Umbach et al., 2005). Earlier work had shown that CSP- mRNA content was increased in lithium-treated PC12 cells that were differentiated using nerve growth factor (Cordeiro et al., 2000a,b). Lithium treatment led to a doubling of cellular CSP- content, and it increased the number of amperometric spikes (triggered by K+ depolarization) without detectably changing spike amplitude or kinetics (Umbach et al., 2005). However, this work did not demonstrate that the change in secretory behavior was due to CSP-, so further work will be needed to understand the basis of this phenomenon. Nevertheless, an investigation of the impact of traumatic brain injury revealed concomitant reductions in SNARE proteins and CSP- that could be significantly ameliorated by lithium (Carlson et al., 2017). Thus, further examination of the CSP-lithium link is certainly warranted. Similarly, it remains to be determined whether there are any functional correlates of the increased CSP- mRNA (and/or protein) content reported for regions of rodent brain after treatment with antidepressants (Yamada et al., 2001) or amphetamine (Bowyer and Davies, 1999). Interestingly, it also has been shown that chronic morphine treatment down-regulates CSP- and Hsc70 expression, but up-regulates Hsc90 levels in rat striatum (Abul-Husn et al., 2011). To date, the impact of these changes on striatal synaptic function have not been assessed. Co-culture of dissociated, Xenopus embryonic spinal cord with myotomes leads to the formation of functional neuromuscular junctions, in vitro (Kidokoro and Yeh, 1982). Infusion of CSP- antibody (but, not control antibody) into the cell body of a motor neuron led to a rapid (3-6 min), and in most cases, complete inhibition of evoked neurotransmitter release (Poage et al., 1999). Spontaneous transmitter release was unaffected throughout the period of antibody application.

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These results were reminiscent of the temperature-dependent block of evoked transmitter release in csp null Drosophila and led to the inference that CSP antibody was either interfering with CSP’s regulation of presynaptic Ca2+ channels or that CSP had a downstream role in the evoked secretory cascade. Because presynaptic Ca2+ currents can be recorded in this system (Yazejian et al., 1997), it clearly would be beneficial to perform experiments to assess the impact on these channels of CSP- antibody and to extend this work to the use of recombinant CSP- constructs (as was done by Chen et al., 2002 using chick nerve endings).

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Xenopus oocytes have also been used to study the role of CSP- in exocytosis. These cells harbor an array of large, cortical granules (1-3 m in diameter) which normally are triggered to expel their contents to prevent polyspermy. A prominent advantage of this system is that cortical granule exocytosis is a one-time exocytotic event (in other words, there is no repeated cycle of exocytosis and endocytosis). Cortical granules are produced by the Golgi apparatus and reside in close proximity to the plasma membrane of mature oocytes where sperm binding initiates the intracellular release of Ca2+ which triggers granule exocytosis. Unexpectedly, it was found that activators of protein kinase C can trigger the Ca2+-independent, cortical granule exocytosis in immature oocytes (Bement and Capco, 1989). Because CSP- is present on cortical granules (Gundersen et al., 2001), we investigated the impact on cortical granule exocytosis of expressing recombinant CSP- constructs (Smith et al., 2005). Not surprisingly, there was no detectable turnover of cortical granules in Xenopus oocytes (at least, on a time scale of days; Gundersen et al., 2001), so we found that over-expressed CSP- accumulated in various other membranes, including the plasma membrane. By the time the CSP- content of an oocyte exceeded control by ~20-fold, there was a marked inhibition (80-100%) of cortical granule exocytosis. We hypothesized that this inhibition of secretion was due to the sequestration of CSP-interacting protein(s) at locations that were unproductive for exocytosis. To test this hypothesis, oocytes were induced to over-express the J domain of CSP. This construct also inhibited cortical granule exocytosis, although not as effectively as full-length CSP. Nevertheless, because the J domain interacts with Hsc70, it was noteworthy that mutations of the highly conserved HPD region of the J domain abolished its inhibitory effect on cortical granule exocytosis. These results had two important implications. First, they showed that CSP, in conjunction with Hsc70, had a key role in the exocytotic cascade in these cells. Second, because there is no Ca2+-dependent step in the secretory response triggered by protein kinase C activators (see discussion in Smith et al., 2005), CSP- must have a role in exocytosis that is independent of the Ca2+-sensing machinery. Later work (Schietroma et al., 2007) revealed that CSP- also interacted with the tail domain of a Type 1 myosin (myosin 1E), and constructs that interfered with this interaction inhibited cortical granule exocytosis. However, the molecular role of the CSP-Hsc70 and CSP-myosin 1E interactions in supporting cortical granule exocytosis has not been resolved.

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CSP- has also been implicated in mucin secretion by airway epithelial cells (Park et al., 2008). These authors reported that CSP formed a complex with Hsc70 and the MARCKS (myristoylated, alanine-rich, C kinase substrate) protein. Knockdown of any one of these proteins (by siRNA) inhibited mucin secretion. They hypothesized that the formation of a ternary complex on the surface of mucin granules was necessary for regulated secretion, and later work implicated a type V myosin in this secretory pathway (Lin et al., 2010). However, the precise sequence of interaction of these proteins in driving mucin secretion remains uncertain. In the exocrine pancreas, administration of the J domain region of CSP- (residues 1-82) in permeabilized cells led to enhanced, Ca2+-dependent secretion of digestive enzymes, while mutations that blocked Hsc70 binding eliminated this effect (Weng et al., 2009). CSP interacted

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with synaptobrevin-8, a component of zymogen granules in these cells, and these authors inferred that this interaction was likely to be involved in mediating CSP function in this system. The drug, quercitin, has been reported to increase the dimerization of CSP- and this effect was correlated with an impairment of synapse formation and a diminution of evoked synaptic transmission in snail neurons (Xu et al., 2010). This dimerization was specific for CSP-, because neither Hsc70 nor another J domain protein showed this effect. Given the major impact of quercitin on synaptic transmission (~80% reduction of the response amplitude at 25 M quercitin), it clearly would be interesting to explore further the mechanism of this effect.

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The insulin-dependent insertion of glucose transporters into cell membranes was shown to be altered in 3T3-L1 adipocytes in which CSP- expression was altered (Jambaldorj et al., 2013). The mechanism of this effect was inferred to reflect CSP’s role in modulating the interaction between synaptobrevin-2 and syntaxin 4 which is necessary for the plasma membrane docking of the vesicles that deliver the glucose transporters. This is a potentially novel role for CSP- in vesicle docking, and it will be interesting to determine whether CSP contributes to this stage of vesicle trafficking in other cells.

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In another study, it was found that CSP antibody or a recombinant construct containing both the J domain and linker region augmented amylase secretion from parotid acinar cells (Shimomura et al., 2013). Separately, the linker region or J domain did not affect secretion. As the authors noted, these results are perplexing (particularly, from the standpoint that they imply an inhibitory role for CSP in exocytosis) and additional work will be needed to reconcile these data.

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Studies of the maturation and trafficking of the cystic fibrosis transmembrane conductance regulator (CFTR) protein of lung epithelium provided evidence that CSP- mediates several important events at the level of the endoplasmic reticulum (Zhang et al., 2002, 2006; Schmidt et al., 2009). Setting the stage for this work, CSP- immunoreactivity was detected both in the endoplasmic reticulum and apical plasma membrane of a lung adenocarcinoma cell line, Calu-3 (Zhang et al., 2002). Using Calu-3 extracts, CSP- co-immunoprecipitated with CFTR, and direct binding studies revealed that CSP- bound to the N-terminus and R-domain of CFTR. Coexpression of CSP- in frog oocytes depressed the levels of CFTR as assessed both functionally and by immunoblot. CSP- with point mutations in the J domain and the linker region did not cause a reduction in CFTR protein or function in the oocyte system. These biochemical results were confirmed in transfected HEK293 cells. Taken together, these data pointed to a role for CSP- in the biogenesis and membrane trafficking of CFTR (Zhang et al., 2002). Pursuing further the CSP-CFTR link (Zhang et al., 2006), it was found that the level of mature CFTR decreased while immature CFTR increased in cells over-expressing CSP-. This effect was blunted by mutations in the J domain that impaired CSP-Hsc70 interaction. The immature CFTR co-localized with the endoplasmic reticulum marker, calnexin, and remained detergent soluble implying that it was not aggregating abnormally. Conversely, a ~40% knockdown of CSP- in 3T3 cells led to a fivefold increase of mature CFTR. Overall, these results were taken to indicate that CSP- had an important role in the exit of CFTR from the endoplasmic reticulum, thereby suppressing the maturation and trafficking of this protein to the plasma membrane. The latest insight into this process came with the finding that CSP- also is involved in regulating the proteasomal degradation of CFTR (Schmidt et al., 2009). Among the key observations were that CSP bound directly to an Hsc70-interacting protein with ubiquitin ligase activity, and this interaction promoted the Hsc70-dependent ubiquitination of CFTR and reduced the half-life of immature CFTR. Thus, in addition to playing a role in regulating the exit of CFTR from the endoplasmic reticulum, CSP contributes to decisions about protein degradation in these epithelial cells. The parallels between these observations and the recent

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reports that CSP- participates in a protein quality control pathway will become apparent in the discussion in section 5. 4.4. The tangled string: can general conclusions be drawn from the functional studies of CSP ?

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Currently, a justifiable conclusion from the diverse observations concerning CSP- function is that this protein exhibits co-chaperone/chaperone function for regulated secretion at nerve endings and elsewhere. This argument was cogently made in the review by Chamberlain and Burgoyne (2000), and as the preceding discourse has illustrated, much evidence remains consistent with this view. For instance, many studies have reported that CSP’s J domain is important functionally and that interaction with Hsc70 is a pivotal component of this picture. In addition, CSP- has been shown to interact with SNARE proteins and synaptotagmins, and these data suggest that CSP- influences the organization or progression of reactions that are important for regulated exocytosis. However, what remains unclear is whether CSP- has different “priorities” in different cells and different sub-cellular compartments, or whether diverse experimental approaches tend to highlight CSP interactions with certain targets (say, syntaxin or SNAP-25) at the expense of others (like, synaptotagmin, synaptobrevin or SGT). These questions will only be resolved by further investigation. At the same time, it is clear from csp KO fruit flies that CSP is not required for regulated exocytosis, because evoked neurotransmitter release persists at motor nerve terminals of null mutants at permissive temperatures. In hindsight, it seems that additional efforts to determine the molecular basis of the failure of evoked transmitter release in csp nulls at restrictive temperature could be very informative. For instance, although the initial efforts (Arnold et al., 2004) provided some insight into the role of the cysteine string in sub-cellular targeting and rescue from paralysis, little more has been done to understand the purpose of this conserved motif. Thus, we are left with more than a few loose ends as far as the cellular and molecular role of CSP- in regulated exocytosis is concerned. As for functions of CSP- in processes other than regulated exocytosis, the studies of its contributions to CFTR trafficking from the endoplasmic reticulum to the plasma membrane serve as a clear case study that will be extended by the observations in section 5. 5. CSPs and neurodegeneration

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CSPs have been linked to neurodegeneration in model organisms (Section 5.2) and by the identification of an adult-onset lysosomal storage disorder associated with dominant mutations in the human csp- gene (DNAJC5; see Section 5.1). CSP- has also been implicated in the pathology of other lysosomal storage disorders (Sambri et al., 2016). For further discussion and insights into these topics, consult the reviews by Zinsmaier (2010); Donnelier and Braun (2014), Gorenberg and Chandra (2017) and Roosen et al.,(2019). In addition, a decline of CSP- expression in forebrain but not cerebellum of Alzheimer’s disease patients (that did not include a parallel drop in synaptophysin content) has been reported (Tiwari et al., 2015). This finding (that CSP- declined without a similar reduction in synaptophysin) led these authors to infer that CSP- might be among the molecular steps precipitating the onset of pathology in Alzheimer’s disease. And, in an indirect link to CSPs, a decline of Hsc70 expression at nerve terminals has been tied to a defect in synaptic vesicle recycling in several models of amyotrophic lateral sclerosis (Coyne et al., 2017). But, the most provocative development has been the reports from two groups indicating that an endosomal pool of CSP- is part of the machinery underlying an unconventional pathway for the export from cells of mis-folded proteins (Fontaine et al., 2016; Lee et al., 2016). These results are discussed in Section 5.3. 5.1. Human disease linked to the DNAJC5 gene: ANCL

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Beginning in 2011, several groups converged on the finding that mutations in the csp- gene (DNAJC5; also referenced as the CLN4 gene) led to adult-onset, autosomal-dominant, neuronal ceroid lipofuscinosis (ANCL, ; Benitez et al., 2011; Noskova et al., 2011; Velinov, et al., 2012; Cadieux-Dion et al., 2013). Clinically, patients with ANCL typically show progressive cognitive decline with dementia, generalized seizures and movement disorders that begin as early as ~25 years of age. Compellingly, all four research teams identified two separate mutations in the dnajC5 gene that were linked to this disorder. In one instance, deletion of three nucleotides leads to the loss of Leu116, and in the other case, a single base change replaces Leu115 with an arginine residue (L115R). These mutations target highly conserved leucine residues that reside just downstream of the first cysteine residue (Cys113) of the cysteine string region. According to Noskova and colleagues (2011), these mutations reduced the amount of CSP- detected in brain and impaired palmitoylation and sorting of CSP-. Further biochemical analysis of the impact of these mutations (Greaves et al., 2012) revealed that they caused mistargeting when expressed in PC12 cells. The mis-targeted, mutant proteins were palmitoylated, membrane-associated and formed SDS-resistant aggregates that could be solubilized after removal of the palmitoyl moieties. Similar SDS-resistant complexes were detected in the brain of a patient with the L115R mutation, and these complexes could be dispersed by depalmitoylation. Interestingly, Zhang and Chandra (2014) reported that these aggregates suppressed the chaperone function of CSP-. Collectively, these results provide useful molecular insights into the underlying pathology in these genetic disorders, and an important step will be to determine whether this pathological process can be interrupted in a clinically beneficial fashion. In a follow-up study, Henderson and colleagues (2016) reported that mutation in the csp- gene profoundly altered the expression and localization of its depalmitoylating enzyme, protein palmitoylthioesterase-1 thereby providing further phenotypic insight into disease pathogenesis. In separate reports, Benitez and colleagues (2015) and Benitez and Sands (2017) concluded that the pathology in this disorder may be linked more acutely to a lysosomal function of CSP- rather than a functional disruption at nerve terminals. In this context, an unexpected observation is that CSP over-expression blunted the secretion of lysosomal enzymes into the medium of ANCL fibroblasts. Further work will be needed to clarify the implications of this result. Complementary work by Sambri and colleagues (2016) argued for a link between lysosomal dysfunction and reduced presynaptic levels of CSP- and -synuclein as being keys to diminished SNARE complex formation in lysosomal storage disorders. And, Jarrett and co-workers (2016) provided evidence for prominent cholinergic dysfunction in the leucine-deletion ANCL mutants. Thus, more work will be needed to reconcile the disparate hypotheses concerning the pathological trajectory in this disorder. 5.2. Neurodegeneration in CSP mutant organisms

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CSP null fruit flies provided the first indication that CSP expression is important for survival and neuroprotection (Zinsmaier et al., 1994). Subsequent work revealed that the fruit fly csp gene is among a cluster of genes that influence synaptic and axonal degeneration (Wishart et al., 2012). However, this system has not been exploited systematically to probe further the intrinsic mechanism of neurodegeneration. In contrast, csp- KO mice have been used extensively to study the progressive neurodegeneration in these animals (Fernandez-Chacon et al., 2004; Chandra et al., 2006; Schmitz et al., 2006). However, as discussed in section 4.1.2, the molecular trajectory underlying neurodegeneration in these mice needs to be re-visited in light of the evidence that these animals do not represent a clean excision of CSPs from brain. In a separate contribution to this topic, Kashyap et al., (2014) studied the phenotype of nematode worms with deletions in the csp (dnj-14) locus. As noted in 4.1.3, mutant worms showed delayed development, reduced lifespan, mild motor problems and age-dependent

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degeneration of sensory neurons. Remarkably, a chemical screen revealed that resveratrol ameliorated the phenotypic defects in the mutants, and this effect was shared by the phosphodiesterase inhibitor, rolipram. A follow-up to this work (Chen et al., 2015) indicated that ethosuximide more effectively blunted the neurodegenerative phenotype in the worm csp mutants, and this effect was linked to the ethosuximide-dependent up-regulation of DAF16/FOXO target genes. Clearly, it will be interesting to determine whether these agents exhibit efficacy in delaying/preventing neurodegeneration in other csp mutant organisms (including, humans).

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Finally, in a parallel context, investigation of the molecular perturbations accompanying (and, possibly contributing to) neurodegeneration in a mouse model of prion disease, led to the finding that hippocampal CSP- content declined by >50% by 21 weeks after infection with the murine prion agent, ME7 (Gray et al., 2009). This decline in CSP- included less-severe reductions in two other synaptic vesicle proteins, synaptophysin and synaptobrevin. However, the level of synaptotagmin-1 did not change significantly (Gray et al., 2009). A follow-up to this work (Davies et al., 2015) concluded that the reduced expression of CSP- did not exacerbate the neurodegeneration in experimentally induced prion disease in mice. Thus, it remains of considerable interest to clarify the mechanism underling the selective reduction of some, but not all synaptic vesicle proteins in this model system.

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5.3 The link between CSP- and an unconventional pathway for protein secretion (UPS)

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As background for this section, it was noted earlier (section 2.4) that Eberle and colleagues (1998) systematically analyzed CSP expression in Drosophila tissues in which csp KOs served as negative controls. This study concluded that although CSP was prominently expressed at nerve terminals and in several cells of the male and female reproductive organs, it showed low level expression in virtually all cell types. The finding that CSP- is present in Xenopus oocytes at the earliest stage of oocyte development (Gundersen et al., 2001) underscores this point. The implication of these results is that CSP subserves one or more functions that operate in all cells. In this regard, the investigation of CSP’s role in CFTR trafficking in the endoplasmic reticulum (section 4.3) may serve as one index of its contribution to events that are separate from its participation in regulated exocytosis. The work of Fontaine and colleagues (2016), Lee and colleagues (2016) and Xu et al. (2018) provide another glimpse into the diversity of CSP functions by revealing that an endosomal pool of CSP- is important for the export of mis-folded proteins from cells. Because the clients for this pathway include proteins (like, tau and synuclein) recognized for their link to neurodegeneration, these papers have spurred further interest into the possible involvement of CSP- in disease etiology.

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In their seminal report, Fontaine et al. (2016) observed that over-expression of CSP- in HEK 293 cells (human embryonic kidney), M17 neuroblastoma cells or in primary cultures led to enhanced secretion of co-expressed tau (a microtubule-binding protein), -synuclein and TDP43 (trans-activation response element DNA-binding protein). This secretory pathway was compromised in neurons from csp- KO mice, and it relied on functional Hsc70, because various protocols that interfered with Hsc70 blocked this pathway. Finally, these authors presented evidence that SNAP-23 rather than SNAP-25 was involved in this pathway, because knockdown of SNAP-23 expression compromised client secretion. Collectively, these results identified a novel pathway by which disease-related proteins could be exported from cells. Lee and colleagues (2016) reported results complementary to those of Fontaine et al., (2016). In their study, it was initially observed that GFP (especially, misfolded GFP) was secreted from cells that over-expressed an endoplasmic reticulum-affiliated, de-ubiquitylase (USP19).

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Abundant evidence supported the conclusion that UPS served a key role in recruiting clients for internalization within an endoplasmic reticulum- associated endosomal compartment. In addition, this group found that -synuclein was exported via this pathway, but unlike Fontaine et al. (2016), they did not detect tau secretion. Nevertheless, taken together, these reports clearly established the presence within several cell types of a novel, protein-secretion pathway.

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Extending the 2016 report, Xu and colleagues (2018) reported that the USP19 de-ubiquitylase co-operated with Hsc70 and CSP- in the UPS pathway. They also documented the presence of endosomal and lysosomal pools of CSP- and made the unexpected observation that proteins secreted via UPS could be taken up by endocytosis and degraded. These results suggest that this sequence of events may enable cells with protein quality-control problems to export mis-folded proteins for degradation in surrounding cells. But, as the authors of these reports noted, this pathway also has more-ominous implications, namely, that it may be a mechanism for exposing surrounding cells to potentially toxic agents. This proposal will clearly be subjected to further scrutiny.

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Taken together, the preceding observations regarding UPS clearly implicate CSP- and Hsc70 in a previously uncharted pathway. This work also leaves a number of unanswered questions: for instance, we are just beginning to understand how client proteins are internalized within endosomes; moreover, besides the apparent role for SNAP-23, the rest of the machinery that mediates secretion also remains unclear. And, it obviously would be interesting to determine whether CSP- or - can replace CSP- in UPS. Further adding to the complexity of these observations, Deng and colleagues (2017) independently implicated CSP- in an exosomedependent pathway for removing unfolded protein from cells. Thus, it will be important to discern the relative contributions of these pathways to proteostasis within neurons.

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6. Conclusions

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This review has sought to identify areas of consensus in the study of CSPs, as well as the myriad questions that remain to be answered about these proteins. Abundant research indicates that CSP- contributes to one or more steps of the regulated secretory cascade. However, the specific molecular role(s) of CSP- remain(s) to be clarified. At the same time, it is clear that CSP- functions in other cellular processes as exemplified by its participation in the life cycle of CFTR and its involvement in UPS. Among the outstanding issues that still need attention are the biology of CSPs- and -. Their expression in reproductive tissue is unquestioned, but additional work is needed to further our understanding of their structure, cellular distribution and function. At the same time, CSP- appears to have a multi-faceted role in neurodegenerationneuroprotection. While specific mutations in CSP- are causal for a late-onset form of ANCL, genetic deletion of the csp- gene uncovers a separate neurodegenerative pathway. And, while this later result led to the notion that CSP- is “neuroprotective”, the recent findings concerning UPS suggest that neuroprotection may be balanced by a separate role for CSP- that has the potential to exacerbate neurodegeneration. Collectively, future studies of CSPs promise to illuminate areas ranging from structural biology, cell biology and enzymology to disease etiology. Acknowledgements: The author thanks Konrad Zinsmaier, who provided extensive, critical comments concerning this manuscript. Alessandro Mastrogiacomo kindly assisted with the figures and offered helpful suggestions. Thanks to Michael Phelps for resources.

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References

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Figure Legends:

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Figure 1: Drosophila CSP sequence and domain structure. A) This is the sequence of one of the two longest splice variants (isoform B) of the Drosophila protein. The cysteine string is highlighted in red. Note the 11 consecutive C residues. CSPs typically range from ~175-250 amino acids residues. The sequence coloring used a program described by: Combet C., Blanchet C., Geourjon C. and Deléage G. (2000) TIBS 291:147-150. B) Generic domain structure of cysteine string proteins. CSPs contain a J domain separated from the cysteine string by a linker and a C-end with a strongly acidic pI in CSPs- and . The reader is encouraged to consult the paper by Patel and colleagues (2016) for the structure of the native and Ser10 phosphorylated N-terminal half of CSP-.

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Figure 2: A) Clustal alignment of CSPs  and . The sequences are of the human proteins and identify identical (*) and evolutionarily conserved (:) residues. B) Clustal alignment of csps from invertebrate and vertebrate species with the same designation for identical and conserved residues. Accession numbers are: nematode worm (C. elegans: NP_001257015); flatworm (Schistosoma japonicum: CAX74426.1); honeybee (Apis cerana: AEY59388.1); mosquito (Anopheles darling: ETN61409.1); zebrafish (Danio rerio: NP_001002464.1); frog (Xenopus laevis: O42196.1); chicken (Gallus gallus: NP_001264938); cow (Bos taurus: AAI20235.1). Note that the sequence conservation up to and through the cysteine string is greater than in the carboxyl terminal region of these proteins in which only 3 residues remain identical. Nevertheless, these proteins all share a cysteine-rich cysteine string and a J domain.

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MNSDGLREAEEGRTSGGASPREESPAADHSHDPKKGLHLYNVLGIQKNATDDEIKKAYRK MPP-------------DSSRGDNSKNKGGQKSSKENINLYAILEVDRNATAEEIRKSYRR --------------------------MDKRKMSTAGDSLYQILEIPKTATPEEIKRTYRK --------------------------MDKRKLSTSGDTLYQTLGLQKTATADEIKKTYRK ----------------------MAEQQRQRSLSTSGESLYHVLGVDKVATVDDIKKSYRK -----------------------MADQRQRSLSTSGESLYHVLGLDKNATTDDIKKCYRK -----------------------MADQRQRSLSTSGESLYHVLGLDKNATSDDIKKSYRK -----------------------MADQRQRSLSTSGESLYHVLGLDKNATSDDIKKSYRK . . ** * : : ** ::*:: **:

60 47 34 34 38 37 37 37

nematode flatworm honeybee mosquito zebrafish frog chicken cow

LALRYHPDKNLDGDPEKTEMFKEINYANAVLSNPNKRRVYDEMGETGLKLMEQFGEDEKI LALKYHPDKNVK-DPGASEKFKEINRAHSILANEQKRKLYDRYGSLGIYVAEHIDEEDWK LALKYHPDKNPN-NPEAAEKFKEINRAHAILTDLTKRNIYDNYGSLGLYVAEQFGEENVN LALKYHPDKNPN-NPDAADKFKEVNRAHSILSDLTKRNIYDNYGSLGLYIAEQFGEENVN LALKYHPDKNPD-NPEAADKFKEINNAHAILNDPTKRNIYDKYGSLGLYVAEQFGEENVN LALKYHPDKNPD-NPEASEKFKEINNAHGILADSTKRNIYDKYGSLGLYVAEQFGEENVN LALKYHPDKNPD-NPEAAEKFKEINNAHAILTDATKRNIYDKYGSLGLYVAEQFGEENVN LALKYHPDKNPD-NPEAADKFKEINNAHAILTDATKRNIYDKYGSLGLYVAEQFGEENVN ***:****** . :* :: ***:* *:.:* : **.:**. *. *: : *::.*::

120 106 93 93 97 96 96 96

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LQWMLKPWFKWTFFAFGLLTGGFFCCCCGCMCCCQCCCNFCCGKYKPKHDDEFADET--PYLALRNPCVQCLACTCFLLTCCCCC-----LCCCYCCGTLYPRNHPVPDEMNEAFEEPH AYFVVTSGWCKALFIFCSLITACYCC-----CCCCFCCNFCCGKFKPTPPEDSGAYHNLQ AYFVVTSPTCKALFMICGIITGCYCC-----CCCCCCCNFCFGKYKPVPPENAGDYHHLH TYFVLSSWWAKALFIFCGLATGCYFC-----CCLCCCCNCCCGKCKPRPPDRPDPEFYVS

177 161 148 148 152

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nematode flatworm honeybee mosquito zebrafish frog chicken cow

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CLUSTAL O(1.2.4) multiple sequence alignment

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frog chicken cow

TYFVLSSWWAKALFMFCGLITGCYCC-----CCLCCCCNCCCGKCKPRPPEGEDQDIYVS 151 TYFVLSSWWAKALFVFCGLITGCYCC-----CCLCCCCNCCCGKCKPKPPEGEEQEYYVS 151 TYFVLSSWWAKALFIFCGLLTCCYCC-----CCLCCCFNCCCGKCKPKAPEGEETEFYVS 151 : : : * * * . : :* :

nematode flatworm honeybee mosquito zebrafish frog chicken cow

------------------------------------SDGDVIVDQPTASEPMPDTNNRQV FEM-PMNMS---------------------------NAETFVTKQPNPA---P------A RDQNPEANV-------VTNQPRGLVKEEE-------SDDEAITAQPQGG---PQNNSTNQ RGDGGASSSNAGDATAVTEQPSRREDLADLDDNGGSSAGGPVTAQPQAG---QA--QGAG PEDLE--------------------AQL-QSDERETVGGEPIVLQPSSA---TETTQLTS PEDLE--------------------AQM-QSDERDT-E-GPVLVQPASA---TETTQLTS PEDLE--------------------AQL-QSDEREASD-APIVIQPASA---TETTQLTA PEDLE--------------------AQL-QSDEREAAD-TPIVIQPASA---TETTQLTA : ** .

nematode flatworm honeybee mosquito zebrafish frog chicken cow

PIVIAMPPPPSQKYGD---------------------------VNLYAMPATSSS--------------EFPTDNKQ---------QPIFAMPPPPATVDE---------NTNLNSGAERVIYTTGITST MPVYAMPPPSANPTNPFTGSTATESTGLNTGNQ-PVYTPGR--DGY----HTT-----------YHTDTGFN--------------DS-----HAS-----------YHTD-GFN--------------DS-----HPS-----------YHTD-GFN--------------DS-----HPS-----------YHTD-GFN---------------

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217 204 226 243 202 197 198 198

201 184 191 203 188 185 186 186

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Figure 3: Inter-bilayer topology proposed for the fatty acylated cysteine string of CSP-. This is the amino acid sequence (single letter code) from human CSP- represented at the interface between the hemi-bilayers of a phospholipid membrane. The dark red “squiggles” associated with the C residues are the cysteinyl fatty acyl modifications which project anti-parallel to the membrane phospholipid acyl chains (phospholipids are represented as blue spheres with pink acyl moieties). The lysyl (K) residue near the C-end presumably is post-translationally modified, neutralized by a counter-ion, or is not membrane embedded. In a bona fide biological membrane, the phospholipid packing will be denser than in this representation which is intended to accentuate the proposed location of the cysteine string and the cysteinyl acyl moieties.

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Figure 4: Schematic representation of the two different mechanisms by which complete fatty acylation of a cysteinyl, -strand could be achieved. Each schematic considers 7 consecutive C (cysteine) residues (Drosophila CSP has 11 consecutive Cs) in a -strand. This conformation allows a DHHC palmitoyltransferase initially to fatty acylate (squiggles) all of the C residues that face in the same direction. However, to fatty acylate the remaining C residues (which, in a strand, orient their –SH groups ~180o relative to their fatty acylated neighbors) requires one of these alternatives: A) scenario 1: the C residues are positioned at the hemi-bilayer interface (panel 1) and the DHHC enzyme-palmitoylCoA complex flips (panel 2 to 3) the palmitoyl CoA within the membrane interior to allow the fatty acyl transferase reaction to occur within the enzyme interior represented by the dark orange triangle. B) scenario 2: here, the partially acylated cysteine string initially resides at the membrane-cytosol interface (panel 1). This locus

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requires the palmitoyltransferase to rotate the fatty acyl group of palmitoyl CoA into the aqueous cytosol (panel 2 to panel 3) to effect the fatty acylation of each remaining C residue. In this scenario, the fatty acylated C residue must then become “buried” within the membrane (as illustrated in Fig.3). The reactions represented by both scenarios must be repeated until the appropriate number of string C residues is fatty acylated.

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Fig.5: Expression of CSP- in a kidney epithelial cell line (RPTEC TERT1). This image is from the Human Protein Atlas (see Thul et al., 2017) and used antibody HPA0077389. Blue is nuclei; red structures are microtubules and green is CSP-. This punctate distribution is consistent with a vesicular/granular affiliation of CSP-. The scale bar is 10 m.

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