The ER proteostasis network in ALS: Determining the differential motoneuron vulnerability

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Contents lists available at ScienceDirect

Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet

Review article

The ER proteostasis network in ALS: Determining the differential motoneuron vulnerability Pablo Rozas a,b,c , Leslie Bargsted a,b,c , Francisca Martínez a,b,c , Claudio Hetz a,b,c,d,e,∗ , Danilo B. Medinas a,b,c,∗ a

Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago 8380453, Chile Program of Cellular and Molecular Biology, Institute of Biomedical Sciences, Center for Molecular Studies of the Cell, University of Chile, Santiago 8380453, Chile c Center for Geroscience, Brain Health and Metabolism, University of Chile, Santiago, Chile d Buck Institute for Research on Aging, Novato, CA 94945, USA e Harvard School of Public Health, Boston, MA 02115, USA b

a r t i c l e

i n f o

Article history: Received 4 February 2016 Received in revised form 17 April 2016 Accepted 29 April 2016 Available online xxx Keywords: ALS Proteostasis ER stress Selective vulnerability Chaperones

a b s t r a c t Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by the selective loss of motoneurons. The mechanisms underlying neuronal degeneration in ALS are starting to be elucidated, highlighting abnormal protein aggregation and altered mRNA metabolism as common phenomena. ALS involves the selective vulnerablility of a subpopulation of motoneurons, suggesting that intrinsic factors may determine ALS pathogenesis. Accumulating evidence indicates that alterations to endoplasmic reticulum (ER) proteostasis play a critical role on disease progression, representing one of the earliests pathological signatures of the disease. Here we discuss recent studies uncovering a fundamental role of ER stress as the driver of selective neuronal vulnerability in ALS and discuss the potential of targeting the unfolded protein response (UPR) as a therapeutic strategy to treat ALS. © 2016 Elsevier Ireland Ltd. All rights reserved.

Contents 1. 2. 3. 4. 5.

ALS, an overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 ER proteostasis and motoneuron vulnerability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 ER chaperones and muscle innervation: modeling early ALS stages . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 From selective vulnerability to therapeutic strategies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Concluding remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 00

1. ALS, an overview Amyotrophic lateral sclerosis (ALS) is a fatal late-onset neurodegenerative disease characterized by progressive loss of cortical and spinal motoneurons leading to muscle weakness, twitching, lack of coordination, paralysis, respiratory failure and death, all of these explained by denervation of a subpopulation of selectively vulner-

∗ Corresponding authors at: Biomedical Neuroscience Institute, Faculty of Medicine, University of Chile, Santiago 8380453, Chile. E-mail addresses: [email protected] (C. Hetz), [email protected] (D.B. Medinas).

able motoneurons to their target muscles [1,2]. Among all the cases, around 90% correspond to sporadic form of the disease (sALS) and the other 10% are associated to mutations on more than 40 genes and are termed familial forms of ALS (fALS) [3]. Although the etiology of the disease remains elusive, converging cellular pathways in fALS have shed light on the disease pathogenesis and progression. The most prevalent mutations of fALS are a hexanucleotide expansion in C9orf72 that accounts for around 50% of fALS; whereas point mutations in superoxide dismutase 1 (SOD1) gene explains 20% of fALS and mutations in trans-active response (TAR) DNA-binding protein 43 (TDP-43) and fused in sarcoma (FUS) are observed in 5% of ALS (reviewed in Ref. [4]).

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Please cite this article in press as: P. Rozas, et al., The ER proteostasis network in ALS: Determining the differential motoneuron vulnerability, Neurosci. Lett. (2016), http://dx.doi.org/10.1016/j.neulet.2016.04.066

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Fig. 1. ER folding network and the selective neuronal vulnerability in ALS. Inside the ER lumen, nascent newly translated proteins are bound by BiP/SIL1 chaperone system and undergo the folding process through the calnexin/calreticulin (CNX/CRT) cycle; disulfide bond formation catalyzed by PDIs; and/or chaperone assisted folding. Misfolded proteins, like the prone to aggregation ALS-linked proteins, are targeted to ER-associated degradation (ERAD) mediated by BiP/SIL1. Properly folded proteins traffic through the secretory pathway to their target destination. Altered expression/function of these vulnerability factors (depicted with colors) leads to misfolded protein aggregation, reduced levels of synaptic proteins and, consequently, denervation at the NMJ and motoneuron loss. MMP9 modulates ER stress in vulnerable motoneurons by an unknown mechanism (dashed line). CNX: calnexin; CRT: calreticulin; S1R: Sigma-1 receptor.

ALS-related proteins are involved in diverse cellular processes, highlighting altered RNA metabolism and protein homeostasis [5]. Mutations in RNA binding proteins lead to toxicity due to loss of nuclear function and/or cytoplasmic mislocalization of protein, leading to gain-of-toxic function [6]. In addition, axonal transport impairment possibly due to alterations in cargo binding to molecular motors contributes to ALS [7,8], in addition to cytosolic and mitochondrial calcium dysregulation [9]; and altered protein homeostasis (referred to as proteostasis) [10]. Indeed, abnormal protein folding, oligomerization and aggregation in form of inclusions are common features of ALS [4,11]. Several ALS-related proteins alter the function of the endoplasmic reticulum (ER), generating a stress condition that triggers an adaptive reaction known as the unfolded protein response (UPR) [12]. The UPR is a signal transduction pathway that mediates the recovery of proteostasis, maintaining cell survival under mild stress by improving the folding capacity of the cells and the degradation of abnormal proteins [13]. However, under chronic or irreversible ER stress the UPR triggers cell death by apoptosis [14,15]. Understanding the switch between adaptive to apoptotic UPR programs represents a cornerstone for the development of new therapies to tackle ER proteostasis disturbances in neurodegenerative diseases such as ALS [16,17]. Importantly, signs of ER stress have been extensively reported in post mortem tissue of sALS and fALS patients, in addition to cellular and animal models of the disease [16–19]. Remarkably, ER stress operates as one of the earliest defects observed in fALS motoneurons in vivo (see next sections) [2]. The surrounding cellular context of motoneurons is a relevant factor that modulates disease onset and progression. There is compelling evidence showing that cell-non autonomous effects modulate motoneuron degeneration [20]. For example, astrocytes derived from fALS patients’ iPSC [21] or post mortem spinal cord neural progenitor cells (NPC) from ALS patients have detrimental effects on co-cultured healthy motoneurons [22]. Using fALS mouse models, deletion of the expression of mutant SOD1 in astrocytes [23], oligodendrocytes [24] or microglia [25] promote survival of mutant SOD1 mouse models of ALS mainly by extending the symptomatic stage of the disease. Remarkably, overexpressing mutant SOD1 in skeletal muscle also induces motor deficits, neuromuscular junction (NMJ) disassembly and motoneuron loss in the spinal cord [26]. Even though non-neuronal cells are important contributors to the progression of the disease, the motoneuron vulnerability to intrinsic stress factors is critically determined by cell-autonomous components such as the genetic background and transcriptional and translational profiles [25,27,28]. Indeed, targeting the expres-

sion of mutant SOD1 in motoneurons delays onset and early disease stage [25]. Furthermore, gene therapy to knock down mutant SOD1 in motoneurons also largely delays disease onset [29]. Thus, a complex scenario arises from the cell-autonomous and extracellular factors altering the normal function of motoneurons rendering them susceptible to muscle denervation and death. In ALS a subpopulation of motoneurons located in the same spinal cord region develops early degeneration, whereas other neighbor motoneurons are resistant to the disease, indicating that intrinsic factors may determine the differential vulnerability to undergo neurodegeneration [2,16]. For example, in mutant mouse models of fALS, different subpopulations of motoneurons denervate its target hindlimb muscles at different stages during the disease course [30]. The phasic functional fast fatigable (FF) motoneurons denervate its target muscle fibers first in the presymptomatic stage of the disease. Then, near disease onset, the phasic fast fatigueresistant (FR) become disconnected from the muscle fibers. Finally, at the end stage of the disease, the tonic motoneurons subpopulation (S motoneurons) disassembles from muscle fibers [30]. This intriguing spatial and temporal course of the disease clearly depicts that FF motoneurons have a selective vulnerability given the same genetic mutant background as FR and S motoneurons. Importantly, these observations bring the opportunity to study the differential transcriptional, translational and post-translational profiles that can account for this selective vulnerability in ALS. This information may uncover novel disease modifiers and targets for future therapeutic intervention. How motoneurons become selectively affected from all the possible neuronal subpopulations in the nervous system despite of similar inflammatory and redox environment? What are the molecular factors and/or pathways that account for this selective neuronal vulnerability? In the next sections we discuss the emerging role of the ER proteostasis network in determining the differential motoneuron vulnerability observed in ALS. 2. ER proteostasis and motoneuron vulnerability The search for the molecular factors and/or pathways that account for the selective motoneuron vulnerability (Fig. 1) has uncovered endoplasmic reticulum stress as an early and transversal pathogenic mechanism driving motoneuron loss in ALS (Table 1) [27,28,31]. Interestingly, ER stress has been recently shown to be selectively increased in human motoneurons from patients suffering spinal muscular atrophy (SMA), another motoneuron disease [32]. A pioneer study by Saxena et al. defined the gene expression profile of vulnerable and resistant motoneurons in mutant

Please cite this article in press as: P. Rozas, et al., The ER proteostasis network in ALS: Determining the differential motoneuron vulnerability, Neurosci. Lett. (2016), http://dx.doi.org/10.1016/j.neulet.2016.04.066

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Table 1 ER proteostasis and selective motoneuron vulnerability in ALS. A summary of selected literature related to relevant factors involved in motoneuron vulnerability and their role for ALS pathology. Gene

Protein

Main function

Role in ALS

Reference

HSPA5

BiP

ER resident chaperone involved in protein folding quality control

• Upregulated in postmortem sALS spinal cord. • Loss of function induces late onset motor alterations in mice.

[18,54]

SIL1

SIL1

Adenine nucleotide exchange factor for BiP

• Highly expressed in ALS resistant motoneurons. • Loss of a single SIL1 allele enhances ER stress and ALS pathology in mice. • Overexpression of SIL1 attenuates ER stress and prolongs survival.

[31]

CRT

CRT

Ca2+ binding ER chaperone

• Early reduction of CRT levels in vulnerable SOD1G93A mice motoneurons. • Loss of a single CRT allele enhances ER stress and Ca+2 deregulation leading to motoneuron apoptosis.

[56,57]

SIGMAR1

Sigma-1 Receptor

ER chaperone

• Point mutation causes juvenile ALS. • Lack of Sigma-1 Receptor induces motoneuron hyperexcitability and exacerbates ALS pathology.

[40,42,44]

MIF

MIF

Inflammatory cytokine with intracellular thiol-reductase and ATP-independent chaperone activity.

• Low MIF protein abundance levels in motoneurons correlates with SOD1 aggregation in fALS mouse model. • MIF ATP-independent chaperone activity inhibits mutant SOD1 misfolding in vitro. • MIF overexpression prolongs ALS motoneuron survival.

[48]

MMP9

MMP9

Extracellular matrix metalloproteinase

• Selectively expressed in fast fatigable vulnerable motoneurons. • Lack of MMP9 extends survival and delays muscle denervation in ALS mice. • Overexpression of MMP9 in vulnerable motoneurons enhances muscle denervation.

[49]

PDIA1

PDIA1

ER resident protein disulfide isomerase involved in protein folding

• ALS-linked mutations alter motoneuron connectivity. • Mutant forms of PDI induce motor defects and denervation in zebrafish. • Spinal cord motoneurons have the lowest translational activity for PDI transcripts compared to astrocytes and oligodendrocytes in wild type mice.

[27,64,65]

PDIA3

PDIA3 or ERp57

ER resident protein disulfide isomerase involved in protein folding

• ALS-linked mutations alter motoneuron connectivity. • Mutant forms of ERp57 induce motor problems in zebrafish. • ERp57 deficiency causes aberrant NMJs in mice and altered motor control.

[64,65]

SOD1 mice uncovering ER stress as one of the earliest pathological signatures, even before the first signs of denervation had occurred, underscoring the involvement of proteostasis disturbances in ALS pathogenesis [28]. Basal levels of ER stress and proteostasis alterations are an intrinsic feature of patient iPSCderived human motoneurons [33]. Remarkably, a vicious cycle was also reported between ER stress and the hyperexcitability of ALS motoneurons [34]. According to Saxena and colleagues, the hyperexcitability of vulnerable motoneurons in ALS might represent an initial adaptive response to enhance neuroprotective signaling [35], resulting in neurotoxicity later on due to excitotoxicity [36]. In a recent follow-up study, Saxena’s group investigated the pattern of expression of the ER folding network in vulnerable and resistant motoneurons to understand the molecular basis of subtype-selective ER stress response. Interestingly, the BiP cochaperone SIL1, which catalyzes adenine nucleotide exchange, was mostly expressed in resistant motoneurons [31]. SIL1 deficiency enhanced ALS pathology whereas SIL1 overexpression afforded significant neuroprotection related to improved ER proteostasis and reduced SOD1 aggregation [31]. Previous studies indicated that

mutations in SIL1 triggers spontaneous degeneration associated with degeneration of Purkinje cells and proteostasis alterations in the cerebellum [37]. Importantly, recent studies in ALS patients carrying C9orf72 mutations suggests that cerebellum may be also altered, where ER stress was a major reaction identified by global gene expression analysis [38]. Moreover, SIL1 levels are reduced in motoneurons of the mutant TDP-43A315T transgenic mice and increased in surviving motoneurons of sALS patients [31], thus evidencing a broad role of SIL1 in ALS pathogenesis. Other ER-related factors may also contribute to neurodegeneration in ALS. For instance, altered calcium metabolism has been implicated in many neurodegenerative diseases including ALS. The ER operates as a major intracellular calcium reservoir and maintains a constant calcium flux to mitochondria through the mitochondriaassociated membranes or MAMs, an important process for energy metabolism [39]. The leak of calcium to the cytosol can disturb neuronal function at many levels and might lower excitability of motoneurons due to hyperpolarization of plasma membrane through calcium-dependent potassium currents [36]. The Sigma1 receptor is an ER chaperone highly expressed in motoneurons

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Fig. 2. Model of ALS progression as an ER stress-driven pathology. Vulnerable motoneurons suffer from early ER stress due their intrinsic low expression of ER chaperones and high expression of MMP9 compared to resistant motoneurons. This event occurs during early pre-symptomatic stages before any denervation is observed in SOD1 mutant mice. Progressive accumulation of toxic aggregates is followed by inflammatory responses. A decrease expression of synaptic proteins levels at the NMJ is observed due to altered folding capacity at the ER. As a consequence, muscle denervation occurs initiating the onset of the disease. At this point, resistant motoneurons become compromised due to neuroinflammation and toxic proteins spreading causing ER stress. Near the end stage of the disease vulnerable motoneuron number is markedly reduced and resistant motoneurons start to denervate the muscles.

[40] that regulates motoneuron excitability [41]. The E102Q autosomal recessive mutation in Sigma-1 receptor causes juvenile ALS [42] and leads to deficient mitochondrial ATP production in cell culture. Interestingly, Sigma-1 receptor interacts with IP3 receptor and promotes the flux of calcium from ER to mitochondria [43]. Surprisingly, Sigma-1 receptor deficient mice have only a mild motor phenotype [40], even though the deletion of Sigma-1 receptor exacerbates experimental ALS [44]. The relationship between the control of calcium flux by Sigma-1 receptor and ER stress in vulnerable motoneurons warrants further investigation. Regarding the integrity of ER and mitochondria membranes in motoneurons, misfolded SOD1 associates to the protein components facing the cytoplasmic face of the membrane, disturbing normal organelle function [45,46]. For instance, misfolded SOD1 binds to Derlin-1, a component of the ERAD system involved in the translocation of unfolded proteins from the ER lumen to the cytosol for degradation by the proteasome-ubiquitin system [47]. Also, a recent study identified macrophage migration inhibitory factor (MIF) as a critical chaperone regulating the interaction of misfolded SOD1 to ER and mitochondria membranes [48]. MIF is capable of shielding misfolded SOD1 through its chaperone activity, thus inhibiting SOD1 association to ER and mitochondria membranes [48]. Despite the high levels of MIF mRNA in motoneurons relative to astrocytes and oligodendrocytes, its protein content in motoneuron cell bodies is remarkably low and correlates with the accumulation of misfolded SOD1 [48]. The molecular basis for reduced MIF levels in motoneurons is currently unknown and might involve its increased degradation or secretion. Remarkably, extracellular MIF is an inducer of matrix metalloproteinase 9 (MMP9), which has been identified as a major factor accounting for vulnerability of motoneuron in ALS [49]. The MMP9 is robustly expressed in the FF motoneurons that denervate earlier in the disease course [49]. Targeting MMP9 in mutant SOD1 mice reduces denervation and extended survival [49]. Furthermore, the MMP9-null vulnerable motoneurons presented delayed ER stress response in the disease course that was comparable to resistant motoneurons. In summary, motoneuron vulnerability appears to

arise from interrelated pathways that converge into the ER proteostasis network.

3. ER chaperones and muscle innervation: modeling early ALS stages Several proteomic studies have shown that the upregulation of ER stress-inducible chaperones is a hallmark of ALS. For example, a proteomic analysis of spinal cord tissue of ALS mouse models uncovered major changes in the levels of two protein disulfide isomerase (PDI) family members, known as PDIA1 (also referred to as PDI) and ERp57 (also known as PDIA3 or Grp58) [50,51]. This observation was also confirmed in cerebrospinal fluid [52] and blood from sALS patients [53]. ERp57 is an ER-resident foldase that promotes disulfide bond formation of a subset of glycoproteins and is a key component in calnexin (CNX) and calreticulin (CRT) cycle (Fig. 1). The incremented levels of this chaperone in body fluids may be due to an initial UPR adaptive response aimed to preserve motoneuron viability and function. Importantly, a recent study using cell-type specific ribosomal profiling in ALS mice indicated that ER stress is a major pathological signature observed in motoneurons and not astrocytes or oligodendrocytes [27]. Recent functional evidence using mouse models suggests that the ER folding network has a relevant role in ALS. For example, the characterization of a knock-in mouse for mutant BiP lacking the ER retrieval sequence generated age-related motor problems. This phenotype involved the selective vulnerability of motoneuron including motoneuron loss and the aggregation of wild-type SOD1 [54] reminiscent of sALS [55]. Additionally, another report indicated that reduced levels of CRT are observed in vulnerable but not resistant motoneurons of ALS mice [56]. Importantly, genetic targeting of CRT expression in mutant SOD1 mice accelerated the progression of the disease, specifically altering muscle innervation and not the late phase of motoneuron apoptosis [57]. These observations suggest that alterations on ER folding capacity may underlay early cellular processes (presymptomatic) initiating the progression of the disease (Fig. 2).

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The protein folding in the ER relies on catalytic redox cycles that introduce disulfide bonds in the maturing polypeptides [58]. The PDI family constitutes a group of oxidoreductase chaperones that catalyze the oxidation, reduction and isomerization of disulfide bonds in the ER [58]. PDI family members contain two thioredoxin-like catalytic domains termed a and a’, in addition to two intercalated non-catalytic domains b and b’ involved in substrate recognition [59]. The active site is comprised of two reactive cysteines separated by glycine and histidine residues (Cys-GlyHis-Cys) [59]. PDIA1 has been shown to co-localize with protein aggregates containing mutant SOD1, TDP-43, and FUS [50,60,61]. The contribution of PDIs in ALS etiology has been highlighted by the identification of single nucleotide polymorphisms (SNPs) in intronic regions of the PDIA1 gene as risk factors for ALS [62,63]. More recently, we have discovered mutations in both PDIA1 and ERp57 as risk factors to develop ALS [64], suggesting that alteration on the ER folding capacity may be part of the etiology of the disease. To assess the relevance of PDI mutants to ALS, we have performed a thorough characterization of possible detrimental effects of the PDIA1 and ERp57 mutants to motoneuron function [65]. Using motoneuron cultures we determined that mutations in PDIA1 and ERp57 adversely affect motoneuron neuritogenesis, leading to reduced neurite length and number [65]. Furthermore, PDI mutants were shown to have detrimental effects on motoneuron branching in zebrafish, causing motor problems [65]. Additionally, biochemical analysis revealed that ERp57 mutants have aberrant interaction with CNX and CRT, which could negatively impact the folding of synaptic proteins. To determine the relevance of PDIs in the nervous system, we generated a conditional knockout of ERp57 which developed motor deficits reminiscent of early ALS symptoms associated with altered NMJ [65]. We also recently showed that ERp57 assists the folding of other disease-related proteins including PrP [66], and its expression enhances axonal regeneration in the peripheral nervous system [67]. In sharp contrast, the overexpression of ERp57 in transgenic mice did not affect the susceptibility of dopaminergic neurons to a Parkinson-inducing neurotoxin [67], suggesting that ERp57 may underlay the vulnerably of certain specific neuronal populations to degeneration. Since muscle denervation is an early event in disease, we propose here that control of proteostasis by ER chaperones and foldases may play an important role in determining motoneuron vulnerability in ALS.

4. From selective vulnerability to therapeutic strategies Recent advances towards understanding motoneuron vulnerability has set the stage for the development of more effective therapeutic approaches to the disease. The only approved drug for ALS treatment, riluzole, inhibits motoneuron hyperexcitability by blocking sodium channels [68] but confers minimal protection [69,70]. Mexiletine, another sodium channel blocker with different pharmacological properties, has also shown protective effects in an in vitro model of motoneuron death induced by astrocyte conditioned media derived from SOD1 mutant mice [68]. Importantly, changes in the electrical activity of ALS neurons have been reported very early in mouse models [71]. Since hyperexcitability and ER stress are directly related in human motoneurons, targeting the ERstress response may offer a complementary approach for the current therapy. Several new drugs are now available to target the UPR in different diseases [72]. In the context of neurodegeneration, compelling evidence indicates that depending on the disease context and the UPR signaling components involved, distinct and even opposite effects may be observed (reviewed in Refs. [73,74]). One feasible

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therapeutic strategy is to enforce UPR adaptive mechanisms by exposing cells to low and non-toxic ER stress levels to enhance its buffering capacity [75]. This mild stress has been successfully proved in models of Parkinson’s disease [76]. The concept of “ER hormesis” has been proposed as a strategy to precondition the cell and resist chronic ER stress by exposing them to a sublethal stimulus that triggers protein misfolding [77]. Several studies have investigated the therapeutic potential of prolonging translational repression by administering inhibitors of eIF2␣ phosphatases to treat ALS. Remarkably, independent groups have reported beneficial effects of administering inhibitors of eIF2␣ phosphatases such as salubrinal, guanabenz, and sephin 1 to mutant SOD1 mice [28,34,78], in addition to worm and zebrafish overexpressing mutant TDP-43 [79]. However, a recent report obtained conflicting results indicating that guanabenz treatment actually accelerates ALS [80]. In SMA mouse model and patientderived motoneurons, guanabenz has been shown efficacy at extending motoneuron survival [32]. The genetic manipulation of ER stress in ALS models revealed a complex scenario where different branches of the UPR could have opposing effects on disease course [73]. A comprehensive understanding of the contribution of different UPR components to disease outcome might enable the formulation of pharmacological cocktails for the finetuning of ER proteostasis in ALS. Interestingly, the identification of SIL1 deficiency in vulnerable motoneurons has opened a new avenue of therapeutic possibilities based on enhancement of SIL1BiP system. Notably, the enforced overexpression of SIL1 through adeno-associated virus (AAV) administration afforded protection against muscle denervation and extended survival of mutant SOD1 mice [31]. UPR based gene therapy has been tested successfully in PD [81] and retinitis pigmentosa [82] mediated by the overexpression of BiP or the enforced expression of the transcription factor XBP1 in various experimental systems [73]. We described that AAV-XBP1s gene therapy provides protection in models of Huntington [83], Parkinson [84], spinal cord injury [85] and peripheral nerve degeneration [86]. In addition, AAV-XBP1 can modulate neuronal plasticity and behavior in mice [87]. It remains to be determined if this strategy alleviates ALS in vivo. Regarding ER chaperones, there is a report of beneficial effects of delivery of adenovirus (AV) expressing PDIA1 in cardiomyocyte ischemia [88]. Targeting MMP9 has been tested as a therapeutic strategy to alleviate ALS [49]. Treating mutant SOD1 mice with MMP9 inhibitor or viruses to knockdown its expression leads to protection against the disease [49,89]. A genetic screening in zebrafish identified Epha4, a receptor in the ephrin axonal repellent system, as a disease modifier [90]. Genetic or pharmacological inhibition of Epha4 signaling resulted in increased survival of mouse and rat models of ALS, correlating with increased muscle innervation [90]. Chemical chaperones are small molecules that can stabilize protein conformations, reducing ER stress levels in various disease settings [72], where we highlight the use of the bile acid TUDCA, phenylbutyrate (4-PBA) and the disaccharide trehalose. TUDCA was recently shown to reduce ER stress levels on cellular models of C9orf72 pathology [91], and also in ALS patients as reported recently on a small clinical trial [92]. 4-PBA has been shown to extend life span in mutant SOD1 mice [93], and is currently being tested in ALS patients [94]. Finally, others and we reported that trehalose treatment reduces SOD1 aggregation and delay ALS in mouse models [95–97]. Interestingly, trehalose may also enhance autophagy levels, an efficient catabolic pathway for the clearance of abnormal protein aggregates and damaged organelles [98]. Thus, therapeutic strategies to reduce ER stress levels offer new avenues for disease intervention.

Please cite this article in press as: P. Rozas, et al., The ER proteostasis network in ALS: Determining the differential motoneuron vulnerability, Neurosci. Lett. (2016), http://dx.doi.org/10.1016/j.neulet.2016.04.066

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5. Concluding remarks Overall, accumulating evidence suggest that the perturbations to the ER proteostasis network may contribute to early events in ALS pathogenesis, driving neuromuscular dysfunction. Since the capacity of the ER to buffer fluctuations of proteostasis decline with age [99], we speculate that alterations in chaperone function and the activity of the UPR may have more drastic effects in neuronal connectivity during aging, and together with other ALS risk factors could translate into an irreversible damage to motoneuron function and the manifestation of the disease. As discussed here, the ER proteostasis network may operate as an important (i) protective component mediating adaptation to initial ER stress that may translate into (ii) a chronic ER stress stage during the symptomatic period driving motoneuron loss. ER stress may also underly (iii) structural and functional changes to the neuromuscular synapse due to persistent ER stress, causing in the long term a progressive disturbance of neuronal connectivity, and (iv) it may also explain the differential neuronal vulnerability observed on specific subpopulations in ALS. The discovery of genetic associations between mutation in components of the ER proteostasis network in ALS and most recent literature have provided a proof-of-concept for one possible mechanism underlying motoneuron degeneration in ALS. Hence, ER folding components, the UPR and quality control mechanisms are novel elements in ALS pathogenesis that may ultimately represent an important pathway for future therapeutic intervention.

Acknowledgements This work was funded by FONDECYT 11150579 (DM), Millennium Institute No. P09-015-F, and FONDAP 15150012, the Frick Foundation, ALS Therapy Alliance 2014-F-059, Muscular Dystrophy Association 382453, CONICYT-USA2013-0003, Michael J Fox Foundation for Parkinson´ıs Research, COPEC-UC Foundation, Ecos-Conicyt C13S02and FONDECYT no. 1140549, Office of Naval Research-Global (ONR-G) N62909-16-1-2003 and CDMRP Amyotrophic Lateral Sclerosis Research Program (ALSRP) Therapeutic Idea Award AL150111 (C.H.). PR and LB are funded by a CONICYT fellowship.

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