Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS-FTD-linked UBQLN2 mutations.
ABSTRACT: Missense mutations in ubiquilin 2 (UBQLN2) cause ALS with frontotemporal dementia (ALS-FTD). Animal models of ALS are useful for understanding the mechanisms of pathogenesis and for preclinical investigations. However, previous rodent models carrying UBQLN2 mutations failed to manifest any sign of motor neuron disease. Here, we show that lines of mice expressing either the ALS-FTD-linked P497S or P506T UBQLN2 mutations have cognitive deficits, shortened lifespans, and develop motor neuron disease, mimicking the human disease. Neuropathologic analysis of the mice with end-stage disease revealed the accumulation of ubiquitinated inclusions in the brain and spinal cord, astrocytosis, a reduction in the number of hippocampal neurons, and reduced staining of TAR-DNA binding protein 43 in the nucleus, with concomitant formation of ubiquitin+ inclusions in the cytoplasm of spinal motor neurons. Moreover, both lines displayed denervation muscle atrophy and age-dependent loss of motor neurons that correlated with a reduction in the number of large-caliber axons. By contrast, two mouse lines expressing WT UBQLN2 were mostly devoid of clinical and pathological signs of disease. These UBQLN2 mouse models provide valuable tools for identifying the mechanisms underlying ALS-FTD pathogenesis and for investigating therapeutic strategies to halt disease.
Project description:Mutations in the gene encoding ubiquilin-2 (UBQLN2) are linked to amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). UBQLN2 plays a central role in ubiquitin proteasome system (UPS) and UBQLN2 up-regulation exacerbates TDP-43 cytoplasmic aggregates. To analyze interaction between UBQLN2 and TDP-43 and to produce a relevant ALS animal model, we have generated a new transgenic mouse expressing UBQLN2<sup>P497H</sup> under the neurofilament heavy (NFH) gene promoter. The UBQLN2<sup>P497H</sup> mice were then bred with our previously described TDP-43<sup>G348C</sup> mice to generate double-transgenic UBQLN2<sup>P497H</sup>; TDP-43<sup>G348C</sup> mice. With low-expression levels of UBQLN2, the double-transgenic mice developed TDP-43 cytosolic accumulations in motor neurons starting at 5 months of age. These double-transgenic mice exhibited motor neuron loss, muscle atrophy, as well as motor and cognitive deficits during aging. The microglia from double-transgenic mice were hyperresponsive to intraperitoneal injection of lipopolysaccharide (LPS). In vivo and in vitro analyses suggested that extra UBQLN2 proteins can exacerbate cytoplasmic TDP-43 accumulations by competing with the UPS for binding to ubiquitin. Thus, increasing the pool of ubiquitin promoted the UPS function with ensuing reduction of TDP-43 cytosolic accumulations. In conclusion, the double-transgenic UBQLN2<sup>P497H</sup>; TDP-43<sup>G348C</sup> mice provides a unique mouse model of ALS/FTD with enhanced TDP-43 pathology that can be exploited for drug testing.
Project description:Members of the conserved ubiquilin (UBQLN) family of ubiquitin (Ub) chaperones harbor an antipodal UBL (Ub-like)-UBA (Ub-associated) domain arrangement and participate in proteasome and autophagosome-mediated protein degradation. Mutations in a proline-rich-repeat region (PRR) of UBQLN2 cause amyotrophic lateral sclerosis (ALS)/frontotemporal dementia (FTD); however, neither the normal functions of the PRR nor impacts of ALS-associated mutations within it are well understood. In this study, we show that ALS mutations perturb UBQLN2 solubility and folding in a mutation-specific manner. Biochemical impacts of ALS mutations were additive, transferable to UBQLN1, and resulted in enhanced Ub association. A Drosophila melanogaster model for UBQLN2-associated ALS revealed that both wild-type and ALS-mutant UBQLN2 alleles disrupted Ub homeostasis; however, UBQLN2ALS mutants exhibited age-dependent aggregation and caused toxicity phenotypes beyond those seen for wild-type UBQLN2. Although UBQLN2 toxicity was not correlated with aggregation in the compound eye, aggregation-prone UBQLN2 mutants elicited climbing defects and neuromuscular junctions (NMJ) abnormalities when expressed in neurons. An UBA domain mutation that abolished Ub binding also diminished UBQLN2 toxicity, implicating Ub binding in the underlying pathomechanism. We propose that ALS-associated mutations in UBQLN2 disrupt folding and that both aggregated species and soluble oligomers instigate neuron autonomous toxicity through interference with Ub homeostasis.
Project description:Missense mutations in UBQLN2 cause X-linked dominant inheritance of amyotrophic lateral sclerosis with frontotemporal dementia (ALS/FTD). UBQLN2 belongs to a family of four highly homologous proteins expressed in humans that play diverse roles in maintaining proteostasis, but whether one isoform can substitute for another is not known. Here, we tested whether overexpression of UBQLN1 can alleviate disease in the P497S UBQLN2 mouse model of ALS/FTD by crossing transgenic (Tg) mouse lines expressing the two proteins and characterizing the resulting genotypes using a battery of pathologic and behavioral tests. The pathologic findings revealed UBQLN1 overexpression dramatically reduced the burden of UBQLN2 inclusions, neuronal loss and disturbances in proteostasis in double Tg mice compared to single P497S Tg mice. The beneficial effects of UBQLN1 overexpression were primarily confirmed by behavioral improvements seen in rotarod performance and grip strength in male, but not female mice. Paradoxically, although UBQLN1 overexpression reduced pathologic signatures of disease in P497S Tg mice, female mice had larger percentage of body weight loss than males, and this correlated with a corresponding lack of behavioral improvements in the females. These findings lead us to speculate that methods to upregulate UBQLN1 expression may reduce pathogenicity caused by UBQLN2 mutations, but may also lead to gender-specific outcomes that will have to be carefully weighed with the therapeutic benefits of UBQLN1 upregulation. Electronic supplementary material The online version of this article (10.1186/s40478-020-01039-9) contains supplementary material, which is available to authorized users.
Project description:Mutations in <i>UBQLN2</i> cause amyotrophic lateral sclerosis (ALS), frontotemporal dementia (FTD), and other neurodegenerations. However, the mechanism by which the UBQLN2 mutations cause disease remains unclear. Alterations in proteins involved in autophagy are prominent in neuronal tissue of human ALS <i>UBQLN2</i> patients and in a transgenic P497S UBQLN2 mouse model of ALS/FTD, suggesting a pathogenic link. Here, we show UBQLN2 functions in autophagy and that ALS/FTD mutant proteins compromise this function. Inactivation of UBQLN2 expression in HeLa cells reduced autophagic flux and autophagosome acidification. The defect in acidification was rescued by reexpression of wild type (WT) UBQLN2 but not by any of the five different UBQLN2 ALS/FTD mutants tested. Proteomic analysis and immunoblot studies revealed P497S mutant mice and UBQLN2 knockout HeLa and NSC34 cells have reduced expression of ATP6v1g1, a critical subunit of the vacuolar ATPase (V-ATPase) pump. Knockout of UBQLN2 expression in HeLa cells decreased turnover of ATP6v1g1, while overexpression of WT UBQLN2 increased biogenesis of ATP6v1g1 compared with P497S mutant UBQLN2 protein. In vitro interaction studies showed that ATP6v1g1 binds more strongly to WT UBQLN2 than to ALS/FTD mutant UBQLN2 proteins. Intriguingly, overexpression of ATP6v1g1 in UBQLN2 knockout HeLa cells increased autophagosome acidification, suggesting a therapeutic approach to overcome the acidification defect. Taken together, our findings suggest that UBQLN2 mutations drive pathogenesis through a dominant-negative loss-of-function mechanism in autophagy and that UBQLN2 functions as an important regulator of the expression and stability of ATP6v1g1. These findings may have important implications for devising therapies to treat <i>UBQLN2</i>-linked ALS/FTD.
Project description:Mutations of Ubiquilin 2 (UBQLN2) or TANK-binding kinase 1 (TBK1) are associated with amyotrophic lateral sclerosis and frontotemporal degeneration (ALS/FTD). However, the mechanisms whereby UBQLN2 or TBK1 mutations lead to ALS and FTD remain unclear. Here, we explored the effect of UBQLN2 on TBK1 in HEK-293T cells or in CRISPR-Cas9-mediated IRF3 and IRF7 knockout (KO) cells. We found an interaction between TBK1 and UBQLN2, which was affected by ALS/FTD-linked mutations in TBK1 or UBQLN2. Co-expression of UBQLN2 with TBK1 elevated the protein level of TBK1 as well as the phosphorylation of TBK1 and IRF3 in a UBQLN2 dose-dependent manner, and this phosphorylation was reduced by mutant UBQLN2. In addition, the cellular production of IFN1 and related pro-inflammatory cytokines was substantially elevated when UBQLN2 and TBK1 were co-expressed, which was also decreased by mutant UBQLN2. Functional assay revealed that mutant UBQLN2 significantly reduced the binding affinity of TBK1 for its partners, including IRF3, (SQSTM1)/p62 and optineurin (OPTN). Moreover, complete loss of IRF3 abolished the induction of IFN1 and related pro-inflammatory cytokines enhanced by UBQLN2 in HEK-293T cells, whereas no significant change in IRF7 knockout cells was observed. Thus, our findings suggest that UBQLN2 promotes IRF3 phosphorylation via TBK1, leading to enhanced IFN1 induction, and also imply that the dysregulated TBK1-IRF3 pathway may play a role in UBQLN2-related neurodegeneration.
Project description: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 but about 5-10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of 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, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin?2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin?2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin?2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin?2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.
Project description:Proteasomal shuttle factor UBQLN2 is recruited to stress granules and undergoes liquid-liquid phase separation (LLPS) into protein-containing droplets. Mutations to UBQLN2 have recently been shown to cause dominant X-linked inheritance of amyotrophic lateral sclerosis (ALS) and ALS/dementia. Interestingly, most of these UBQLN2 mutations reside in its proline-rich (Pxx) region, an important modulator of LLPS. Here, we demonstrated that ALS-linked Pxx mutations differentially affect UBQLN2 LLPS, depending on both amino acid substitution and sequence position. Using size-exclusion chromatography, analytical ultracentrifugation, microscopy, and NMR spectroscopy, we determined that those Pxx mutants that enhanced UBQLN2 oligomerization decreased saturation concentrations needed for LLPS and promoted solid-like and viscoelastic morphological changes to UBQLN2 liquid assemblies. Ubiquitin disassembled all LLPS-induced mutant UBQLN2 aggregates. We postulate that the changes in physical properties caused by ALS-linked Pxx mutations modify UBQLN2 behavior in vivo, possibly contributing to aberrant stress granule morphology and dynamics, leading to formation of inclusions, pathological characteristics of ALS.
Project description:Mutations in the gene encoding ubiquilin2 (UBQLN2) cause amyotrophic lateral sclerosis (ALS), frontotemporal type of dementia, or both. However, the molecular mechanisms are unknown. Here, we show that ALS/dementia-linked UBQLN2(P497H) transgenic mice develop neuronal pathology with ubiquilin2/ubiquitin/p62-positive inclusions in the brain, especially in the hippocampus, recapitulating several key pathological features of dementia observed in human patients with UBQLN2 mutations. A major feature of the ubiquilin2-related pathology in these mice, and reminiscent of human disease, is a dendritic spinopathy with protein aggregation in the dendritic spines and an associated decrease in dendritic spine density and synaptic dysfunction. Finally, we show that the protein inclusions in the dendritic spines are composed of several components of the proteasome machinery, including Ub(G76V)-GFP, a representative ubiquitinated protein substrate that is accumulated in the transgenic mice. Our data, therefore, directly link impaired protein degradation to inclusion formation that is associated with synaptic dysfunction and cognitive deficits. These data imply a convergent molecular pathway involving synaptic protein recycling that may also be involved in other neurodegenerative disorders, with implications for development of widely applicable rational therapeutics.
Project description:Mutations in the UBQLN2 gene, which encodes a member of the ubiquitin-like protein family (ubiquilin-2), have been identified in patients with dominant X-linked amyotrophic lateral sclerosis (ALS) and ALS with frontotemporal dementia (FTD). We analyzed mutations in the UBQLN2 gene in a Chinese cohort of 515 patients with sporadic ALS (sALS). A novel missense mutation (p.M392V) was detected in one sALS patient. The p.M392V mutation substitutes a highly conserved residue, has not been reported in the population databases, and previously, at the same residue, a missense mutation p.M392I was detected in two Turkey ALS patients and was considered to be pathogenic, so the M392V is a variant of uncertain significance (VOUS) for ALS. We also found a deletion mutation (p.P500_G502del), which seems to be benign. In conclusion, our data suggest that mutations in the UBQLN2 gene are rare in Chinese sALS patients.
Project description:Amyotrophic lateral sclerosis (ALS) is a degenerative disorder affecting upper and lower motor neurons, but it is increasingly recognized to affect other systems, with cognitive impairment resembling frontotemporal dementia (FTD) in some patients. We report clinical and pathologic findings of a family with ALS due to a truncating mutation, p.Gly141X, in copper/zinc superoxide dismutase (SOD1). The proband presented clinically with FTD and later showed progressive motor neuron disease, while all other family members had early-onset and rapidly progressive ALS without significant cognitive deficits. Pathologic examination of both the proband and her daughter revealed degeneration of corticospinal tracts and motor neurons in brain and spinal cord compatible with ALS. On the other hand, the proband also had neocortical and limbic system degeneration with pleomorphic neuronal cytoplasmic inclusions. Extramotor pathology in her daughter was relatively restricted to the hypothalamus and extrapyramidal system, but not the neocortex. The inclusions in the proband and her daughter were immunoreactive for SOD1, but negative for TAR DNA-binding protein of 43 kDa (TDP-43). In the proband, a number of the neocortical inclusions were immunopositive for ?-internexin, initially suggesting a diagnosis of atypical FTLD, but there was no evidence of fused in sarcoma (FUS) immunoreactivity, which is often detected in atypical FTLD. Analogous to atypical FTLD, neuronal inclusions had variable co-localization of SOD1 and ?-internexin. The current classification of FTLD is based on the major constituent protein: FTLD-tau, FTLD-TDP-43, and FTLD-FUS. The proband in this family indicates that SOD1, while rare, can also be the substrate of FTLD, in addition to the more common presentation of ALS. The explanation for clinical and pathologic heterogeneity of SOD1 mutations, including the p.Gly141X mutation, remains unresolved.