Viral expression of ALS-linked ubiquilin-2 mutants causes inclusion pathology and behavioral deficits in mice

Abstract

BackgroundUBQLN2 mutations have recently been associated with familial forms of amyotrophic lateral sclerosis (ALS) and ALS-dementia. UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like protein family which facilitates delivery of ubiquitinated proteins to the proteasome for degradation. To study the potential role of ubiquilin-2 in ALS, we used recombinant adeno-associated viral (rAAV) vectors to express UBQLN2 and three of the identified ALS-linked mutants (P497H, P497S, and P506T) in primary neuroglial cultures and in developing neonatal mouse brains.ResultsIn primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates. Intracerebroventricular injection of FVB mice at post-natal day 0 with rAAV2/8 expressing wild type or mutant UBQLN2 resulted in widespread, sustained expression of ubiquilin-2 in brain. In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil. Ubiquilin-2 inclusions co-localized with ubiquitin, p62/SQSTM, optineurin, and occasionally TDP-43, but were negative for α-synuclein, neurofilament, tau, and FUS. Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates. Mice expressing mutant forms of UBQLN2 variably developed a motor phenotype at 3–4 months, including nonspecific clasping and rotarod deficits.ConclusionsThese findings demonstrate that UBQLN2 mutants (P497H, P497S, and P506T) induce proteinopathy and cause behavioral deficits, supporting a “toxic” gain-of-function, which may contribute to ALS pathology. These data establish also that our rAAV model can be used to rapidly assess the pathological consequences of various UBQLN2 mutations and provides an agile system to further interrogate the molecular mechanisms of ubiquilins in neurodegeneration.

Motor neuron disease, TDP-43 pathology, and memory deficits in mice expressing ALS–FTD-linked UBQLN2 mutations

“…We speculate that this susceptibility is because they are vulnerable to disturbances in proteostasis, based on the defective properties of mutant UBQLN2 proteins and by extrapolation from other studies. First, our results, like those of others studies, clearly show the UBQLN2 mutants disrupt proteostasis, leading to accumulation of misfolded proteins (13,(28)(29)(30)(31). Second, accumulating evidence suggests disturbance in proteostasis, particularly induction of the UPR, as central in disease pathogenesis caused by ALS mutations in SOD1 and C9ORF72 (45)(46)(47)(48)(49).…”

“…There is variance as to whether TDP-43 is present in the UBQLN2 inclusions that form in the brain in rodent models of UBQLN2, with two reports demonstrating it is not present (13,27) and one that showed it was present (31). Using a C-terminal TDP-43-specific antibody we did not detect any evidence for TDP-43 in the UBQLN2 inclusions that decorate the dentate gyrus of the brains in our mutant UBQLN2 mice.…”

“…In another study where mouse lines were not generated, WT and mutant UBQLN2 proteins were ectopically expressed in mice following intracerebroventricular injection of recombinant AAV (31). Several of the injected mice developed UBQLN inclusions in the brain and those injected with constructs encoding P497S and P506T mtUBQLN2 proteins had compromised rotarod performance at 3 mo of age compared with mice injected with constructs encoding WT or P497H UBQLN2 proteins.…”

“…Indeed, overexpression of the mutant UBQLN2 (mtUBQLN2) proteins in cells and animals support both possibilities. For example, overexpression of ALS mtUBQLN2 proteins caused interference in proteasomal degradation, including perturbation of endoplasmic reticulum-associated degradation (ERAD) (13,(28)(29)(30)(31). The interference in degradation appears to stem from an inability of the mutants to deliver polyubiquitinated proteins to the proteasome for degradation (30,32).…”

“…A P520T knockin mouse model, carrying the equivalent P506T UBQLN2 mutation in humans, developed cognitive deficits and UBQLN2 inclusions in the brain, but did not develop motor neuron disease (19). Other studies showed that mice injected with adeno-associated virus (AAV) expressing the P497S and P506T UBQLN2 mutations perform worse on the rotarod than mice expressing WT UBQLN2 or the P497H mutation (31). However, the underlying reason for the variation in their motor performance was not described.…”