Mutant Ataxin-3 with an Abnormally Expanded Polyglutamine Chain Disrupts Dendritic Development and Metabotropic Glutamate Receptor Signaling in Mouse Cerebellar Purkinje Cells

Abstract

Spinocerebellar ataxia type 3 (SCA3) is caused by the abnormal expansion of CAG repeats within the ataxin-3 gene. Previously, we generated transgenic mice (SCA3 mice) that express a truncated form of ataxin-3 containing abnormally expanded CAG repeats specifically in cerebellar Purkinje cells (PCs). Here, we further characterize these SCA3 mice. Whole-cell patch-clamp analysis of PCs from advanced-stage SCA3 mice revealed a significant decrease in membrane capacitance due to poor dendritic arborization and the complete absence of metabotropic glutamate receptor subtype1 (mGluR1)-mediated retrograde suppression of synaptic transmission at parallel fiber terminals, with an overall preservation of AMPA receptor-mediated fast synaptic transmission. Because these cerebellar phenotypes are reminiscent of retinoic acid receptor-related orphan receptor α (RORα)-defective staggerer mice, we examined the levels of RORα in the SCA3 mouse cerebellum by immunohistochemistry and found a marked reduction of RORα in the nuclei of SCA3 mouse PCs. To confirm that the defects in SCA3 mice were caused by postnatal deposition of mutant ataxin-3 in PCs, not by genome disruption via transgene insertion, we tried to reduce the accumulation of mutant ataxin-3 in developing PCs by viral vector-mediated expression of CRAG, a molecule that facilitates the degradation of stress proteins. Concomitant with the removal of mutant ataxin-3, CRAG-expressing PCs had greater numbers of differentiated dendrites compared to non-transduced PCs and exhibited retrograde suppression of synaptic transmission following mGluR1 activation. These results suggest that postnatal nuclear accumulation of mutant ataxin-3 disrupts dendritic differentiation and mGluR-signaling in SCA3 mouse PCs, and this disruption may be caused by a defect in a RORα-driven transcription pathway.

Experience of mGLuR1 Gene Therapy in Transgenic Models of SCA1 Mice

“…SCA1 transgenic (B05) heterozygous mice which are selectively express expanded polyglutamine chain in PCs were used in this study [9]. According to the previous works [5], impairment of mGluR1 signaling in SCA model mice seems to be a hallmark of motor dyscoordination in the early stage of disease. To rescue SCA1 phenotype, we produced lentiviral and adenoviral vectors expressing mGluR1 and GFP proteins in a head of L7-4mCMV and MSCV promoters, respectively.…”

“…We made intracortical injection of adenoviral vectors into the mature cerebella (5-week-old mice) and lentiviral vectors to immature cerebellum of P6 SCA1 transgenic mice. According to our previous works connected with lentiviral expression of different gene constructs [5] the lentiviral injection to adult cerebellum caused the formation the small expression area limited of one-two lobes close to injection field. For this reason we used P6 SCA1 transgenic mice to increase the expression area.…”

“…Also, the LTD of fast EPSCs at PF-PC synapses requires postsynaptic mGluR1 signaling in PCs [7,21]. In our previous work we found impaired LTD in PCs of 12 week old SCA1 transgenic mice [5]. We therefore examined LTD in PCs adenovirally and lentivirally express transgenic mGluR1 protein.…”

“…We examined this type of short-term plasticity, termed SSE, by monitoring AMPA receptor-mediated fast EPSCs at PF-PC synapses after PF burst stimulation to induce SSE [4]. In our previous study we show that the functional impairment of mGluRs and SSE disruption are prominent at 12 weeks of age in SCA1 transgenic mice [5]. The SSE measured in WT mice treated with PBS and MSCV-GFP-P2A-mGluR1-WPRE was similar ( Figure 5 (a)), whereas in SCA1 transgenic mice it was reviled restoration of SSE in PCs adenovirally and lentivirally expressing mGluR1 protein.…”

“…Our previous study showed that metabotropic glutamate receptor type 1 (mGluR1)-mediated signaling at cerebellar parallel fiber (PF)-Purkinje cell (PC) synapses is completely disrupted in homozygous 'staggerer' mutant and partially disrupted in transgenic SCA1 mice [4,5], which is essential for proper motor coordination and learning [6,7]. In the present study, we tried to rescue…”