Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model

Human Molecular Genetics, 2015, Vol. 24, No. 17 4780–4791 doi: 10.1093/hmg/ddv202 Advance Access Publication Date: 1 June 2015 Original Article ORIGINAL ARTICLE Loss of MyD88 alters neuroinflammatory response and attenuates early Purkinje cell loss in a spinocerebellar ataxia type 6 mouse model 1 Center for Brain Integration Research, 2Department of Bioinformatics, Medical Research Institute, 3Department of Neurology and Neurogical Science, Tokyo Medical and Dental University, Tokyo 113-8510, Japan, 4 Core Research for Evolutional Science and Technology (CREST) of the Japan Science and Technology (JST), Tokyo 102-8666, Japan, 5Center for Genomic and Regenerative Medicine, Juntendo University, Tokyo 113-0033, Japan, 6 Department of Social Services and Healthcare Management, International University of Health and Welfare, Tochigi 324-8501, Japan and 7National Center Hospital, National Center of Neurology and Psychiatry, Tokyo 187-8551, Japan *To whom correspondence should be addressed at: Center for Brain Integration Research, Tokyo Medical and Dental University, 1-5-45, Yushima, Bunkyo, Tokyo 113-8510, Japan. Tel: +81 358034716; Fax: +81 358034716; Email: Abstract Spinocerebellar ataxia type 6 (SCA6) is dominantly inherited neurodegenerative disease, caused by an expansion of CAG repeat encoding a polyglutamine (PolyQ) tract in the Cav2.1 voltage-gated calcium channel. Its key pathological features include selective degeneration of the cerebellar Purkinje cells (PCs), a common target for PolyQ-induced toxicity in various SCAs. Mutant Cav2.1 confers toxicity primarily through a toxic gain-of-function mechanism; however, its molecular basis remains elusive. Here, we studied the cerebellar gene expression patterns of young Sca6-MPI118Q/118Q knockin (KI) mice, which expressed mutant Cav2.1 from an endogenous locus and recapitulated many phenotypic features of human SCA6. Transcriptional signatures in the MPI118Q/118Q mice were distinct from those in the Sca1154Q/2Q mice, a faithful SCA1 KI mouse model. Temporal expression profiles of the candidate genes revealed that the up-regulation of genes associated with microglial activation was initiated before PC degeneration and was augmented as the disease progressed. Histological analysis of the MPI118Q/118Q cerebellum showed the predominance of M1-like pro-inflammatory microglia and it was concomitant with elevated expression levels of tumor necrosis factor, interleukin-6, Toll-like receptor (TLR) 2 and 7. Genetic ablation of MyD88, a major adaptor protein conveying TLR signaling, altered expression patterns of M1/M2 microglial phenotypic markers in the MPI118Q/118Q cerebellum. More importantly, it ameliorated PC loss and partially rescued motor impairments in the early disease phase. These results suggest that early neuroinflammatory response may play an important role in the pathogenesis of SCA6 and its modulation could pave the way for slowing the disease progression during the early stage of the disease. Received: April 8, 2015. Revised and Accepted: May 26, 2015 © The Author 2015. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/ licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact 4780 Tomonori Aikawa1,4, Kaoru Mogushi2,4,5, Kumiko Iijima-Tsutsui2,4,6, Kinya Ishikawa3,4, Miyano Sakurai1, Hiroshi Tanaka2,4, Hidehiro Mizusawa1,3,4,7 and Kei Watase1,4, * Human Molecular Genetics, 2015, Vol. 24, No. 17 Introduction in the cerebellum of two Sca6 KI models, initiation of microglial activation preceded the onset of PC loss. Moreover, modulation of the neuroinflammtory response by genetically ablating myeloid differentiation factor 88 (MyD88), a major adaptor molecule essential for Toll-like receptor (TLR) signaling pathway, significantly delayed the progression of PC degeneration in the MPI118Q/118Q mice. Together, our data suggest a role of innate immune response in the pathogenesis of SCA6 as well as a novel mechanism that could be targeted to curb the disease progression. Results Cerebellar gene expression changes in early symptomatic MPI118Q/118Q mice First, we surveyed transcriptional changes in the MPI118Q/118Q mouse cerebellum using the Affymetrix GeneChip Mouse Genome 430 2.0 Array. The experiments were conducted at an early symptomatic disease stage (6 weeks) when the mutant animals developed mild ataxia, and PC loss was yet to be observed (6). Comparison of the gene expression patterns between the MPI118Q/118Q mice (n = 3) and their wild-type (WT) littermates (n = 3) revealed that 150 genes were up-regulated and 364 genes were downregulated in the MPI118Q/118Q mouse cerebellum (based on the cutoff values of P < 0.01 and fold change >1.5) (Fig. 1A and B and Supplementary Fig. S1). To exclude, the changes that were possibly caused by inserting a splice mutation and/or ‘humanization’ of exon 47 (6), we additionally compared the expression profiles of the 6-week-old MPI11Q/11Q KI mice (n = 3) with their WT littermates (n = 3). Both MPI11Q and MPI118Q alleles carry similar splice acceptor site mutations and humanized exon 47; however, the CAG repeat tract in the former has a normal (11 pure CAG repeats) size. In this analysis, we found that a total of 110 genes (15 up-regulated and 95 down-regulated genes) were differentially expressed (Fig. 1A and B and Supplementary Fig. S1). Of these, three genes (one up-regulated and two down-regulated genes) were commonly misregulated in both MPI11Q/11Q KI and MPI118Q/118Q mouse cerebella (Fig. 1A and B), suggesting that these changes were not specific to the MPI118Q/118Q mice. Therefore, we determined the up-/ down-regulation of the remaining 149/362 genes as candidate changes caused by the expansion of a PolyQ tract in the channel. To confirm the microarray data, quantitative real-time PCR (qPCR) analysis of these candidate genes was performed using the RNAs derived from another batch of 6-week-old MPI118Q/118Q mice and their WT littermates (n = 3, each). Of the 186 genes analyzed, the analysis successfully validated differential expression in the same direction for 150 genes (80.6%) (Supplementary Fig. S2A and B). To examine the overall difference among the gene expression profiles of the MPI118Q/118Q, MPI11Q/11Q and WT mice, we performed a principal component analysis (PCA). The gene expression profiles of the MPI118Q/118Q mice were clearly distinguished from those of the WT and MPI11Q/11Q mice by the first principal component (Fig. 1C). On the other hand, the MPI11Q/11Q and WT mice showed gene expression changes based on the second principal component (Fig. 1C). Therefore, the first principal component would reflect MPI118Q/118Q-specific gene expression patterns. Comparison of cerebellar gene expression patter (...truncated)