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Overexpression of UBQLN1 reduces neuropathology in the P497S UBQLN2 mouse model of ALS/FTD

ABSTRACT: 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.

SUBMITTER: Wang S

PROVIDER: S-EPMC7539388 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Project description:Accumulating evidence suggests X-linked dominant mutations in UBQLN2 cause amyotrophic lateral sclerosis (ALS) with frontotemporal dementia (FTD) through both loss- and gain-of-function mechanisms. However, the mechanisms by which the mutations cause disease are still unclear. The goal of the study was to uncover the possible pathomechanism(s) by which UBQLN2 mutations cause ALS/FTD. An analysis of proteomic changes in neuronal tissue was used to identify proteins with altered accumulation in the P497S UBQLN2 transgenic mouse model of ALS/FTD. We then used immunocytochemistry and biochemical techniques to confirm protein changes in the mutant P497S mice. Additionally, we used cell lines inactivated of UBQLN2 expression to determine whether its loss underlies the alteration in the proteins seen in P497S mice. The proteome screen identified a dramatic alteration of serine protease inhibitor (serpin) proteins in the mutant P497S animals. Double immunofluorescent staining of brain and spinal cord tissues of the mutant and control mice revealed an age-dependent change in accumulation of Serpin A1, C1, and I1 in puncta whose staining colocalized with UBQLN2 puncta in the mutant P497S mice. Serpin A1 aggregation in P497S animals was confirmed by biochemical extraction and filter retardation assays. A similar phenomenon of serpin protein aggregation was found in HeLa and NSC34 motor neuron cells with inactivated UBQLN2 expression. We found aberrant aggregation of serpin proteins, particularly Serpin A1, in the brain and spinal cord of the P497S UBQLN2 mouse model of ALS/FTD. Similar aggregation of serpin proteins was found in UBQLN2 knockout cells suggesting that serpin aggregation in the mutant P497S animals may stem from loss of UBQLN2 function. Because serpin aggregation is known to cause disease through both loss- and gain-of-function mechanisms, we speculate that their accumulation in the P497S mouse model of ALS/FTD may contribute to disease pathogenesis through similar mechanism(s).

Project description:BackgroundAmyotrophic Lateral Sclerosis (ALS) is a fatal and progressive neurodegenerative disorder with identified genetic causes representing a significant minority of all cases. A GGGGCC hexanucleotide repeat expansion (HRE) mutation within the C9ORF72 gene has recently been identified as the most frequent known cause of ALS. The expansion leads to partial heterochromatinization of the locus, yet mutant RNAs and dipeptide repeat proteins (DPRs) are still produced in sufficient quantities to confer neurotoxicity. The levels of these toxic HRE products positively correlate with cellular toxicity and phenotypic severity across multiple disease models. Moreover, the degree of epigenetic repression inversely correlates with some facets of clinical presentation in C9-ALS patients. Recently, bacterial artificial chromosomes (BAC) have been used to generate transgenic mice that harbor the HRE mutation, complementing other relevant model systems such as patient-derived induced pluripotent stem cells (iPSCs). While epigenetic features of the HRE have been investigated in various model systems and post-mortem tissues, epigenetic dysregulation at the expanded locus in C9-BAC mice remains unexplored.Methods and resultsHere, we sought to determine whether clinically relevant epigenetic perturbations caused by the HRE are mirrored in a C9-BAC mouse model. We used complementary DNA methylation assessment and immunoprecipitation methods to demonstrate that epigenetic aberrations caused by the HRE, such as DNA and histone methylation, are recapitulated in the C9-BAC mice. Strikingly, we found that cytosine hypermethylation within the promoter region of the human transgene occurred in a subset of C9-BAC mice similar to what is observed in patient populations. Moreover, we show that partial heterochromatinization of the C9 HRE occurs during the first weeks of the mouse lifespan, indicating age-dependent epigenetic repression. Using iPSC neurons, we found that preventing R-loop formation did not impede heterochromatinization of the HRE.ConclusionsTaken together, these observations provide further insight into mechanism and developmental time-course of epigenetic perturbations conferred by the C9ORF72 HRE. Finally, we suggest that epigenetic repression of the C9ORF72 HRE and nearby gene promoter could impede or delay motor neuron degeneration in C9-BAC mouse models of ALS/FTD.

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Overexpression of UBQLN1 reduces neuropathology in the P497S UBQLN2 mouse model of ALS/FTD

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