Wildtype s 1 receptor and the receptor agonist improve ALS-associated mutation-induced insolubility and toxicity

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding s -1 receptor ( s 1R). We previously reported that s 1RE102Q elicits toxicity in cells. The s 1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALS-related mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with s 1R-mCherry (mCh), s 1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. s 1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2- a kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with s 1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased s 1RE102Q-mCh insolubility and inhibited apoptosis. Whereas s 1R-mCh formed monomers and dimers, s 1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The s 1RE102Q insolubility was diminished by s 1R-mCh co-expression. These results suggest that the agonist and WT s 1R modify the detergent insolubility, toxicity, and oligomeric state of s 1RE102Q, which may lead to promising new treatments for s 1R-related ALS.

Genetic mutations related to ALS, a progressive neurological disease, have been discovered in the gene encoding s-1 receptor (s1R). We previously reported that s1RE102Q elicits toxicity in cells. The s1R forms oligomeric states that are regulated by ligands. Nevertheless, little is known about the effect of ALSrelated mutations on oligomer formation. Here, we transfected NSC-34 cells, a motor neuronal cell line, and HEK293T cells with s1R-mCherry (mCh), s1RE102Q-mCh, or nontagged forms to investigate detergent solubility and subcellular distribution using immunocytochemistry and fluorescence recovery after photobleaching. The oligomeric state was determined using crosslinking procedure. s1Rs were soluble to detergents, whereas the mutants accumulated in the insoluble fraction. Within the soluble fraction, peak distribution of mutants appeared in higher sucrose density fractions. Mutants formed intracellular aggregates that were co-stained with p62, ubiquitin, and phosphorylated pancreatic eukaryotic translation initiation factor-2-a kinase in NSC-34 cells but not in HEK293T cells. The aggregates had significantly lower recovery in fluorescence recovery after photobleaching. Acute treatment with s1R agonist SA4503 failed to improve recovery, whereas prolonged treatment for 48 h significantly decreased s1RE102Q-mCh insolubility and inhibited apoptosis. Whereas s1R-mCh formed monomers and dimers, s1RE102Q-mCh also formed trimers and tetramers. SA4503 reduced accumulation of the four types in the insoluble fraction and increased monomers in the soluble fraction. The s1RE102Q insolubility was diminished by s1R-mCh co-expression. These results suggest that the agonist and WT s1R modify the detergent insolubility, toxicity, and oligomeric state of s1RE102Q, which may lead to promising new treatments for s1R-related ALS.
The s1 receptor (s1R) was initially identified as the binding site (s) of SKF-10047, a benzomorphine-related compound, and has since been classified as a nonopioid receptor according to its pharmacological properties (1)(2)(3). Recently, s1R has been revealed to be a type II transmembrane protein localized in the endoplasmic reticulum (ER) membrane with its C-terminal chaperone domain toward the ER lumen (4,5). s1R is unique in its involvement in diverse cellular functions like calcium transport, stress response, lipid metabolism, regulation of neuronal activity, and RNA transcription by binding to various proteins in ER, plasma membrane, and nuclear envelope (6,7). A number of physiological and pharmacological studies have been conducted to reveal that s1R acts as the binding target not only for SKF-10047 but also for intrinsic molecules, several drugs, and other compounds (8)(9)(10)(11).
Recent investigations have uncovered the genetic mutations of the human SIGMAR1 gene in motor neuron diseases like ALS, distal hereditary motor neuropathy, and Silver-like syndrome (12)(13)(14)(15)(16)(17)(18)(19)(20). These mutations cause single amino acid substitutions or various deletions in the C-terminal chaperone domain; however, it remains unclear how these mutations affect motor neurons and lead to the onset of these disorders. We previously reported how the variation (E102Q) identified in juvenile ALS showed abnormal localization in neuroblastoma Neuro2A cells (21). The transient expression of this mutation (s1RE102Q) caused deficits in mitochondrial calcium transport and function, leading to a decrease in proteasomal activity and cell death. Abnormal protein aggregation is the prominent pathological feature in neurodegenerative diseases (22,23). TAR DNA binding protein-43 and other proteins of which genetic mutations have been identified in familial ALS form subcellular inclusions containing ubiquitin and p62 proteins in motor neurons (23). This suggests that the deficit in the clearance of toxic proteins and aggregates by the ubiquitin-proteasome system and autophagy is associated with the pathology of ALS. Mutations in genes involved in the ubiquitin-proteasome system and autophagy are related to ALS and frontotemporal dementia with ALS (24)(25)(26)(27)(28).
s1R localizes preferentially to the mitochondria-associated ER membranes (MAMs) or ER-associated lipid droplets (ER-LDs), which are highly resistant to detergents such as Triton X-114 in various types of cultured cells (29)(30)(31). Hayashi et al. (31) proposed that ER-LDs are detergent-resistant membranes (DRMs) that contain MAMs and are enriched in cholesterol and sphingolipids tethering s1R to these microdomains. They also reported s1R to be localized to DRMs, which were separated in the light fraction of sucrose density gradient centrifugation (i.e. third to fifth of 12 or 13 fractions) and that the agonist (1)-pentazocine caused s1R disassociation from the ER-LDs (29,32); however, it remains uncertain how ALS-related mutations influence the characteristics of s1R such as detergent solubility/insolubility and subcellular dynamics in motor neurons.
Size exclusion chromatography and FRET analyses revealed that s1R forms oligomeric states ranging from dimers to octamers and even higher oligomeric forms (33)(34)(35). Radiographic structural analysis confirmed the formation and trimeric structure of this receptor (4). Further studies elucidated how s1R agonists and antagonists affect the oligomeric states (7,36). When inactivated, s1R forms a complex with the ER chaperone protein immunoglobulin heavy chain-binding protein/glucose-regulated protein (BiP/GRP)78. Binding of agonists causes s1R to dissociate from BiP/GRP78, thereby enabling its translocation to intracellular domains where it can interact with protein substrates as a chaperone protein. On the other hand, whereas agonists stabilize monomers and dimers and thus increase activity of the receptor, antagonists can stabilize higher-order oligomers and consequently negatively affect its activity (7,36).
Here, we found that the s1R mutant fused with the red fluorescent protein (RFP) mCherry (s1RE102Q-mCh) showed abnormal insolubility to detergents, subcellular distribution, and aggregations. These features were distinct from conventional MAMs or ER-LDs. Treatment with s1R agonist SA4503 abrogated the insolubility and toxicity of the ALS mutant. Whereas s1R-mCh formed exclusively monomers and dimers, s1RE102Q-mCh also formed trimers and tetramers in NSC-34 cells, which could be partly reversed by SA4503. Co-expression of s1R-mCh was found to inhibit s1RE102Q fractionation in the insoluble fraction. These findings indicate that the agonist and WT s1R can rescue the aberrant characteristics of this ALS-related s1R mutant.

ALS-related mutant shows abnormal insolubility to detergents
We transfected mouse motor neuron-like hybrid NSC-34 cells with WT s1R and the ALS-related mutant E102Q, which were C-terminally fused with the RFP mCherry (s1R-mCh and s1RE102Q-mCh). Successful expression was confirmed using an anti-RFP antibody (Fig. 1A, top). Cells were lysed in buffer containing 1% NP-40 and separated by ultracentrifugation to soluble supernatants and insoluble pellets. Insoluble pellets were mixed with SDS lysis buffer and sonicated. Under these conditions, BiP/GRP78, the ER stress sensor inositol requiring (IRE)1a, and voltage-dependent anion channel, which were reported to interact with s1R (30,37,38), were fractionated exclusively in the soluble fraction (Fig. 1B). s1R-mCh was also fractionated in the soluble fraction; in contrast, s1RE102Q-mCh was mainly separated in the insoluble fraction (Fig. 1C). This abnormality was also evident by a significantly higher ratio of protein level in the insoluble fraction relative to its soluble counterpart (Fig. 1D). Similar results were obtained using other detergents such as 0.5% Triton X-100 and 20 mg/ml CHAPS (Fig. S1, A-C), which is consistent with previous reports for WT s1R (29,31). Consistently, s1RE102Q-mCh showed similar insolubility to 1% Nonidet P-40 (NP-40) in HEK293T cells (Fig.  S1, D and E). These results demonstrate that s1RE102Q-mCh shows abnormal properties regarding detergent insolubility.
ALS-related mutant shows that altered subcellular distribution and p62-and ubiquitin-positive structures which are correlated with ER stress induction in NSC-34 cells To evaluate the subcellular distribution of s1RE102Q-mCh, we further separated both fractions by sucrose density gradient centrifugation ( Fig. 2A). Western blotting analysis following the collection of 12 fractions from the top showed a wide range of distribution of s1R-mCh, which partially overlapped with that of BiP/GRP78 ( Fig. 2A, fractions 2-6). The distribution peak of s1RE102Q-mCh within the soluble fraction changed slightly into the latter fractions (Fig. 2B, s1R-mCh: fractions 5 and 6, s1RE102Q-mCh: fractions 7 and 8). Within the insoluble fraction, s1RE102Q-mCh was most abundant in the bottom portion together with p62 (Fig. 2, A and B). These results were distinct from the distribution of s1R in conventional ER-LDs or DRMs, which are separated in the light fractions (29,32). Furthermore, we performed the same fractionation with nontagged forms to exclude the possibility that the fluorescent tag influences the distributions and mutation-induced alteration (Fig. 2, C and D). As a result, transfected s1R in the soluble fraction was A, representative images of expression of WT s 1 R and ALS-related mutant (s 1 R E102Q ) C-terminally fused with mCherry (mCh) in total fraction. B, representative images of s 1 R-mCh, s 1 R E102Q -mCh, and marker proteins for cellular organelles in each fraction. C, quantitative analysis for relative levels of s 1 R. Each bar shows mean 6 S.D. (error bars). Statistical significance was tested using two-way ANOVA followed by Bonferroni post hoc test. n = 6. F (1, 20) = 79.6 (WT/E102Q), 49.4 (soluble/insoluble) and 344.6 (interaction). **p , 0.01 versus soluble. ##p , 0.01 versus s 1 R-mCh. D, quantitative analysis for the ratio of insoluble to soluble fraction. Each bar shows mean 6 S.D. (error bars). Statistical significance was tested using unpaired t test. n = 6. t (10) = 13.0. **p , 0.01 versus s 1 R-mCh.
preferentially separated in the front half of 12 fractions. This result was similar to endogenous s1R observed in mock-transfected cells. However, s1RE102Q was additionally fractionated in the 12th fraction with a slight decrease in mobility in SDS-PAGE (Fig. 2C, third panel, arrow, s1RE102Q; arrowhead, endogenous s1R) when PAGE was performed with longer time exceptionally to distinguish them. Simultaneously, s1RE102Q in the insoluble fraction was exclusively collected in the 12th fraction (Fig. 2, C and D).
We further performed immunocytochemistry to analyze the cellular distribution of s1RE102Q-mCh in NSC-34 cells (Fig.  3). When cells were labeled with an antibody against s1R, the appearance of red fluorescence of mCherry (magenta) and detected signal (green) were slightly different in the signal:noise ratio; nonetheless, the localization patterns were indeed similar (Fig. 3, A and B). s1RE102Q-mCh showed abnormal aggre-gate-like structures as we reported previously using Neuro2A cells (Fig. 3B) (21). BiP/GRP78 was found to be partially colocalized with both s1R-mCh and s1RE102Q-mCh, suggesting that a portion of these proteins do interact (Fig. 3, C and D). The immunostained signal for p62 did not colocalize with s1R-mCh ( Fig. 3E) but colocalized with the aggregate-like structures of s1RE102Q-mCh (Fig. 3F, inset and arrows). We observed an aberrant accumulation of ubiquitin, which colocalized with s1RE102Q-mCh aggregates (Fig. 3, G and H). As previously reported, s1RE102Q-mCh aggregates were costained especially with phosphorylated pancreatic eukaryotic translation initiation factor-2-a kinase (pPERK), an ER stress sensor in NSC-34 cells (Fig. 3, I and J) (39). The aggregates of s1RE102Q-mCh did not show colocalization with inositol 1,4,5-triphosphate receptor type 2 (IP3R2), a major subtype of IP3Rs expressed in motor neurons (Fig. 3, K and L) (40). . E102Q mutation changes s 1 R distribution in sucrose gradient density fractionation. A, representative images of WT s 1 R-mCh in the soluble fraction and s 1 R E102Q -mCh mutant in the soluble and insoluble fraction followed by sucrose gradient fractionation. Twelve fractions were collected from the top. B, quantification of relative abundance of s 1 R-mCh in the 12 fractions. C, representative images of nontagged s 1 R in the soluble fraction and E102Q mutant in the soluble and insoluble fraction followed by sucrose gradient fractionation. Twelve fractions were collected from the top. Third panel, arrow and arrowhead indicate transfected mutant and endogenous (endo.) s 1 R, respectively. D, quantification of relative abundance of s 1 R in the twelve fractions. The quantification of s 1 R E102Q in soluble fractions was simultaneously performed with lower bands of endogenous s 1 R.
The localization of translocase of outer mitochondrial membrane (TOM)20, a protein in the mitochondrial outer membrane, showed partial colocalization with tubular structures of s1R-mCh and s1RE102Q-mCh (Fig. 3, M and N). In contrast, TOM20 did not colocalize with aggregate-like structures of s1RE102Q-mCh (Fig. 3N, inset). The analyzed images were also processed and converted into binary images to visualize MAMs (Fig. S2). It showed similar localizations of s1R-mCh and s1RE102Q-mCh in these structures.
Acute SA4503 treatment does not affect intracellular dynamics of s1RE102Q-mCh tubular structures and aggregates Next, we performed fluorescence recovery after photobleaching (FRAP) analysis to evaluate potential changes in the intracellular dynamics of s1RE102Q-mCh tubular and aggregate-like structures (Fig. 4). FRAP analysis enables the assessment of molecular mobility in the cells using fluorescence. The rigid aggregate-like structures elicit the decline of protein penetration toward aggregates and therefore lead to slow fluorescent recovery after bleaching (41, 42). The fluorescence of the s1R-mCh tubular structures recovered just after bleaching within 39 s (0.38 6 0.08 in recovery) (Fig. 4, A, C, and E and Movie S1); however, the fluorescence of the s1RE102Q-mCh aggregate-like structures did not recover (0.09 6 0.05 in recovery) (Fig. 4, B, D, and E and Movie S2). We calculated the normalized intensity in bleached regions (Fig. 4, A and B, black circles) in control and SA4503-treated cells (Fig. 4, C and D).
The recovery derived from the normalized intensity was significantly lower in s1RE102Q-mCh aggregate-like structures than in both s1R-mCh and s1RE102Q-mCh tubular structures (Fig.  4E). This suggests that the abnormal structures were indeed intracellular aggregates. SA4503 treatment for 1 h decreased the recovery of s1R-mCh and s1RE102Q-mCh tubular structures slightly, but there was no statistically significant difference (p = 0.13 for s1R-mCh, p = 0.10 for s1RE102Q-mCh).

Prolonged SA4503 treatment decreases s1RE102Q-mCh insolubility and cell apoptosis
Based on our observations that a short period of treatment did not have any effect on intracellular dynamics, we next treated cells with SA4503 for 48 h after transfection. SA4503 treatment decreased the ratio of s1RE102Q-mCh protein levels in insoluble versus soluble fractions significantly (Fig. 5A, top panel, and Fig. 5B). We next evaluated the concentration dependence by treatment at various conditions (0, 0.2, 1, 5, and 20 mM) (Fig. 5, C and D). As a result, the ratio of s1RE102Q-mCh protein levels gradually decreased: there was statistically significant difference at 1, 5, and 20 mM (p , 0.01). Because abnormal Pharmacological/genetic effect on ALS-related s1R features aggregates were generally considered to be degraded by autophagy, we analyzed the expression levels of marker proteins reflecting autophagic flux in total fractions (Fig. S3). The ratio of microtubule-associated protein 1 light chain 3 type II/I (LC3-II/I) decreased slightly in SA4503-treated cells; however, expression of p62, which is degraded by autophagy, was not altered by SA4503 treatment. Hence, we concluded that binding of SA4503 to s1RE102Q-mCh changed its solubility directly but did not affect the autophagic machinery or cause the degradation of aggregates. Cells transfected with s1RE102Q-mCh, but not with s1R-mCh, were immunostained with an antibody against activated cas-pase-3 (Fig. 5, E and F). Remarkably, the percentage of cells with nuclei featuring a hazy outer boundary in a DNA diffusion assay increased significantly in s1RE102Q-transfected cells (Fig.  5, G and H). These results suggest that the ALS-associated mutant of s1R induced apoptotic cell death, which could be rescued by treatment with the s1R agonist SA4503 for 48 h.

Nontagged mutant showed similar aberrant aggregates in NSC-34 cells, but tagged mutant failed in HEK293T cells
We further evaluated the fluorescent study of s1RE102Q with nontagged form in NSC-34 cells and with tagged form in HEK293T cells (Fig. 6). Nontagged WT s1R localized in diffuse pattern in the cytosol (Fig. 6, A, C, E, and G); however, s1RE102Q caused aberrant aggregates which were co-stained with p62, ubiquitin, and pPERK (Fig. 6, B, D, and F). Cells expressing s1RE102Q were stained with activated caspase-3 (Fig. 6H). s1R-mCh showed similar patterns in HEK293T cells (Fig. 6, I, K, M, and O), whereas s1RE102Q-mCh showed large aggregates (Fig. 6, J, L, N, and P). These aggregates were stained with ubiquitin but not with p62 or pPERK antibodies. Nevertheless, cells highly expressing s1RE102Q-mCh localized in abnormal structure were stained with activated caspase-3 (Fig. 6P).
SA4503 reduces s1RE102Q-mCh monomer-tetramer formation in the insoluble fraction but increases monomer formation in the soluble fraction We hypothesized that SA4503 stabilizes and thus increases monomers/dimers, causing the observed changes in abnormal insolubility and aggregations of s1RE102Q-mCh (36). To test this, we subjected cells to a crosslinking procedure with the uncleavable crosslinker disuccinimidyl suberate (DSS) following transfection and treatment. We analyzed the oligomeric state in soluble and insoluble fractions by Western blotting (Fig. 7). s1R-mCh primarily formed monomers and dimers in the soluble fraction (Fig. 7A), whereas s1RE102Q-mCh was not only found to form monomers/dimers but also trimers/tetramers in both the soluble and insoluble fractions (Fig. 7, A and B). Quantitative analysis showed that SA4503 treatment increased s1RE102Q-mCh monomer levels in the soluble fraction and decreased monomer/dimer/trimer/tetramer levels in the insoluble fraction (Fig. 7, C and D). The level of s1RE102Q-mCh dimers in the soluble fraction was slightly higher in cells treated with SA4503, although the difference was not statistically significant (Fig. 7C, p = 0.15). There was a smear pattern but no apparent specific band with a higher molecular weight of s1RE102Q-mCh in the insoluble fraction (Fig. S4). Co-expression of s1R-mCh diminishes s1RE102Q insolubility in NSC-34 cells We further analyzed whether WT s1R could influence s1RE102Q-mCh insolubility (Fig. 8). We transfected NSC-34 cells with s1R C-terminally fused with GFP (s1R-GFP) together with s1R-mCh or s1RE102Q-mCh and then performed co-immunoprecipitation using an antibody against GFP (Fig.  8A). As a result, s1R-mCh and s1RE102Q-mCh were immunoprecipitated with s1R-GFP (upper panel, lanes 7 and 8) but not with GFP (upper panel, lanes 3 and 4). This indicates that the ALS-associated mutant can form chimeric oligomers with WT s1R. Co-expression of s1R-mCh reduced s1RE102Q fractionation in the insoluble fraction and the ratio (insoluble/soluble) significantly (Fig. 8, B and C). In the regular time-course of PAGE, endogenous s1R and transfected s1RE102Q could not be separated. This seems to cause the apparent soluble s1R level to appear higher and the ratio to appear lower compared with the mCh-tagged form (Fig. 1, B and D and Fig. 8, B and C).
These results indicate that WT s1R could bind to and abrogate the insolubility of the ALS-related s1R mutant and its aggregates.

Discussion
In the present study we have demonstrated that the ALSrelated mutation of s1R, s1RE102Q, affected the solubility and oligomer formation of this receptor, which induced toxicity in NSC-34 cells. The smear pattern of s1RE102Q-mCh with a higher molecular weight, which appeared in the insoluble fractions (Fig. S4), suggests a correlation between the aggregation and its insolubility (Fig. 9). The aggregates of s1RE102Q-mCh colocalized with pPERK but not with IP3R2. This suggests that s1RE102Q-mCh aggregation leads to a dysregulation of calcium homeostasis and ER stress, eventually resulting in cell death as previously reported (21,39). In spinal motor neurons of ALS model mice, ER stress and unfolded protein response were induced in degenerating neurons but not in preserved neurons in the period of onset (43), suggesting the progressive role in the disease. Co-expression of s1R-mCh inhibited s1RE102Q insolubility, which was possibly caused by direct interaction. Nontagged s1RE102Q showed similar p62-, ubiquitin-, and pPERK-positive aggregates in NSC-34 cells (Fig. 6,  A-H). However, in HEK293T cells, s1RE102Q-mCh provoked ubiquitin-positive and p62-and pPERK-negative aggregates, although an extremely highly expressed cell was stained with anti-activated caspase-3 antibody (Fig. 6, I-P). These results might suggest the vulnerability of motor neurons to toxicity from ER stress and the usefulness of NSC-34 cells as a motor neuron model.
Hayashi et al. (29,31) reported that s1R localizes preferentially in ER-LDs, which are resistant to Triton X-114 and show a ring-like structure in neuroblastoma NG-108 cells. They also reported that overexpression of a tagged s1R targets enlarged ER-LDs (29,32). The appearance of s1RE102Q-mCh aggregates was similar to these large ER-LDs (Fig. 3), with the following differences: 1) s1RE102Q-mCh localized in a tubular rather than a ring-like structure in NSC-34 cells; 2) whereas ER-LDs tend to be fractionated in light fractions in sucrose density gradient centrifugation, the mutation changed the distribution peak of tagged form from the fifth and sixth to the seventh and eighth in the soluble fraction, mainly to the bottom in the insoluble fraction (Fig. 2, A and B). This result was identical in nontagged s1R regarding the altered direction (Fig. 2, C and D); and 3) in contrast to ER-LDs, which contain enriched MAMs, both s1R-mCh and s1RE102Q-mCh showed similar colocalization with TOM20 (Fig. 3, M and N and Fig. S2). We therefore conclude that the fractionation of s1RE102Q-mCh in the insoluble fraction did not indicate a localization in ER-LDs or MAMs in NSC-34 cells. Indeed, sucrose density gradient centrifugation confirmed that s1R-mCh and s1RE102Q-mCh appeared only in the bottom fractions (Fig. S5). A common feature between our study and that of Hayashi et al. (32) is that Figure 8. Effect of co-expression of s 1 R-mCh on s 1 R E102Q insolubility. A, cell lysates were co-immunoprecipitated with an anti-GFP antibody, and representative images after detection using anti-RFP (mCh) and GFP antibodies are shown for immunoprecipitated samples (Co-IP) and total lysates (Input). Blue arrowheads show mCh-and GFP-tagged s 1 R, and black arrowheads show mCh and GFP. Asterisk indicates the immunoglobulin heavy chain used for co-immunoprecipitation. B, representative images of s 1 R and s 1 R E102Q transfected with s 1 R-mCh in soluble and insoluble fractions. C, quantitative analysis for the ratio of insoluble to soluble fractions. Each bar shows mean 6 S.D. (error bars). Statistical significance was tested using two-way ANOVA followed by Bonferroni post hoc test. n = 4. F (1, 12) = 26.6 (WT/E102Q), 18.6 (mCh/s 1 R-mCh) and 11.6 (interaction). **p , 0.01 versus s 1 R control (mCh). ##p , 0.01 versus s 1 R E102Q control (mCh).
Pharmacological/genetic effect on ALS-related s1R features (1)-pentazocine dissociated s1R from ER-LDs and that SA4503 changed s1RE102Q-mCh fractionation from the insoluble to the soluble fraction (Fig. 5B). Furthermore, a glycosylphosphatidylinositol-anchored protein (alkaline phosphatase) was reported to become insoluble to detergents when binding with cholesterol/sphingolipids (44,45). These molecules might alter the insoluble/soluble property of s1R, although we at least failed to confirm s1RE102Q localization in ER-LDs (Fig. S5). Further studies will be required to clarify any discrepancies and to fully understand s1R's role in this context.
Our FRAP analysis demonstrated that recovery of s1RE102Q-mCh in tubular structures was identical to that of s1R-mCh, which was significantly faster than that of s1RE102Q-mCh in aggregates (Fig. 4). Treatment with SA4503 for 1 h failed to influence the recovery. The intracellular dynamics of s1R, including the effects of the mutation and agonist/antagonist, were already investigated by two other groups recently (39,46). Dreser et al. (39) reported that a yellow fluorescent protein-tagged version of s1RE102Q (s1RE102Q-YFP) shows slower recovery compared with WT in COS-7 cells, which is consistent with our observations. Wong et al. (46) found that s1R-YFP in reticular structures exhibits a significantly lower recovery than BiP-mCh in mouse embryonic fibroblasts. The s1R agonist SKF-10047 (100 mM, 1 h) increased the recovery of s1R-YFP in reticular structures but not s1RE102Q-YFP, which had a faster recovery. The speed of recovery of both s1Rand s1RE102Q-YFP in puncta structures was slow and similar to that of s1R-YFP in reticular structures. Although we cannot explain the discrepancy between these reports and our results, s1R may have different characteristics according to different cell types because the localization in mouse embryonic fibroblasts showed much more diffuse structures, which were not identical to the tubular structures seen in NSC-34 cells (Fig.  4) (46). We hypothesized that monomers and dimers of s1RE102Q-mCh would be soluble to detergents, whereas higher-order oligomers such as tetramers would not be. However, interestingly, s1RE102Q-mCh monomers, dimers, trimers, and tetramers were fractionated in both soluble and insoluble fractions (Fig. 7). This suggests that the oligomeric state is not sufficient to determine the vulnerability of s1RE102Q to detergents. SA4503 treatment decreased the formation of these four oligomeric states in the insoluble fraction but increased the accumulation of monomers in the soluble fraction. The effects of agonists and antagonists on the oligomeric state of s1R were recently investigated by another group (47)(48)(49)(50), who transfected HEK293 cells with nontagged or tagged forms of s1R and performed normal SDS-PAGE or PAGE with mild detergents (perfluorooctanoic acid or sodium lauroyl sarcosinate). They found that most of the agonists decrease multimer formation with a molecular weight of over 130 kDa, whereas antagonists increase such formations. Their results also showed that s1RE102Q reduces multimer or high-order oligomers over dimers (49,50). In our study, s1R-mCh formed exclusively monomers and dimers, whereas s1RE102Q-mCh was also observed in trimers and tetramers (Fig. 7, A and B). Although we cannot explain this discrepancy, it suggests that these cell lines have no appropriate properties including protein expressions or lipid composition that might require s1R oligomerization. It is not fully demonstrated how the oligomer forms and is regulated in the cells/during extraction. Considering this, our procedure (DSS 1 SDS-PAGE) might be useful to assess the intracellular behavior of the receptor. The smear pattern of s1RE102Q-mCh with higher molecular weight especially in the insoluble fraction (Fig. S4) suggests a correlation of the insolubility and aggregate formation. Furthermore, in the spinal cord, s1R is reportedly enriched in the post-synaptic terminal in the cell soma, axons, and dendrites of motor neurons (51,52). However, it is not determined that NSC-34 cells can form the synapse between cells. Further studies are required for assessing the influence of synaptic formation on s1R mutant features.
We performed co-immunoprecipitation and revealed that s1R exerted homomeric interactions in NSC-34 cells (Fig. 8A). This result supports previous reports using size exclusion chromatography and FRET analysis (4,(33)(34)(35). We also revealed that s1RE102Q preserves the interaction with WT s1R. The co-expression of s1R-mCh with the mutant inhibited the aberrant insolubility (Fig. 8, B and C). The inheritance of most genetic mutations associated with familial ALS identified so far are autosomal or X-linked dominant (24,25,(53)(54)(55)(56)(57)(58)(59), suggesting a high toxicity of those mutations. The s1RE102Q mutation causes juvenile ALS, thus also evidencing high toxicity (12). However, the inheritance of this variation is autosomal recessive, with the homozygous mutation affecting motor neurons severely, whereas the heterozygous mutation has no effect. Our finding that WT s1R-mCh abolished the abnormal insolubility of s1RE102Q may explain this discrepancy.
In conclusion, the s1RE102Q mutation caused abnormal insolubility to detergents, which correlated with aggregation, although these were distinct from the conventional ER-LDs or MAMs. Treatment with the s1R agonist SA4503 and WT s1R Lower, s 1 R E102Q forms not only monomers/dimers but also trimers/tetramers and aggregates, which are detergent-insoluble. Aggregation of s 1 R E102Q correlates with the induction of ER stress and might result in cell death. s 1 R agonist SA4503 and WT co-expression rescued these mutationinduced abnormalities.
co-expression could abrogate the aberrant oligomer formation caused by the mutation. In the future, pharmacological treatment with SA4503 and genetic therapy using WT s1R expression may be promising for the treatment of s1R mutationrelated ALS.
Cell culture, transfection, and treatment NSC-34 cells, a mouse motor neuron-like cell line, were a kind gift from Dr. Yasuhiro Kosuge (Nihon University) (62). HEK293T cells (CRL-3216) were purchased from American Type Culture Collection (63). The cells were maintained in DMEM supplemented with 10% FBS (Biowest, Nuaillé, France) and penicillin/streptomycin (Thermo Fisher Scientific) in a 5% CO 2 incubator at 37°C. The cells were transfected with plasmids encoding WT or ALS-related mutant (E102Q) of s1R Cterminally fused with mCherry or GFP, nontagged forms or control (pcDNA3.1, mCherry or GFP) for 6 h using Lipofectamine LTX Reagent (Thermo Fisher Scientific) (21). After transfection, the cells were cultured in complete DMEM and incubated with 0, 0.2, 1, 5, and 20 mM SA4503 for 48 h, then used for further experiments. For FRAP, SA4503 was added only 1 h before analysis. SA4503 was dissolved in sterilized double distilled water (ddw) at 20 mM stock solutions and stored at -20°C. For analysis of concentration dependence, stock solutions were sequentially diluted with sterilized ddw before use.

Detergent-soluble/insoluble fractionation
Detergent-soluble/insoluble fractionation was performed according to previous reports with slight modifications (31,64). The cells were washed with PBS, harvested in lysis buffer containing 1% NP-40 (20 mM HEPES-NaOH, pH 7.4, 100 mM KCl, 1.5 mM MgCl 2 , 250 mM sucrose, 1 mM EDTA, and 1 mM EGTA), and gently agitated at 4°C for 0.5 h. The lysates were centrifuged at 10,000 3 g for 10 min, and the supernatant was collected in a new tube. The samples were further centrifuged at 100,000 3 g for 1 h, and the supernatant was collected as a soluble fraction. The pellets were washed with homogenizing buffer followed by adding half the volume of SDS lysis buffer (50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate, and 1% glycerol) and sonication as with the insoluble fraction. For further investigation of the insoluble features of s1RE102Q, the cells were homogenized in lysis buffer containing 0.5% Triton X-100 or 20 mg/ml CHAPS (10 mM Tris-HCl, pH 7.4, 150 mM NaCl, and 5 mM EDTA) as previously reported (31). Following agitation at 4°C for 0.5 h, the cells were centrifuged at 12,000 3 g for 20 min. The supernatants were further separated into soluble and insoluble fractions by ultracentrifugation as described above. For total fractions, the cells were harvested in SDS lysis buffer, sonicated, and centrifuged at 10,000 3 g for 10 min before the supernatant was collected. Protease/protein phosphatase inhibitors (50 mg/ ml trypsin inhibitor, 0.1 mM leupeptin, 1 mM DTT, 75 mM pepstatin A, and 25 nM calyculin A) were added to the lysis buffer. For Western blotting, each fraction was boiled at 100°C for 3 min following the addition of 63 Laemmli SDS sample buffer.

Sucrose density gradient centrifugation
Sucrose density gradient centrifugation was performed according to a previous study (30). Insoluble fractions were adjusted to 250 mM sucrose, which was identical to the concentration of the soluble fraction. Both fractions were laid on top of 40-80% sucrose density gradients (5 volumes) and centrifuged at 100,000 3 g for 17 h. Twelve fractions were collected from the top, and the proteins were precipitated by adding an equal volume of 40% TCA. After centrifugation, the pellets were washed with ethanol, followed by adding SDS lysis buffer and sonication. Proteins were boiled with 63 Laemmli SDS sample buffer for Western blotting.
Another condition of sucrose density gradient centrifugation was used to investigate the localization of s1R-mCh and s1RE102Q-mCh in ER-LDs or MAMs (29). Briefly, transfected cells were extracted with lysis buffer containing 0.5% Triton X-100, and the lysates were separated by 0-35% sucrose density gradient using centrifugation at 141,000 3 g for 24 h. Eleven fractions were collected from the top, and the proteins were precipitated by TCA and denatured with SDS sample buffer.

Crosslinking procedure
The cells were washed twice and harvested in PBS. The cells were added with DSS crosslinker (final 2 mM; Thermo Fisher Scientific) and incubated for 0.5 h at room temperature. The reaction was stopped by adding Tris-HCl (pH 7.5, final 50 mM) and incubation at room temperature for 15 min. The cells were subjected to detergent-soluble/insoluble fractionation as described above.

Western blotting analysis
Western blotting analysis was performed as previously reported (21). Briefly, an equal number of samples were applied Pharmacological/genetic effect on ALS-related s1R features to SDS-PAGE with 5-12/15% separated polyacrylamide gels or 2-15% gradient gels (MULTIGEL II mini, Cosmo Bio, Tokyo, Japan), followed by transfer to polyvinylidene fluoride membranes (Merck). The membranes were agitated in 5% skim milk in TBS with Tween 20 for 1 h, then shaken with primary antibody (1:1,000 dilution except for the antibody against b-tubulin (1:2,000)) as described above at 4°C overnight. After three washes with TBS with Tween 20, the membranes were shaken with goat anti-rabbit or mouse IgG antibody conjugated with horseradish peroxidase (1:5,000 dilution; Southern Biotech, Birmingham, Alabama, USA). Chemiluminescent signal was detected by imaging analyzer (LAS4000 Mini; FUJIFILM, Tokyo, Japan). Densitometry analysis was visualized using Multi Gauge Software (FUJIFILM).

Immunocytochemistry
The cells were cultured on poly-L-lysine-coated cover glass in a 12-well multiplate. Immunocytochemistry was performed at room temperature unless otherwise specified. After fixation with 4% paraformaldehyde in PBS for 10 min, the cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min. The cells were incubated with 1% BSA in PBS for 0.5 h for blocking, then probed with the primary antibody in BSA solution (1:200 or 1:500) at 4°C overnight. After three washes, the cells were incubated with a secondary antibody of Alexa Flour 488-conjugated donkey anti-rabbit, mouse IgG antibody, or Alexa Flour 594-conjugated donkey anti-mouse IgG antibody (1:500; Thermo Fisher Scientific). After washing, the nucleus was stained with 49,6-diamidino-2-phenylindole (Merck), and the cells were immersed in Vectashield (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were obtained by confocal microscopy (TCS SP8, Leica Microsystems, Wetzlar, Germany). For immunostaining proteins in ER using antibodies against s1R, BiP/GRP78, pPERK, and IP3R2, antigen retrieval was performed by incubating cells in 6 M urea in 0.1 M Tris-HCl, pH 9.5, at 80°C for 10 min after fixation (65). The fluorescent images of mCherry and TOM20 were automatically adjusted in intensity, the background signal was subtracted, and the threshold was determined and converted into binary images using Image J software to visualize the MAMs (66). Merged images with these binary images show the colocalization as MAMs.
Fluorescence recovery after photobleaching FRAP was performed for analyzing the molecular dynamics within cells using LAS X Software (Leica Microsystems). The cells were cultured in a poly-lysine-coated glass-bottom dish (Matsunami Glass Ind., Osaka, Japan) for 2 days after transfection. The cells were stimulated with SA4503 (20 mM) for 1 h, then washed with and incubated in Hank's Balanced Salt Solution, and red fluorescence of mCherry was observed in live imaging using the confocal microscope. Imaging settings were as follows: 512 3 512 pixels, 633 magnification lens, 33 zoom, and scan speed 400 Hz. In this setting, 40 images were taken in ;1.3 s/image with photobleaching just after taking the 10th image. The fluorescent intensity of regions of interest was measured using Image J software, and the normalized intensity was calculated compared with the intensity in the first image. The difference between intensity in the final and the 11th images was calculated as the index of recovery.

Immunoprecipitation
Immunoprecipitation was performed as previously reported (67). Briefly, the anti-GFP antibody was mixed with Protein A-Sepharose beads (GE Healthcare) and gently agitated at 4°C for 0.5 h. Cell lysates were added to the beads and rotated at 4°C for 2 h. The beads were washed with homogenizing buffer and denatured for Western blotting.

DNA diffusion assay
Cell apoptosis was determined by DNA diffusion assay (68). All procedures were performed in the dark as much as possible to avoid DNA damage and at room temperature unless otherwise specified. The cells were transfected with nontagged s1R and s1RE102Q or control vector (pcDNA3.1) to avoid the fluorescence of mCherry and collected in PBS 2 days after transfection. Then, the cells were mixed with 0.5% low melting temperature agarose (Cambrex, East Rutherford, NJ, USA) in PBS at 37°C. The mixture lay on 0.5% normal melting temperature agarose (Nippon Gene, Tokyo, Japan)coated slide glass and covered with a cover glass. The gels were kept at 4°C until getting solid for 3 min. The cover glass was removed, and the top layer of 2% low melting temperature agarose was made in the same procedure. The gels were immersed in chilled lysis solution (10 mM Tris-NaOH, pH 10, 2.5 M NaCl, 1% Triton X-100, and 2 mM EDTA) and kept at 4°C for 1 h. Then, the gels were immersed in alkaline solution (0.3 N NaOH, pH . 13.5, and 0.2% DMSO) for 10 min to denature the genomic DNA and then dipped into precipitation solution (1 M CH3COONH4 in ethanol) for 15 min. After washing with 75% ethanol, the gels were air-dried and incubated in prestaining solution (10 mM NaH 2 PO 4 , 5% DMSO, and 5% sucrose) for 5 min and stained with 20 mg/ml ethidium bromide (Nacalai Tesque) for 5 min. The gels were washed with ddw, immersed in Vectashield, and observed using the confocal microscope. The cells with a round appearance with clear borders were counted as nonapoptotic cells, and the cells with a halo of granular DNA with a hazy outer boundary were counted as apoptotic cells (68). Over 100 cells in 10 randomly selected regions were analyzed, and the percentages of apoptotic cells were calculated. Counting and analysis was performed in a blinded manner.

Statistical analysis
Data were expressed as mean 6 S.D. with individual plots. Unpaired t test for two groups and one-way or two-way analysis of variance (ANOVA) followed by Bonferroni post hoc test for more than two groups were performed using Graph-Pad Prism version 8.4.2 (GraphPad Software, San Diego, CA, USA). t values in t test and F values in ANOVA are described in the Fig. legends. p , 0.05 was considered statistically significant.

Data availability
Data are to be shared upon request.
Acknowledgments-We thank Dr. Yasuhiro Kosuge of Nihon University, School of Pharmacy, Laboratory of Pharmacology, for kindly providing NSC-34 cells. We thank Dr. Chen Zhang of Zhejiang University, College of Pharmaceutical Sciences, for kindly synthesizing SA4503.
Author contributions-Y. S. writing-original draft; Y. S. and K. F. writing-review and editing; Y. S., Y. H., and K. A. performed experiments and analysis. Conflict of interest-The authors declare that they have no conflicts of interest with the contents of this article.