Background
Several mutations in the
UBQLN2 gene have recently been identified and associated with X-linked familial ALS and ALS-dementia [
1‐
3].
UBQLN2 encodes ubiquilin-2, a member of the ubiquitin-like family of proteins that facilitate delivery of polyubiquitinated proteins to the proteasome for degradation [
1]. In humans there are at least 4 ubiquilins. Each is widely expressed, except for ubiquilin-3 which is testes specific [
4]. Ubiquilins are characterized by an N-terminal ubiquitin-biding domain (UBA), a variable number of Sti1-like repeats, and a C-terminal ubiquitin-like domain (UBL) that associates with the proteasome. Identified ALS-linked mutations (P497S/H, P506TS/T, and P525S) are primarily located in a C-terminal proline-rich domain that contains 12 PXX repeats [
1]; however, 3 have been identified outside this region [
2]. Recently, another mutation was identified within the proline-rich region in
UBQLN2 and linked to familial ALS (c.1490C > T, p.P497L) [
3]. Mutations in ubiquilin-2 have been proposed to alter proteasome mediated protein clearance, suggesting a loss-of-function and possible cause for abnormal protein accumulation and deposition [
1]. However, ubiquilins have also been implicated in ER-associated protein degradation and autophagy [
5‐
7]. Examination of protein inclusions in pathological tissue from both sporadic ALS and ALS-dementia demonstrate the presence of ubiquilin-2 in inclusions and co-localization with other proteins such as ubiquitin and p62/SQSTM1, further suggesting a role for ubiquilin-2 in proteinopathy and in ALS pathology [
1,
8,
9]. Few studies to date, however, have examined the role of ubiquilin-2 and consequence of identified mutations—so far limited to P497H mutant—on the development of ALS pathology [
10,
11].
To determine the pathological consequences of
UBQLN2 mutants, we developed rAAV 2/8 vectors to compare the effects of overexpression of wild type (WT) and three of the recently identified ALS-mutant ubiquilins in primary neuroglial cultures and in the developing mouse brain. In mice we utilized “somatic brain transgenesis” (SBT) to rapidly introduce and express
UBQLN2 mutants in throughout the brain. Although having more limited and variable expression compared to traditional transgenic models, SBT still allows for rapid, widespread expression and screening of genes of interest before expending the time and expense developing traditional transgenic models [
12,
13]. Our findings demonstrate that overexpression of pathological forms of mutant ubiquilin-2 compared to WT all develop widespread inclusion pathology, including amyloid-like aggregates, that persists over 6 months and which is associated with mild, early motor deficits. These studies provide further insight into the
in vivo effects of expression of ALS-linked mutant forms of ubiquilin-2 in mice. Furthermore, our SBT mouse models demonstrate a powerful and complementary approach to traditional transgenics that will allow further dissection of pathological mechanisms of ubiquilin-2 mutants and their role in development of ALS and ALS-dementia.
Discussion
UBQLN2 mutations have recently been added to the list of potential genes that cause familial ALS and ALS-FTD [
1,
2].
UBQLN2 encodes for ubiquilin-2, a member of the ubiquitin-like family of proteins that facilitate transport of ubiquitinated proteins to the proteasome for degradation. Although evidence to date suggests that ALS-linked ubiquilin-2 mutants have reduced proteasomal function and cause a potential loss-of-function [
1], the role of ubiquilin-2 in ALS pathology remains unclear. To determine the functional consequences of ALS-linked
UBQLN2 mutations, we developed rAAV vectors to express WT and three of the identified ubiquilin-2 mutants (P497, P497H, and P506T) in primary neuronal cells and in the developing mouse brain. In primary cultures we found that viral overexpression of ubiquilin-2 resulted in large intracellular accumulations that were more prominent and distributed along neuronal processes for mutant forms than for WT ubiquilin-2. Fractionated lysates from these cultures demonstrated also that mutant ubiquilin-2, but not WT, were present in TX-insoluble (SDS soluble) fractions, suggesting tendency for mutant forms of ubiquilin-2 to form insoluble aggregates. To determine whether viral expression ALS-linked mutant ubiquilin-2 could induce pathological and behavioral abnormalities in mice, we developed a model system using somatic brain transgenesis, or SBT, to widely and rapidly overexpress ubiquilin-2 in the developing mouse nervous system. We demonstrate herein that mice injected i.c.v. with rAAV-ubiquilin-2 mutants and aged up to 6 months develop early, widespread neuronal inclusion pathology, dystrophic neurite changes, and motor deficits.
To date few studies have examined the
in vivo consequences of ALS-linked ubquilin-2 in brain and spinal cord. Recently, Gorrie et al. [
10] published the first findings from transgenic mice that express one of the ALS-linked mutant ubquilin-2 (P497H) under the direction of the
UBQLN2 promoter. Progressive ubiquilin-2 pathology was observed in these mice and particularly prominent in the hippocampal gyrus, but also in the frontal and temporal lobes with increasing age, similar to that seen in human ALS tissues [
1]. Abundant ubiqulin-2-positve neuropil aggregates in gray matter, but not in white matter, were noted [
10] and similar to that observed in our mouse brains transduced with rAAV-UBQLN2 mutants. These findings suggest that ubiquilin-2 aggregates are localized to dendrites rather than axons. Indeed, electron microscopy studies indicate primary somatodendritic aggregates which are prominent in dendritic spines in hippocampal and cortical tissues and which may contribute to altered spine density and plasticity [
10]. The findings from our mouse models are complimentary and together these models indicate that expression of ALS-linked ubiquilin-2 mutants cause progressive ubiquilin-2 pathology involving aggregate formation and proteinopathy. However, the link between these findings, neurodegeneration, and development of ALS remains unclear. In both our mouse SBT model and the
UBQLN2
P497H transgenic mice, neuronal loss and neurodegeneration have not been observed. However, more recently, Wu et al. in a similar transgenic model in rats did show neuronal loss proceeded by formation of ubiquilin-2 aggregates and evidence of impaired autophagy and endosomal function [
11]. Lack of evidence for neurodegeneration in our model may possibly be explained by relative low viral transduction of neurons (estimated at 30–40 %, greatest in regions near the ventricles); however, detailed analyses with both tunnel and caspase 3/7 were unrevealing. Nevertheless, in our study SBT mice expressing mutant UBQLN2 variably developed clasping and rotarod deficits as early as 3–4 months, which although nonspecific may indicate progressive pathology and possible later development of a more disease-relevant motor phenotype. This finding is in contrast to recent transgenic P497H models that report evidence for cognitive rather than motor deficits [
10,
11], which may have relevance to ALS-FTD and other neurodegenerative dementias. To fully determine the utility of our novel rAAV model system, we will need to further establish the effects of mutant ubiquilin-2 expression in mouse brain and spinal cord beyond 6 months to determine whether we can induce pathological and phenotypic changes, such as paresis, expected for ALS/ALS-FTD.
Evidence to date indicates that ubiquilins play important roles in multiple protein recycling and degradation pathways, including the UPS, ERAD, and autophagy [
17]. Although the function of ubiquilin-2 remains unclear, its homology to ubiquilin-1 suggests a similar function and role in the UPS and degradation of proteins. Identified ALS-linked mutations in ubiquilin-2 all localize to a proline-rich (PXX repeat) region that is distinct from either the N-terminal UBL (ubiquitin-like) domain that interacts with the proteasome or the C-terminal UBA (ubiquitin-associated) domain that associates with ubiquitinated proteins, suggesting that ALS mutants may leave these functional domains intact. ALS-linked mutations in ubiquilin-2 have been shown
in vitro to impair proteasomal degradation and these findings appear consistent with its primary function in the UPS [
1]. Recently
in vivo data from bigenic mice expressing both
UBQLN2
P497H and the ubiquitinated protein substrate,
Ub
G67V-GFP, appears to support these findings.
Ub
G67V-GFP accumulated in the brain of bigenic mice expressing
UBQLN2
P497H suggesting impaired UPS function [
10]. Furthermore, ubiquilin-2 deposits in brain sections from these mice colocalized with antibodies to proteasome subunits. These findings appear to indicate that mutant ubiquilin-2(P497H) may still function to bring ubiquitinated proteins to the proteasome, but somehow interferes with proteasomal degradation, leading to accumulation and abnormal deposition proteins. Our data indicate that ALS-linked ubiquilin-2 variants may have differential effects on UPS function. Indeed the P497H mutant had little effect on d2EGFP levels and was similar to WT, whereas expression of both the P497S and P506T mutants impaired d2EGFP metabolism (Fig.
7). These data suggest that alternative protein degradation mechanisms may be involved such as the autophagy-lysosomal system to explain the effects of these ubiquilin-2 mutants on proteinopathy seen in our models.
Recent studies also implicate ubiquilin-2 in macroautophagy. Early studies demonstrated that ubiquilin-1 binds the target of rapamycin (mTOR) kinase in mammalian cells, a critical regulator of macroautophagy [
18]. Both ubiquilin-1 and 2 have also been shown to colocalize with the microtubule-associated protein 1 light chain 3 (LC3), a membrane component of autophagosomes, and have been implicated in the maturation of autophagic vesicles [
5]. Notably, knockdown of ubiquilin-2 (and 1) rendered cells expressing either a Alzheimer’s-related presenilin mutant or a huntingtin polyglutamine expansion more susceptible to starvation-induced death, whereas overexpression is protective, further supporting a role in autophagy and neurodegenerative disease [
5]. The effects of ALS-linked mutations on ubiquilin-2 function in macroautophagy have not been explored and remain unclear. We hypothesize that expression of ubiquilin-2 mutants may impair macroautophagy, as well as UPS function, disrupting proteostasis and contributing to protein accumulation, aggregate formation, cell stress and cytotoxicity.
To date, few studies have identified protein interactors with ubiquilin-2 or ALS-linked mutants. UBA and UBL domains in ubiquilins are known to interact with polyubiquitinated proteins and the proteasome, respectively, consistent with their function in the UPS [
4]. In addition, ubiquilins have been shown to interact with components of the ERAD including Erasin and p97/VCP (valosin-containing protein) that form a complex at the ER membrane to direct degradation of misfolded protein as part of the unfolded protein response [
6]. More recently, ubiquilin-2 was shown to interact with the ubiquitin regulatory X domain-containing protein 8 (UBXD8), which mediates translocation of ERAD substrates such as p97/VCP, and this interaction was impaired by the ubiquilin-2 mutant (P497) [
19]. Although ubiquilin-2 has been colocalized with several other proteins
in vitro and
in vivo including LC3 [
5], p62/SQSTM1, ubiquitin [
1], and optineurin [
10], direct interactions have not been demonstrated. Our data indicate colocalization of ubiquilin-2 inclusions with cytoplasmic, phospho-TDP-43 in mice expressing the P506T mutant ubiquilin-2. Recent evidence suggests that ubiquilin-2 binds to C-terminal fragments of TDP-43 [
1,
20]. TDP-43 and in particular mislocalization and aggregation of C-terminal fragments of TDP-43 have been implicated in both ALS and FTD pathology. Together, these data provide an incomplete picture of proteins that may interact with ubiquilin-2 or ALS-linked mutants that may be critical to understanding both the normal function of ubiquilin-2 as well as how identified mutations alter its function and may influence development of ALS/ALS-FTD pathology.
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Competing interests
The authors declare that they have no competing interest.
Authors’ contributions
CCD carried out the molecular studies, immunoassays, animal procedures, behavioral testing, histology, analysis and drafting of the manuscript. AMR generated the virus, performed histochemistry, and participated in animal procedures and testing. HJP performed the proteasomal assays. PC participated in the study design and animal procedures. AS assisted with animal procedures. PEC cloned the molecular and viral constructs. ZS performed the histopathology. NL assisted with animal procedures and histology. CM contributed to the histopathology. NR participated in the histology. TEG participated in overall design and conception of the study, and manuscript preparation and editing. NRM participated in the experimental design, coordination, interpretation, drafting and editing of the manuscript. All authors read and approved the final manuscript.