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. 2012 Aug 24;287(35):29949-57.
doi: 10.1074/jbc.M112.371724. Epub 2012 Jul 11.

Dysfunction of the ubiquitin ligase Ube3a may be associated with synaptic pathophysiology in a mouse model of Huntington disease

Megha Maheshwari  1 , Ananya Samanta, Swetha K Godavarthi, Rajarshi Mukherjee, Nihar Ranjan Jana
Affiliations

Dysfunction of the ubiquitin ligase Ube3a may be associated with synaptic pathophysiology in a mouse model of Huntington disease

Megha Maheshwari et al. J Biol Chem. .

Abstract

Huntington disease (HD) is a hereditary neurodegenerative disorder characterized by progressive cognitive, psychiatric, and motor symptoms. The disease is caused by abnormal expansion of CAG repeats in the gene encoding huntingtin, but how mutant huntingtin leads to early cognitive deficits in HD is poorly understood. Here, we demonstrate that the ubiquitin ligase Ube3a, which is implicated in synaptic plasticity and involved in the clearance of misfolded polyglutamine protein, is strongly recruited to the mutant huntingtin nuclear aggregates, resulting in significant loss of its functional pool in different regions of HD mouse brain. Interestingly, Arc, one of the substrates of Ube3a linked with synaptic plasticity, is also associated with nuclear aggregates, although its synaptic level is increased in the hippocampus and cortex of HD mouse brain. Different regions of HD mouse brain also exhibit decreased levels of AMPA receptors and various pre- and postsynaptic proteins, which could be due to the partial loss of function of Ube3a. Transient expression of mutant huntingtin in mouse primary cortical neurons further demonstrates recruitment of Ube3a into mutant huntingtin aggregates, increased accumulation of Arc, and decreased numbers of GluR1 puncta in the neuronal processes. Altogether, our results suggest that the loss of function of Ube3a might be associated with the synaptic abnormalities observed in HD.

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Figures

FIGURE 1.
FIGURE 1.
Recruitment of Ube3a into huntingtin nuclear aggregates. A, representative immunohistochemical staining of Ube3a in different brain regions of wild-type and R6/2 mice. Brain sections (20-μm thickness) collected from 10–12-week-old mice were used for staining. B, double immunofluorescence staining of Ube3a and ubiquitin in the hippocampal region of R6/2 mouse brain. Scale bars = 10 μm. Arrows indicate the localization of Ube3a in nuclear aggregates.
FIGURE 2.
FIGURE 2.
Protein level of Ube3a is significantly reduced in different brain regions of R6/2 mice. A, immunoblot analysis of the levels of Ube3a in the cortices, striatums, and cerebellums of wild-type and R6/2 mice (10–12 weeks of age). B, the band intensities of Ube3a collected from five different mice in each group were quantitated using NIH ImageJ analysis software and normalized against β-tubulin. *, p < 0.01 in comparison with wild-type mice. C and D, brain samples (cortex and cerebellum) were collected from wild-type and R6/2 mice and processed for semiquantitative RT-PCR (C) and quantitative real-time RT-PCR (D). R6/2 mouse cortex and cerebellum did not exhibit any significant decrease in Ube3a mRNA levels compared with wild-type mouse cortex and cerebellum (p > 0.2). Values are means ± S.D. (n = 4). E, the total brain lysates collected from wild-type and R6/2 mice were subjected to filter trap assay on cellulose acetate membrane and blotted with anti-huntingtin (Htt), anti-Ube3a, and anti-Arc antibodies. Nitrocellulose membrane was placed at the bottom of the cellulose acetate membrane in the filter trap assay and probed with the abovementioned antibodies to monitor the equal loading of the samples. The negative control (NC) was developed without primary antibody.
FIGURE 3.
FIGURE 3.
R6/2 mouse brain exhibits altered levels and localization of Arc. A, representative immunohistochemical staining of Arc in the cortices, hippocampuses, and cerebellums of wild-type and R6/2 mouse brains (12 weeks old). Scale bar = 10 μm. Arrows indicate the localization of Arc in nuclear aggregates, and arrowheads point to neuritic Arc (increased levels in R6/2 brain). B, immunoblot analysis of Arc in different brain regions of wild-type and R6/2 mice (6 and 12 weeks old).
FIGURE 4.
FIGURE 4.
Analysis of levels of Ube3a, Arc, and GluR1 in synaptosomal fractions. A, representative immunoblot of Ube3a, Arc, GluR1, and synaptophysin in the synaptosomal preparations obtained from wild-type and R6/2 mouse (12 weeks old) total brain. B, quantification of the band intensities of Arc, Ube3a and GluR1 shown in A. *, p < 0.01 in comparison with wild-type mice (n = 3). C, co-immunoprecipitation (IP) of Arc in the synaptosomal preparation. Blots were probed with anti-Arc and anti-ubiquitin antibodies. IB, immunoblot; IgGH, IgG heavy chain.
FIGURE 5.
FIGURE 5.
A, localization of Arc with mutant huntingtin aggregates in HD150Q cells. HD16Q and HD150Q cells were plated onto 2-well tissue culture chamber slides and induced with 1 μm ponasterone A for 48 h. Cells were then processed for immunofluorescence staining using anti-Arc antibody. Rhodamine-conjugated secondary antibody was used to detect Arc. Arrows indicate the localization of Arc in the huntingtin aggregates. Scale bar = 20 μm. B, Arc levels are increased in HD150Q cells. HD16Q and HD150Q cells were left uninduced or induced with ponasterone A for 48 h, and the cell lysates were made and subjected to immunoblot analysis using antibodies against Arc, Ube3a, GAPDH, and GFP (to detect 16Q and 150Q proteins).
FIGURE 6.
FIGURE 6.
Expression of GluR1 and GluR2 is decreased in R6/2 mouse brain. A and B, representative immunohistochemical staining of GluR1 and GluR2 in the cortices and hippocampuses of wild-type and R6/2 mice. Scale bars = 10 μm. C, immunoblot analysis of GluR1 and GluR2 in the cortex and hippocampal (Hippo) regions of wild-type and R6/2 mouse brains (10–12 weeks old). D and E, GluR1 and GluR2 blots obtained from four different mice in each group were quantitated using NIH ImageJ analysis software and normalized against β-tubulin. *, p < 0.05 in comparison with wild-type mice.
FIGURE 7.
FIGURE 7.
Decreased levels of synapsin-1 and PSD95 in R6/2 mouse brain. A, immunoblot analysis of PSD95, synapsin-1, synaptophysin, and β-tubulin in cortices, striatums, and cerebellums of wild-type and R6/2 mice. B and C, the band intensities of PSD95 and synapsin-1, respectively, collected from four different mice in each group were quantitated using NIH ImageJ analysis software and normalized against β-tubulin. *, p < 0.01 in comparison with wild-type mice. D, representative immunohistochemical staining of PSD95 and synapsin-1 in the hippocampuses of wild-type and R6/2 mice. Scale bar = 10 μm.
FIGURE 8.
FIGURE 8.
Immunofluorescence staining of Ube3a, Arc, and GluR1 in primary cortical neuronal culture. A, primary cortical neurons (after 4 days of culture) were transiently transfected with tNhtt-150Q-EGFP constructs for 36 h and then subjected to immunofluorescence staining using antibodies against Ube3a, Arc, and GluR1. Rhodamine-conjugated secondary antibody was used to detect Ube3a, Arc, and GluR1. Nuclei were counterstained with DAPI. Arrows indicate the localization of Arc in the mutant huntingtin aggregates; arrowheads points to GluR1 puncta. Scale bar = 10 μm. The tNhtt-150Q-transfected cells showed significantly reduced numbers of neuronal processes compared with the control cells: control, 15.25 ± 3.6; and tNhtt-150Q, 4.64 ± 1.52 (p < 0.01). Neuronal branching of untransfected and transfected cells was calculated using NeuronStudio (Beta) software. At least nine cultured neurons in each group were used for analysis. B, double immunofluorescence staining of Arc/Ube3a and GluR1/Ube3a in primary cortical neuronal cultures (5 days) obtained from wild-type (m+/p+) and maternal Ube3a-deficient (m−/p+) mouse brains. Rhodamine-conjugated secondary antibody was used to detect Ube3a (mouse-specific), whereas FITC-conjugated secondary antibody was used to label Arc and GluR1 (rabbit-specific). Nuclei were counterstained with DAPI in overlaid images. Scale bars = 10 μm.
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