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Comparative Study
. 2015 Oct:82:281-288.
doi: 10.1016/j.nbd.2015.06.017. Epub 2015 Jun 30.

Differential recruitment of UBQLN2 to nuclear inclusions in the polyglutamine diseases HD and SCA3

Li Zeng  1 , Bo Wang  1 , Sean A Merillat  1 , Eiko N Minakawa  2 , Matthew D Perkins  3 , Biswarathan Ramani  1 , Sara J Tallaksen-Greene  1 , Maria do Carmo Costa  1 , Roger L Albin  4 , Henry L Paulson  5
Affiliations
Comparative Study

Differential recruitment of UBQLN2 to nuclear inclusions in the polyglutamine diseases HD and SCA3

Li Zeng et al. Neurobiol Dis. 2015 Oct.

Abstract

Accumulation of mutant polyglutamine proteins in intraneuronal inclusions is a hallmark of polyglutamine diseases. Impairment of protein clearance systems and sequestration of clearance-related proteins into inclusions occur in many protein folding diseases, including polyglutamine diseases. The ubiquitin-binding and proteasome adaptor protein UBQLN2 participates in protein homeostasis and localizes to inclusions in various neurodegenerative diseases. Employing mouse models and human brain tissue of Huntington's disease (HD) and spinocerebellar ataxia type 3 (SCA3), we show that UBQLN2 is selectively recruited to inclusions in HD but not SCA3. Consistent with this result, in a cell-based system mutant HTT interacts with UBQLN2 through the UBA domain while the SCA3 disease protein ATXN3, a deubiquitinating enzyme, does not interact with UBQLN2. Differential recruitment of UBQLN2 to aggregates in HD and SCA3 underscores the heterogeneity of inclusions in polyglutamine diseases and suggests that components of neuronal protein quality control may be differentially perturbed in distinct polyQ diseases.

Keywords: Huntington's disease; Polyglutamine; SCA3; UBA; UBL; UBQLN2.

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Figures

Fig.1
Fig.1
UBQLN2 localizes to polyglutamine inclusions in HD-KI (Q200) mice but not SCA3-KI mice. Immunohistochemistry with anti-HTT (A-D) or anti-UBQLN2 (E-H, Q-T) antibody was performed on brain sections from 75-week-old HD-KI (Q200) mice (A-H) or WT littermates (Q-T). HD-KI (Q200) mice show robust inclusions in the indicated brain regions that often co-immunostain positively for UBQLN2. Immunohistochemistry with anti-ATXN3 1H9 (I-L) or anti-UBQLN2 (M-P) antibody was performed on brain sections from 52-week-old SCA3 KI mice. ATXN3 inclusions in SCA3-KI mice do not coimmunostain for UBQLN2. Scale bars = 20 μm.
Fig.2
Fig.2
UBQLN2 localizes to inclusions in mouse models of HD but not SCA3. (A-C) Brain sections from 75-week-old heterozygous HD-KI (Q200) mice were double immunolabeled for (A) HTT and UBQLN2, (B) HTT and ubiquitin or (C) HTT and p62. UBQLN2, ubiquitin and p62 all localize to HTT intranuclear inclusions in the hippocampus. Scale bars = 10 μm. (D-F) Brain sections from 52-week-old homozygous SCA3-KI mice were double immunolabeled for (D) ATXN3 and UBQLN2, (E) ATXN3 and ubiquitin, or (F) ATXN3 and p62. UBQLN2 does not localize to SCA3 nuclear inclusions in the hippocampus whereas ubiquitin and p62 do. (G) Double labeling of HTT (green) and UBQLN2 (red) shows colocalization in nuclear inclusions in the striatum of a second HD model, the HD-KI (Q150) mouse, at 70 weeks of age. (H) Double labeling of ATXN3 (green) and UBQLN2 (red) indicates UBQLN2 is not recruited into SCA3 nuclear inclusions in the cortex of a second SCA3 model, homozygous SCA3 YAC mice, at 45 weeks of age. Scale bars = 10 μm.
Fig.3
Fig.3
Early recruitment of UBQLN2 to inclusions of HD-KI (Q200) mice but not SCA3-KI mice. (A) Double immunostaining for ubiquitin (green) and UBQLN2 (red) was performed in hippocampus of 19, 45 and 75 week old heterozygous HD-KI (Q200) mice. The presence of ubiquitin and UBQLN2 immunoreactivity within HTT nuclear inclusions paralleled the timing of HTT inclusion formation in the hippocampus. Scale bars = 10 μm. (B) Double immunostaining for ATXN3 (green) and UBQLN1/2 (red) was performed in hippocampus of 50 and 100-week-old homozygous SCA3-KI mice; the anti-UBQLN antibody used in B recognizes both UBQLN1 and UBQLN2. Even in 2-year old SCA3-KI mice, UBQLN1 and UBQLN2 do not localize to SCA3 intranuclear inclusions. (C) Double immunofluorescence for ATXN3 (green) and UBQLN2 (red) in the CA1 of hippocampus of 100-week-old SCA3-KI mice and age-matched wild type mice showing that UBQLN2 is not recruited into SCA3 nuclear inclusions even at an advanced age. Scale bars = 10 μm. (D) Double immunofluorescence for ATXN3 (green) and UBQLN2 (red) in the striatum radiatum (SR) of the hippocampus from 100-week-old SCA3-KI mice and age-matched wild type mice showing that UBQLN2 is not recruited into large extranuclear ATXN3 inclusions.
Fig.4
Fig.4
Accumulation of UBQLN2 in inclusions of human HD brain but not SCA3 brain. (A) Sections from HD basal ganglia were double immunolabeled for HTT and ubiquitin, ubiquitin and UBQLN2 or ubiquitin and p62. UBQLN2, ubiquitin and p62 colocalize with HTT nuclear inclusions (N=3). B) Brain sections from SCA3 pons were double immunolabeled for ATXN3 and ubiquitin, ATXN3 and UBQLN2, or ATXN3 and p62. Ubiquitin and p62, but not UBQLN2, colocalize to SCA3 nuclear inclusions (N=1). Scale bars = 10 μm.
Fig. 5
Fig. 5
UBQLN2 selectively interacts with mutant HTT through the UBA domain. (A) Diagram of UBQLN2, HTT and ATXN3 expression constructs. (B-C) In HEK293 cells, flag-tagged UBQLN2, ΔUBA or ΔUBL was co-expressed with GFP-HTT25Q or GFPHTT103Q (B) or with GFP-ATXN3-28Q or GFP-ATXN3-84Q (C). HTT or ATXN3 in lysates from transfected cells was immunoprecipitated (IP) with anti-GFP antibody (B) or anti-1H9 antibody (C). Co-precipitated UBQLN2 was detected by IB with anti-FLAG antibody (B-C), shown on top panels. Input levels of the expressed proteins are shown on the bottom. UBQLN2 selectively interacts with an N-terminal HTT fragment with a 103Q expansion but not with normal or expanded ATXN3; deletion of the UBA domain of UBQLN2 attenuates interaction with HTT-103Q. Lysates and IP samples were also probed with anti-ubiquitin antibody, which did not reveal specific ubiquitinated forms of HTT or ATXN3. In B-C, vector represents co-transfected empty vector plasmid.
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