Endoplasmic reticulum and mitochondria in diseases of motor and sensory neurons: a broken relationship?

Recent progress in the understanding of neurodegenerative diseases revealed that multiple molecular mechanisms contribute to pathological changes in neurons. A large fraction of these alterations can be linked to dysfunction in the endoplasmic reticulum (ER) and mitochondria, affecting metabolism and secretion of lipids and proteins, calcium homeostasis, and energy production. Remarkably, these organelles are interacting with each other at specialized domains on the ER called mitochondria-associated membranes (MAMs). These membrane structures rely on the interaction of several complexes of proteins localized either at the mitochondria or at the ER interface and serve as an exchange platform of calcium, metabolites, and lipids, which are critical for the function of both organelles. In addition, recent evidence indicates that MAMs also play a role in the control of mitochondria dynamics and autophagy. MAMs thus start to emerge as a key element connecting many changes observed in neurodegenerative diseases. This review will focus on the role of MAMs in amyotrophic lateral sclerosis (ALS) and hereditary motor and sensory neuropathy, two neurodegenerative diseases particularly affecting neurons with long projecting axons. We will discuss how defects in MAM signaling may impair neuronal calcium homeostasis, mitochondrial dynamics, ER function, and autophagy, leading eventually to axonal degeneration. The possible impact of MAM dysfunction in glial cells, which may affect the capacity to support neurons and/or axons, will also be described. Finally, the possible role of MAMs as an interesting target for development of therapeutic interventions aiming at delaying or preventing neurodegeneration will be highlighted.


Facts
Defects in endoplasmic reticulum and mitochondria are observed in multiple forms of neurodegenerative diseases. Sites of contacts between endoplasmic reticulum and mitochondria at MAMs play a critical role in normal function of both of these organelles.
Alteration of MAMs lead to many of the pathophysiological changes observed in neurodegenerative diseases. Mutations in genes encoding proteins implicated in MAM function have a causal role in ALS and HMSN. Modulation of MAM function can alleviate some symptoms of neurodegeneration.

Open Questions
How is the assembly and maintenance of MAMs controlled? How do different defects affecting MAMs (e.g. mutation in genes encoding different components of MAMs) lead to alteration in ER/mitochondria function?

Introduction
Neuronal function relies on synaptic transmission, which is based on the propagation of action potentials along axons and neurotransmitter release. As the majority of biosynthetic pathways take place in the neuron soma, axons and distal synaptic contacts need efficient axonal transport for the supply of organelles and vesicles. Axonal transport is driven by motor proteins, which consume substantial amounts of energy. Sensory neurons and motoneurons have axons up to 1 m in length. Their extreme dendrite/cell-body/axon polarization and their large soma make these neurons highly demanding in energy to function properly. It was estimated that the anterograde transport of one vesicle along the 1 m long axon of a human motoneuron requires approximately 1.25 × 10 8 adenosine tri-phosphate (ATP) molecules 1 . High metabolic demand requires a tight coordination between protein secretion, organelle biogenesis, and degradation processes that avoid accumulation of defective components. Long axons are therefore particularly vulnerable to conditions of suboptimal energy supply 2 . The axonal compartment often degenerates first in diseases affecting long-projection neurons, such as amyotrophic lateral sclerosis (ALS) and hereditary motor and sensory neuropathies (HMSNs) also known as Charcot-Marie-Tooth diseases (CMTs) 2,3 .
Maintenance of ionic gradients, as well as the mobilization and cycling of synaptic vesicles in the axons, are mechanisms that are energetically demanding 4 and require controlled intracellular calcium signaling 5 . This is partly achieved by compartmentalizing biochemical reactions in pools of specialized organelles. The endoplasmic reticulum (ER) is the main site for protein and lipid biosynthesis and intracellular calcium storage, while mitochondria generate most of neuron's ATP via oxidative phosphorylation. Importantly, the interorganelle communication is essential to coordinate these activities. Mitochondrial ATP production depends on calcium concentration, which is controlled by the ER 6 . Juxtapositions of ER and mitochondrial membranes, called mitochondria-associated membranes (MAMs), represent one of the most specialized sites for interorganelle membrane interactions. ER and mitochondria become dysfunctional early during neuronal degeneration 7,8 . Therefore, defects at the level of MAMs could be among the initial triggers of the disease. For some of the genes linked to neurodegenerative diseases, the encoded proteins are located at MAMs 9 . Furthermore, MAM dysregulation occurs in several neurological pathologies including Alzheimer's disease, Parkinson's disease, and motoneuron diseases 10-12 . In this review, we will summarize the general function of MAMs with a particular attention on their role in neuronal degeneration in ALS and HMSN diseases, and discuss the contribution of MAMs in other cell types that support neuronal function in the central (CNS) and peripheral nervous system (PNS). We will also evoke possible outcomes of this research in terms of therapies targeting MAMs in neurodegenerative diseases.

Mitochondria-ER contacts
Tight contacts between the ER and the mitochondria were originally observed in 1956 on electron micrographs of the rat liver 13 . The characterization of the MAMs has been refined and now relies, in addition to electron microscopy, on biochemical methods, such as cell fractionation 14,15 , and fluorescent microscopy, in particular using super-resolution light microscopy 16 . Fluorescence resonance energy transfer-based indicators of ER-mitochondria proximity 17 and in situ proximity ligation assays 18,19 have been extensively used to characterize MAMs. Contact sites between ER and mitochondria are defined by an intermembrane distance of 10-30 nm, which depends on cell type and the level of stress to which the cells are exposed 20 . It is estimated that 15-20% of the mitochondrial surface is connected to the ER 21 .
MAMs control various critical cellular functions including lipid exchange, calcium homeostasis, autophagy, and mitochondrial dynamics, and contribute to regulation of inflammation and cell death 10,12 .
In mammalian cells, four types of connectors between ER and mitochondria have been identified. We have summarized their implications into MAMs signaling and ALS/HMSN in Tables 1 and 2 and Fig. 1: (1) Initially observed at the mitochondria, mitofusin 2 (MNF2) is a dynamin-related GTPase identified at the ER surface almost 10 years ago 22 . MFN2 contributes to ER and mitochondria tethering either by homologous interaction between ER-associated MFN2 and mitochondrial MFN2 or by heterologous interaction with mitofusin 1 (MFN1), a homolog protein only located at the outer mitochondrial membrane 22 .
(3) The subtype 3 of the 1,4,5-triphosphate receptor (IP3R3) forms a complex with the 75 kDa glucoseregulated protein (GRP75) and the mitochondrial voltage-dependent anion channel 1 (VDAC1), which serves as a calcium exchange platform between ER and mitochondria. These three proteins are referred to as tethering and calcium signaling proteins at MAMs 24 .  The role of these factors in disease affecting motor or sensory neurons remains to be explored (4) The ER-resident protein BAP31 (B-cell receptorassociated protein 31), the mitochondrial fission protein FIS1 (fission 1 homolog) and the phosphofurin acidic cluster sorting protein-2 (PACS-2) are MAM connectors involved in the induction of apoptosis 25,26 .

MAM dysfunction in ALS and HMSN diseases
Owing to their central position between ER and mitochondria, there is an increasing interest in the possible role of MAMs in neurodegenerative diseases. In the CNS, changes in MAMs were reported in multiple types of common neurodegenerative diseases, including Alzheimer's disease, Parkinson's disease, and spastic paraplegia 31 . In the PNS, motor and sensory neurons have particularly long axons. They represent extreme examples of how important the maintenance of ER and mitochondrial functions can be to sustain high axonal metabolic demand 3 . We will focus on ALS and HMSN, diseases affecting neurons projecting to the periphery, to discuss the implications of dysregulated MAMs in neuronal/ axonal pathology 16,32 (Fig. 2).

Disturbance of ER-mitochondria connections in ALS and HMSN
Missense mutations in VAPB and SIGMAR1 (sigma non-opioid intracellular receptor 1) can lead to inheritable forms of ALS and HMSN, whereas missense mutations in MFN2 cause a form of HMSN described as CMT2A (see Box 1). These genes provide the most direct evidence for the role of MAMs in neuron function.
De Vos et al. 23 were the first to demonstrate MAM dysfunction in ALS. They observed that P56S-mutated VAPB, which is linked to ALS8, has increased binding capacity to PTPIP51. Other studies identified loss-offunction homozygous mutations of SIGMAR1 in familial cases of juvenile ALS (ALS16) and HMSN [33][34][35][36] . Downregulation or loss of SIGMAR1 in neurons decreases ER-mitochondria association 18,36 . The loss of contacts between ER and mitochondria is due to the inability of mutated SIGMAR1 to properly bind IP3R3, causing IP3R3 destabilization 18,36 . SIGMAR1 is expressed throughout the CNS, with highest levels in motoneurons of the brainstem and spinal cord 37 . In alpha motoneurons, SIGMAR1 is mainly expressed in subsurface cisternae formed by the ER in postsynaptic densities associated with cholinergic synapses (C terminals). In ALS tissues, SIG-MAR1 is abnormally distributed and accumulates in large C terminals, cytoplasm, and proximal axons 38 . Consistent with the possible pathogenic effects of SIGMAR1 loss-offunction, the knockdown of Sigmar1 in mice leads to a reduction in ER-mitochondria contacts in motoneurons, concomitant with impaired locomotion and motoneuron degeneration 18 . The most common axonal form of HMSN, referred to as CMT2A, is linked to mutations in the MFN2 gene [39][40][41][42] . Although the loss of MFN2 perturbs the number of ER-mitochondria contacts 17,22,[43][44][45] , it is unclear what are the effects of CMT2A-causing dominant mutations on the formation of MAMs. Expression of mutated R94Q MFN2 in MFN2-null cells rescues mitochondrial morphology by complementing the effects of MFN2 on mitochondrial dynamics 22 . However, no changes in the number of MAMs were observed in these cells despite the loss of MFN2 22 . This observation suggests that this CMT2A-causing mutation is unlikely to affect mitochondrial dynamics, whereas pathogenic effects on MAM function remain entirely possible. Overexpression of the homologous protein MFN1 in primary sensory neurons partially decreased axonal degeneration induced by R94Q MFN2 46 . As MFN1 is localized on the mitochondria and not on the ER, it is likely that MFN1 cannot fully restore MAM function, which may explain why the rescue is only partial. Further studies in models of CMT2A will help to determine how MFN2 mutations affect MAM function.
In 2013, Poston et al. 9 performed a proteomic analysis of MAMs isolated from the mouse brain and identified more than one thousand associated proteins. Among them, ganglioside-induced differentiation-associated protein 1 (GDAP1), originally known to be located on the outer membrane of mitochondria, was found enriched in MAMs. Mutations in GDAP1 are linked to CMT, which are either recessive and demyelinating (CMT4A), or dominant/ recessive and axonal (CMT2K) [47][48][49][50] . Gdap1 knockout mice develop peripheral neuropathy 51 . Although there is evidence that the lack of GDAP1 can perturb calcium homeostasis and affect both ER and mitochondria, the role of GDAP1 at the level of MAMs remains unclear 52 .
Other proteins more commonly implicated in ALS, such as superoxide dismutase 1 (SOD1), TAR DNAbinding protein 3 (TDP43), and fused in sarcoma (FUS) (Box 1), do not have any established physiological role in the MAMs. However, pathogenic mutations can confer novel properties to these proteins that can affect MAM function. Compared to their wild-type counterparts, mutated forms of SOD1, TDP43, or FUS proteins tend to accumulate in ectopic compartments in disease conditions, and can perturb MAM function 36,53,54 .
By performing subcellular fractionation in samples from a neuronal cell line overexpressing mutated SOD1 and from the spinal cord of SOD1 ALS mice, Watanabe et al. 36 found different forms of mutated SOD1 enriched in both the mitochondrial and MAMs fractions, which was not observed for wild-type SOD1 36 . Moreover, the ERassociated mitochondrial surface was decreased in ALS 36 . Previous studies have shown that misfolded mutated SOD1 could bind VDAC1 in spinal cord tissue of ALS rats, reducing the activity of this mitochondrial anion

4.
PACS-2 Fig. 1 Overview of proteins required for ER-mitochondria tethering. The figure summarizes the four main tethers at MAMs. (1) Besides its main location at the outer mitochondrial membrane, a small portion of MFN2 can be found at ER. ER-MFN2/mitochondria-MFN2 homodimers and ER-MFN2/mitochondria-MFN1 heterodimers participate in the tethering of these two organelles. (2) VAPB located at the ER surface interacts with PTPIP51 located at the mitochondrial outer membrane. (3) The IP3R3 at the ER makes close connections with the mitochondrial channel VDAC1 owing to the chaperone GRP75, which interacts directly with the two proteins. At the ER, SIGMAR1 is chaperoning IP3R3. This complex regulates calcium transfer between the two organelles.(4) ER-resident protein BAP31 interact with the mitochondrial fission protein FIS1. With PACS-2, this MAM complex regulates ER-mitochondria coupling and apoptosis.    33,36 and also in patients affected with distal HMN (dHMN) 34,35 . SIGMAR1 is an ER chaperone protein present in MAMs and strongly expressed in motoneurons 38,160 . The mutated SIGMAR1 protein is unstable and non-functional, probably acting in a loss-of-function manner 36 . MFN2 (mitofusin-2) mutations (>60 were identified) are linked to the development of classical HMSN (also known as CMT2A) 39,42,138 . Mutated MFN2 accounts for~30% of axonal CMTs. CMT2A patients develop the channel 55 . It remains to be explored whether this interaction also prevents complex formation with the grp75 chaperone and IP3R3 at the level of the ER. Wild-type or mutated forms of TDP43 and FUS overexpressed in cell lines reduce ER-mitochondria contacts by disrupting VAPB-PTPIP51 binding 53,54 . In mice, overexpression of wild-type FUS leads to an ALS phenotype, with progressive hindlimb paralysis and death at 3 months of age 56 . In this model, the number of ER-mitochondria contacts is reduced and VAPB-PTPIP51 binding decreased. However, there is no reported direct binding of TDP43 and FUS to VAPB or PTPIP51 which may explain these effects 53,54 .

A. Physiological role of MAMs
Previous work suggested that both TDP43 and FUS activate the glycogen synthase kinase-3b (GSK3B) 57 . GSK3B activity is mainly regulated by inhibitory phosphorylation at serine 9. As phosphorylation controls protein-protein interaction, it was proposed that FUS and TDP43 may regulate VAPB-PTPIP51 dissociation by modulating GSK3B activity, via changes in the level of serine 9 phosphorylation 53,54 . However, the role of GSK3B in MAMs is still unknown. Moreover, these observations did not refer to any specific toxic effect of ALS-causing mutations, as the wild-type forms of TDP43 and FUS also affect MAM formation. Nevertheless, TDP43 and FUS are predominantly localized in the nucleus in physiological conditions, whereas only a small fraction of these proteins reside in the cytoplasm [58][59][60][61] . Pathogenic conditions including ALS-causing mutations increase the cytoplasmic expression of TDP43 and FUS 62 , which may cause toxicity at MAMs. Indeed, forced expression of wild-type FUS in the cytoplasm worsened its effect on the MAMs 54 .
Overall these studies highlight MAMs as a common starting point in motoneuronal degeneration caused by several genes associated to ALS and HMSN. A recent interactome study performed in a neuronal cell line expressing wild-type chromosome 9 open reading frame 72 (C9orf72) shows its enrichment in the mitochondria-enriched fraction 63 . It was found to interact with several proteins located on the mitochondrial outer membrane, including known components of the MAMs. Future research should address whether hexanucleotide repeat extensions in the C9ORF72 gene, which cause about 30% of familial ALS, may also affect MAM function.

Pathophysiological consequences of disrupted MAMs
MAMs participate in the regulation of crucial cellular functions such as calcium homeostasis, lipid production, autophagy, mitochondrial dynamic, and motility, as well as axonal maintenance. These functions are often defective in ALS/HMSN, hence contributing to motoneuron dysfunction and degeneration. Here, we review these findings, further underlining the possible contribution of MAMs to these processes (Fig. 2).

Axonal degeneration and MAMs
Since most of the studies focused on the role of MAMs in the cell body, there is scarce information regarding their role in the axonal compartment. With respect to motoneuron disease, it is unknown if MAM dysfunction may specifically affect the axons since most studies have used cell lines with no or limited axonal compartment. Furthermore, it is technically challenging to visualize MAMs in axons, where the interaction between the ER and the mitochondria are likely to be highly dynamic, to allow for effective axonal transport of mitochondria. Nevertheless, both the rough and smooth ER as well as mitochondria can be visualized by electron microscopy and immunofluorescence in the axoplasm of sciatic nerve 64,65 . Using electron microscopy combined with three-dimensional reconstruction, Villegas et al. 66 noticed the presence of mitochondria closely associated to the smooth ER in sciatic nerve explants. Using the sciatic nerve explant as an in vitro model of injury-induced axonal degeneration, they found that modulating calcium transfer from the ER towards the mitochondria protects against axonal degeneration 66 . disease with an early or late onset. The sooner symptoms will appear the more severe they will be. Most of CMT2A patients are severely affected and they become non-ambulatory before the age of 20 50 . More rarely, MFN2 mutations can also lead to optic atrophy and CNS impairment. GDAP1 (ganglioside-induced differentiation-associated protein 1) mutations are linked to both axonal and demyelinating CMT known as CMT2K (autosomal dominant, axonal CMT) 49 , CMT4A (autosomal recessive, demyelinating CMT), and AR-CMT2K (autosomal recessive, axonal CMT) 47,48 . The onset and symptoms are variable and can involve classical CMT symptoms with variable severity, as well as vocal cord paresis or pyramidal involvement 50 . GDAP1 protein regulates mitochondrial network by promoting mitochondrial fission. INF2 (inverted-formin 2) mutations cause intermediate Charcot-Marie-Tooth disease E (CMTDIE), which can be associated with glomerulopathy 141 . One isoform of INF2 localizes at the ER and at MAMs. INF2 protein regulates actin polymerization 31,140 .
In context of ALS and HMSN, mutations in the MAMassociated proteins SIGMAR1 and MFN2 induced axonal degeneration, suggesting that MAMs are indeed important for axon maintenance 18,46 . Moreover, inhibition and deletion of SIGMAR1 similarly decreased MAMs in the axon and soma of neurons 18 . Importantly, axonal degeneration precedes cell body degeneration in models of ALS and HMSN that are related to MAMs defects 18,46,67 . Therefore, one can hypothesize that in long-projection motoneurons, the axonal compartment is more vulnerable to MAM dysfunction than the neuronal cell body. However, further research is warranted to address this question.

Calcium dyshomeostasis
Dysregulation of calcium homeostasis is thought to play a key role in neuronal degeneration in both ALS and HMSN diseases. Local calcium concentrations are tightly regulated inside the cell to avoid calcium overload in the ER and mitochondria, which can trigger apoptosis 68 . By bringing ER and mitochondria in close proximity, MAM function as a platform for the exchange of calcium between these two organelles. MAMs have an important role in controlling calcium levels in the ER and also in mitochondria necessary to produce ATP.
Disruption of key factors, including VAPB-PTPIP51, SIGMAR1, and MFN2, correlates with perturbations of calcium homeostasis 18,23,46,69 . Downregulating either VAPB or PTPIP51 by RNA interference in cell lines reduces the number of ER-mitochondria contacts, which affects the mitochondrial uptake of calcium released from ER stores 23 . Unlike wild-type VAPB, expression of P56Smutated VAPB reinforces ER-mitochondria interactions, which causes an increase in calcium transferred to the mitochondria 23 . In primary cortical neurons, overexpression of P56S VAPB also perturbs resting cytosolic calcium levels, which affects the anterograde transport of the mitochondria 69 . It is however unclear if changes at the level of ER-mitochondria contacts are induced in the latter case.
Similarly, changes in SIGMAR1 activity affect the cytosolic and mitochondrial levels of calcium in vitro 18,36,38 . The physiological activity of SIGMAR1 regulates intracellular calcium levels in motoneurons. In embryonic motoneurons, inhibition of SIGMAR1 with the selective antagonist NE-100 increases intracellular resting calcium levels and prolongs the time needed for basal calcium levels to recover after potassium-evoked depolarization 18 . On the other hand, overexpression of SIG-MAR1 facilitates mitochondrial calcium uptake 36 . However, similar to SIGMAR1 downregulation by siRNA, the expression of two ALS-linked SIGMAR1 mutants (E102Q, L95fs) in neuronal cell lines causes an increase in cytoplasmic calcium levels and reduces mitochondrial calcium levels following ATP stimulus 36,38 . These results indicate that the loss of SIGMAR1 activity due to ALSassociated mutations may affect motoneuron function and survival via perturbations of calcium homeostasis at the level of MAMs.
Abnormal calcium handling was also linked to HMSN. Despite the implication of MFN2 in HMSN, most of the results on the role of MFN2 in MAM function were obtained by modulating its expression in cell lines. Only few experiments have been performed in neurons 22 . Recent studies have used lentivirus-transduced cultures of sensory neurons to compare the effects of wild-type MFN2 and the CMT2A-associated R94Q mutant following overexpression. Overexpression of mutated MFN2 caused axonal degeneration, which was concomitant with an axonal calcium rise 46 . However, it remains to be determined whether elevated calcium levels induced by mutated MFN2 are due to MAM dysfunction.
Conversely, the level of intracellular calcium can also affect ER-mitochondria interactions. Massively releasing calcium from the ER compartment using thapsigargin leads to ER mitochondria detachment, which may avoid further rise of calcium inside the mitochondria and prevent pro-apoptotic effects 70 .

MAM dysfunction affects the transport and dynamics of mitochondria
In neurons, communication between the cell body and synaptic terminals is based on axonal transport. In particular, efficient axonal transport is key for the mitochondria to reach specialized sites with high-energy demand, such as the nodes of Ranvier or the synapses 71 . In models of motoneuron diseases, defects of mitochondrial axonal transport have been observed both in vitro and in vivo 18,46,[72][73][74] . Interactions between ER and mitochondria are likely to occur in these compartments, and defects in several MAM-associated proteins have been shown to impair axonal transport.
Morotz et al. 69 demonstrated that overexpression of the ALS-associated P56S VAPB mutant affects the anterograde axonal transport of mitochondria in neurons. Concomitant to these defects, the level of tubulinassociated MIRO1 is reduced. MIRO1/2 are calciumsensitive Rho-like GTPases located at MAMs, linking mitochondria to kinesin 74 (see Table 2). P56S VAPB leads to an increase in cytoplasmic calcium levels, which could release the MIRO1/trafficking kinesin protein 1/kinesin-1 complex from the microtubules and decouple mitochondria from axonal transport 75 . Importantly, when MIRO1 is overexpressed in neurons expressing P56S VAPB, the association of MIRO1 with tubulin is rescued and mitochondrial transport restored 69 .
Mitochondrial axonal transport defects were also reported in primary sensory neurons following either Mfn2 deletion or overexpression of the CMT2Aassociated R94Q mutant 46,74,76 . Both anterograde and retrograde transports were slower, with mitochondria spending more time paused 74 . These defects are similar to the ones observed following knockdown of MIRO2, suggesting that MIRO2 function may be altered in CMT2A neurons. Despite the fact that the R94Q MFN2 may still interact with the MIRO-associated transport machinery, calcium handling could be altered in these axons, causing pathological changes in the association of MIRO proteins to the microtubule tracts 46,74 . In transgenic mice overexpressing R94Q MFN2, mitochondria accumulate in the distal part of the nerve, reinforcing the notion that the axonal transport of mitochondria could be affected 77 . However, other studies suggest that general axonal transport defects are not the sole cause of HMSN. When decreasing general axonal transport by overexpressing syntaphilin, a protein docking mitochondria to microtubules, sensory neurons do not undergo axonal degeneration 46 . Other CMT2A-associated MFN2 mutations also failed to induce similar axonal transport defects in sensory and motoneurons. In particular, sensory neurons isolated from homozygous or heterozygous R94W Mfn2 knock-in mice show mitochondria velocities comparable to their wild-type counterparts 78 . Two recent studies characterized motoneurons differentiated from iPS cells derived from CMT2A-R94Q patients 79,80 . Surprisingly, mitochondrial trafficking was only mildly affected in these motoneurons and neither axonal elongation nor motoneuron survival were reduced 79,80 , suggesting that mutated MFN2 may cause other pathogenic effects than changes in axonal transport. Moreover, MFN2 mutations could differentially impact on sensory and motor neurons 3 .
The main function of mitochondrial MFN2 is to control the fusion of outer mitochondrial membranes. In neurons overexpressing mutated MFN2, there is an overrepresentation of smaller mitochondria, which may indicate defective fusion 46,81 . The balance between organelle fission and fusion is important for mitochondrial health, and alterations of these dynamic processes can also impact on mitochondrial transport.
Finally, mitochondrial axonal transport is also affected in motoneurons deleted for SIGMAR1 or treated with a selective SIGMAR1 antagonist. Loss of SIGMAR1 specifically affects the retrograde transport of mitochondria, leading to an accumulation of mitochondria in the distal part of axons 18 . It also leads to an increase of mitochondrial length, which contrasts with the effects of R94Q MFN2, but may indicate defects in mitochondrial dynamics. As mentioned above, MAMs have been identified as sites promoting mitochondrial fission. By disrupting ER-mitochondria contacts, the loss of SIGMAR1 could therefore prevent mitochondrial fission.

MAMs regulate ER function in ALS/HMSN
In addition to their effects on MAMs, loss of MFN2 or SIGMAR1 and overexpression of P56S VAPB affect ER morphology 22,23,82 . As previously discussed, alteration of MAMs can affect ER calcium homeostasis depending on the rate of calcium transfer between the two organelles. Consequently, the overloading/depletion of calcium inside the ER can negatively impact on protein folding, leading to ER stress associated to the activation of the unfolded protein response (UPR). UPR-mediated intracellular signaling coordinates the suppression of protein synthesis with a transcriptional response enhancing protein folding and ERassociated protein degradation (ERAD) to re-establish ER homeostasis 83 . Alternatively, UPR can also lead to apoptosis if ER function is not restored. Several studies demonstrated the activation of ER stress and UPR in ALS, which was proposed to play a key role in motoneuron degeneration 8,18,[84][85][86] . The depletion of the MAM proteins MFN2, SIGMAR1, and PACS-2, as well as the expression of mutated VAPB, induced UPR in various cell types 18,26,38,[87][88][89] . MFN2, SIGMAR1, and VAPB modulate, by direct interaction, UPR sensors including activating transcription factor 6, inositol-requiring enzyme-1, and protein kinase RNA-like endoplasmic reticulum kinase (PERK) 88,90,91 .
Several components of the UPR machinery and ER chaperones are enriched at MAMs 89,92 . Previous studies have shown that ER and mitochondria can reciprocally regulate their morphology and function via the MAMs. MFN2 acts upstream of PERK by inhibiting this UPR sensor through physical interaction 88 . Conversely, the loss of MFN2 promotes constitutive activation of PERK and associated cell death. Silencing of PERK restores mitochondrial calcium levels as well as mitochondrial elongation. Also, by alleviating ER stress with Salubrinal, it is possible to rescue mitochondrial dynamics following deletion or inhibition of SIGMAR1 and thereby prevent motoneuron degeneration 18 .

MAMs and the control of autophagy and mitophagy in models of ALS/HMSN
Autophagy plays an important role in the maintenance of cellular homeostasis and function, by allowing the degradation of long-lived proteins and organelles through lysosomal activity. Autophagy is initiated by the formation of a phagophore, which elongates into a doublemembrane structure to form the autophagosome. ER-mitochondria contacts are critical sites for the emergence of autophagosomes, a process controlled by VAPB-PTPIP51, PACS-2, and MFN2 93,94 . However, these factors appear to have different effects in cellular models of autophagy induction. The loss of either VAPB or PTPIP51 leads to the loss of MAMs while stimulating autophagic flux, whereas the downregulation of MFN2 or PACS-2, which also leads to the loss of MAMs, prevents autophagosome formation 93,94 . This apparent discrepancy could be due to different cellular responses depending upon autophagy inducers. However, it may as well indicate that differences exist in the role of these proteins as regulators of autophagy at MAMs.
Similarly, several HMSN-related genes are also known to perturb the autophagy pathway 100 . Loss of SIGMAR1 in the motoneuronal cell line NSC-34 leads to accumulation of autophagic vacuoles, together with markers of autophagy initiation and endolysosomal pathway 82 . Experiments on human fibroblasts demonstrated that lipid rafts at MAMs serve as a platform to induce autophagosome formation 101 . Interestingly, SIGMAR1 depletion can destabilize lipid rafts 82 , which could explain how this factor affects autophagy.
Rizzo et al. 79 observed a global reduction of the mitochondria content in induced pluripotent stem cell-derived CMT2A motoneurons, with increased expression of mitophagy genes, such as PTEN-induced putative kinase 1 (PINK1), Parkin RBR E3 ubiquitin protein ligase (PARK2), BCL2 interacting protein 3 (BNIP3), and a splice variant of beclin 1 (BECN1). These results indicate a potential imbalance in mitochondrial homeostasis, with an enhancement of mitophagy, which is a selective form of autophagy 79 . PINK1/Parkin-dependent ubiquitination of proteins located on the outer mitochondrial membrane targets dysfunctional mitochondria for degradation. Parkin was reported to be located at MAMs, where it promotes calcium transfer by increasing contacts between the two organelles 102 . Parkin interacts with GRP75 and MFN2, and ubiquitinates MFN 31,103,104 . Further studies will be required to determine whether this is affected by MFN2 mutations causing CMT2A.
Additional factors present at MAMs regulate mitophagic activity. PINK1 and BECN1 have been found localized at MAMs after induction of mitophagy in a neuroblastoma cell line 105 . They both promote ER-mitochondria interactions supporting the formation of omegasomes (membrane extensions within the ER), and subsequently autophagosomes 105 . Moreover, PINK1 level is increased in a calcium-dependent manner following mitophagy induction 106 . As calcium is transferred between organelles at MAMs, it is possible that local rises in calcium levels induce PINK1 expression and promote mitophagy. Both Parkin and PINK1 were found to be deregulated in tissues from ALS mice and/or patients [107][108][109] . Together, there is accumulating evidence for the role of MAMs in controlling mitophagy, which could be related to both ALS and HMSN.

Role of MAMs in non-neuronal cells potentially implicated in ALS/HMSN
Multiple studies have highlighted the role of nonneuronal cells in both ALS and HMSN pathologies 110,111 . Besides physical support to neurons, glial cells such as astrocytes, the myelinating oligodendrocytes (OL) and Schwann cells (SCs), also provide metabolic and trophic support [112][113][114][115][116] . It is therefore plausible that the dysregulation of MAM function in glial cells also contributes to pathophysiology of ALS and HMSN. OL and SC produce myelin, which is enriched in lipids that are produced by the ER, such as galactosylceramide 117,118 . The MAM protein SIGMAR1 has been detected in progenitors and mature OL in the rat brain, as well as in SC in the rat sciatic nerve 119,120 . In primary rat OL, the level of SIG-MAR1 expression affects OL differentiation by regulating the compartmentalization and transport of lipids 118 . However, the role of SIGMAR1 in OL has not been explored in the context of ALS. Downregulation of either SIGMAR1 or PACS-2 leads to degeneration of neurons and astrocytes, which indicates that MAM integrity is essential for the survival of both cell types 121 . Although these results are intriguing, additional studies are required to determine whether MAM dysfunction in glial cells may directly contribute to the pathogenesis of ALS and HMSN.

Therapeutic strategies targeting MAMs
Factors modulating MAMs could provide effective targets for therapeutic intervention, by synergistically impacting on the function of both ER and mitochondria. Notably, an agonist of SIGMAR1 (Pre-084) was shown to improve muscle activity and motor performance in presymptomatic ALS SOD1 mice 122 . This compound protects motoneurons and extends survival of the treated mice. The same treatment also preserves motoneuron survival and motor performance in the wobbler mice, a model of spontaneous motoneuron degeneration 123 . This finding suggests that pharmacological modulation of MAMs could be beneficial to motoneurons also in models that are not related to mutated SOD1 pathology, and opens therapeutic perspectives for ALS/HMSN diseases involving MAM dysfunction. Recently, another SIGMAR1 agonist, SA4503, was found to reduce cytosolic calcium transients and improve cytoplasmic calcium clearance in cultures of ALS SOD1 motoneurons 124 .
Defects in calcium homeostasis possibly related to MAM dysfunction are a prominent feature in ALS and HMSN diseases 36,46,52,68,125 . Therefore, molecules modulating calcium transfer between ER and mitochondria by activating or blocking IP3R or Ryanodine receptors could be considered as therapeutic agents. However, such an approach would need to be tightly targeted, since an increase in cytosolic calcium can be detrimental. Notably, overexpression of the type 2 IP3R in ALS SOD1 mice has been shown to shorten lifespan of these mice 126 . The use of ER stress inhibitors, such as Salubrinal, could also be a strategy to counteract defects at the level of MAMs. In addition to reducing ER stress, Salubrinal can restore calcium homeostasis in conditions of SIGMAR1 dysfunction 18 . Importantly, daily injections of Salubrinal in ALS SOD1 mice improve their motor functions and extend their survival 86 .

Conclusion/perspectives
Long-projection neurons such as motoneurons and sensory neurons are particularly affected by ALS and HMSN. This is consistent with the notion that these cells are highly sensitive to any stress, including energy deprivation, which may primarily lead to the degeneration of their axons 2,3,86 . The interplay between ER and mitochondria undeniably plays a pivotal role in maintaining cellular functions essential to neuronal cells. Disruptions of MAM function provoke ER and/or mitochondria stress, with perturbations in calcium homeostasis and energy supply. It is therefore tempting to speculate that perturbations at the level of MAMs could be a starting point for axonal degeneration. However, most of the studies that explored the role of MAM-associated factors linked to ALS and HMSN have used cell lines as model system. Further experiments using primary cultures of sensory or motor neurons as well as in vivo models of axonopathies are needed to clarify the role of MAMs in the cell types that are directly relevant to disease.