UBQLN2 undergoes a reversible temperature-induced conformational switch that regulates binding with HSPA1B: ALS/FTD mutations cripple the switch but do not destroy HSPA1B binding

Here we present evidence, based on alterations of its intrinsic tryptophan fluorescence, that UBQLN2 protein undergoes a conformational switch when the temperature is raised from 37 °C to 42 °C. The switch is reset on restoration of the temperature. We speculate that the switch regulates UBQLN2 function in the heat shock response because elevation of the temperature from 37 °C to 42 °C dramatically increased in vitro binding between UBQLN2 and HSPA1B. Furthermore, restoration of the temperature to 37 °C decreased HSPA1B binding. By comparison to wild type (WT) UBQLN2, we found that all five ALS/FTD mutant UBQLN2 proteins we examined had attenuated alterations in tryptophan fluorescence when shifted to 42 °C, suggesting that the conformational switch is crippled in the mutants. Paradoxically, all five mutants bound similar amounts of HSPA1B compared to WT UBQLN2 protein at 42 °C, suggesting that either the conformational switch is not instrumental for HSPA1B binding, or that, although damaged, it is still functional. Comparison of the poly-ubiquitin chain binding revealed that WT UBQLN2 binds more avidly with K63 than with K48 chains. The avidity may explain the involvement of UBQLN2 in autophagy and cell signaling. Consistent with its function in autophagy, we found UBQLN2 binds directly with LC3, the autophagosomal-specific membrane-tethered protein. Finally, we provide evidence that WT UBQLN2 can homodimerize, and heterodimerize with WT UBQLN1. We show that ALS mutant P497S-UBQLN2 protein can oligomerize with either WT UBQLN1 or 2, providing a possible mechanism for how mutant UBQLN2 proteins could bind and inactivate UBQLN proteins, causing loss of function.     

Ubiquilin-2 (UBQLN2) binds with high affinity to the C-terminal region of TDP-43 and modulates TDP-43 levels in H4 cells: Characterization of inhibition by nucleic acids and 4-aminoquinolines

Recently, it was reported that mutations in the ubiquitin-like protein ubiquilin-2 (UBQLN2) are associated with X-linked amyotrophic lateral sclerosis (ALS), and that both wild-type and mutant UBQLN2 can co-localize with aggregates of C-terminal fragments of TAR DNA binding protein (TDP-43). Here, we describe a high affinity interaction between UBQLN2 and TDP-43 and demonstrate that overexpression of both UBQLN2 and TDP-43 reduces levels of both exogenous and endogenous TDP-43 in human H4 cells. UBQLN2 bound with high affinity to both full length TDP-43 and a C-terminal TDP-43 fragment (261–414 aa) with K_D values of 6.2 nM and 8.7 nM, respectively. Both DNA oligonucleotides and 4-aminoquinolines, which bind to TDP-43, also inhibited UBQLN2 binding to TDP-43 with similar rank order affinities compared to inhibition of oligonucleotide binding to TDP-43. Inhibitor characterization experiments demonstrated that the DNA oligonucleotides noncompetitively inhibited UBQLN2 binding to TDP-43, which is consistent with UBQLN2 binding to the C-terminal region of TDP-43. Interestingly, the 4-aminoquinolines were competitive inhibitors of UBQLN2 binding to TDP-43, suggesting that these compounds also bind to the C-terminal region of TDP-43. In support of the biochemical data, co-immunoprecipitation experiments demonstrated that both TDP-43 and UBQLN2 interact in human neuroglioma H4 cells. Finally, overexpression of UBQLN2 in the presence of overexpressed full length TDP-43 or C-terminal TDP-43 (170–414) dramatically lowered levels of both full length TDP-43 and C-terminal TDP-43 fragments (CTFs). Consequently, these data suggest that UBQLN2 enhances the clearance of TDP-43 and TDP-43 CTFs and therefore may play a role in the development of TDP-43 associated neurotoxicity.     

Interactions between FUS and the C-terminal Domain of Nup62 are Sufficient for their Co-phase Separation into Amorphous Assemblies

Deficient nucleocytoplasmic transport is emerging as a pathogenic feature of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD), including in ALS caused by mutations in Fused in Sarcoma (FUS). Recently, both wild-type and ALS-linked mutant FUS were shown to directly interact with the phenylalanine-glycine (FG)-rich nucleoporin 62 (Nup62) protein, where FUS WT/ Nup62 interactions were enriched within the nucleus but ALS-linked mutant FUS/ Nup62 interactions were enriched within the cytoplasm of cells. Nup62 is a central channel Nup that has a prominent role in forming the selectivity filter within the nuclear pore complex and in regulating effective nucleocytoplasmic transport. Under conditions where FUS phase separates into liquid droplets in vitro, the addition of Nup62 caused the synergistic formation of amorphous assemblies containing both FUS and Nup62. Here, we examined the molecular determinants of this process using recombinant FUS and Nup62 proteins and biochemical approaches. We demonstrate that the structured C-terminal domain of Nup62 containing an alpha-helical coiled-coil region plays a dominant role in binding FUS and is sufficient for inducing the formation of FUS/Nup62 amorphous assemblies. In contrast, the natively unstructured, F/G repeat-rich N-terminal domain of Nup62 modestly contributed to FUS/Nup62 phase separation behavior. Expression of individual Nup62 domain constructs in human cells confirmed that the Nup62 C-terminal domain is essential for localization of the protein to the nuclear envelope. Our results raise the possibility that interactions between FUS and the C-terminal domain of Nup62 can influence the function of Nup62 under physiological and/or pathological conditions.     

Proteasome Assembly Influences Interaction with Ubiquitinated Proteins and Shuttle Factors

A major fraction of intracellular protein degradation is mediated by the proteasome. Successful degradation of these substrates requires ubiquitination and delivery to the proteasome followed by protein unfolding and disassembly of the multiubiquitin chain. Enzymes, such as Rpn11, dismantle multiubiquitin chains, and mutations can affect proteasome assembly and activity. We report that different rpn11 mutations can affect proteasome interaction with ubiquitinated proteins. Moreover, proteasomes are unstable in rpn11-1 and do not form productive interactions with multiubiquitinated proteins despite high levels in cell extracts. However, increased levels of ubiquitinated proteins were found associated with shuttle factors. In contrast to rpn11-1, proteasomes expressing a catalytically inactive mutant (rpn11^AXA) were more stable and bound very high amounts of ubiquitinated substrates. Expression of the carboxyl-terminal domain of Rpn11 partially suppressed the growth and proteasome stability defects of rpn11-1. These results indicate that ubiquitinated substrates are preferentially delivered to intact proteasome.     

The E2 ubiquitin-conjugating enzyme HIP2 is a crucial regulator of quality control against mutant SOD1 proteotoxicity

Mutations in superoxide dismutase 1 (SOD1) leading to the formation of intracellular protein aggregates cause amyotrophic lateral sclerosis (ALS), a fatal neurodegenerative disorder characterized by a selective degeneration of motor neurons. The ALS-linked mutant SOD1 emerged as a possible target for ubiquitin–proteasome system (UPS)-mediated degradation. We aimed to elucidate the role of huntingtin interaction protein 2 (HIP2), an E2 ubiquitin-conjugating enzyme, in the proteotoxicity of mutant SOD1 aggregates. We found that HIP2 interacts with mutant SOD1, but not wild-type SOD1, and is upregulated in response to mutant SOD1 expression. Upregulation of HIP2 protein was observed in the spinal cord of 16-week-old SOD1-G93A transgenic mice. HIP2 further modified mutant SOD1 proteins via K48-linked polyubiquitination and degraded mutant SOD1 proteins through the UPS. Upregulation of HIP2 protected cells from mutant SOD1-induced toxicity. Taken together, our findings demonstrate that HIP2 is a crucial regulator of quality control against the proteotoxicity of mutant SOD1. Our results suggest that modulating HIP2 may represent a novel therapeutic strategy for the treatment of ALS.     

Intracellular conformational alterations of mutant SOD1 and the implications for fALS-associated SOD1 mutant induced motor neuron cell death

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disorder characterized by the selective death of motor neurons. Approximately 10% of ALS cases are familial (fALS) and about 25% of fALS patients inherit autosomal dominant mutations in the gene encoding copper-zinc superoxide dismutase (SOD1). Over 90 different SOD1 mutations have been identified in fALS patients. It has been established that the ALS-linked SOD1 mutations provoke a new toxic function, the nature of which remains unclear. In vitro studies using various biophysical techniques have demonstrated that the SOD1 mutants share a reduced conformational stability. However, conformational alterations of the ALS mutants have not been directly demonstrated in vivo. We employed an SOD1-GFP fusion protein system in this study to monitor the intracellular protein conformation. We demonstrate that the ALS-linked SOD1 mutants adopt different conformations from the wild-type (WT) protein in living cells. Moreover, the conformational alterations of mutant SOD1 render the mutants susceptible to the formation of high-molecular-weight complexes prior to the appearance of detergent-resistant aggregates. Finally, we show that the motor neuron-like cells expressing mutant SOD1 are more susceptible to H2O2 induced cell death compared to the cells expressing WT SOD1. This study provides direct evidence of in vivo conformational differences between WT and mutant SOD1. In addition, the SOD1-GFP system can be exploited in future studies to investigate how conformational alterations of mutant SOD1 lead to protein aggregation and to study the potential toxicity of such aggregates in familial ALS.     

A new Drosophila model of Ubiquilin knockdown shows the effect of impaired proteostasis on locomotive and learning abilities

Ubiquilin (UBQLN) plays a crucial role in cellular proteostasis through its involvement in the ubiquitin proteasome system and autophagy. Mutations in the UBQLN2 gene have been implicated in amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal lobar dementia (ALS/FTLD). Previous studies reported a key role for UBQLN in Alzheimer's disease (AD); however, the mechanistic involvement of UBQLN in other neurodegenerative diseases remains unclear. The genome of Drosophila contains a single UBQLN homolog (dUbqn) that shows high similarity to UBQLN1 and UBQLN2; therefore, the fly is a useful model for characterizing the role of UBQLN in vivo in neurological disorders affecting locomotion and learning abilities. We herein performed a phenotypic and molecular characterization of diverse dUbqn RNAi lines. We found that the depletion of dUbqn induced the accumulation of polyubiquitinated proteins and caused morphological defects in various tissues. Our results showed that structural defects in larval neuromuscular junctions, abdominal neuromeres, and mushroom bodies correlated with limited abilities in locomotion, learning, and memory. These results contribute to our understanding of the impact of impaired proteostasis in neurodegenerative diseases and provide a useful Drosophila model for the development of promising therapies for ALS and FTLD.     

The specificity of ubiquitin binding to ubiquilin-1 is regulated by sequences besides its UBA domain

UBQLN proteins regulate proteostasis by facilitating clearance of misfolded proteins through the proteasome and autophagy degradation pathways. Consistent with its proteasomal function, UBQLN proteins contain both UBL and UBA domains, which bind subunits of the proteasome, including the S5a subunit, and ubiquitin chains, respectively. Conclusions regarding the binding properties of UBQLN proteins have been derived principally through studies of its individual domains, not the full-length (FL) proteins. Here we describe the in vitro binding properties of FL-UBQLN1 with the S5a subunit of the proteasome and two different lysine-linked (K48 or K63) ubiquitin chains. We show that in contrast to its isolated UBA domain, which binds almost equally well with both K48 and K63 ubiquitin chains, FL UBQLN1 binds preferentially with K63 chains. Furthermore, we show that deletion of the UBL domain from UBQLN1 abrogates ubiquitin binding. Taken together these results suggest that sequences outside of the UBA domain in UBQLN1 function to regulate the specificity and binding with different ubiquitin moieties. We also show that the UBL domain of UBQLN1 is required for S5a binding and that its binding to UBQLN1, in turn, enhances K48 ubiquitin chain binding to the complex. We discuss the implications of our findings with the known function of UBQLN proteins in protein degradation.     

Accumulation of wildtype and ALS-linked mutated VAPB impairs activity of the proteasome

Cellular homeostasis relies on a tight control of protein synthesis, folding and degradation, in which the endoplasmic reticulum (ER) quality control and the ubiquitin proteasome system (UPS) have an instrumental function. ER stress and aberrant accumulation of misfolded proteins represent a pathological signature of amyotrophic lateral sclerosis (ALS), a fatal paralytic disorder caused by the selective degeneration of motoneurons in the brain and spinal cord. Mutations in the ER-resident protein VAPB have been associated with familial forms of the disease. ALS-linked mutations cause VAPB to form cytoplasmic aggregates. We previously demonstrated that viral-mediated expression of both wildtype and mutant human VAPB (hVAPB) leads to an ER stress response that contributes to the selective death of motoneurons. However, the mechanisms behind ER stress, defective UPS and hVAPB-associated motoneuron degeneration remain elusive. Here, we show that the overexpression of wildtype and mutated hVAPB, which is found to be less stable than the wildtype protein, leads to the abnormal accumulation of ubiquitin and ubiquitin-like protein conjugates in non-human primate cells. We observed that overexpression of both forms of hVAPB elicited an ER stress response. Treatment of wildtype and mutated hVAPB expressing cells with the ER stress inhibitor salubrinal diminished the burden of ubiquitinated proteins, suggesting that ER stress contributes to the impairment of proteasome function. We also found that both wildtype and mutated hVAPB can associate with the 20S proteasome, which was found to accumulate at the ER with wildtype hVAPB or in mutant hVAPB aggregates. Our results suggest that ER stress and corruption of the proteasome function might contribute to the aberrant protein homeostasis associated with hVAPB.     

ALS-linked FUS mutations dysregulate G-quadruplex-dependent liquid–liquid phase separation and liquid-to-solid transition

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the accumulation of protein aggregates in motor neurons. Recent discoveries of genetic mutations in ALS patients promoted research into the complex molecular mechanisms underlying ALS. FUS (fused in sarcoma) is a representative ALS-linked RNA-binding protein (RBP) that specifically recognizes G-quadruplex (G4)-DNA/RNAs. However, the effects of ALS-linked FUS mutations on the G4-RNA-binding activity and the phase behavior have never been investigated. Using the purified full-length FUS, we analyzed the molecular mechanisms of multidomain structures consisting of multiple functional modules that bind to G4. Here we succeeded to observe the liquid–liquid phase separation (LLPS) of FUS condensate formation and subsequent liquid-to-solid transition (LST) leading to the formation of FUS aggregates. This process was markedly promoted through FUS interaction with G4-RNA. To further investigate, we selected a total of eight representative ALS-linked FUS mutants within multidomain structures and purified these proteins. The regulation of G4-RNA-dependent LLPS and LST pathways was lost for all ALS-linked FUS mutants defective in G4-RNA recognition tested, supporting the essential role of G4-RNA in this process. Noteworthy, the P525L mutation that causes juvenile ALS exhibited the largest effect on both G4-RNA binding and FUS aggregation. The findings described herein could provide a clue to the hitherto undefined connection between protein aggregation and dysfunction of RBPs in the complex pathway of ALS pathogenesis.     

Treatment with a Global Methyltransferase Inhibitor Induces the Intranuclear Aggregation of ALS-Linked FUS Mutant In Vitro

FUS/TLS (fused in sarcoma/translocated in liposarcoma) encodes a multifunctional DNA/RNA binding protein with non-classical carboxy (C)-terminal nuclear localization signal (NLS). A variety of ALS-linked mutations are clustered in the C-terminal NLS, resulting in the cytoplasmic mislocalization and aggregation. Since the arginine methylations are implicated in the nuclear-cytoplasmic shuttling of FUS, a methylation inhibitor could be one of therapeutic targets for FUS-linked ALS. We here examined effects of methylation inhibitors on the cytoplasmic mislocalization and aggregates of ALS-linked C-terminal FUS mutant in a cell culture system. Treatment with adenosine dialdehyde (AdOx), a representative global methyltransferase inhibitor, remarkably mitigated the cytoplasmic mislocalization and aggregation of FUS mutant, which is consistent with previous reports. However, AdOx treatment of higher concentration and longer time period evoked the intranuclear aggregation of the ectopic expressed FUS protein. The pull down assay and the morphological analysis indicated the binding between FUS and Transportin could be potentiated by AdOx treatment through modulating methylation status in RGG domains of FUS. These findings indicated the treatment with a methylation inhibitor at the appropriate levels could alleviate the cytoplasmic mislocalization but in excess this could cause the intranuclear aggregation of FUS C-terminal mutant.

     

Aggregation of ALS-linked FUS mutant sequesters RNA binding proteins and impairs RNA granules formation

Protein aggregate/inclusion is one of hallmarks for neurodegenerative disorders including amyotrophic lateral sclerosis (ALS). FUS/TLS, one of causative genes for familial ALS, encodes a multifunctional DNA/RNA binding protein predominantly localized in the nucleus. C-terminal mutations in FUS/TLS cause the retention and the inclusion of FUS/TLS mutants in the cytoplasm. In the present study, we examined the effects of ALS-linked FUS mutants on ALS-associated RNA binding proteins and RNA granules. FUS C-terminal mutants were diffusely mislocalized in the cytoplasm as small granules in transiently transfected SH-SY5Y cells, whereas large aggregates were spontaneously formed in ∼10% of those cells. hnRNP A1, hnRNP A2, and SMN1 as well as FUS wild type were assembled into stress granules under stress conditions, and these were also recruited to FUS mutant-derived spontaneous aggregates in the cytoplasm. These aggregates stalled poly(A) mRNAs and sequestered SMN1 in the detergent insoluble fraction, which also reduced the number of nuclear oligo(dT)-positive foci (speckles) in FISH (fluorescence in situ hybridization) assay. In addition, the number of P-bodies was decreased in cells harboring cytoplasmic granules of FUS P525L. These findings raise the possibility that ALS-linked C-terminal FUS mutants could sequester a variety of RNA binding proteins and mRNAs in the cytoplasmic aggregates, which could disrupt various aspects of RNA equilibrium and biogenesis.     

Stress signaling from the endoplasmic reticulum: A central player in the pathogenesis of amyotrophic lateral sclerosis

Amyotrophic lateral sclerosis (ALS) is a devastating neurodegenerative disease characterized by the misfolding and aggregation of distinct proteins in affected tissues, however, the pathogenic cause of disease remains unknown. Recent evidence indicates that endoplasmic reticulum (ER) stress plays a central role in ALS pathogenesis. ER stress activates the unfolded protein response (UPR), a homeostatic response to misfolded proteins. The UPR is initially protective by up-regulation of specific ER stress-regulated genes and inhibition of general protein translation. However, long-term ER stress leads to cell death via apoptotic signaling, thus providing a link to neurodegeneration. Activation of the UPR is one of the earliest events in affected motor neurons of transgenic rodent models expressing ALS-linked mutant superoxide dismutase 1 (SOD1). Recently, genetic manipulation of ER stress in several different SOD1 mouse models was shown to alter disease onset and progression, implicating an active role for the UPR in disease mechanisms. Furthermore, mutations to vesicle-associated membrane protein-associated protein B (VAPB), an ER transmembrane protein involved in ER stress regulation, also cause some cases of familial ALS. ER stress also occurs in spinal cord tissues of human sporadic ALS patients, and recent evidence suggests that perturbation of the ER could occur in ALS cases associated with TAR DNA binding protein 43 (TDP-43), fused in sarcoma (FUS) and valosin containing protein (VCP). Together these findings implicate ER stress as a potential upstream mechanism involved in both familial and sporadic forms of ALS.     

Pathogenic mutation of UBQLN2 impairs its interaction with UBXD8 and disrupts endoplasmic reticulum‐associated protein degradation

Protein aggregation is a common feature of several neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration. How protein aggregates are formed and contribute to neurodegeneration, however, is not clear. Mutation of Ubiquilin 2 (UBQLN2) has recently been linked to ALS and frontotemporal lobar degeneration. Therefore, we examined the effect of ALS‐linked UBQLN2 mutation on endoplasmic reticulum‐associated protein degradation (ERAD). Compared to its wild‐type counterpart, mutated UBQLN2 caused greater accumulation of the ERAD substrate Hong Kong variant of α‐1‐antitrypsin, although ERAD was disturbed by both UBQLN2 over‐expression and knockdown. Also, UBQLN2 interacted with ubiquitin regulatory X domain‐containing protein 8 (UBXD8) in vitro and in vivo, and this interaction was impaired by pathogenic mutation of UBQLN2. As UBXD8 is an endoplasmic membrane protein involved in the translocation of ubiquitinated ERAD substrates, UBQLN2 likely cooperates with UBXD8 to transport defective proteins from the endoplasmic reticulum to the cytosol for degradation, and this cell‐protective function is disturbed by pathogenic mutation of UBQLN2.

     

Disease-Associated Mutations of TDP-43 Promote Turnover of the Protein Through the Proteasomal Pathway

TAR DNA-binding protein (TDP-43) is a major component of most ubiquitin-positive neuronal and glial inclusions of amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). A number of missense mutations in the TARDBP gene have been identified in patients with familial and sporadic ALS, as well as familial FTLD with ALS. In the diseased states, TDP-43 proteins exhibit characteristic alterations, including truncation, abnormal phosphorylation, and altered subcellular distribution. However, the mechanisms by which TDP-43 mutations induce neurodegeneration remain unclear at present. In the current study, we analyzed protein turnover and subcellular distribution of wild-type TDP-43 and two disease-associated mutants (G298S and A382T) in human neuroblastoma SH-SY5Y cells stably expressing TDP-43 with a C-terminal tag. Cycloheximide chase experiments revealed more rapid turnover of TDP-43 mutant proteins than their wild-type counterpart. The decrease in the TDP-43 level after cycloheximide treatment was partially recovered upon co-treatment with the proteasome inhibitor, epoxomicin, but not the lysosomotropic agent, chloroquine, suggesting involvement of the proteasomal pathway in TDP-43 degradation. Analysis of the subcellular distribution of TDP-43 revealed predominant localization in the nuclear fraction, whereas the relative level in the cytoplasm remained unaltered in cells expressing either mutant protein, compared with wild-type protein. Our results suggest that higher turnover of disease-associated mutant TDP-43 proteins through the ubiquitin proteasome system is pathogenetically relevant and highlight the significance of proteolysis in the pathogenetic mechanism of TDP-43 proteinopathy.     

Dimerization,Oligomerization, and Aggregation of Human Amyotrophic Lateral Sclerosis Copper/Zinc Superoxide Dismutase 1 Protein Mutant Forms in Live Cells

More than 100 copper/zinc superoxide dismutase 1 (SOD1) genetic mutations have been characterized. These mutations lead to the death of motor neurons in ALS. In its native form, the SOD1 protein is expressed as a homodimer in the cytosol. In vitro studies have shown that SOD1 mutations impair the dimerization kinetics of the protein, and in vivo studies have shown that SOD1 forms aggregates in patients with familial forms of ALS. In this study, we analyzed WT SOD1 and 9 mutant (mt) forms of the protein by non-invasive fluorescence techniques. Using microscopic techniques such as fluorescence resonance energy transfer, fluorescence complementation, image-based quantification, and fluorescence correlation spectroscopy, we studied SOD1 dimerization, oligomerization, and aggregation. Our results indicate that SOD1 mutations lead to an impairment in SOD1 dimerization and, subsequently, affect protein aggregation. We also show that SOD1 WT and mt proteins can dimerize. However, aggregates are predominantly composed of SOD1 mt proteins.     

Proteins that bind to misfolded mutant superoxide dismutase-1 in spinal cords from transgenic amyotrophic lateral sclerosis (ALS) model mice

Mutant superoxide dismutase-1 (SOD1) has an unidentified toxic property that provokes ALS. Several ALS-linked SOD1 mutations cause long C-terminal truncations, which suggests that common cytotoxic SOD1 conformational species should be misfolded and that the C-terminal end cannot be involved. The cytotoxicity may arise from interaction of cellular proteins with misfolded SOD1 species. Here we specifically immunocaptured misfolded SOD1 by the C-terminal end, from extracts of spinal cords from transgenic ALS model mice. Associated proteins were identified with proteomic techniques. Two transgenic models expressing SOD1s with contrasting molecular properties were examined: the stable G93A mutant, which is abundant in the spinal cord with only a tiny subfraction misfolded, and the scarce disordered truncation mutant G127insTGGG. For comparison, proteins in spinal cord extracts with affinity for immobilized apo G93A mutant SOD1 were determined. Two-dimensional gel patterns with a limited number of bound proteins were found, which were similar for the two SOD1 mutants. Apart from neurofilament light, the proteins identified were all chaperones and by far most abundant was Hsc70. The immobilized apo G93A SOD1, which would populate a variety of conformations, was found to bind to a considerable number of additional proteins. A substantial proportion of the misfolded SOD1 in the spinal cord extracts appeared to be chaperone-associated. Still, only about 1% of the Hsc70 appeared to be associated with misfolded SOD1. The results argue against the notion that chaperone depletion is involved in ALS pathogenesis in the transgenic models and in humans carrying SOD1 mutations.