Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic (SALS), but about 5–10% are familial (FALS). Mutations in superoxide dismutase 1 (SOD1) 1,2, TAR DNA-binding protein (TDP43) 3,4 and fused in sarcoma/translated in liposarcoma (FUS/TLS) 5,6 account for approximately 30% of classic FALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes 7–15. The causes for the rest of familial ALS and the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type (FTD), may occur in some ALS cases. It is unclear if ALS and dementia share common etiology and pathogenesis in ALS/dementia. Here, we show that mutations in UBQLN2, which encodes a ubiquitin-like protein, ubiquilin2, cause dominantly inherited chromosome X-linked ALS and ALS/dementia. We describe novel ubiquilin2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin2 is a member of the ubiquilin family (ubiquilins), which regulate degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Our findings, therefore, link abnormalities in ubiquilin2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, implying a common pathogenic mechanism that can be exploited for therapeutic intervention.
We identified a five-generation family (Family #186) with ALS, including 19 affected individuals (Supplementary information). The disease is transmitted in a dominant fashion with reduced penetrance in females. Mutations in the known ALS-linked genes were excluded. No evidence of genetic linkage was found with genome-wide set of autosomal microsatellite markers. There was no evidence of male-to-male transmission of the disease, so we screened the family with markers from the X chromosome. Linkage was established with several X chromosome microsatellite markers, with the highest two-point LOD score of 5.0 with marker DXS9736 at θ=0 (Supplementary Table 1). Detailed mapping with dense microsatellite markers and Illumina’s Sentrix HumanHap300 Genotyping BeadChip defined the disease-causing gene in a 21.3Mb minimum candidate region (MCR) between markers rs6417786 and DXS1275, which is located in the pericentric region from Xp11.23 to Xq13.1.
No additional large ALS families without male to male transmission were available to us to narrow down the MCR. We therefore focused on finding the causative gene in Family #186. Of the 206 genes in this MCR, 191 genes were protein coding. Genes in this MCR were analyzed based on their expression profile, function, structure and potential relevance of their encoded proteins to disease. Forty-one genes were sequenced and a unique mutation in UBQLN2 was identified. This mutation, c.1490C>A, is predicted to result in an amino acid substitution of proline by histidine at codon 497 (P497H) (Fig. 1a). The c.1490C>A mutation co-segregated with the disease in this large X-ALS pedigree (Fig. 1a). This mutation was not present in the SNP database nor was it present in 928 ethnically matched control samples (representing 1332 X chromosomes).
UBQLN2 is an intronless gene. To test if mutations of UBQLN2 are causative for other ALS patients, we analyzed 188 probands from families with ALS or ALS/dementia, but without male-to-male transmission. Mutations in SOD1, TDP43 and FUS were excluded in this cohort. The sequenced region covered the entire coding sequence (Materials and Methods). We found four other UBQLN2 mutations in four unrelated families, including c.1489C>T (p.P497S), c.1516C>A (p.P506T), c.1525C>T (p.P509S) and c.1573C>T (p.P525S) (Fig. 1 and Supplementary Fig.1). All the amino acids residues at the mutated sites are conserved (Fig. 1c). None of these mutations were present in the SNP database and 928 control samples. Remarkably, all the five ALS-linked UBQLN2 mutations identified in this study involved proline residues within a unique PXX repeat region (Fig. 1c and 1d).
Clinical data were obtained from 40 individuals in the five families with UBQLN2 mutations, including 35 patients and five obligate carriers. We estimated a penetrance of approximately 90% by the age of 70 years. The age of onset of the disease ranged from 16 to 71 years. A significant difference in age at onset was noted between male and female patients, with male patients having earlier age of onset (33.9 ± 14.0 vs. 47.3 ± 10.8 years, p=0.003, two-tailed Student t-test) (Supplementary Table 2). However, differences in the duration of the disease were not statistically significant (43.1 ± 42.1 vs. 48.5 ± 19.9 months, p=0.61). Eight patients with both ALS and dementia were identified. Dementia in these patients was similar to FTD type, including abnormalities in both behavior and executive function. The dementia was progressive, and eventually global in most ALS/dementia patients. In some cases the dementia preceded motor symptoms, but all patients eventually developed motor disability. Pathological analysis of spinal cord autopsy samples from two patients with either P497H or P506T mutation revealed axonal loss in the corticospinal tract, loss of anterior horn cells, and astrocytosis in the anterior horn of the spinal cord (Supplementary Fig. 2).
Protein aggregates/inclusions have been recognized as a pathological hallmark in several neurodegenerative disorders, such as extracellular amyloid-beta plaques and intracellular tau neurofibrillary tangles in Alzheimer disease (AD), and α-synuclein-containing Lewy bodies in Parkinson disease (PD) 16. In ALS protein aggregates/inclusions are most common in spinal motor neurons, and are typically skein-like in morphology. These ubiquitin-positive inclusions among others are considered to be a hallmark of ALS pathology. Notably, several proteins that are mutated in a small subset of ALS, such as SOD1, TDP43, FUS and optineurin (OPTN) are prominent components of these inclusions 6,12,17–20. To test if ubiquilin2 is present in the characteristic skein-like inclusions, we performed immunohistochemical analysis of the postmortem spinal cord sections from two patients with a P497H or P506T mutation. Two different ubiquilin2 antibodies were used. One was a commercially available mouse monoclonal antibody raised with a polypeptide of 71 amino acids (aa) at the C-terminus (554aa-624aa, ubiquilin2-C). The other was a rabbit polyclonal antibody that we generated using a polypeptide of 17aa at the N-terminus (8aa-24aa, ubiquilin2-N). The polypeptide of 17aa is unique to ubiquilin2 and not present in other members of the ubiquilin family or any known protein. The ubiquilin2-N antibody recognized human and mouse ubiquilin2 (Supplementary Fig. 3). We also detected a single band of the expected size by Western blots using ubiquilin2-N and ubiquilin2-C antibodies with human spinal cord autopsy tissues (Supplementary Fig. 3). Using immunohistochemistry, we observed skein-like inclusions that were immunoreactive with both ubiquilin2-C and ubiquilin2-N antibodies (Supplementary Fig. 4), suggesting that ubiquilin2 is involved in inclusion formation in X-ALS. We then examined if the inclusions in X-ALS cases were also immunoreactive with antibodies against other proteins that are known to be involved in the formation of the inclusions in other types of ALS. We found that the skein-like inclusions in the X-ALS patients were also immunoreactive with antibodies to ubiquitin, p62, TDP43, FUS and OPTN (Fig. 2a-c and Supplementary Figures 4 and 5), but not SOD1.
Mutations in TDP43, FUS or optineurin occur in a small fraction of FALS, but these proteins have been found in the inclusions of a wide spectrum of ALS 6,12,17,18,20. To test if ubiquilin2 is involved in inclusion formation of other types of ALS, we examined 47 post-mortem spinal cord samples, including SALS (n=23), FALS without mutations in SOD1, TDP43 and FUS (n=5), ALS with dementia (n=5), FALS with SOD1 mutations [n=7, (A4V, n=4; G85R, n=2; E100G, n=1)], FALS with a G298S mutation in TDP43 (n=1), and controls without ALS (n=6). We observed ubiquilin2-positive and skein-like inclusions in all ALS cases (Supplementary Figures 6 and 7), suggesting that ubiquilin2 is a common component in the skein-like inclusions of a wide variety of ALS as well.
Dementia was a prominent feature in eight UBQLN2-linked cases. To examine if ubiquilin2-immunoreactive inclusions are present in brain, and to explore the potential link between ubiquilin2 inclusions and dementia, we analyzed brain autopsy samples from two patients with the P506T mutation. We observed ubiquilin2 pathology, which was most prominent in the hippocampus (Fig. 2d–g and Supplementary Fig. 8). Small ubiqulin2 inclusions were predominantly situated in the neuropil (1–5 μm in diameter). The fascia dentata presented with a band of radially oriented dendritic and neuropil inclusions in the intermediate region of the molecular layer (Supplementary Fig. 8). In addition to the small neuropil inclusions, large inclusions (up to 20 μm in diameter) were observed in some pyramidal neurons, especially those in the CA3 and CA1 (Fig. 2f and 2g, and Supplementary Fig. 8). Co-localization of ubiquilin2 and ubiquitin in these inclusions were confirmed with confocal microscopy (Supplementary Fig. 8). This type of hippocampal pathology has not been previously observed in any other neurodegenerative disorder. The ubiquilin2/ubiquitin positive inclusions did not appear to be co-localized with major glial markers (Supplementary Fig. 9). In addition, we also observed a novel membrane-bound perikaryal structure, which contained eosinophilic granules of varying sizes in some hippocampal pyramidal neurons. These structures were strongly immunoreactive for ubiquilin2 (Supplementary Fig. 10).
To test if ubiquilin2 pathology is present in the hippocampus of ALS/dementia cases without UBQLN2 mutations, and to explore the correlation of ubiquilin2 pathology with dementia in ALS, we further examined hippocampal sections of 15 pathologically characterized ALS cases without UBQLN2 mutation, including five cases of ALS/dementia with pathological signatures corresponding to frontotemporal lobar degeneration of motor neuron disease type (FTLD-MND/FTLD-U). We found prominent ubiquilin2 pathology in the hippocampus of all five cases with ALS/dementia (Supplementary Fig. 11). Similar to the ubiquilin2 inclusions in UBQLN2-linked ALS/dementia cases, the ubiquilin2 inclusions in these non-UBQLN2-linked ALS/dementia cases were also positive with ubiquitin and p62 (Supplementary Fig. 11), but negative with FUS. Although there was no apparent TDP43 neuritic pathology in the dentate molecular layer, we observed variable numbers of cytoplasmic TDP43 inclusions in dentate granule cells, which have been previously shown in ALS/dementia 18 (Supplementary Fig. 11). However, we noted that a significant number of the ubiquilin2/ubiquitin/p62 inclusions were negative for TDP43 (Supplementary Figures 11 and 12). The presence of TDP43-negative inclusions was further confirmed with an antibody specific to phosphorylated TDP43 that is only present in the cytoplasmic inclusions 18 (Supplementary Fig. 13). We also observed that the ubiquilin2/ubiquitin/p62 inclusions were largely negative for TDP43 in the CA regions in the non-UBQLN2-linked ALS/dementia cases (Supplementary Fig. 12). We did not observe the ubiquilin2 pathology in the hippocampus of the 10 ALS cases without dementia. The correlation of the hippocampal ubiquilin2 pathology and dementia in ALS cases with or without UBQLN2 mutations suggest that ubiquilin2 is widely involved in ALS-related dementia, even without UBQLN2 mutations.
TDP43 inclusions have been observed in dentate granule cells of the hippocampus in most of the cases with FTLD 18. FUS inclusions have been shown in most of the TDP43-negative FTLD cases 21,22. To test if ubiquilin2 co-aggregates with these two known ALS- and dementia-linked proteins in vitro, we generated 10 expression constructs (Supplementary information) and co-transfected Neuro2a cells with different combinations. Both wild-type and mutant ubiquilin2 were largely distributed in the cytosol. We did not observe obvious differences in the distribution between the wild type and mutant ubiquilin2. The wtFUS and wtTDP43 were located almost exclusively in the nuclei (Fig. 3 and Supplementary Fig. 14); whereas, mutant FUS showed prominent cytoplasmic distribution (Supplementary Fig. 13) and the C-terminal fragment (218–414, C-TDP43) of TDP43 that has been linked to ALS and FTLD 18,23 was almost exclusively located in the cytosol (Fig. 3). We did not observe cytoplasmic inclusions in cells transfected with wtFUS and mutant FUS (Supplementary Fig. 14) or wtTDP43 (Fig. 3). However, cytoplasmic inclusions were observed in cells expressing either wild-type or mutant ubiquilin2. Notably, the C-TDP43 was co-localized with either wild-type or mutant ubiquilin2 in the cytoplasmic inclusions (Fig. 3). We obtained consistent data using two expression systems, either tagged ubiquilin2 or tag-free ubiquilin2 (Fig. 3 and Supplementary Figures 14 and 15). These data suggest that both ALS- and dementia-linked ubiquilin2 and TDP43 are prone to co-aggregation. We also noted that inclusion formation was apparently dose-dependent, because the cells with low expression of wild-type and mutant ubiquilin2, or C-TDP43 did not show cytoplasmic inclusions. Moreover, ubiquilin2-positive, but C-TDP43-negative inclusions were frequently observed in cells with relatively lower levels of expression (Fig. 3). This phenomenon suggests that ubiquilin2 may be more prone to aggregation than TDP43. This notion is consistent with the pathology observed in ALS/dementia cases, where the ubiquilin2-containing inclusions in the molecular layer and in some dentate granule cells were TDP43-negative.
Ubiquilin2 is a member of the ubiquitin-like protein family (ubiquilins). Humans have four ubiquilin genes, each encoding a separate protein. Ubiquilins are characterized by the presence of a N-terminal ubiquitin-like (UBL) domain and a C-terminal ubiquitin-associated (UBA) domain (Fig. 1d). The middle part of ubiquilins is highly variable. This structural organization is characteristic of proteins that function to deliver ubiquitinated proteins to the proteasome for degradation. In accordance with this function, the UBL domain of the ubiquilins binds subunits of the proteasome, and its UBA domain binds to polyubiquitin chains that are typically conjugated onto proteins marked for degradation by the proteasome 24. In addition to the UBL and UBA domains that are shared by all ubiquilins, ubiquilin2 has a unique repeat region containing 12 PXX tandem repeats (Fig.1d). Remarkably, all the five ALS-linked mutations identified in this study involve proline residues within this short PXX repeat region (Fig. 1c and 1d), suggesting that these mutations may confer on ubiquilin2 a common property that may be related to the pathogenic mechanism of the disease.
Based on the involvement of ubiquilin2 in the protein degradation pathway, we then investigated the functional consequences of mutant ubiquilin2 in protein degradation through the UPS. We used the UPS reporter substrate UbiquitinG76V-Green Fluorescent Protein (UbG76V-GFP) 25 to test the effects of mutant ubiquilin2 on ubiquitin-mediated protein degradation. Two mutations at two different sites were tested (P497H and P506T) using the UbG76V-GFP reporter system. The G76V substitution prevents removal of the N-terminal-fused ubiquitin by cellular de-ubiquitinating enzymes, leading to efficient proteasomal degradation of the UbG76V-GFP reporter 25. We first tested the transfection efficiency of wild-type and mutant ubiquilin2 constructs, and observed similar levels of exogenous ubiquilin2 expression (Supplementary Fig. 16). We also tested the functionality of the UbG76V-GFP reporter system using proteasomal inhibitor MG-132 in transiently transfected cells. As expected, incubation with MG-132 resulted in remarkable accumulation of the UbG76V-GFP signal (Supplementary Figure 17). We then examined the accumulation of UbG76V-GFP reporter in Neuro2a cells transiently transfected with either wild-type (WT) or mutant ubiquilin2 constructs. Expression of mutant ubiquilin2 resulted in significantly higher accumulation of UbG76V-GFP than WT-ubiquilin2 (Fig. 4a). Similar data were obtained using SH-SY5Y cells (Supplementary Fig. 18).
We further analyzed the dynamics of UbG76V-GFP reporter degradation after new protein synthesis was blocked with cycloheximide for 0, 2, 4, and 6 hours in Neuro2a cells. We found that the rates of reporter degradation were significantly slower in both ubiquilin2-P497H and ubiquilin2-P506T mutants when compared to wild-type ubiquilin2 at 4 hours (p <0.05) and 6 hours (p <0.001) (Fig. 4b), further supporting the notion that the ubiquilin2 mutants impair the protein degradation pathway.
It is interesting to note that all the five ALS-linked UBQLN2 mutations identified in the present study involve four proline residues in the PXX region. Proline is a unique amino acid in that it has a side-chain cyclized onto the backbone nitrogen atom, leading to sterical restriction of its rotation, and thus, hindering the formation of major known secondary structures. Moreover, among the primary structures of many ligands for protein-protein interactions, a proline residue is often critical 26. Some protein-protein interaction domains, such as SH3, prefer ligand sequences containing tandem PXXP motifs, as noted in the PXX domain of ubiquilin2, for high affinity and selectivity of such interactions 27. Further studies of the consequences of proline mutations in ubiquilin2 may shed light on the understanding of the pathogenic mechanism.
The exact function of ubiquilin2 is not well understood. However, increasing lines of evidence have shown that ubiquilins, together with their interactions with other proteins, are involved in a broad spectrum of neurodegenerative disorders. Ubiquilin1, another member of the ubiquilins, is associated with AD and it interacts with presenilins 1 and 2 28, and TDP43 29. We observed that ubiquilin2 formed cytoplasmic inclusions with ALS- and FTLD-linked TDP43, implicating that an interaction of TDP43 and ubiquilin2 underlies the pathogenesis of ALS and ALS/dementia, and possibly other neurodegenerative disorders as well.
The removal of misfolded or damaged proteins is critical for optimal cell functioning. In the cytosol and the nucleus, a major proteolytic pathway to recycle misfolded or damaged proteins is the UPS. Though impaired UPS is thought to be associated with the formation of proteinaceous inclusions in many neurodegenerative disorders, direct evidence of mutations in the UPS pathway has been limited 30. In this study, we show mutations of ubiquilin2, a ubiquitin-like protein in five families with ALS and ALS/dementia. We also show that ubiquilin2-containing inclusions are a common pathological feature in a wide spectrum of ALS and ALS/dementia. Functional studies indicate an impairment of ubiquitin-mediated proteasomal degradation in cells expressing mutant ubiquilin2. These data provide robust lines of evidence for an impairment of protein turnover in the pathogenesis of ALS and ALS/dementia, and possibly for other neurodegenerative disorders as well. Further elucidation of these processes may be central to the understanding of pathogenic pathways. These pathways should provide novel molecular targets for designing rational therapies for these disorders.