Protein palmitoylation in the development and plasticity of neuronal connections

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ScienceDirect Protein palmitoylation in the development and plasticity of neuronal connections Andrea K Globa and Shernaz X Bamji Protein palmitoylation, or the reversible addition of the fatty acid, palmitate, onto substrate proteins, can impact the structure and stability of proteins as well as regulate protein– protein interactions and the trafficking and localization of proteins to cell membranes. This posttranslational modification is mediated by palmitoyl-acyltransferases, consisting of a family of 23 zDHHC proteins in mammals. This review focuses on the subcellular distribution of zDHHC proteins within the neuron and the regulation of zDHHC trafficking and function by synaptic activity. We review recent studies identifying actin binding proteins, cell adhesion molecules and synaptic scaffolding proteins as targets of palmitoylation, and examine the implications of activity-mediated palmitoylation in the establishment and plasticity of neuronal connections. Address Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, British Columbia V6T 1Z3, Canada Corresponding author: Bamji, Shernaz X ([email protected])

Current Opinion in Neurobiology 2017, 45:xx–yy This review comes from a themed issue on Molecular neuroscience Edited by Susumu Tomita and Brenda Bloodgood

http://dx.doi.org/10.1016/j.conb.2017.02.016 0959-4388/ã 2017 Elsevier Ltd. All rights reserved.

Introduction Protein palmitoylation is the most common form of protein S-acylation in eukaryotic cells and involves the reversible addition of the fatty acid, palmitate, to cysteine residues of the substrate protein. This lipid modification is mediated by a family of multi-pass transmembrane proteins containing a conserved aspartate-histidine-histidine-cysteine (DHHC) motif required for its palmitoylacyltransferase (PAT) activity [1,2]. The DHHC catalytic motif is located within a cysteine-rich, zinc finger-like domain, resulting in the current, standard ‘zDHHC’ nomenclature. It is important to note that DHHC and zDHHC nomenclatures are not interchangeable, and that some clone numbers initially collected in Fukata et al. [3] using the ‘DHHC’ nomenclature are different from the ‘zDHHC’ nomenclature. For clarity, the ‘zDHHC’ www.sciencedirect.com

nomenclature will be used in this review. To date, 23 mammalian zDHHC proteins have been identified with the majority of them being validated as having PAT function in yeast [4] and in mammalian cells [3]. As palmitoylation is a reversible modification, the enzymes responsible for depalmitoylation are also of great interest to researchers. All palmitoyl-protein thioesterases (PPTs) identified to date contain a/b-hydrolase domains (ABHD proteins) [5]; however, the search for other families of enzymes with PPT activity continues. Palmitoylation/depalmitoylation cycles vary greatly between substrates [6,7,8,9–11,12,13]. Rapid palmitate turnover is likely responsible for regulating local, dynamic cellular events such as activity-mediated protein trafficking in neurons [6,7,8,9,11,12], whereas slower palmitate turnover has been observed in the long-term static targeting of proteins to cell membranes [10,13]. It stands to reason that PATs and PPTs that rapidly palmitoylate/depalmitoylate proteins are localized in close proximity to their substrates including specific subcellular regions within axons and dendrites. In contrast, PATs that mediate the long-term static targeting of proteins to cell membranes are typically localized to the somatic golgi [10,13–15]. Detailed discussion of the palmitoylation of glutamate receptors [16] neuronal kinases [17], and presynaptic vesicle machinery [18] has been reviewed elsewhere. Here we review recent studies identifying actin binding proteins, cell adhesion molecules and synaptic scaffolding proteins as targets of palmitoylation, and discuss how activity-mediated palmitoylation of these substrates can regulate the establishment and plasticity of neuronal connections.

Subcellular localization of zDHHC proteins in neurons According to the Allen Brain Atlas almost half of all zDHHCs are detectable in the brain [19] (Table 1). Although some PATs, including zDHHCs 5, 9 and 17, are ubiquitously expressed in the brain, a subset of PATs exhibit highly specific expression patterns including high expression of zDHHCs 2 and 7 in CA1 hippocampal pyramidal neurons, and strong expression of zDHHCs 5 and 7 in cerebellar Purkinje cells. Work from the Barres lab has examined zDHHC mRNAs in specific cell types derived from mouse cortical samples, providing a deeper understanding of zDHHC expression in the brain [20] (Table 1). Current Opinion in Neurobiology 2017, 45:1–11

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zDHHC

Brain expression pattern a

Cell type b

Subcellular localization c

zDHHC1

zDHHC2

Medium: CA1 hippocampus [56]

Medium: cortical astrocytes, cortical neurons, stellate/basket cells, corticostriatal neurons Low: cortical oligodendrocytes, cortical microglia

Neurochondrin [24]

High: cortex, CA1 hippocampus [57] Medium: hypothalamus, olfactory bulb, pallidum Low: striatum

High: cortical neurons, cortical oligodendrocyte precursor cells, cortical newly formed oligodendrocytes Medium: cortical astrocytes, cortical myelinating oligodendrocytes, cerebellar granule cells, striatal cholinergic neurons, cortical olig2+ oligodendrocytes Low: cerebellar olig2+ oligodendrocytes, motor neurons Medium: cortical oligodendrocyte precursor cells, cortical endothelial cells Low: cortical astrocytes, cortical neurons, cortical newly forming oligodendrocytes, cortical myelinating oligodendrocytes, cortical microglia, cerebellar granule cells, Drd1 medium spiny neurons

Yeast: ER Neurons: dendrites, early endosomes when overexpressed [24] Yeast: ER/Golgi Neurons: dendrites; activity dependent movement from shaft to spine after activity blockade [8,25]

Yeast: Golgi Neurons: somatic golgi [9,25]

PSD95, SNAP25, SNAP23, Gas, Gaq, Gai2, CSP, GABAAg2, eNOS, GluA1/2, GAD65, STREX, Fyn, BACE1, NDE1, NDEL1, NCAM140f, CaMKIg, NR2A/B, Neurochondrin [24] BACE1

zDHHC3 (GODZ)

zDHHC4

zDHHC5

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zDHHC6

Ubiquitous Expression. High: cortex, olfactory bulb, hippocampus, pallidum, thalamus, hypothalamus, pons, medulla, cerebellum (specifically Purkinje cell layer) Medium: striatum, midbrain. Low: cortex, olfactory bulb

Medium: cortiacal neurons, cortical astrocytes, cortical oligodendrocytes, cortical microglia, cortical endothelial cells Low: Drd2 medium spiny neurons, striatal cholinergic neurons, motor neurons High: Corticostriatal neurons Medium: cortical neurons, cortical oligodendrocyte precursor cells, cortical microglia, cortical endothelial cells Low: cortical astrocytes, cortical newly formed oligodendrocytes, cortical myelinating oligodendrocytes, Purkinje cells, stellate/basket cells

Medium: cortical oligodendrocyte precursor cells, cortical endothelial cells Low: cortical neurons, cortical astrocytes, cortical newly formed oligodendrocytes, cortical myelinating oligodendrocytes, cortical microglia, Drd2 medium spiny neurons, striatal cholinergic neurons, forebrain cholinergic neurons, corticospinal neurons, corticostriatal neurons

Yeast: Golgi

Substrates d

Disease associations e

PSD95, SNAP25, SNAP23, eNOS, Fyn, NDE1, NDEL1, CD151, CKAP4, ABCA1, GAP43, Tetraspanins CD9/ CD151, CKAP4/p63

Yeast: Plasma Membrane Neurons: dendrites, excitatory and inhibitory synapses. Activitydependent movement from PM to recycling endosomes [7] but others found stronger localization in dendritic shaft [9]

STREX, flotillin-2 [58], GRIP1 [9], d-catenin [6], somatostatin receptor 5 [59]

Yeast: ER

Calnexin [60], Inositol 1,4,5triphosphate receptor [61]

Schizophrenia [51]

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Localization, known substrates and disease associations of zDHHCs in the brain

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Table 1

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zDHHC

Brain expression pattern a

Cell type b

Subcellular localization c

Substrates d

zDHHC7

High: cortex, olfactory bulb, CA1 hippocampus Medium: striatum, pallidum, pons medulla, cerebellum (specifically Purkinje cell layer) Low: midbrain

High: cortical newly formed oligodendrocyte Medium: cortical oligodendrocyte precursor cells, cortical microglia, cortical endothelial cells Low: cortical neurons, cortical astrocytes, cortical myelinating oligodendrocytes, Drd1 medium spiny neurons, corticostriatal neurons

Yeast: Golgi Neurons: somatic golgi [9]

zDHHC8

High: cortex, olfactory bulb, hippocampus

Yeast: Golgi Neurons: dendrites, at synapses [9]

zDHHC9

Ubiquitous expression. High: cortex Medium: hippocampus, striatum, pallidum, thalamus, midbrain, pons, medulla, cerebellum, olfactory bulb Low: hypothalamus

Medium: cortical neurons, corticostriatal neurons Low: cortical astrocytes, cortical oligodendrocytes, cortical microglia, cortical endothelial cells, Purkinje neurons, cerebellar granule cells, stellate/basket cells, corticothalamic neurons, corticospinal neurons, cortical cortistatin neurons, cortical CCK neurons High: cortical newly formed oligodendrocytes, cortical myelinating oligodendrocytes, cortical endothelial cells, cortical cmtm5+ oligodendrocytes Medium: cortical neurons, cortical astrocytes, cortical oligodendrocyte precursor cells, cortical microglia Low: cerebellar olig2+ oligodendrocytes, striatal cholinergic neurons, corticothalamic neurons, corticospinal neurons, corticostriatal neurons, cortical cortistatin neurons cortical olig2+ oligodendrocytes High: cortical newly formed oligodendrocytes, cerebellar astrocytes, cortical astrocytes Medium: cortical myelinating oligodendrocytes, cerebellar olig2+ oligodendrocytes, corticostriatal neurons, cortical cmtm5+ and olig2+ oligodendrocytes Low: cortical neurons, cortical astrocytes, cortical oligodendrocyte precursor cells, cortical microglia, cortical endothelial cells, Drd1 medium spiny neurons, motor neurons, cortical cortistatin neurons Low: cortical neurons, cortical astrocytes, cortical oligodendrocytes, cortical microglia, cortical endothelial cells, corticothalamic neurons, corticospinal neurons, corticostriatal neurons

PSD95, GAP43, SNAP25, SNAP23, Gas, Gaq, Gai2, CSP, GABAAg2, eNOS, STREX, Fyn, BACE1, NDE1, NDEL1, NCAM140, sortillin, PDE10A2, CSP, GABAAg2, eNOS, PSD95, NCAM, Neurochondrin [24], APP [52] eNOS, SNAP25, paralemmin-1, GAD65, PSD95, PSD93, ABCA1, PICK1 [33], GRIP1 [9]

zDHHC12

zDHHC13 (DHHC22, HIP14L)

High: CA1 hippocampus [56]

Yeast: ER/Golgi

Cofactor GCP16 required for enzymatic activity STREX, H-Ras, NRas,

Yeast: ER/Golgi Neurons: Somatic Golgi, golgi outposts (when overexpressed) [12]

ABCA1, APP, Gephyrin [12]

Yeast: ER/Golgi

Disease associations e Alzheimer’s disease [52]

22q11 deletion syndrome (includes schizophrenia, autism, attention deficit disorder, mood disorders) X-linked mental retardation

Alzheimer’s Disease [53] (but see Ref. [52])

Huntington’s disease

Palmitoylation in neuronal connectivity Globa and Bamji 3

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Table 1 (Continued )

zDHHC14

High: CA1 hippocampus [56]

zDHHC15

Low: CA1 hippocampus [56]

zDHHC16

zDHHC17 (Hip14)

Ubiquitous expression. High: cortex, olfactory bulb, hippocampus, striatum, pallidum, thalamus, hypothalamus, midbrain Medium: pons, medulla, cerebellum

zDHHC18

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zDHHC20

Medium: cortex, hippocampus, pallidum, hypothalamus, midbrain, medulla, pons

zDHHC21

Low: CA1 hippocampus [56]

zDHHC22

High: thalamus Medium: medulla Low: midbrain, cortex, hypothalamus, CA1 hippocampus [56] Low: hippocampus (not present in dentate gyrus) [56]

zDHHC23 (DHHC11)

Cell type b High: cortical newly formed oligodendrocytes, purkinje cells Medium: cortical oligodendrocyte precursor cells, cortical myelinating oligodendrocytes, corticospinal neurons, cortical cortistatin neurons, cortical CCK neurons Low: cortical neurons, cortical microglia, cerebellar golgi cells, stellate/basket cells, corticothalamic neurons, corticostriatal neurons Medium: cerebellar olig2+ oligodendrocytes Low: cortical neurons, cortical astrocytes, cortical oligodendrocyte precursor cells, cortical newly forming oligodendrocytes, cortical endothelial cells

Subcellular localization c

Substrates d

Yeast: ER

Yeast: Golgi

PSD95, GAP43, SNAP25b, CSP, GABAAg2, Fyn, BACE1, CD151, CIMPR, sortillin

X-linked mental retardation

Huntington’s disease

Medium: cortical neurons, cortical astrocytes, cortical olig2+ oligodendrocytes, cortical microglia, cortical endothelial cells High: cortical astrocytes, cortical newly formed oligodendrocytes Medium: cortical neurons, cortical oligodendrocyte precursor cells, cortical myelinating oligodendrocytes Low: cortical microglia, cortical endothelial cells

Yeast: ER

Medium: cortical neurons, cortical astrocytes, cortical oligodendrocyte precursor cells, cortical microglia, cortical endothelial cells Low: cortical newly formed oligodendrocytes, cortical myelinating oligodendrocytes High: cortical endothelial cells Medium: cortical astrocytes, cortical oligodendrocyte precursor cells, cortical newly formed oligodendrocytes Low: cortical neurons, cortical myelinating oligodendrocytes, cortical microglia High: cortical astrocytes Medium: cortical neurons Low: cortical oligodendrocytes, endothelial cells High: cortical neurons Low: cortical oligodendrocyte precursor cells, cortical newly formed oligodendrocytes

Yeast: Golgi

Lck, SNAP25, SNAP23, CSP, huntingtin, GluA1/2, GAD65, STREX, PSD-95, synaptotagmin I, ClipR-59 [62], MPP1/ p55 [63], JNK [64,65] Lck, H-Ras

Yeast: Plasma Membrane

Fyn, BACE1, ABCA1

Yeast: Plasma Membrane

Fyn, eNOS, Lck, ABCA1, APP [52]

Low: cortical neurons, cortical oligodendrocyte precursor cells, cortical newly formed oligodendrocytes

Disease associations e

Yeast: Golgi Neurons: presynaptic terminals in drosophila [22,23]

BK Channels [66]

Yeast: ER

BK Channels [66]

Alzheimer’s Disease [52]

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Brain expression pattern a

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Table 1 (Continued ) zDHHC

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a Brain expression pattern according to Allen Brain Atlas in situ hybridization studies [19]. Expression was defined as low, medium or high based on reported raw expression values (0–1.5 low, 1.5–3 medium, >3 high). zDHHCs written in blue did not exhibit observable expression in the Allen Brain Atlas, but have been reported in the brain in other studies [20,56,57]. b Cell expression pattern according to cortical RNA-seq transcriptome and splicing database [20] and whole brain translating ribosome affinity purification (TRAP) database [67]. These data were generated by isolating neurons, astrocytes, microglia endothelial cells, and oligodendrocytes from mouse cortical cells [20] or by isolating 24 distinct neural and glial cell populations from cerebellum, spinal cord, striatum, basal forebrain, brainstem and cortex of mouse brain [67]. Expression was defined as low, medium or high based on reported fragments per kilobase of transcript sequence per million mapped fragments (FPKM) values (1–10 low, 11–20 medium, >20 high) [20], or based on IP/UB ratios, which were calculated using the enrichment for each mRNA immunoprecipitated from the targeted cell type (IP) versus the expression in the tissue sample dissected (UB; 1–4 low, 5–9 medium, >10 high) [67]. Although not listed in the table, RNA-seq expression studies within the hippocampus have also identified the expression of zDHHCs in distinct subregions [57]. c Subcellular localization in yeast as described by Ref. [68], subcellular localization in neurons as described by cited papers. d Known substrates of zDHHC protein (reviewed in Refs. [54,69,70], unless otherwise cited). e Known neurological disease associations (as reviewed in Refs. [54,55], unless otherwise cited).

Yeast: ER Low: cortical neurons, cortical astrocytes, cortical oligodendrocyte precursor cells, cortical newly formed oligodendrocytes, cortical myelinating oligodendrocytes, cortical microglia, cortical endothelial cells zDHHC24 (DHHC13)

Table 1 (Continued )

zDHHC

Brain expression pattern a

Cell type b

Subcellular localization c

Substrates d

Disease associations e

Palmitoylation in neuronal connectivity Globa and Bamji 5

Over 40% of all known synaptic proteins are substrates for palmitoylation, with 419 of the 1028 known synaptic proteins being identified as substrates for palmitoylation [21]. Many synaptic proteins exhibit a rapid rate of palmitate turnover [6,7,9,11], suggesting that zDHHC proteins are expressed in axons and dendrites. To date, only zDHHC17 has been detected in axons [22,23], while zDHHCs 1 [24], 2 [8,25], 5 [7,9], 8 [9], and 12 [12] have been detected in dendrites. zDHHCs 2, 5 and 8 are all prominently expressed at the plasma membrane and appear to localize to a subset of postsynaptic compartments. Whereas Thomas et al. reported that zDHHC8 but not zDHHC5 is highly localized to synapses [9], Brigidi et al. observed that zDHHC5 is localized to 80% of excitatory and 47% of inhibitory synapses [7]. This discrepancy may be due to culture conditions and the overall activity of neurons in the culture (see section below). zDHHC2 also localizes to postsynaptic compartments to varying degrees depending on neuronal activity [25]. zDHHCs 2, 5 and 8 colocalize with the transferrin receptor, (TfR) [7,8,9], indicating that they are trafficked to and from the postsynaptic membrane on recycling endosomes (RE) [9,7,8,26]. zDHHC12 colocalizes with the golgi marker, giantin, in dendrites indicating its localization at golgi outposts [12]. Additional analysis of the subcellular localization of zDHHC proteins will provide further clues about their function in neurons.

Activity-mediated regulation of zDHHC trafficking A number of synaptic proteins have been shown to be palmitoylated in response to changes in synaptic activity; however the molecular mechanism(s) underlying activityinduced protein palmitoylation is less well understood. One mechanism involves activity-induced trafficking of zDHHC proteins and alterations in their proximity to their substrates. Previous work has shown that prolonged TTX treatment promotes the recruitment of zDHHC2 to the post-synaptic density, where it increasingly palmitoylates its substrate, PSD-95, and promotes PSD-95’s association with the post-synaptic density and its ability to cluster postsynaptic receptors [25] (and reviewed in Ref. [27]). Brigidi et al. have demonstrated that the subcellular trafficking of zDHHC5 can also be regulated by synaptic activity (Figure 1). Under basal conditions, zDHHC5 is localized to the plasma membrane and interacts with both PSD-95 and Fyn kinase through zDHHC5’s C-terminal PDZ binding motif and polyproline repeats, respectively [7]. zDHHC5 is maintained at the plasma membrane through Fyn kinase-mediated phosphorylation of tyrosine residue 533, which lies within the endocytic motif of zDHHC5. Increasing neuronal activity using an established chemical long-term potentiation (cLTP) protocol leads to the rapid dissociation of the zDHHC5/Fyn/ PSD-95 complex, resulting in the dephosphorylation of Current Opinion in Neurobiology 2017, 45:1–11

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Figure 1

zDHHC5

N-cadherin

zDHHC2

AMPAR

zDHHC12

NMDAR

PSD-95

GABAAR

δ-catenin

Fyn Kinase

cLTP

a

1 6

zDHHC5

δ

Fyn PSD95

5

d Ca2+

δ

c 2

4

Gephyrin

AKAP

AKAP79/150

AK AP

δ

GABAAR activation

D

A b

3

C zDHHC2

δ

δ

AKAP

AKAP

B

zDHHC12

Gephyrin

Brigidi et al., 2014, 2015 6,7

Dejanovic et al., 2014 12

(1) Enhanced synaptic activity (2) Internalization of DHHC5 from dendritic spines to shafts on REs (3) Palmitoylation of δ-catenin by DHHC5 (4) Trafficking of DHHC5 and δ-catenin to spines and insertion into membrane (5) δ-catenin binding to cadherin (6) Stabilization of cadherin and AMPAR at the membrane

(A) Activation of GABAAR (B) Palmitoylation of gephyrin by DHHC12 at Golgi outposts (C) Recruitment of palmitoylated gephyrin to the membrane (D) Recruitment of GABAAR

Woolfrey et al., 2015 8 (a) Enhanced synaptic activity (b) Palmitoylation of AKAP by DHHC2 (c) AKAP-mediated exocytosis of RE (d) Increased localization of AMPAR at the postsynaptic membrane Current Opinion in Neurobiology

Role of palmitoylation on activity-mediated synaptic plasticity. Schematic depicting activity-mediated trafficking of zDHHC proteins and palmitoylated substrates and their impact on the clustering of postsynaptic receptors and synapse plasticity.

zDHHC5 and its translocation from dendritic spines to shafts on REs. Although it is still unclear which phosphatase dephosphorylates zDHHC5 to enable its association with endocytic proteins, one candidate includes striatal enriched phosphatase (STEP) 61, a phosphatase known to dephosphorylate both Fyn [28] as well as its substrates [29], in an activity-dependent manner [30]. The dynamic, activity-regulated localization of zDHHC5 suggests this PAT may differentially palmitoylate substrates upon fluctuation in synaptic activity.

Activity-mediated regulation of protein palmitoylation Palmitoylation of soluble proteins

zDHHC5 has been shown to palmitoylate both d-catenin, a cadherin binding protein enriched in dendritic Current Opinion in Neurobiology 2017, 45:1–11

shafts, and GRIP1B, a glutamate receptor interacting protein shown to localize to postsynaptic spines, under basal conditions [31]. Activity-dependent trafficking of zDHHC5 from postsynaptic spines to dendritic shafts increases the palmitoylation of d-catenin, resulting in the recruitment of d-catenin to cadherin adhesion complexes at postsynaptic spines and the stabilization of N-cadherin and a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors (AMPARs) at the postsynaptic membrane [7] (Figure 1). Inhibiting zDHHC5 internalization following increased synaptic activity abolishes the palmitoylation of d-catenin, its recruitment to postsynaptic spines, and the strengthening of synaptic connections [7]. Importantly, increased d-catenin palmitoylation is observed following the acquisition of a fear memory, suggesting that d-catenin www.sciencedirect.com

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palmitoylation is a key event in synapse plasticity associated with learning events. Palmitoylation of GRIP1B by zDHHC5 and 8 has been shown to increase AMPAR turnover [9]. It is therefore tempting to speculate that zDHHC5 trafficking from spines to shafts enhances AMPAR stabilization by both increasing d-catenin palmitoylation and decreasing GRIP1B palmitoylation. However, palmitoylation of GRIP1B is not altered in response to synaptic activity [9,25]. This may be due to the fact that GRIP1B can also be palmitoylated by zDHHC8, or because the trafficking of zDHHC5 to and from spines occurs within 20 minutes [7], a timeframe shorter than the reported turnover rate of GRIP1B (half-time [T1/2] approximately 35 min) [9]. Palmitoylation of scaffold proteins

Activity-induced palmitoylation of synaptic scaffolding proteins can modulate synaptic plasticity by regulating the localization of AMPAR and g-aminobutyric acid type A receptors (GABAARs) at the postsynaptic membrane [8,9,12,32,33]. PSD-95 is the best studied synaptic scaffold protein and Fukata et al. have made significant headway in understanding how palmitoylation of PSD95 regulates the localization of AMPAR and synaptic plasticity [3,25,26,34,35] (and reviewed in Ref. [27]). Moreover, this group recently demonstrated that a/b-hydrolase domain-containing protein 17(ABHD17) is the PPT responsible for the depalmitoylation of PSD-95 [36]. Changes in activity have been shown to influence PSD-95 palmitoylation, with prolonged TTX treatment enhancing PSD-95 palmitoylation and promoting PSD-95’s recruitment to the postsynaptic density where it increases postsynaptic receptor clustering [25]. Recent work from Johannes Hell’s lab demonstrates that the influx of Ca2+ increases Ca2+/calmodulin (CaM) binding to the N-terminal domain of PSD-95, and blocks the palmitoylation of PSD-95 [37]. The authors postulate that impairment of PSD-95 palmitoylation by Ca2+/CaM disrupts PSD-95 binding to CDKL5. This is of interest as CDKL5 binds to PSD-95 in a palmitoylation-dependent manner [38] and proper CDKL5 localization at postsynaptic sites positively regulates synapse strength [39]. Together, Zhang et al. conclude that the binding of Ca2+/CaM to the N-terminus of PSD-95 inhibits PSD-95 palmitoylation and subsequently its postsynaptic accumulation, its binding to CDLK5, and its ability to enhance postsynaptic strength [37]. The scaffold protein, A-Kinase anchor protein 79/150 (AKAP79/150), regulates the phosphorylation and trafficking of AMPA receptors by recruiting protein kinase A (PKA), protein kinase C (PKC) and calcineurin-PP2B (CaN) to the postsynaptic density (reviewed in Ref. [40]). Palmitoylation of AKAP79 is required for its www.sciencedirect.com

localization to REs and for its activity mediated recruitment to post-synaptic spines [32] (Figure 1). Recently, Woolfrey et al. demonstrated that the palmitoylation of AKAP79 is mediated by zDHHC2 in an activity-dependent manner [8]. Knockdown of zDHHC2 in cultured hippocampal neurons abrogated activity-induced recruitment of AKAP79 to spines, RE exocytosis, the enlargement of dendritic spines and activity-induced synapse strengthening. Interestingly, expression of myristoylated AKAP79 (analogous to a constitutively palmitoylated protein) in a zDHHC2 knock down background, restored activity-induced RE exocytosis and synapse strengthening, suggesting that the targeting of AKAP79 to membranes is necessary and sufficient for its ability to regulate activity-induced synapse plasticity. Gephyrin is a post-synaptic scaffold protein that has been shown to organize inhibitory synapses by recruiting and clustering glycine and GABAARs [41,42]. Work by Dejanovic et al. [12] has shown that gephyrin is palmitoylated on cysteine residues 212 and 284 by zDHHC12, and that the palmitoylation of gephyrin is essential for its clustering at GABAergic synapses (Figure 1). Indeed, palmitoylation-deficient gephyrin was unable to localize to synaptic membranes, whereas zDHHC12 overexpression enhanced the size of synaptic gephyrin clusters. Increasing gephyrin clustering at inhibitory synapses through the overexpression of zDHHC12 resulted in an increase in the amplitude of mini inhibitory postsynaptic currents, suggesting that gephyrin palmitoylation enhances GABAAR recruitment to inhibitory synapses. Interestingly, reducing GABAAR activity for one hour with the GABAAR antagonist, bicuculline, decreased gephyrin palmitoylation, whereas one-hour GABA treatment enhanced palmitoylation. Together, this demonstrates a feed forward loop where enhanced GABAAR activity increases gephyrin palmitoylation, its clustering and association with the membrane, as well as gephyrinmediated clustering of GABAAR. Further work is required to determine how GABAAR activation enhances DHHC12-mediated palmitoylation of gephyrin, and to determine the role of other PATs that are localized at inhibitory synapses including zDHHC5 [7]. Palmitoylation of transmembrane proteins

Work from the Diaz lab has demonstrated that the AMPAR accessory protein, synapse differentiationinduced gene 1 (SynDIG1) is palmitoylated in an activity-dependent manner [43]. SynDIG1 plays an important role in regulating AMPAR content at excitatory synapses, and loss of SynDIG1 significantly attenuates excitatory synapse number as well as AMPAR localization at synapses [44]. More recent work by Kaur et al. demonstrates that palmitoylation of SynDIG1 on cysteines 191 192 is required for its stability as well as its clustering and localization in dendrites. While wildtype SynDIG1 was distributed throughout the dendrites, Current Opinion in Neurobiology 2017, 45:1–11

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palmitoylation-deficient SynDIG1was restricted to the cell soma and proximal dendrites [43]. Blocking synaptic activity with TTX enhanced SynDIG1 palmitoylation, resulting in increased recruitment to postsynaptic spines. Although specifically not shown by Kaur et al., it is tempting to speculate that TTX-mediated recruitment of SynDIG1 to spines increases AMPAR content at synapses, thereby providing an additional mechanism for the regulation of homeostatic plasticity. Additional work is required to identify which PAT(s) regulate SynDIG1 palmitoylation. Interestingly, prolonged TTX treatment also promotes the recruitment of zDHHC2 to postsynaptic spines, where it enhances AMPAR clustering by palmitoylating PSD-95 [25] suggesting that multiple, parallel pathways may exist to regulate activity-induced protein palmitoylation and the clustering of postsynaptic receptors. Together, it appears that activity-mediated palmitoylation of synaptic proteins such as d-catenin [6], GRIP1B [9], SynDIG1 [43], PSD-95 [25], AKAP79/150 [32] and gephyrin [12] impacts their subcellular localization and their ability to recruit and cluster postsynaptic receptors, leading to changes in synapse strength. A complete list of zDHHC proteins that mediate activity-induced palmitoylation is still lacking, as well as an understanding of whether these PATs localize to the same synapses or different synapses to mediate their effects.

Palmitoylation and actin remodelling in synapse formation and plasticity Palmitoylation of the actin binding protein, LIM Kinase 1 (LIMK1), has recently been shown to play an important role in mediating the formation of synaptic connections [45]. Both LIMK1 and 2 phosphorylate and inactivate members of the ADF/cofilin family of actin binding and filament severing proteins [46]; however, only LIMK1 has been shown to be a substrate for palmitoylation [45]. A recent study by George et al. demonstrates that LIMK1 palmitoylation results in its recruitment to dendritic spines where it acts to stabilize spines and promote the formation of synapses. Palmitoylation-mediated recruitment of LIMK1 to dendritic spines is required for the phosphorylation and activation of LIMK1 by its upstream regulator p21-activated kinase (PAK), that is itself enriched in spines. LIMK1 activation, in turn, mediates the phosphorylation and inactivation of cofilin, resulting in a net increase in actin polymerization and the enlargement and stabilization of dendritic spines [45] (also reviewed in Ref. [17]). Disrupting LIMK1 palmitoylation abolishes activity-induced spine enlargement consistent with the importance of actin polymerization in this process. Given the known changes in spine number and size in response to synaptic activity, it is tempting to speculate that synaptic activity may drive the palmitoylation of LIMK1 and its recruitment to postsynaptic spines. However, further work is required to test this hypothesis, as

Figure 2

1

FGFR1 ?

Src

FGFR1

A NCAM140/180

FGF2 zDHHC3 Fyn Kinase

Fyn

5

B

2 Y 18

YY 295/297

3

C

PKC

PKC Lipid Raft

4 Neurite Outgrowth

6

Src Kinase NCAM140/180

Lievens et al., 2016 14 (1) FGFR1activation (2) Phosphorylation of zDHHC3 at Y18 (3) Increased palmitoylation of NCAM (4) Trafficking of NCAM to lipid rafts 13 (5) Activation of Fyn and PKC signaling pathways 13 (6) Increased neurite outgrowth13 (A) Src kinase activation (B) Phosphorylation of zDHHC3 at Y295/297 (C) Reduced zDHHC3 autopalmitoylation and NCAM palmitoylation

Current Opinion in Neurobiology

Dual phosphorylation of DHHC3 regulates the palmitoylation of NCAM. Schematic depicting the dual phosphorylation of zDHHC3 by FGFR1 and Src kinase, and opposing effects on zDHHC3-mediated palmitoylation of NCAM. This work was done in N2a cells, SYF / cells, and primary hippocampal cultures. Current Opinion in Neurobiology 2017, 45:1–11

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Please cite this article in press as: Globa AK, Bamji SX: Protein palmitoylation in the development and plasticity of neuronal connections, Curr Opin Neurobiol (2017), http://dx.doi.org/10.1016/j. conb.2017.02.016

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Palmitoylation in neuronal connectivity Globa and Bamji 9

well as determine zDHHC enzyme(s) responsible for the palmitoylation of LIMK1.

Palmitoylation of cell adhesion molecules in neurite outgrowth Cell adhesion molecules are common substrates for palmitoylation [35,47,48], and have been shown to regulate neurite outgrowth and targeting, synapse formation and synaptic plasticity (reviewed in Ref. [49]). Palmitoylation of the neural cell adhesion molecule (NCAM) by zDHHCs 3 and 7 results in the targeting of NCAM to lipid rafts where it can activate downstream signalling pathways [48] and promote neurite outgrowth [13]. Recent work by Lievens et al. has demonstrated that the palmitoylation function of zDHHC3 is regulated by the phosphorylation of zDHHC3 by fibroblast growth factor receptor 1 (FGFR1) and Src kinase [14] (Figure 2). FGFR1-mediated phosphorylation of zDHHC3 at tyrosine residue 18 was shown to increase the palmitoylation of NCAM, while Src kinase-mediated phosphorylation of tyrosine residues 295 and 297 reduced NCAM palmitoylation, demonstrating dual control of zDHHC3 enzymatic activity through differential phosphorylation of tyrosine residues. Phosphorylation of tyrosine residues 295 and 297 decreased zDHHC3 autopalmitoylation [14] and its association with NCAM, providing some mechanistic explanation for this result. Further work is required to identify the kinase that mediates Y18 phosphorylation upon FGFR1 activation, and to determine how FGFR1 activation and Src kinase have opposing effects on zDHHC3 function, as FGFR signalling can also modulate Src kinase activity [50]. As the majority of these experiments were performed in neuroblastoma N2a cell lines and mouse embryonic fibroblast cell lines deficient in Src family kinases (SYF / ), it will be of interest to determine the extent to which these mechanisms are conserved in primary neurons [14].

Conclusions Understanding the role of zDHHC proteins in the development and function of the nervous system is still in its infancy. Here we review work showing that synaptic activity can alter the palmitoylation of synaptic proteins. This can occur through altered trafficking of PATs such as zDHHCs 2 [8,25] and 5 [7], through altered interactions of zDHHCs with other proteins [7], through altered posttranslational processing of zDHHCs such as phosphorylation [14] or through altered zDHHC enzymatic activity [14]. In several cases, these activitydependent changes in zDHHC localization and function can be linked to NMDA receptor-mediated calcium signaling [7,8,37]; however further investigation is required to determine which additional pathways may be involved in regulating these events. Interestingly, 9 out of the 23 zDHHCs have been associated with neurological diseases [51–55] (Table 1). A complete list www.sciencedirect.com

of substrates for each zDHHC protein as well as a better understanding of the PPTs that depalmitoylate substrates is required to not only deepen our understanding of protein palmitoylation, but also the molecular mechanisms of neurological disease.

Conflict of interest statement The authors declare no conflict of interest.

Acknowledgement This work was supported by grants from Canadian Institutes of Health Research MOP-142721 (to SXB) and GSD-140360 (to AKG).

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Please cite this article in press as: Globa AK, Bamji SX: Protein palmitoylation in the development and plasticity of neuronal connections, Curr Opin Neurobiol (2017), http://dx.doi.org/10.1016/j. conb.2017.02.016