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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

Viral expression of human ubiquilin-2 in whole brain lysates. Western blots of brain lysates from 3 and 6 month animals demonstrate sustained expression of WT ubiquilin-2 and mutants. In the Trition-X100 soluble fraction (a) three bands are seen for ubiquilin-2: top is mouse UBQLN2 whereas middle and lower (truncated?) bands represent human UBQLN2. b) Only mutant forms of human UBQLN2 are seen in the Triton insoluble fractions. c) Graph of human vs endogenous mouse UBQLN2 expression in whole brain (Triton soluble) lysates. N = 2–3 sample each with mean ± SD ratio shown
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Fig3: Viral expression of human ubiquilin-2 in whole brain lysates. Western blots of brain lysates from 3 and 6 month animals demonstrate sustained expression of WT ubiquilin-2 and mutants. In the Trition-X100 soluble fraction (a) three bands are seen for ubiquilin-2: top is mouse UBQLN2 whereas middle and lower (truncated?) bands represent human UBQLN2. b) Only mutant forms of human UBQLN2 are seen in the Triton insoluble fractions. c) Graph of human vs endogenous mouse UBQLN2 expression in whole brain (Triton soluble) lysates. N = 2–3 sample each with mean ± SD ratio shown

Mentions: To investigate the role of UBLQN2 and ALS-liked mutations in pathology, we used somatic brain transgenesis with rAAV serotype 2/8 to express either EGFP-control, WT or one of three different mutant forms of ubiquilin-2 (P497S, P497H, and P506T) in the developing mouse brain. Non-transgenic FVB mice all received bilateral i.c.v. injections of virus at P0. Mice injected with rAAV2/8-UBQLN2 wild type and ALS-linked mutants all demonstrated widespread neuronal (specific) expression of ubiquilin-2 in the olfactory bulb, cortex, hippocampus, thalamus, striatum, brainstem, and cerebellum as early as 1 month post-injection, and maintained at both 3 and 6 month time points (Fig. 2). In sites near to the injection such as cortex, hippocampus, thalamus and striatum, nearly 30–40 % neurons were transduced. Western blots of whole brain tissue lysates similarly indicated sustained ubiquilin-2 expression through the 6 month time point with levels reaching 10–40 % that of endogenous mouse ubiquilin-2 (Fig. 3). Transduced neurons expressing human ubiquilin-2, however, were easily identified by immunohistochemistry relative to background endogenous mouse ubiquilin-2, suggesting several-fold overexpression. Expression of WT ubiquilin-2 in neurons was diffuse, involving the soma and proximal dendrites, and included few small punctate cytoplasmic accumulations (see Fig. 4, confocal images). In contrast, expression of each of the mutant forms of ubiquilin-2 resulted in large intracellular neuronal inclusions and extensive neuropil aggregates in the surrounding gray matter, similar to that recently described by Gorrie et al. in transgenic mice with the P497H mutant ubiquilin-2 [10]. Whereas WT ubiquilin-2 was mainly cytoplasmic and diffuse, mutant ubiquilin-2 expression also appeared to have more prominent nuclear localization. As early as 1 month dystrophic changes were also seen in the dendritic arbors of purkinje cells expressing mutant ubiquilin-2, which appeared to have reduced branching architecture (Fig. 2). Glial markers showed only a rare ubiquilin-2-positive astrocyte in areas of abundant viral expression (Fig. 4). Despite the presence of abundant large inclusion seen in mice expressing mutant forms of ubiquilin-2, there was no apparent neurodegeneration or cell loss even in 6 month mice. Tissues were immunostained for apoptotic cell markers including caspase-3/7 and tunnel stain, and both negative (data not shown). Examination of hematoxylin & eosin stained sections also showed no evident cell loss or degeneration of brain regions overexpressing ubiquilin-2.Fig. 2


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)

Viral expression of human ubiquilin-2 in whole brain lysates. Western blots of brain lysates from 3 and 6 month animals demonstrate sustained expression of WT ubiquilin-2 and mutants. In the Trition-X100 soluble fraction (a) three bands are seen for ubiquilin-2: top is mouse UBQLN2 whereas middle and lower (truncated?) bands represent human UBQLN2. b) Only mutant forms of human UBQLN2 are seen in the Triton insoluble fractions. c) Graph of human vs endogenous mouse UBQLN2 expression in whole brain (Triton soluble) lysates. N = 2–3 sample each with mean ± SD ratio shown
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Fig3: Viral expression of human ubiquilin-2 in whole brain lysates. Western blots of brain lysates from 3 and 6 month animals demonstrate sustained expression of WT ubiquilin-2 and mutants. In the Trition-X100 soluble fraction (a) three bands are seen for ubiquilin-2: top is mouse UBQLN2 whereas middle and lower (truncated?) bands represent human UBQLN2. b) Only mutant forms of human UBQLN2 are seen in the Triton insoluble fractions. c) Graph of human vs endogenous mouse UBQLN2 expression in whole brain (Triton soluble) lysates. N = 2–3 sample each with mean ± SD ratio shown
Mentions: To investigate the role of UBLQN2 and ALS-liked mutations in pathology, we used somatic brain transgenesis with rAAV serotype 2/8 to express either EGFP-control, WT or one of three different mutant forms of ubiquilin-2 (P497S, P497H, and P506T) in the developing mouse brain. Non-transgenic FVB mice all received bilateral i.c.v. injections of virus at P0. Mice injected with rAAV2/8-UBQLN2 wild type and ALS-linked mutants all demonstrated widespread neuronal (specific) expression of ubiquilin-2 in the olfactory bulb, cortex, hippocampus, thalamus, striatum, brainstem, and cerebellum as early as 1 month post-injection, and maintained at both 3 and 6 month time points (Fig. 2). In sites near to the injection such as cortex, hippocampus, thalamus and striatum, nearly 30–40 % neurons were transduced. Western blots of whole brain tissue lysates similarly indicated sustained ubiquilin-2 expression through the 6 month time point with levels reaching 10–40 % that of endogenous mouse ubiquilin-2 (Fig. 3). Transduced neurons expressing human ubiquilin-2, however, were easily identified by immunohistochemistry relative to background endogenous mouse ubiquilin-2, suggesting several-fold overexpression. Expression of WT ubiquilin-2 in neurons was diffuse, involving the soma and proximal dendrites, and included few small punctate cytoplasmic accumulations (see Fig. 4, confocal images). In contrast, expression of each of the mutant forms of ubiquilin-2 resulted in large intracellular neuronal inclusions and extensive neuropil aggregates in the surrounding gray matter, similar to that recently described by Gorrie et al. in transgenic mice with the P497H mutant ubiquilin-2 [10]. Whereas WT ubiquilin-2 was mainly cytoplasmic and diffuse, mutant ubiquilin-2 expression also appeared to have more prominent nuclear localization. As early as 1 month dystrophic changes were also seen in the dendritic arbors of purkinje cells expressing mutant ubiquilin-2, which appeared to have reduced branching architecture (Fig. 2). Glial markers showed only a rare ubiquilin-2-positive astrocyte in areas of abundant viral expression (Fig. 4). Despite the presence of abundant large inclusion seen in mice expressing mutant forms of ubiquilin-2, there was no apparent neurodegeneration or cell loss even in 6 month mice. Tissues were immunostained for apoptotic cell markers including caspase-3/7 and tunnel stain, and both negative (data not shown). Examination of hematoxylin & eosin stained sections also showed no evident cell loss or degeneration of brain regions overexpressing ubiquilin-2.Fig. 2

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