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

Ceballos-Diaz C, Rosario AM, Park HJ, Chakrabarty P, Sacino A, Cruz PE, Siemienski Z, Lara N, Moran C, Ravelo N, Golde TE, McFarland NR - Mol Neurodegener (2015)

Bottom Line: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates.In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil.Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, 1275 Center Dr, PO Box 100159, Gainesville, FL, 32610, USA.

ABSTRACT

Background: UBQLN2 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.

Results: In 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.

Conclusions: These 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.

No MeSH data available.


Related in: MedlinePlus

Mutant ubiquilin-2 expression induces ThioS-positive inclusions. Photos show Thioflavin-S staining that colocalizes (arrows; yellow in merged images) with intracellular ubiquilin-2-positive inclusions seen in mice injected with rAAV expressing mutant but not WT ubiquilin-2. Images shown are from mice aged 6 months, but similar findings were seen also in younger mice. (bar = 50 μm)
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Fig7: Mutant ubiquilin-2 expression induces ThioS-positive inclusions. Photos show Thioflavin-S staining that colocalizes (arrows; yellow in merged images) with intracellular ubiquilin-2-positive inclusions seen in mice injected with rAAV expressing mutant but not WT ubiquilin-2. Images shown are from mice aged 6 months, but similar findings were seen also in younger mice. (bar = 50 μm)

Mentions: As ubiquilin-2 has been found colocalized with other proteins in ALS inclusions, such as ubiquitin, p62, and FUS [1, 14], we examined brain tissue from mice for co-localization of these and several other neuropathological proteins including pSer129-synuclein, tau, phospho-tau, and TDP-43, which is found both in frontotemporal dementia (FTLD-U) and ALS brains. Minimal differences were noted in expression patterns between 1, 3, and 6 month mice. As expected, ubiquilin-2-positive inclusions and aggregates co-stained for ubiquitin, p62, and optineurin (Fig. 5). However, ubiquilin-2 inclusions did not colocalize with FUS (except for that within nuclei) or phosphorylated α-synuclein using the pSer129/81A antibody, which has recently shown also to bind phosphorylated neurofilament subunit L, or NFL [15] (data not shown). Inclusions also did not colocalize with tau, consistent with published data that indicate no correlation of ubiquilin-2 with tau pathology [16]. However, in mice expressing the mutant ubuiqilin-2(P506T) cytoplasmic TDP-43 aggregates, immunostained with antibodies to either phospho-TDP-43 (403–404) or (409–410) epitopes, were associated with ubiquilin-2-positive inclusions (Fig. 6). These findings suggest that ubiquilin-2(P506T) may be more prone than the P497S/H mutants to cause proteinopathy involving TDP-43 pathology that is seen in frontotemporal dementia (FTD). Interestingly, expression of mutant forms and not WT, of ubiquilin-2 also resulted in inclusions or aggregates that stained positive for ThioflavinS suggesting induction of amyloid pathology (Fig. 7) further supporting the notion that ALS-linked ubiquilin-2 mutants induce proteinopathy via misfolding and aggregation of proteins.Fig. 5


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

Ceballos-Diaz C, Rosario AM, Park HJ, Chakrabarty P, Sacino A, Cruz PE, Siemienski Z, Lara N, Moran C, Ravelo N, Golde TE, McFarland NR - Mol Neurodegener (2015)

Mutant ubiquilin-2 expression induces ThioS-positive inclusions. Photos show Thioflavin-S staining that colocalizes (arrows; yellow in merged images) with intracellular ubiquilin-2-positive inclusions seen in mice injected with rAAV expressing mutant but not WT ubiquilin-2. Images shown are from mice aged 6 months, but similar findings were seen also in younger mice. (bar = 50 μm)
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495639&req=5

Fig7: Mutant ubiquilin-2 expression induces ThioS-positive inclusions. Photos show Thioflavin-S staining that colocalizes (arrows; yellow in merged images) with intracellular ubiquilin-2-positive inclusions seen in mice injected with rAAV expressing mutant but not WT ubiquilin-2. Images shown are from mice aged 6 months, but similar findings were seen also in younger mice. (bar = 50 μm)
Mentions: As ubiquilin-2 has been found colocalized with other proteins in ALS inclusions, such as ubiquitin, p62, and FUS [1, 14], we examined brain tissue from mice for co-localization of these and several other neuropathological proteins including pSer129-synuclein, tau, phospho-tau, and TDP-43, which is found both in frontotemporal dementia (FTLD-U) and ALS brains. Minimal differences were noted in expression patterns between 1, 3, and 6 month mice. As expected, ubiquilin-2-positive inclusions and aggregates co-stained for ubiquitin, p62, and optineurin (Fig. 5). However, ubiquilin-2 inclusions did not colocalize with FUS (except for that within nuclei) or phosphorylated α-synuclein using the pSer129/81A antibody, which has recently shown also to bind phosphorylated neurofilament subunit L, or NFL [15] (data not shown). Inclusions also did not colocalize with tau, consistent with published data that indicate no correlation of ubiquilin-2 with tau pathology [16]. However, in mice expressing the mutant ubuiqilin-2(P506T) cytoplasmic TDP-43 aggregates, immunostained with antibodies to either phospho-TDP-43 (403–404) or (409–410) epitopes, were associated with ubiquilin-2-positive inclusions (Fig. 6). These findings suggest that ubiquilin-2(P506T) may be more prone than the P497S/H mutants to cause proteinopathy involving TDP-43 pathology that is seen in frontotemporal dementia (FTD). Interestingly, expression of mutant forms and not WT, of ubiquilin-2 also resulted in inclusions or aggregates that stained positive for ThioflavinS suggesting induction of amyloid pathology (Fig. 7) further supporting the notion that ALS-linked ubiquilin-2 mutants induce proteinopathy via misfolding and aggregation of proteins.Fig. 5

Bottom Line: In primary cultures rAAV2/8-mediated expression of UBQLN2 mutants resulted in inclusion bodies and insoluble aggregates.In contrast to wild type, mutant UBQLN2 expression induced significant pathology with large neuronal, cytoplasmic inclusions and ubiquilin-2-positive aggregates in surrounding neuropil.Mutant UBLQN2 expression also resulted in Thioflavin-S-positive inclusions/aggregates.

View Article: PubMed Central - PubMed

Affiliation: Center for Translational Research in Neurodegenerative Disease, Department of Neuroscience, University of Florida, 1275 Center Dr, PO Box 100159, Gainesville, FL, 32610, USA.

ABSTRACT

Background: UBQLN2 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.

Results: In 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.

Conclusions: These 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.

No MeSH data available.


Related in: MedlinePlus