Altered localization, abnormal modification and loss of function of ...

Human Molecular Genetics, 2013, Vol. 22, No. 8 doi:10.1093/hmg/ddt008 Advance Access published on January 11, 2013

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Altered localization, abnormal modification and loss of function of Sigma receptor-1 in amyotrophic lateral sclerosis J. Prause1,{, A. Goswami1,∗ , {, I. Katona1, A. Roos1, M. Schnizler3, E. Bushuven1, A. Dreier1, S. Buchkremer1, S. Johann2, C. Beyer2, M. Deschauer4, D. Troost5 and J. Weis1 1

Institute of Neuropathology and 2Institute of Neuroanatomy, RWTH Aachen University and JARA Brain Translational Medicine, Pauwelsstr. 30, 52074 Aachen, Germany, 3Institute of Physiology, RWTH Aachen University, Pauwelsstr. 30, 52074 Aachen, Germany, 4Department of Neurology, Martin-Luther-University Halle-Wittenberg, 06120 Halle, Germany and 5Division of Neuropathology, Department of Pathology, Academic Medical Centre, 1105 AZ Amsterdam, The Netherlands Received November 22, 2012; Revised and Accepted January 7, 2013

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patient’s fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

INTRODUCTION Abnormal accumulations of mutant misfolded proteins are the pathological traits of many late-onset neurodegenerative diseases, such as Alzheimer’s disease (AD), polyglutamine diseases, Parkinson’s disease (PD) and amyotrophic lateral sclerosis (ALS) (1– 3). Mutant proteins that do not fold correctly or that are abnormally modified after translation are degraded by the ubiquitin proteasome pathway (4 – 6). However, if abnormal proteins overcome the degradation capacity of the proteasome, they may aggregate and form ubiquitinated cellular inclusions. Such aggregates or inclusion bodies

have toxic properties (5 – 8). In both sporadic (s) and familial (f) ALS cases, protein inclusions in motor neurons are a constant and pathognomonic feature. Mutations causing fALS account for 5 – 10% of all ALS cases and share pathological features with sALS cases (9,10). To date, numerous mutations have been identified in several genes which give rise to fALS: superoxide dismutase (SOD1), vesicle-associated membrane protein associated protein B (VAPB), senataxin (SETX), alsin (ALS2), dynactin (DCTN1), TAR DNA-binding protein 43 (TARDBP) and RNAbinding protein FUS (FUsed in Sarcoma, FUS), as well as in the open reading frame C90ORF (9,11– 14). These discoveries

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To whom correspondence should be addressed. Tel: +49 2418089399; Fax: +49 2418082416; Email: [email protected] These authors contributed equally to this work.

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# The Author 2013. Published by Oxford University Press. All rights reserved. For Permissions, please email: [email protected]

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have shed light on the importance of oxidative stress, the unfolded protein response (UPR), protein aggregation and abnormal RNA processing in the pathogenesis of ALS (11,12). Recently, Luty et al. (15) described variants in the 3′ untranslated regions (UTR) of Sigma receptor 1 (SIGMAR1, SigR1) in three ALS-FTLD/FTLD families with an autosomal dominant mode of inheritance. Shortly thereafter, Al-Saif et al. (16) described a novel mutation in a highly conserved amino acid located in the transmembrane domain of SigR1 as the cause of a juvenile form of ALS. Interestingly, the mutated proteins showed an aberrant sub-cellular distribution in transfected NSC34 cells and reduced resistance to apoptosis induced by endoplasmic reticulum (ER) stress (16). These results suggest a critical role of SigR1 in maintaining neuronal function and homeostasis. Sigma receptors (SigR1 and SigR2) are unique non-opioid, non-phencyclidine receptors (17). SigR1 has no homology to any other mammalian protein, but shares a 30% identity with a yeast C8-C7 sterol isomerase (18). The spectrum of biological functions of SigR1 has only been partially defined so far. However, it has become clear that SigR1 is an ER chaperone that binds a wide range of ligands, including benzomorphans (e.g. (+) pentazocine), steroids and an array of psychotropic drugs (19). In the nervous system, SigR1 has been shown to regulate K+ channels, IP3R-mediated Ca2+ signaling, neuritogenesis and aspects of memory and learning, and is also involved in drug addiction (20– 22). Glutamate excitotoxicity contributes to motor neuron degeneration (23). Ligands of SigR1 have been shown to prevent neuronal death associated with glutamate toxicity in vitro and in cerebral ischemia models (24,25). SigR1 agonists have also been demonstrated to inhibit b-amyloid- or ischemia-induced neurodegeneration (26). For example, in a model of glutamate toxicity in organotypic slice culture of spinal cord and in explanted dorsal root ganglia, a selective ligand of SigR1, 2-(4-morpholinethyl) 1-phenylcyclohexanecarboxylate (PRE-084), prevented neuronal death and supported neurite outgrowth (27). More recently, neuroprotective effects of SigR1 agonists during oxidative stress as well as reduced cognitive impairment in neuropsychiatric diseases have been demonstrated (28,29). SigR1 knockout mice have a shorter latency period in rotarod experiments with motor deficits, suggesting a crucial role of SigR1 in maintaining motor function (30). Notably, shRNA knockdown of SigR1 has been shown to induce apoptosis, as have SigR1 antagonists (31). Such effects can be blocked either by caspase-3 inhibitors or by the application of SigR1 agonists (32). Taken together, these results suggest that SigR1 is important for neuroprotection, neuronal survival and maintenance. It is largely unclear so far, however, whether SigR1 protein is abnormally modified or its functions are altered, and if so, how these alterations or abnormal modifications of SigR1 contribute to the pathogenesis of ALS. We therefore studied the expression of SigR1 in post mortem spinal cord tissues from sporadic and familial ALS cases, in SOD1 transgenic mice, in NSC34 motor neuron-like cells and in skin fibroblasts obtained from a recently identified ALS-8 case (33,34) expressing mutant P56S-VAPB. We found that SigR1 was abnormally accumulated in enlarged C-terminals and in ER structures of alpha motor neurons of ALS patients; these abnormal accumulations were associated with the 20s proteasome component.

Consistently, we detected similar accumulations in cultured human ALS-8 skin fibroblasts, in SOD1 transgenic mouse alpha motor neurons and in cultured NSC34 cells. In order to understand the functional role of SigR1, we used a loss of function approach. shRNA knockdown of SigR1 expression in the NSC34 cell line gave rise to a severe phenotype with induction of the UPR followed by inhibition of the ubiquitin proteasome system (UPS) and cell death. Electron microscopy revealed deformation of the ER and ER-derived vacuoles associated with autophagy in these cells. Finally, we show that pharmacological activation of SigR1 was neuroprotective in a cell culture model of ALS-8 (P56S-VAPB NSC34 cells). Taken together, our results suggest that alteration and pathogenic modification of SigR1 contributes to neurodegeneration in ALS and that SigR1 agonists have therapeutic potential for this disease.

RESULTS SigR1 is abnormally redistributed in a-motor neurons of ALS patients Several proteins have been shown to play crucial roles in ALS pathogenesis, including TDP43, FUS/TLS, p62 and SOD1. These proteins are also major components of inclusion bodies in motor neurons of ALS patients (11,14,35). Inclusion bodies are aggregates of (often ubiquitinated) proteins which are thought to be central to the pathogenesis of neurodegeneration (3,6,36). We first investigated the involvement and distribution of components of the UPS and of chaperones in motor neurons of a group of sporadic and familial ALS cases (Supplementary Material, Table S1). Serial sections of the lumbar spinal cord of these patients were subjected to immunohistochemical staining using antibodies against ubiquitin, HSP70, GRP78, 20s proteasome and p62. Immunohistochemistry revealed strongly labeled ubiquitinated aggregates (Supplementary Material, Fig. S1A) of variable morphology including scale and dot like or larger globoid shapes confirming the diagnosis of ALS. The immunohistochemical profiles indicate that these inclusion bodies consist of abnormal proteins that are targeted for refolding and clearance. A number of proteins mutated in familial ALS including SOD1 and ubiquilin-2 aggregate in affected neurons. These proteins have also been identified in intracellular inclusions in sALS cases (9,10). We therefore hypothesized that wildtype SigR1 protein might be misfolded and aggregated in sALS as well and searched for the presence of SigR1 within the above-described aggregates. Surprisingly, abnormal p62-immunoreactive aggregates of SigR1 were not found in a-motor neurons of the ALS cases investigated (Supplementary Material, Fig. S1Cc). Instead, the distribution of SigR1 was frequently altered in other ways. In a-motor neurons, SigR1 is normally located in postsynaptic densities associated with cholinergic synapses known as C-terminals (30,37). In our control cases, SigR1 was predominantly localized to such densities at the plasma membrane. In the ALS cases, the SigR1-immunoreactive C-terminals were significantly larger in diameter (Fig. 1; Supplementary Material, Fig. S1B). In addition, cytoplasmic accumulations associated with the ER were prominent in ALS a-motor neurons. Intriguingly, abnormal SigR1 accumulations were also frequent


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Figure 1. Altered distribution and accumulation of SigR1 in ALS. (A) Schematic representation of SigR1 localization in motor neurons. The numbers correspond to SigR1 distribution in various compartments within the neuron. (B) Quantification of distribution and localization of SigR1 protein in ALS patient lumbar a-motor neurons when compared with normal controls based on the above scheme. Control vs. sALS: P ¼ 8.165e209, control vs. fALS: P ¼ 1.182e208, sALS vs. fALS: P ¼ 0.2256 (see Materials and Methods for details). For the morphological studies n ¼ 18 sALS cases; n ¼ 2 fALS cases; n ¼ 8 control cases. For the analysis shown in Figure 1B, five sections each, n ¼ 12 sALS; n ¼ 2 fALS; n ¼ 4 control cases were investigated; no. of motor neurons: sALS ¼ 213, fALS ¼ 38, controls ¼ 93. The asterisks denote significant differences (∗∗ P , 0.005). (C) Immunohistochemical staining of lumbar a-motor neurons for SigR1 (brown) in ALS when compared with normal controls. Arrowheads are placed to mark some of the accumulations of SigR1 immunoreactivity (see Results for description). Paraffin sections, scale bar ¼ 10 mm.

in the proximal axon (Fig. 1; Supplementary Material, Fig. S1Bb, h). Consistent with these results, a similar abnormal staining and clearly distinct pattern of SigR1 was

observed in lumbar spinal cord a-motor neurons of G93A SOD1 mice (Supplementary Material, Fig. S1Cd). To verify the abnormal nature of the strongly labeled, large

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intraneuronal SigR1 accumulations, we co-stained SigR1 with components of degradation pathways, 20S proteasome and p62. The SigR1 accumulations were co-localized with 20s proteasome (Supplementary Material, Fig. 1Cb). In contrast, as described above for human ALS a-motor neurons, we did not observe any SigR1 co-localized with p62 aggregates (Supplementary Material, Fig. 1Cc). SigR1 forms ubiquitinated aggregates and aberrantly interacts with VAPB in ALS-8 To strengthen the point that SigR1, similar to other crucial proteins such as FUS/TLS, TDP43 and SOD1, is commonly affected in ALS (12,35), we chose ALS-8 as a paradigmatic disorder. This disease is caused by mutation of another ER protein, VAPB (vesicle-associated membrane protein-associated protein B; OMIM 605704), leading to prominent UPR. ALS-8 is a rare hereditary form of motor neuron disease initially found in Brazilian families with an autosomal dominant mutation (P56S) in the VAPB gene (33,34). Subsequently, the same mutation was discovered in a German ALS family (34) and in patients of Japanese and French ancestry (13). The P56SVAPB point mutation translates into a heavily misfolded protein which forms cytoplasmic inclusion bodies (Supplementary Material, Fig. S2a, c). As both SigR1 and VAPB have been shown to be expressed ubiquitously (38,39), we took advantage of the rather easily accessible skin fibroblasts from our ALS-8 patient (34). Consistent with the above-described results in motor neurons, abnormal accumulation of SigR1 was evident in these fibroblasts when compared with normal control fibroblasts from a healthy individual (Fig. 2A – D). Furthermore, these SigR1 accumulations were again co-localized with the 20s component of the proteasome (Fig. 2C), and ubiquitin immunoreactivity indicated that they were targeted to ubiquitinmediated degradation pathways (Fig. 2D). We then asked whether SigR1 accumulates together with these mutant VAPB aggregates. Interestingly, we detected significant co-localization of VAPB and SigR1 (Fig. 2B) in the ALS8 patient’s fibroblasts and obtained analogous results in HeLa cells transfected with P56S-VAPB (Supplementary Material, Fig. S2c). Finally, we found that the SigR1-immunoreactive inclusions in lumbar a-motor neurons of G93A SOD1 transgenic mice were also stained after incubation with VAPB antibodies (Supplementary Material, Fig. S2b). Overall, these results suggest that SigR1 is a major constituent of the abnormal intracellular protein deposits in ALS-8 and associates with VAPB in ALS. SigR1 expression is deregulated in ALS and ALS-8 To further assess the expression pattern of SigR1 in ALS, we performed immunoblots of frozen lumbar spinal cord specimens from ALS patients and controls. Interestingly, overall SigR1 protein levels were significantly reduced in these samples. Furthermore, there was a prominent induction of UPR signaling parameters. The elevation of GRP78, PDI and GADD all indicated the induction of UPR followed by ER overload response in these patients (Fig. 3A). The reduced overall levels of soluble SigR1 in ALS spinal cord despite the focal accumulation in surviving a-motor neurons are not surprising,

because the loss of motor neurons in ALS spinal cord (Supplementary Material, Fig. S1A-c) is expected to contribute to the decrease in overall soluble SigR1 protein levels. Moreover, SigR1 has already been reported to be decreased in CNS tissue of early stage PD patients and in early AD (38,40,41). Finally, NSC34 cells transfected with P56S-VAPB formed VAPB and SigR1-immunoreactive aggregates (Supplementary Material, Fig. S2c), along with a significant reduction in soluble SigR1 protein levels and a persistently increased level of misfolded protein stress as well as UPS inhibition leading to accumulation of the UPS substrate d1EGFP (Supplementary Material, Fig. S3c-d). To further confirm these findings, we analyzed skin fibroblast from the ALS-8 patient. Here, we found elevated levels of soluble SigR1 together with many small and few larger cytoplasmic VAPB and SigR1-immunoreactive aggregates. Furthermore, there was an up-regulation of UPR markers and of cytosolic chaperones (Fig. 3A– D). The elevated levels of soluble SigR1 in these cells probably reflect the successful (see Discussion) cellular defense response with constitutive activation of the UPR in ALS-8 patient fibroblasts. Alternatively, elevated levels of SigR1 might be a consequence of inhibition of UPS as evidenced by ubiquitin blot (Fig. 3C), because UPS inhibition also leads to an induction of various chaperones (42) and activation of the UPR (43). Apparently, the aggregation of insoluble SigR1 in these cells (Fig. 2A) is not sufficient to lead to a net reduction in soluble SigR1 levels. Taken together, these results suggest that accumulation of misfolded protein including abnormal VAPB in the ER induces the UPR and leads to SigR1 accumulation in response to ER stress to handle the misfolded protein. This is also in line with other studies which showed that the UPR and other stress responses are activated in ALS (44– 46). As GADD is up-regulated in response to DNA damage and cell death signals, we examined protein levels of the cleaved form of caspase-3 as well as of LC3. These proteins are indicators of apoptosis and autophagy (45,47,48). We found a significant elevation of cleaved caspase-3 and conversion of LC3-I to LC3-II reflecting the concurrence of apoptosis and autophagy in the ALS tissue samples (Fig. 3A). Up-regulation of HSP70, a marker of persistent misfolded protein stress, was also evident in these samples (Fig. 3A, B) supporting earlier reports (42). Similarly, G93A SOD1 transgenic mouse lumbar spinal cord homogenates showed a consistent up-regulation of ER stress markers and of ER stress-mediated induction of autophagy (Supplementary Material, Fig. S3a-b). shRNA knockdown of SigR1 induces UPR, inhibition of UPS and caspase-3 mediated apoptosis We established that the accumulation of SigR1 along with the misfolded protein P56S-VAPB in the ER gives rise to ER stress. Conversely, prolonged UPR or inhibition of UPS apparently leads to the up-regulation and accumulation of SigR1 protein (Supplementary Material, Fig. S2c) representing a vicious pathogenic cycle. In order to confirm the functional relevance of these findings, we knocked down endogenous levels of SigR1 in NSC34 cells using custommade shRNA. Knockdown of SigR1 for 48 h lead to the increase of the ER stress markers GRP78, peIF2alfa and pPERK indicating a profound activation of the UPR


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Figure 2. SigR1 forms ubiquitinated aggregates in ALS-8. (A) Globular structure of SigR1 aggregates in cultured ALS-8 patient fibroblast when compared with the normal control, lower panel (arrowheads). Note the preferential aggregation associated with the ER; scale bar ¼ 5 mm. (B) Co-localization (yellow) of SigR1 aggregates with VAPB aggregates in ALS-8 fibroblasts (yellow punctate staining); scale bar ¼ 5 mm. (C) Immunofluorescence staining showing the co-localization of SigR1 aggregates with accumulated 20s subunit of the proteasome in ALS-8 fibroblasts (arrowheads); scale bar ¼ 5 mm. (D) Ubiquitin immunoreactivity of SigR1 aggregates in ALS-8 fibroblasts (arrowheads); scale bar ¼ 5 mm.

(Fig. 4A). These results were confirmed by functional experiments using the known ER stressors and proteasome inhibitors thapsigargin and MG132 as controls along with or without the knockdown of SigR1 to evaluate the exact contribution of the knockdown to the induction of the UPR. The treatments combined with the knockdown had an even more severe effect as demonstrated by western blot analysis (Fig. 4B– C) revealing a consistent reactive induction of ER stress markers.

Heavily misfolded proteins are either targeted to the refolding machinery (to reinstate their normal conformation) or to the degradation machinery (for complete degradation and recycling). Failure of these processes can result in massive accumulation of misfolded ubiquitinated proteins (4,49). Immunoblotting with ubiquitin antibodies revealed accumulation of ubiquitinated proteins (Fig. 4D and E) when compared with the MG132 control, confirming again the deleterious effect of the knockdown of SigR1.

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Figure 3. SigR1 expression is deregulated in sALS and ALS-8. (A) Western blot analysis of SigR1 and of established ER stress markers in lumbar spinal cord lysates of ALS patients (n ¼ 10) and normal controls (n ¼ 4). (B) Quantification of the band intensities against a-tubulin was performed using Photoshop CS5. Values are means + SD of three independent analyses each. ∗ P , 0.05, ∗∗ P , 0.005. (C) Immunoblot analysis of SigR1 and of established ER stress markers in ALS8 patient fibroblasts when compared with a normal control. (D) Quantitative analysis.

Because prolonged UPR is known to compromise UPS function (43), we suspected that SigR1 knockdown might have a similar effect. For this study, we used a model substrate

of the proteasome, a destabilized enhanced green fluorescence protein (d1EGFP) containing a PEST (proline, glutamate, serine and threonine) sequence at its C-terminus. The half-life


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Figure 4. shRNA knockdown of SigR1 induces UPR. (A) NSC34 motor neuron-like cells were transfected with SigR1 shRNA for 48 h and analyzed for UPR induction by immunoblotting. Fold change on the left side represents the quantification of band intensities normalized against a-tubulin. Values are means + SD of three independent experiments. ∗ P , 0.05, ∗∗ P , 0.005. (B) NSC34 cells transfected with SigR1 shRNA for 48 h, with or without 24 h treatment with the established UPR inducer thapsigargin (2 mM) and the UPS inhibitor MG132 (2 mM). Cell lysates were subjected to immunoblot analysis for UPR induction. (C) Quantification of immunoblot analyses depicted in (B). Values are means + SD of three independent experiments each. ∗ P , 0.05, ∗∗ P , 0.005. (D, E) Ubiquitin immunoblot analysis of above samples. Quantifications of immunoblots in (E) are shown in lower panel.

of this protein is 1h. Proteasomal malfunction will increase the stability of d1EGFP leading to enhanced fluorescence intensity of GFP (5,50). NSC34 cells were transfected with

shRNA constructs of SigR1 or control shRNA along with pd1EGFP plasmid for 48 h. SigR1 knockdown lead to increased GFP fluorescence indicating proteasome dysfunction

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(Fig. 5A). These SigR1 knockdown cells also exhibited increased levels of d1EGFP protein as shown by immunoblotting (Fig. 5B and C). To further confirm UPS malfunction, we directly measured cellular proteasome activity in the treated cells. The SigR1 knockdown cells were exposed to thapsigargin and MG132 or left untreated for 24 h. Chymotrypsin protease activity of the proteasome was significantly inhibited by SigR1 knockdown when compared with the MG132 and thapsigargin controls (Fig. 5D and E). A similar inhibitory effect of SigR1 knockdown on proteasomal activity was also observed in HeLa cells (data not shown). Proteasomal dysfunction induces apoptosis and Caspase-3 is the main mediator of the apoptotic pathway (5,50,51). SigR1 knockdown for 48 h significantly increased the activation of caspase-3 as shown by immunoblot analysis and caspase-3 protease activity assay (Fig. 5F and G). Knockdown of SigR1 increases intracellular Ca21 levels from IP3R The physical association between the ER and mitochondria, which is known as the mitochondria-associated ER membrane (MAM), is implicated in various cellular ‘housekeeping’ functions including the non-vesicular transport of phospholipids (52). It has recently become clear that the MAM also enables highly efficient transmission of Ca2+ from the ER to mitochondria to stimulate oxidative metabolism and, conversely, might enable the metabolically energized mitochondria to regulate ER Ca2+ homeostasis (39). Thus, alterations in the expression of the ER protein SigR1 might disturb ER Ca2+ homeostasis. We therefore assessed the effect of SigR1 knockdown on calcium mobilization in NSC34 cells. Intracellular calcium concentration [Ca2+]i was measured using a conventional Fura-2 technique (53) and data were presented as 340/380 ratios. Knockdown of SigR1 leads to a significant increase in [Ca2+]i through IP3R3 when stimulated with bradykinin (BDK) (Fig. 6A). On the other hand, there was less Ca2+ stored in the ER after stimulating cells with ionomycin, suggesting improper calcium handling and storage (Fig. 6B). Knockdown of SigR1 disturbs the mitochondrial membrane potential followed by cytochrome-C release and cell death It is well established that excessive intracellular Ca2+ is efficiently taken up by mitochondria via the mitochondrial Ca2+ uniporter/channel (MCU) located in the mitochondrial inner membrane (54) followed by mitochondrial membrane depolarization and cytochrome-C release (5,55). Therefore, we asked whether disturbed intracellular Ca2+ signaling in SigR1 knockdown cells is linked to such mitochondrial disturbances that should alter the mitochondrial membrane potential. After 48 h of SigR1 knockdown, NSC34 cells were either subjected to JC-1 staining to study changes of mitochondrial membrane potential or processed for immunofluorescence staining and immunoblot analysis of cytochrome-C to study its release into the cytosol from mitochondria. The fluorescent potentiometric dye JC-1 exists as a green monomer at low concentrations or at low (Dc) membrane potential. At higher concentrations or at higher (Dc) membrane potentials, JC-1

forms red fluorescent ‘J-aggregates’ that can be excited at 590 nm. A decrease in red fluorescence indicated lowering of the membrane potential when cells were treated with the known ER stressor thapsigargin (Supplementary Material, Fig. S4). Knockdown of SigR1 significantly decreased the mitochondrial membrane potential in a very similar way as indicated by the disappearance of the red-dotted color in the mitochondria (Fig. 7A, right panel). A small increase in cytochrome-C release already occurred in the SigR1 knockdown cells when compared with control transfected cells; however, this increase was less prominent compared with the cells treated with thapsigargin (Fig. 7B). These results were consistent with the increment of intracellular Ca2+ levels (Fig. 6A), and were further verified by the Alamar blue cell viability assay (56) which measures mitochondrial activity of living cells. Alamar blue activity was decreased by 30– 40% after SigR1 knockdown (Fig. 7C). Based on these results, we conclude that activation of an intrinsic apoptotic pathway has been initiated by the release of cytochrome-C into the cytoplasm from the intra-mitochondrial space (57), followed by binding to apoptosis protease activating factor-1 to induce apoptotic cell death. These results are in agreement with recent findings suggesting that mitochondrial energy depletion and ER stress mediate apoptosis upon expression of a truncated form of SigR1 (58). Knockdown of SigR1 disturbs ER integrity and lipid transport and induces abnormal vacuolization Next we studied the consequences of SigR1 dysfunction at the ultrastructural level. Electron microscopy following SigR1 knockdown in NSC34 cells revealed a widened ER and numerous, probably ER-derived vacuoles, some of which were filled with membranous material indicating autophagy (Fig. 8A, B). Interestingly, NSC34 cells lacking SigR1 (but not control transfected cells) often showed protrusions of nuclear material at sites of apparent instability of the nuclear envelope (Fig. 8E). Cytoplasmic membrane-bound vacuoles and alterations of the nuclear-cytoplasmic interface were also found in SigR1 knockdown transfected Hek293 cells. Nuclear protrusions and invaginations were even more pronounced in transfected Hek293 cells than in transfected NSC34 cells. We also consistently observed Golgi dispersion as well as focal dilatation and distortion of Golgi complex tubules associated with membranous accumulations of phospholipids in knockdown NSC34 cells, again indicating disturbances in lipid metabolism during prolonged ER stress (Supplementary Material, Fig. S5). Altogether, these results suggest that SigR1 is important for the maintenance of ER structural integrity and that it plays a role in the formation of ER-derived vesicles, either for maintaining autophagy or for vesicular transport. Pharmacological activation of SigR1 decreases mutant VAPB aggregation and has a neuroprotective effect Activation of SigR1 by selective ligands is known to be neuroprotective in various neurological diseases. We observed co-aggregation of SigR1 along with P56S-VAPB (Fig. 2; Supplementary Material, Fig. S2c) suggesting a failure of SigR1


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Figure 5. shRNA knockdown of SigR1 causes proteasome impairment and induces apoptosis. (A) HeLa cells were transiently transfected with SigR1 shRNA along with pd1EGFP plasmid (1 mg/ml of media). Forty-eight hours after transfection, cells were processed for immunofluorescence. Representative images depict the accumulation of d1EGFP as a reporter for UPS inhibition. (B) NSC34 cells were transiently transfected as described above. Immunoblot analysis was performed using a GFP antibody. Values are mean + SD of three independent experiments. ∗∗ P , 0.005. (C) NSC34 cells were transiently transfected as described above with or without the treatment with the UPR inducer thapsigargin (2 mM) and the UPS inhibitor MG132 (2 mM) and incubated for 24 h. Cell lysates were prepared and subjected to immunoblot analysis using the GFP antibody. Quantifications of immunoblots are shown in lower panel. Values are means + SD of three independent experiments. ∗ P , 0.05, ∗∗ P , 0.005. (D, E). NSC34 cells were transiently transfected with SigR1 shRNA for 48 h, with or without the treatment with thapsigargin (2 mM) and tunicamycin (5 mg/ml) and MG132 (2 mM) and incubated for 24 h. Chymotrypsin-like proteasomal activity assays were performed as described in Material and Methods. Means + SD of three independent experiments each performed in triplicate. ∗ P , 0.05, ∗∗ P , 0.005. (F) NSC34 cells were transiently transfected with SigR1 shRNA and treated as described above. Cell lysates were subjected to caspase-3 western blot and (G) caspase-3 protease activity assay. Values are means + SD of three independent experiments each performed in triplicate. ∗ P , 0.05, ∗∗ P , 0.005.

chaperoning and maybe other chaperones to refold mutant misfolded VAPB. Hence, we next asked whether we can exploit the chaperone activity of SigR1 by its activation through its selective agonist to reduce mutant VAPB aggregation. In order to test our hypothesis, we used PRE-084, a

selective agonist of SigR1, for the rescue experiments. Treatment with PRE-084 significantly reduced mutant VAPB aggregation in a dose-dependent manner (Fig. 9A) and facilitated the degradation of soluble mutant VAPB without affecting the normal level of wild-type proteins. This suggests

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Figure 6. shRNA knockdown of SigR1 disturbs Ca2+ homeostasis. (A) Transient intracellular calcium signaling evoked from the ER store through IP3R by BDK was moderately increased by knockdown of SigR1 for 48 h. NSC34 cells were transiently transfected with SigR1 shRNA for 48 h and were loaded with fura-2AM for 30 min and then stimulated with 10 mM BDK. Representative traces of the BDK-induced increase in [Ca2+]i in the knockdown cells when compared with control cells are shown in the pictograph. Results are expressed as means + SEM of 30 cells. Mean change in peak [Ca2+]i measured in the above groups (right panel). The asterisk denotes a significant difference (∗ P , 0.05). (B) Transient intracellular Ca2+ signaling evoked from the ER store by ionomycin was decreased by shRNA knockdown of SigR1. Representative traces of [Ca2+] as a function of time recorded in the fura-2 loaded NSC34 cells either transfected with control shRNA or SigR1 shRNA. Cells were stimulated with ionomycin (5 mM) in Ca2+-free HEPES buffer solution. All measurements were performed in Ca2+-free HEPES buffer solution with EGTA at 258C. Results are expressed as means + SEM (as above) of 30 cells. Mean change in peak [Ca2+] measured in the above transfected groups (right panel). Asterisks denote significant differences (∗ P , 0.05).

the specificity of the drug for the mutant protein (Fig. 9). Treatment with PRE-084 was also able to restore mitochondrial activity (Fig. 9B) and alleviated the ER stress exerted by mutant P56S-VAPB and its insoluble aggregates (Fig. 9C, D). These results are in agreement with the recent description of the neuroprotective effect PRE-084 in G93A SOD-1 mice (59).

DISCUSSION SigR1 alterations have been found in various neurodegenerative diseases including AD (40,60– 62) and PD (38), and pathogenic mutations in SigR1 were recently discovered in ALS-FTLD/FTLD and in juvenile ALS cases (15,16). Moreover, there is abundant evidence for neuroprotective effects

of SigR1 in a wide range of neurological disorders (24,25,27,59,62– 64). Conversely, shRNA knockdown of sigma receptors in human cells has been shown to induce apoptosis (31). We therefore hypothesized that SigR1 is crucial for neuronal survival and maintenance and that it might be altered in ALS. To follow up on this hypothesis, we first analyzed autopsy material from sALS and fALS patients. In motor neurons of ALS patients, the localization of SigR1 was significantly altered. The SigR1 protein accumulations were ubiquitinated and associated with the 20s proteasome subunit. They were not co-localized with p62 aggregates suggesting that the UPS pathway rather than macro-autophagy degrades such abnormal ER proteins. We confirmed and extended these results by immunohistochemistry of G93A SOD1 mouse a-motor neurons.


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Figure 7. shRNA knockdown of SigR1 disrupts the mitochondrial membrane potential and leads to cytochrome-C release. (A) NSC34 cells were either transfected with control shRNA or SigR1 shRNA. Forty-eight hours later, transfected cells were subjected to either JC-1 staining to study the changes in mitochondrial membrane potential or (B) to cytochrome-C immunostaining to study cytochrome-C release, scale bars ¼ 10 mm. (C) NSC34 cells were either transfected with control shRNA or SigR1 shRNA with or without thapsigargin. These samples were processed for cytochrome-C analysis as described in Materials and Methods. Purified cytoplasmic fractions were subjected to western blot analysis for release of cytochrome-C; Coomassie blue staining was used as a loading control. (D) Cell lysates were analyzed for mitochondrial activity by Alamar blue assay. Asterisks denote significant difference (∗ P , 0.05).

VAPB is another ER protein, the mutation of which has been shown to cause familial ALS. Therefore, we investigated NSC34 motor neuron-like cells transfected with the ALS-8 P56S-VAPB mutant and found significant accumulation of SigR1-associated VAPB aggregates (Supplementary Material, Fig. S2c). Similarly, such accumulations of ubiquitinated SigR1 in association with the 20s UPS component and mutant VAPB aggregates were detected in cultured skin fibroblasts from an ALS-8 patient (Fig. 2) in which mutant VAPB forms ubiquitinated aggregates. Immunoblotting revealed that soluble SigR1 protein levels were decreased in ALS and G93A SOD1 mouse lumbar spinal cord (Fig. 3; Supplementary Material, Fig. S3). This reduction might be due both to the reduction in total motor neuron number during the course of ALS and to lowered levels of soluble SigR1 because of the build-up of insoluble SigR1 accumulations in the remaining neurons. Similarly, in NSC34 cells transfected with P56S-VAPB, large aggregates of insoluble VAPB and SigR1 formed with a reduction in soluble SigR1 (Supplementary Material,

Fig. S3). Furthermore, the level of insoluble VAPB and SigR1 increased (Supplementary Material, Fig. S2c) when cells were treated with the proteasome inhibitor MG132. In contrast, only small aggregates that were immunoreactive for VAPB and SigR1 were observed in primary ALS-8 patient fibroblasts expressing P56S-VAPB along with increased levels of soluble SigR1. In these cells, SigR1 expression might be up-regulated due to UPR activation following mutant VAPB aggregation. However, this aggregation is only minor and dispersed, not leading to larger aggregates of insoluble protein and major cell damage. Thus, up-regulation of SigR1 can be regarded as a compensatory phenomenon which—in case of the fibroblasts in ALS-8—might protect these cells from degeneration. This is in line with the phenotype in ALS-8 patients who show a selective neuronal degeneration despite the fact that the mutated gene is present in all cells. This hypothesis is also supported by several recent reports describing neuroprotective effects of SigR1 under various conditions (20,24,27,28,59).

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Figure 8. shRNA knockdown of SigR1 alters the ER ultrastructure. NSC34 cells or Hek293 cells were either transfected with control shRNA or SigR1 shRNA. Forty-eight hours later, transfected cells were fixed with 2.5% buffered glutaraldehyde and processed for electron microscopy. (A) NSC34 cell transfected with control shRNA show normal ER ultrastructure; scale bar ¼ 2 mm. (B) NSC34 cell transfected with SigRl shRNA shows membrane-bound vacuolar structures, at least some of which correspond to enlarged ER profiles; scale bar ¼ 2 mm. (C) Hek293 cell transfected with control shRNA shows normal ultrastructure. (D) Hek293 cell transfected with SigR1 shRNA shows a vacuolated cytoplasm with enlarged ER profiles and autophagic material (arrowhead). (E) Enlarged view of SigR1 shRNA-transfected NSC34 cells. (a) Large vacuoles, some of which contain membranous autophagic material (arrowhead). (b) Nuclear protrusion (arrow) associated with nuclear envelope widening. (F) (a and b) Nuclear protrusions (arrowheads). (c) Swollen mitochondria (arrowhead). (d and e) Membranous autophagic material. Scale bar ¼ 0.5 mm.

Altered intracellular localization of SigR1: significance to ALS SigR1 is predominantly localized to cholinergic postsynaptic densities, also known as C terminals, and is co-localized with

type-2 muscarinic receptors at these sites. Ultrastructural analysis of the neurons indicates that SigR1 is located in ER-derived subsurface cisternae close to but separate from the plasma membrane (30,37). These cisternae are a prominent feature of cholinergic postsynaptic densities (30). We observed


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Figure 9. Treatment with SigR1 agonist PRE-084 reduces mutant VAPB aggregation. (A) NSC34 cells were differentiated and either transfected with RFP, wtVAPB or P56S-VAPB; after 4 h the medium was replaced by media containing PRE-084 at varying concentrations as indicated for 24 h. Cells were fixed with 4% PFA and processed for immunofluorescence staining. (B, D) NSC34 cells were seeded on 24-well plates (for Alamar blue assay) or 60 mm plates (for western blots), differentiated and transfected with GFP, wt VAPB or mutant P56S-VAPB; after 4 h, media were replaced by fresh media supplemented with 20 mM PRE-084. After 24 h, cells were processed for either Alamar blue assay to measure mitochondrial activity or for western blot analysis. Note the reduction of soluble P56S-VAPB and high molecular weight VAPB with the treatment. Values are means + SD of three independent experiments. ∗ P , 0.05, ∗∗ P , 0.005. (C) NSC34 cells were transfected with P56S-VAPB, incubated with and without 20 mM PRE-084 and processed for western blotting.

a similar focal sub-surface SigR1 immunoreactivity in normal human a-motor neurons and in ALS patient a-motor neurons. The sub-plasmalemmal SigR1-immunoreactive zones were significantly increased in size in ALS a-motor neurons. These results are consistent with studies by Pullen and colleagues which suggested an apparent increase in size of Cterminals in ALS patient a-motor neurons (65,66). A recent report from the same group confirms the selective compensatory growth of C-terminal synapses within spinal motor neurons in ALS/MND patients and describes the increase in presynaptic territory of C-terminals in lumbar motor neurons of G93A SOD1 mice during disease progression (66). These authors hypothesized that the increase in the size of

C-terminals is due to an elevation in synaptic drive from parent interneurons to compensate for the substantial loss of synaptic input from other sources in order to protect the surviving neurons (66). In this context, our finding of elevated SigR1 immunoreactivity in C-terminal territories and altered localization suggests that the surviving motor neurons attempt to preserve functions of SigR1 at these sites. The defects in regulating ion channel conduction, synaptic transmission, axonal transport, lipid metabolism, IP3R-mediated Ca2+ signaling and aspects of memory and learning, as a part of SigR1 function which deteriorates as ALS progresses, might reflect the consequences of altered localization and abnormal modifications of SigR1 over a long period of time.

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SigR1-mediated pathology: ER chaperone function vs. regulation of Ca21 homeostasis ER stress-mediated apoptosis with persistent involvement and sequestration of molecular chaperons in insoluble aggregates within motor neurons is one of the major pathogenic mechanisms in ALS (45,67,68). SigR1 is an ER chaperone and is up-regulated in response to ER stress (39). On the other hand, it also associates with IP3 receptors (IP3R) and thereby regulates Ca2+ homeostasis at the MAM (20,21,39,52).

ER-chaperone function To dissect the relevance of these two different functions for motor neuron pathophysiology, we analyzed NSC34 cells in which we knocked down SigR1 expression by shRNA. We detected a massive induction of the UPR (Fig. 4A, B) as well as UPS impairment (Fig. 5A – E) along with an accumulation of misfolded proteins (Fig. 4D, E) and widening of the ER in SigR1-depleted NSC34 and Hek293 cells (Fig. 8). These cells also consistently contained ER-derived vacuoles filled with aggregated material. Furthermore, abnormal outfoldings of the nuclear envelope which is continuous with the ER were a constant feature. These results are in line with the widely accepted notion that an excess of misfolded proteins leads to induction of various chaperons (42), ER stress and inhibition of proteasome function (5,43,69) and also with previous publications (21,39,70), which describe a prominent role of SigR1 in regulating ER-associated degradation (ERAD) and chaperone activity. The UPR is generally activated as a defense mechanism in response to misfolded protein stress in the ER, thereby up-regulating various ER chaperones, including SigR1, to ensure protein quality control. VAPB is also physiologically involved in the IRE-1/XBP-1 signaling of the UPR, but unable to mediate proper UPR signaling when mutated (P56S) and forming insoluble aggregates (13,71,72). Therefore, both SigR1 and VAPB are important for UPR signaling. Our results suggest that mutant P56S-VAPB acts as a client for the SigR1 chaperone and that these proteins co-aggregate in the process of refolding or protein quality control management. Co-aggregation of SigR1 with VAPB then reflects the disturbances in UPR signaling and the attempt of this chaperone system to refold VAPB. Importantly, SigR1 does not interact with aggregates of polyglutamine proteins, such as mutant huntingtin and ataxin-3, which form cytoplasmic or nuclear aggregates (A.G.’s own unpublished observations) rather than aggregates around the ER, suggesting the specificity of the chaperone activity of SigR1 for ER proteins such as VAPB. These observations were also verified by our rescue experiments that revealed reduction of VAPB aggregates and increased degradation of mutant VAPB by activation of SigR1 using a selective agonist (Fig. 9). This is consistent with the activation of SigR1 for the quality control management of its client proteins. Interestingly, the SigR1 mutations that cause familial ALS have been shown to predispose cells to ER stress and subsequent apoptosis (16). Our findings suggest that increased ubiquitination of SigR1 is a generic feature that can be observed in many, if not all ALS patients. We observed accumulation of

ubiquitinated SigR1 associated with 20s proteasome in a-motor neurons of ALS patients and cultured ALS-8 patient skin fibroblasts. Abnormal accumulation and loss of function (knock down) of SigR1 could itself directly impair proteasomal degradation, because SigR1 is known to regulate ERAD (70). Alternatively, UPS impairment could be mediated by co-aggregation of SigR1 with the 20s proteasome. Although the redistribution and accumulation of SigR1 with the proteasome should serve as an adaptive response of the neurons to clear misfolded proteins, the UPR induction and UPS inhibition apparently overwhelms the chaperone and proteasome system during the course of the disease.

Alterations of Ca21 and mitochondrial metabolism Evidence supporting a toxic gain of function is derived from our observation that SigR1 accumulates in ALS (patient and SOD1 mouse) motor neurons and in ALS-8 patient fibroblasts. In ALS-8, P56S-VAPB forms aggregates continuous with the MAM (73,74). As mentioned above, SigR1 is predominantly located at the MAM and regulates calcium homeostasis in association with IP3 receptors besides its chaperone function (21,39). Several lines of evidence suggest that deregulation of calcium homeostasis is another major pathomechanism in ALS (69) and other neurodegenerative disorders (75). Disturbances in calcium homeostasis and mitochondrial energy metabolism have also been described recently in a cell culture model of ALS-8 (74,76) and have been shown to be responsible for inducing apoptosis. Deranged calcium signaling in ALS-8 (76) can be explained by our findings of aberrant co-localization of SigR1 and P56S-VAPB aggregates, which might disturb intracellular calcium handling by SigR1 and also by our measurements of intracellular calcium after SigR1 knockdown, where we found significant changes in intracellular calcium mobilization from the ER through IP3 receptors (IP3R) (Fig. 6). Alternatively, improper calcium handling and calcium storage defects of the ER could also occur because of the loss of ER integrity after the knockdown (Fig. 8). Another mechanism that contributes to cell damage after SigR1 depletion might be related to the abnormal depolarization of mitochondria. This leads to the release of cytochrome-C which induces caspase-3-mediated apoptosis (55,77). Consistent with this, we observed membrane depolarization (Supplementary Material, Fig. S4; Fig. 7) and cell death mediated by cytochrome-C release (Fig. 7) either induced by ER stress through thapsigargin or by shRNA knockdown of SigR1. Moreover, expression of a truncated form of SigR1 induces UPR and promotes mitochondrial energy depletion and ER stress-mediated apoptosis (58). UPS impairment can be aggravated by energy disturbances due to mitochondrial membrane depolarization (Fig. 7), because optimal ATP levels are required for the proper function of the 26s proteasome ATPase subunit (78). Thus, the consequences of shRNA knockdown of SigR1 both on the ER chaperone system and on calcium homeostasis at the MAM can combine to create a vicious circle in which induction of UPR and UPS impairment result in apoptosis and cell death (Figs 5 – 7 and 10).


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Figure 10. Schematic representation of SigR1-mediated pathology in ALS.

SigR1 and lipid transport SigR1 plays an important role in lipid raft assembly (22). We therefore speculated that reduced levels of SigR1 after the knockdown disturb lipid raft homeostasis. Confirming this hypothesis, we found Golgi dispersion as a consequence of the knockdown of SigR1 (Supplementary Material, Fig. S5 and S6). The same effect was achieved by treatment of the cells with thapsigargin, suggesting that the Golgi dispersal is a consequence of ER stress. This also supports the notion that either depletion of SigR1 or pathogenic interaction of SigR1 with the VAPB aggregates affects intracellular vesicular transport. These results are also endorsed by studies demonstrating that VAP family proteins are involved in lipid sensing and vesicular transport (79). In summary (Fig. 10), SigR1 is currently emerging both as an important player in molecular pathophysiology and as an interesting therapeutic target in neurodegenerative diseases. Here, we propose novel mechanisms of the involvement of SigR1 in the pathogenesis of ALS. These mechanisms might be targeted by SigR1-related pharmacological therapy regimens that have already been developed in the context of various other neuropsychiatric diseases (20,29,80).

MATERIALS AND METHODS Human post mortem samples Human spinal cord samples were obtained from the Amsterdam Academic Medical Center (AMC), Institute of Neuropathology Brain Bank following the guidelines of the local ethics committee. The spinal cords of these clinically confirmed sporadic and familial ALS patients, as well as agematched controls had been removed within 6– 12 h of death (for details see Supplementary Material, Table S1). Tissues were used in a manner compliant with the Declaration of

Helsinki. The familial cases did not show C9ORF expansion; no other pathogenic mutation was found so far. All ALS patients had suffered from clinical signs and symptoms of lower and upper motor neuron disease with the eventual involvement of brain stem motor nuclei. Importantly, none of these patients had cognitive impairment or dementia. Although variable from one case to another, the terminal stage of the disease was characterized by predominant bulbar failure, manifested as impaired swallowing and usually complicated by aspiration pneumonia or by respiratory insufficiency. Age-matched control patients did not show any neuropathological anomalies. Frozen samples of the lumbar spinal cord were used for biochemical studies (western blotting). Samples of control and diseased spinal cords were processed in parallel. Experimental animals All procedures were approved by the UK Aachen Institutional Animal Care and Use Committee, and conducted in compliance with the Guide for the Care and Use of Laboratory Animals. Mice expressing high copy numbers of human mutant G93A-SOD1 were obtained from Jackson Laboratories (Bar Harbor ME; strain designation B6SJL-Tg (SOD1 G93A) 1Gur/J). Homozygous SOD1 mice were maintained by mating G93A males with B6SJL/J hybrid females. Transgenic litters were genotyped using PCR for human SOD1 from tail tissue. These animals show progressive motor abnormalities, neurogenic muscular atrophy, reduced life-span and neuropathological changes such as the formation of inclusions in spinal cord motor neurons (81). Severely affected 18-week-old male mice and their corresponding control littermates were used for all experiments (n ¼ 3 each). To ensure optimal quality of SOD1 mutant and control mouse spinal cords for immunohistochemical

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investigation, tissues were fixed by transcardial perfusion with ice-cold 2% paraformaldehyde (PFA) containing 2% picric acid (pH 7.4). The spinal column was post-fixed over night, followed by careful dissection of the spinal cord. Tissue blocks containing the cervical- (C4-T1) and lumbar (L2-S1) expansions were then processed for paraffin embedding.

random fields of view of the ventral horn where motor neurons were visible. The actual counting was performed using Image-J software (Rasband, W.S., ImageJ, US National Institutes of Health, Bethesda, MD, USA, http://imagej.nih. gov/ij/, 1997 – 2012). To compare the distribution of SigR1 immunoreactivity, we used Pearson’s x 2 test.

Reagents

Cell culture, transient transfection and treatments

Fluorescent nucleic acid stain Hoechst 33258 and mitochondrial JC-1 were purchased from Molecular Probes. Alamar Blue was purchased from Invitrogen. MG-132, Thapsigargin, Tunicamycin, Fura-2AM, NaCl, KCl, pluronic acid, CaCl2, MgCl2, Glucose, HEPES 4-(2-hydroxyethyl)1-piperazineethanesulfonic acid, Ionomycin, MTT and BDK were purchased from Sigma-Aldrich. Rabbit polyclonal anti-ubiquitin was purchased from Dako, anti-20S proteasome from Calbiochem, anti-GFP, anti-RFP, anti-LC3 and anti-p62 antibodies from MBL, anti-20 core proteasome, anti-GADD 153, anti-IkBa, anti-SigR1, anti-GRP94, anti-Hsp70 from Santa Cruz Biotechnology, anti-GRP78, antipeIF2a, anti-pPERK, anti-PERK, anti-caspase-3 from Cell Signalling, anti-a tubulin, anti-human specific SigR1 from Sigma-Aldrich and anti-cytochrome-C from BD bioscience. The generation and application of our VAPB antiserum is described elsewhere (82).

Isolation and primary culture of fibroblasts Skin fibroblast cultures were established and maintained from biopsies of normal, control individuals and ALS-8 patients utilizing previously described methods (26).

Immunohistochemistry Transverse paraffin sections (3– 4 mm in thickness) of mouse (cervical and lumbar) and human (lumbar) spinal cord were cut on a microtome. Sections were placed on silane-coated slides, de-waxed, rehydrated and heated in citrate buffer for antigen retrieval. Non-specific binding was blocked by incubation in PBS containing 2% normal goat serum prior to overnight incubation with the primary antibodies diluted in blocking solution (see Supplementary Material, Table S2 for details of antibody dilutions). Appropriate biotinylated secondary antibodies were used (1:200, Vector Laboratories, USA) for 1 h, followed by DAB visualization (DAKO, USA). Sections were photographed using the Axioplan microscope (Zeiss) with an Axio CamHR. Quantification of distribution of SigR1 at C-terminals in lumbar spinal cord a-motor neurons C-terminals define a-motor neurons and are characterized presynaptically by a high packing density of both spherical or slightly flattened synaptic vesicles, an appositional length of up to 3.5 mm and a distinctive postsynaptic ultrastructure comprising a 10– 15 nm wide sub-synaptic ER cistern which underlies most of the length of the terminal. Towards the cell body, the C terminal is juxtaposed to the Nissl body composed of single or several lamellae of rough ER separated by groups of polyribosomes (37,66). Numbers and sizes of SigR1-immunoreactive accumulations at C-terminals in a-motor neurons were analyzed quantitatively on images taken with a Zeiss-Axiocam microscope imaging system with ×20 magnification. Five to ten images were taken per each patient or control lumbar spinal cord choosing

Cell culture and treatment HeLa human epithelial cancer cells, the HEK293 human embryonic kidney cells and NSC34 motor neuron-like cells were maintained in Dulbecco’s modified Eagle’s medium (DMEM, Invitrogen, Carlsbad, CA, USA) supplemented with 10% v/v FBS and 1% anti-biotic/anti-mycotic solution (Invitrogen). Cell cultures were maintained in a humidified incubator at 378C/5% CO2. When differentiation of NSC34 cells was required, cells were plated onto poly-D-lysine-coated tissue culture plastic and grown in differentiation medium containing 1:1 DMEM/Ham’s F12 (Invitrogen) supplemented with 1% FBS, 1% penicillin/streptomycin and 1% modified Eagle’s medium with non-essential amino acids (Invitrogen) for 3 days. Plasmids The construction of plasmids encoding human wt VAPB and P56S mutation has been described previously (82). The reporter plasmid for UPS inhibition, d1EGFP, was purchased from Clontech. RNA interference The HuSH shRNA (pRFPCRS) vector containing SigR1 oligonucleotide sequence for optimal suppression of SigR1 was purchased from Origene. Knockdown by transient transfections of SigR1 shRNA or other constructs into either HeLa cells or NSC34 cells were performed using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s recommendations. Scrambled shRNA was used as a control in every experiment. Western blots were performed to verify the efficiency of the knockdown (see below). Immunocytochemistry HeLa cells were transiently transfected on glass slides with either VAPB constructs (wild-type or mutant) or shRNA SigR1 constructs. The medium was changed after 4 h, after which some samples were treated with the known proteasome inhibitor MG132 or the ER stressor thapsigargin. After 48 h, cells were fixed in 4% PFA, permeabilized with 0.1% Triton-X100 and immunostained with mouse monoclonal antiSigR1 or rabbit anti-VAPB antibodies (see Supplementary Material, Table S2 for antibody dilutions). Secondary


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Alexa-488 or 594 conjugated anti-mouse or anti-rabbit antibodies (Invitrogen) were used for visualization. Nuclei were counterstained with Hoechst 33342 (1 mg/ml). Differentiated NSC34 cells were also transiently transfected and further processed for immunofluorescence (see Supplementary Material, Table S2 for antibodies used and their dilution/concentration). Samples were then cover slipped and visualized using a Zeiss LSM 700 confocal microscope. Resulting images were processed using the Zeiss LSM software and Adobe Photoshop CS5.

and 380 nm (using the Polychrome V monochromator; TILL Photonics) was used to demonstrate intracellular Ca2+ concentrations. Data were expressed as emission ratios in response to 340/380 nm excitation. Whole-cell calcium measurements from SigR1 knockdown and control NSC34 cells were obtained at room temperature (20 –238C). Cells were superfused with a bathing solution containing 100 mM NaCl, 5.4 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM HEPES, 10 MES, 5.5 glucose and pH was adjusted to 7.4.

Analysis of cytochrome-C release and measurement of mitochondrial membrane potential

Transmission electron microscopy (TEM)

To evaluate the release of cytochrome-C from mitochondria by immunoblotting, NSC34 cells were grown on tissue culture dishes, followed by shRNA knockdown of SigR1 for 48 h in differentiating media. The cells were collected by scraping; the samples were then washed in PBS followed by sucrose buffer (20 mM HEPES pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM DTT and 0.1 mM PMSF containing 250 mM sucrose) and then resuspended in the same buffer. After 1 h of incubation on ice, cells were lysed using 30 strokes of the Dounce homogenizer B-type pestle. Homogenates were centrifuged at 750g for 10 min at 48C, and the supernatant re-centrifuged at 10 000g for 15 min at 48C. The final supernatant was used for immunoblotting experiments for the detection of cytochrome-C (see below). Immunocytochemistry was also performed to confirm the immunoblotting results for the detection of mitochondrial cytochrome-C release (data not shown). The fluorescent potentiometric dye, JC-1, exists as green fluorescent monomer at low concentrations or at low membrane potential. However, at higher concentrations or at higher Dc membrane potentials, JC-1 forms red fluorescent ‘J-aggregates’ that exhibit a broad excitation spectrum. This cyanine dye can be used as a sensitive measure of mitochondrial membrane potential. HEK293 cells were seeded on 25-mm cover slips and grown for 24 h. The resting membrane potential was assessed by JC-1 uptake and fluorescence. The cover slips were washed twice and mounted in a cell chamber (ALA Scientific Instruments, Westbury, NY, USA) in HEPES buffer (145 nM NaCl, 3 mM KCl, 2 mM CaCl2, 1 mM MgCl2, 10 mM glucose, 10 mM HEPES). Serial confocal images were taken every 5 min using the Zeiss LSM-700 with excitation at 490 nm and emission at 590 nm before and after JC-1 (5 mg/ml) incubation, respectively. Quantitative analysis of the dynamic change of the mitochondrial membrane potential was performed by measurement of the red fluorescence intensity, using the Zen software (Zeiss). Fura-2 calcium ion imaging The intracellular calcium ion concentration, [Ca2+]i, was measured using a conventional Fura-2 technique (53). After 48 h of SigR1 knockdown, NSC34 cells were loaded with the membrane-permeable AM-form of Fura-2 (1.5 ng/ml; Invitrogen) in the presence of pluronic acid (25%) for 30 min at 378C. Emitted fluorescence at 530 nm (detected using a Sensicam; pco.imaging) in response to alternate excitation at 340

NSC34 and Hek293 cells were transfected with control, scrambled shRNA and SigR1 shRNA as described above. Cells were collected by scraping from the 10 cm tissue plate. Both cell types were washed in 0.1 M phosphate buffer and immediately fixed in 2.5% glutardialdehyde in 0.1 M phosphate buffer for 24 h followed by washing in buffer for a further 24 h. The cell pellets were collected by centrifugation (1000 rpm, 5 min) and embedded in 2% agarose (at 608C; Fluka #05073). Small blocks of embedded cells were sliced and post-fixed in 2.5% glutardialdehyde for 24 h followed by a 24 h washed in 0.1 M phosphate buffer. Agarose blocks were then incubated in 1% OsO4 (in 0.2 M phosphate buffer) for 3 h, washed twice in distilled water and dehydrated with using ascending alcohol concentrations (i.e. 2535, 50, 70, 85, 95, 100%; each step for 5 min). Dehydrated blocks were incubated in propylenoxide followed by a subsequent 20 min incubation in a 1:1 mixture of epon (47.5% glycidether, 26.5% dodenylsuccinic acid anhydride, 24.5% methylnadic anhydride and 1.5% Tris (dimethylaminomethyl)phenol) and propylenoxide,. Prepared samples were incubated in epoxy resin for 1 h at room temperature followed by polymerization (288C for 8 h, 808C for 2.5 h and finally at room temperature for 4 h. Ultra-thin sections (70 nm) were prepared, mounted on grids for electron microscopy and examined using a Philips CM10 transmission electron microscope as described (83).

Assays for protease activity and cell viability NSC34 cells were seeded into a six-well tissue culture plate and, on the following day, were transfected by 48 h incubation with SigR1 shRNA or control, scambled shRNA plasmids as described earlier. At a range of time points after transfection, cells were collected and processed for proteasome or caspase-3 activity assays (84). The proteasome substrates Suc-Leu-Leu-Val-Tyr-MCA and Z-Leu- Leu-Glu-MCA were used to determine chymotrypsin and postglutamyl peptidyl hydrolytic-like activities (7). The caspase-3 substrate Ac-AspGlu-Val-Asp-MCA was used to determine the caspase-3-like protease activity. For the cell viability assay, NSC34 cells were seeded into 24-well tissue culture plates (5 × 103 cells/well), differentiated and transfected with SigR1 shRNA or control shRNA plasmids. Cell viability after transfection was measured using the MTT assay. Mitochondrial activity of the transfected cells was also assessed using Alamar Blue (Invitrogen) in a 24-well format following the manufacturer’s instructions.

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Western blot analysis

FUNDING

Cells were washed twice with ice-cold PBS, scraped and resuspended in lysis buffer (0.5% Triton X-100 in PBS, 0.5 mM PMSF and complete protease inhibitor mixture (Roche Applied Sciences). After incubation on ice for 30 min, lysates were briefly sonicated. Spinal cord tissue of human patients and of mice was homogenized in RIPA buffer followed by sonication. Clear lysates were obtained after brief centrifugation for 5 min at 6000 rpm. Protein concentrations were determined using the BCA method (Molecular Probes). Equal amounts of protein were boiled for 5 min in 2× SDS-sample buffer and subjected to 10 or 12% SDS – PAGE prior to transfer to a polyvinylidene difluoride membrane. The membranes were blocked in 5% skimmed milk in 0.05% Tween 20/Tris-buffered saline (TBS-T) for 30 min prior to incubation with primary antibody. Most primary antibodies were used at 1:1000 dilutions except for anti-SigR1 as well as anti-VAPB (1:500). The membranes were incubated overnight at 48C, then washed three times in TBS-T and incubated for 1 h in appropriate horseradish peroxidase-conjugated secondary antibody (dilution 1:10 000, Thermo Scientific). Immunoreactive proteins were detected by enhanced chemiluminescence (Amersham Biosciences); densitometric quantification of the band intensity was normalized to tubulin levels using Adobe Photoshop CS5.

This work was supported by grants from the German Research Council (DFG) (WE1406/13-1 to J.W.), the German Motor Neuron Disease Network (BMBF MND-Net) to J.W., the Interdisciplinary Centre for Clinical Research (IZKF Aachen) (N1-1) to J.W. and the German Myopathy Society (DGM) to A.G. and J.W.

Statistical analysis We used the unpaired Student’s t-test for comparison between two sample groups. Values were expressed as mean + standard deviation (SD) from three independent experiments. Differences between values were regarded as significant when ∗ P , 0.05, ∗∗ P , 0.005. For Ca2+ measurements, results were expressed as means + SEM of 30 cells. Differences between peak values were regarded as significant when ∗ P , 0.05 and ∗∗ P , 0.005. To compare the distribution of SigR1 immunoreactivity, we used Pearson’s x 2 test.

SUPPLEMENTARY MATERIAL Supplementary Material is available at HMG online.

ACKNOWLEDGEMENTS We sincerely thank R. Tolba and A. Teubner for their support in animal housing and handling. We are grateful to A. Knischewski, H. Wiederholt, E. Pascual and H. Mader (Institute of Neuropathology, RWTH Aachen University), H. Heidtmann (Institute of Physiology, RWTH Aachen University) and J. Aninik (AMC, Amsterdam) for technical support. We also thank Vinod Kumar (International Max Planck Research School, Tuebingen, Germany) for Image analysis and S. Gru¨nder (Institute of Physiology, RWTH Aachen University) for confocal microscopy and calcium imaging. Finally, we thank G. Brook (Nerve Regeneration Group, Institute of Neuropathology, RWTH Aachen University) for his valuable comments and stylistic correction of the manuscript. Conflict of Interest statement. None declared.

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