FRINK Linked Data Fragments server

Matches in SemOpenAlex for { <https://semopenalex.org/work/W3033574098> ?p ?o ?g. }

• W3033574098 endingPage "9785" @default.
• W3033574098 startingPage "9768" @default.
• W3033574098 abstract "Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this “FACS-array” with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis. Huntington disease (HD) is a neurodegenerative disorder caused by expanded CAG repeats in the Huntingtin gene. Results from previous studies have suggested that transcriptional dysregulation is one of the key mechanisms underlying striatal medium spiny neuron (MSN) degeneration in HD. However, some of the critical genes involved in HD etiology or pathology could be masked in a common expression profiling assay because of contamination with non-MSN cells. To gain insight into the MSN-specific gene expression changes in presymptomatic R6/2 mice, a common HD mouse model, here we used a transgenic fluorescent protein marker of MSNs for purification via FACS before profiling gene expression with gene microarrays and compared the results of this “FACS-array” with those obtained with homogenized striatal samples (STR-array). We identified hundreds of differentially expressed genes (DEGs) and enhanced detection of MSN-specific DEGs by comparing the results of the FACS-array with those of the STR-array. The gene sets obtained included genes ubiquitously expressed in both MSNs and non-MSN cells of the brain and associated with transcriptional regulation and DNA damage responses. We proposed that the comparative gene expression approach using the FACS-array may be useful for uncovering the gene cascades affected in MSNs during HD pathogenesis. Huntington disease (HD) is an autosomal-dominant neurodegenerative disease characterized by chorea, psychiatric disturbances, and cognitive dysfunction (1Walker F.O. Huntington’s disease.Lancet. 2007; 369 (17240289): 218-22810.1016/S0140-6736(07)60111-1Abstract Full Text Full Text PDF PubMed Scopus (1351) Google Scholar). Neuropathological changes in HD are most notable in caudate and putamen, with prominent loss of striatal medium spiny neurons (MSNs) (2Ehrlich M.E. Huntington’s disease and the striatal medium spiny neuron: cell-autonomous and non-cell-autonomous mechanisms of disease.Neurotherapeutics. 2012; 9 (22441874): 270-28410.1007/s13311-012-0112-2Crossref PubMed Scopus (84) Google Scholar). Genetically, HD is caused by the expansion of CAG repeats encoding polyglutamine (polyQ) in exon 1 of Huntingtin (HTT) (3Huntington's Disease Collaborative Research Group A novel gene containing a trinucleotide repeat that is expanded and unstable on Huntington’s disease chromosomes.Cell. 1993; 72 (8458085): 971-98310.1016/0092-8674(93)90585-EAbstract Full Text PDF PubMed Scopus (6694) Google Scholar). Extensive experimental evidence shows that the truncated mutant huntingtin protein (mHTT) causes transcriptional dysregulation via disruption of transcriptional regulators, such as transcription factors (4Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.Science. 2001; 291 (11264541): 2423-242810.1126/science.1056784Crossref PubMed Scopus (906) Google Scholar, 5Suhr S.T. Senut M.C. Whitelegge J.P. Faull K.F. Cuizon D.B. Gage F.H. Identities of sequestered proteins in aggregates from cells with induced polyglutamine expression.J. Cell Biol. 2001; 153 (11309410): 283-29410.1083/jcb.153.2.283Crossref PubMed Scopus (174) Google Scholar, 6Yamanaka T. Miyazaki H. Oyama F. Kurosawa M. Washizu C. Doi H. Nukina N. Mutant Huntingtin reduces HSP70 expression through the sequestration of NF-Y transcription factor.EMBO J. 2008; 27 (18288205): 827-83910.1038/emboj.2008.23Crossref PubMed Scopus (88) Google Scholar) and chromatin status changes (7Vashishtha M. Ng C.W. Yildirim F. Gipson T.A. Kratter I.H. Bodai L. Song W. Lau A. Labadorf A. Vogel-Ciernia A. Troncosco J. Ross C.A. Bates G.P. Krainc D. Sadri-Vakili G. et al.Targeting H3K4 trimethylation in Huntington disease.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23872847): E3027-E303610.1073/pnas.1311323110Crossref PubMed Scopus (108) Google Scholar, 8Kim M.O. Chawla P. Overland R.P. Xia E. Sadri-Vakili G. Cha J.H. Altered histone monoubiquitylation mediated by mutant huntingtin induces transcriptional dysregulation.J. Neurosci. 2008; 28 (18400894): 3947-395710.1523/JNEUROSCI.5667-07.2008Crossref PubMed Scopus (51) Google Scholar). Human HD brains exhibit a large number of gene expression changes, and the representative dysregulated genes, such as SCN4B, PENK, RGS4, and CNR1, substantially correspond to those of HD model mice (7Vashishtha M. Ng C.W. Yildirim F. Gipson T.A. Kratter I.H. Bodai L. Song W. Lau A. Labadorf A. Vogel-Ciernia A. Troncosco J. Ross C.A. Bates G.P. Krainc D. Sadri-Vakili G. et al.Targeting H3K4 trimethylation in Huntington disease.Proc. Natl. Acad. Sci. U. S. A. 2013; 110 (23872847): E3027-E303610.1073/pnas.1311323110Crossref PubMed Scopus (108) Google Scholar, 9Hodges A. Strand A.D. Aragaki A.K. Kuhn A. Sengstag T. Hughes G. Elliston L.A. Hartog C. Goldstein D.R. Thu D. Hollingsworth Z.R. Collin F. Synek B. Holmans P.A. Young A.B. et al.Regional and cellular gene expression changes in human Huntington’s disease brain.Hum. Mol. Genet. 2006; 15 (16467349): 965-97710.1093/hmg/ddl013Crossref PubMed Scopus (544) Google Scholar, 10Oyama F. Miyazaki H. Sakamoto N. Becquet C. Machida Y. Kaneko K. Uchikawa C. Suzuki T. Kurosawa M. Ikeda T. Tamaoka A. Sakurai T. Nukina N. Sodium channel β4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington’s disease transgenic mice.J. Neurochem. 2006; 98 (16805843): 518-52910.1111/j.1471-4159.2006.03893.xCrossref PubMed Scopus (73) Google Scholar), suggesting that transcriptional dysregulation is a central machinery of HD pathogenesis. Gene expression profiling can be a potential tool for understanding disease phenotypes and identifying therapeutic targets in HD (9Hodges A. Strand A.D. Aragaki A.K. Kuhn A. Sengstag T. Hughes G. Elliston L.A. Hartog C. Goldstein D.R. Thu D. Hollingsworth Z.R. Collin F. Synek B. Holmans P.A. Young A.B. et al.Regional and cellular gene expression changes in human Huntington’s disease brain.Hum. Mol. Genet. 2006; 15 (16467349): 965-97710.1093/hmg/ddl013Crossref PubMed Scopus (544) Google Scholar, 10Oyama F. Miyazaki H. Sakamoto N. Becquet C. Machida Y. Kaneko K. Uchikawa C. Suzuki T. Kurosawa M. Ikeda T. Tamaoka A. Sakurai T. Nukina N. Sodium channel β4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington’s disease transgenic mice.J. Neurochem. 2006; 98 (16805843): 518-52910.1111/j.1471-4159.2006.03893.xCrossref PubMed Scopus (73) Google Scholar, 11Luthi-Carter R. Hanson S.A. Strand A.D. Bergstrom D.A. Chun W. Peters N.L. Woods A.M. Chan E.Y. Kooperberg C. Krainc D. Young A.B. Tapscott S.J. Olson J.M. Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain.Hum. Mol. Genet. 2002; 11 (12165554): 1911-192610.1093/hmg/11.17.1911Crossref PubMed Scopus (311) Google Scholar, 12Kotliarova S. Jana N.R. Sakamoto N. Kurosawa M. Miyazaki H. Nekooki M. Doi H. Machida Y. Wong H.K. Suzuki T. Uchikawa C. Kotliarov Y. Uchida K. Nagao Y. Nagaoka U. et al.Decreased expression of hypothalamic neuropeptides in Huntington disease transgenic mice with expanded polyglutamine-EGFP fluorescent aggregates.J. Neurochem. 2005; 93 (15836623): 641-65310.1111/j.1471-4159.2005.03035.xCrossref PubMed Scopus (61) Google Scholar, 13Jia H. Morris C.D. Williams R.M. Loring J.F. Thomas E.A. HDAC inhibition imparts beneficial transgenerational effects in Huntington’s disease mice via altered DNA and histone methylation.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (25535382): E56-E6410.1073/pnas.1415195112Crossref PubMed Scopus (70) Google Scholar). Previous transcriptome analysis using whole brains and whole striatal samples revealed a large number of dysregulated genes and affected biological pathways in HD (14Seredenina T. Luthi-Carter R. What have we learned from gene expression profiles in Huntington’s disease?.Neurobiol. Dis. 2012; 45 (21820514): 83-9810.1016/j.nbd.2011.07.001Crossref PubMed Scopus (103) Google Scholar). In the dysregulated genes, more than 80% of down-regulated genes are strongly expressed in striatal MSNs (e.g. D1R, D2R, SCN4B, and PPP1R1B) (15Desplats P.A. Kass K.E. Gilmartin T. Stanwood G.D. Woodward E.L. Head S.R. Sutcliffe J.G. Thomas E.A. Selective deficits in the expression of striatal-enriched mRNAs in Huntington’s disease.J. Neurochem. 2006; 96 (16405510): 743-75710.1111/j.1471-4159.2005.03588.xCrossref PubMed Scopus (107) Google Scholar), and affected biological pathways are related to the function of striatum (e.g. neurotransmitter receptors, calcium signaling, and G-protein signaling) (14Seredenina T. Luthi-Carter R. What have we learned from gene expression profiles in Huntington’s disease?.Neurobiol. Dis. 2012; 45 (21820514): 83-9810.1016/j.nbd.2011.07.001Crossref PubMed Scopus (103) Google Scholar). On the other hand, gene expression alterations in non-MSN cells were also reported. Up-regulation of GFAP expression, an astrocytic inflammatory gene expression, was shown in the brain of HD model mice and HD patients (9Hodges A. Strand A.D. Aragaki A.K. Kuhn A. Sengstag T. Hughes G. Elliston L.A. Hartog C. Goldstein D.R. Thu D. Hollingsworth Z.R. Collin F. Synek B. Holmans P.A. Young A.B. et al.Regional and cellular gene expression changes in human Huntington’s disease brain.Hum. Mol. Genet. 2006; 15 (16467349): 965-97710.1093/hmg/ddl013Crossref PubMed Scopus (544) Google Scholar, 11Luthi-Carter R. Hanson S.A. Strand A.D. Bergstrom D.A. Chun W. Peters N.L. Woods A.M. Chan E.Y. Kooperberg C. Krainc D. Young A.B. Tapscott S.J. Olson J.M. Dysregulation of gene expression in the R6/2 model of polyglutamine disease: parallel changes in muscle and brain.Hum. Mol. Genet. 2002; 11 (12165554): 1911-192610.1093/hmg/11.17.1911Crossref PubMed Scopus (311) Google Scholar). In addition, transcriptional activation of pro-inflammatory genes in microglia occurs in the brain of HD mice and HD patients (16Crotti A. Benner C. Kerman B.E. Gosselin D. Lagier-Tourenne C. Zuccato C. Cattaneo E. Gage F.H. Cleveland D.W. Glass C.K. Mutant Huntingtin promotes autonomous microglia activation via myeloid lineage-determining factors.Nat. Neurosci. 2014; 17 (24584051): 513-52110.1038/nn.3668Crossref PubMed Scopus (182) Google Scholar). These results suggest that gene expression profiling using whole brains or whole striatal samples is affected by glial cell responses, and that critical genes contributing to MSN degeneration may be masked in common profiling assays. Here, we report comparative analysis of gene expression profiling between purified MSNs and whole striatal samples derived from presymptomatic R6/2 mice, which are the well-characterized and widely used HD model mice (17Mangiarini L. Sathasivam K. Seller M. Cozens B. Harper A. Hetherington C. Lawton M. Trottier Y. Lehrach H. Davies S.W. Bates G.P. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice.Cell. 1996; 87 (8898202): 493-50610.1016/S0092-8674(00)81369-0Abstract Full Text Full Text PDF PubMed Scopus (2477) Google Scholar). The R6/2 expresses the 5′ end of the human HD gene (HTT) containing an ∼120-CAG repeat expansion. The transgene expression is driven by the human HTT promoter, and the expression levels of transgene are around 75% of the endogenous levels. R6/2 mice display loss of body weight and progressive neurological phenotypes, such as motor deficits and tremor (17Mangiarini L. Sathasivam K. Seller M. Cozens B. Harper A. Hetherington C. Lawton M. Trottier Y. Lehrach H. Davies S.W. Bates G.P. Exon 1 of the HD gene with an expanded CAG repeat is sufficient to cause a progressive neurological phenotype in transgenic mice.Cell. 1996; 87 (8898202): 493-50610.1016/S0092-8674(00)81369-0Abstract Full Text Full Text PDF PubMed Scopus (2477) Google Scholar). To avoid incorporation of non-MSN cells, striatal MSNs were genetically labeled (18Miyazaki H. Oyama F. Inoue R. Aosaki T. Abe T. Kiyonari H. Kino Y. Kurosawa M. Shimizu J. Ogiwara I. Yamakawa K. Koshimizu Y. Fujiyama F. Kaneko T. Shimizu H. et al.Singular localization of sodium channel β4 subunit in unmyelinated fibres and its role in the striatum.Nat. Commun. 2014; 5 (25413837): 552510.1038/ncomms6525Crossref PubMed Scopus (41) Google Scholar) and purified by FACS (19Lobo M.K. Karsten S.L. Gray M. Geschwind D.H. Yang X.W. FACS-array profiling of striatal projection neuron subtypes in juvenile and adult mouse brains.Nat. Neurosci. 2006; 9 (16491081): 443-45210.1038/nn1654Crossref PubMed Scopus (304) Google Scholar, 20Ena S.L. De Backer J.-F. Schiffmann S.N. de Kerchove d'Exaerde A. FACS array profiling identifies Ecto-5′ nucleotidase as a striatopallidal neuron-specific gene involved in striatal-dependent learning.J. Neurosci. 2013; 33 (23678122): 8794-880910.1523/JNEUROSCI.2989-12.2013Crossref PubMed Scopus (33) Google Scholar, 21Bye C.R. Jonsson M.E. Bjorklund A. Parish C.L. Thompson L.H. Transcriptome analysis reveals transmembrane targets on transplantable midbrain dopamine progenitors.Proc. Natl. Acad. Sci. U. S. A. 2015; 112 (25775569): E1946-E195510.1073/pnas.1501989112Crossref PubMed Scopus (39) Google Scholar). To identify differentially expressed probes/genes (DEPs/DEGs) from purified MSNs and whole striatal samples, we performed microarray analysis using these two samples (FACS-array and STR-array). We identified a number of FACS-enriched DEPs/DEGs showing enhanced detection in FACS-array compared with STR-array. FACS-enriched DEPs/DEGs contained genes that were ubiquitously expressed in the brain rather than specifically expressed in the MSNs. Those FACS-enriched DEPs/DEGs could be masked in a common profiling assay by the changes of their expression in non-MSN cells. Gene ontology (GO) enrichment analysis revealed that FACS-enriched DEPs/DEGs were associated with “transcriptional regulation” and “DNA damage” that were distinct from the results of other gene sets (FACS-nonenriched DEPs/DEGs, STR-enriched DEPs/DEGs, STR-nonenriched DEPs/DEGs). Thus, the novel gene set, generated from contrast analysis between FACS-array and STR-array, provided masked disease cascade in a common profiling assay. We propose that the study of vulnerable cell-specific transcriptome analysis provides information valuable for understanding pathological cascades in neurodegenerative disorders. To label MSNs in R6/2 mice with Venus fluorescence protein genetically, we crossed R6/2 with a Scn4b-Venus mouse expressing Venus in MSNs under the control of a Scn4b-promoter (18Miyazaki H. Oyama F. Inoue R. Aosaki T. Abe T. Kiyonari H. Kino Y. Kurosawa M. Shimizu J. Ogiwara I. Yamakawa K. Koshimizu Y. Fujiyama F. Kaneko T. Shimizu H. et al.Singular localization of sodium channel β4 subunit in unmyelinated fibres and its role in the striatum.Nat. Commun. 2014; 5 (25413837): 552510.1038/ncomms6525Crossref PubMed Scopus (41) Google Scholar). Double staining using anti-GFP, which recognizes Venus protein, and anti-DARPP-32, an established marker of MSNs, showed colocalization of these two signals (Fig. S1). This result indicates that Venus was definitely expressed in the MSNs of Scn4b-Venus mice. Next, we examined mRNA and protein levels of Venus in R6/2;Scn4b-Venus mice. Because Venus expression is driven by the promoter of Scn4b, which is one of the early down-regulated genes in HD (10Oyama F. Miyazaki H. Sakamoto N. Becquet C. Machida Y. Kaneko K. Uchikawa C. Suzuki T. Kurosawa M. Ikeda T. Tamaoka A. Sakurai T. Nukina N. Sodium channel β4 subunit: down-regulation and possible involvement in neuritic degeneration in Huntington’s disease transgenic mice.J. Neurochem. 2006; 98 (16805843): 518-52910.1111/j.1471-4159.2006.03893.xCrossref PubMed Scopus (73) Google Scholar), it is possible that Venus expression is also affected by mHTT. The results of in situ hybridization (ISH) showed that, in addition to Scn4b mRNA, mRNA levels of Venus in MSNs of 4-week-old R6/2;Scn4b-Venus mice were already down-regulated to half of control levels (Fig. 1A), suggesting that Venus expression is affected by mHTT through the promoter. Consistent results were obtained by quantitative PCR (qPCR) using whole striatal samples (Fig. 1B). On the other hand, the immunohistochemical analysis showed that protein levels of Venus were mostly preserved in MSNs of 4-week-old R6/2;Scn4b-Venus mice, although the Venus protein expression was clearly reduced in 8- and 12-week-old R6/2;Scn4b-Venus mice compared with control (Fig. 1, C and D). These results indicate that even though Venus mRNA expression was affected by mHTT, protein levels of Venus were preserved in MSNs until 4 weeks in R6/2;Scn4b-Venus. Double staining with anti-GFP and anti-Htt (EM48), generated against the first 256 amino acids of truncated huntingtin protein (22Gutekunst C.A. Li S.H. Yi H. Mulroy J.S. Kuemmerle S. Jones R. Rye D. Ferrante R.J. Hersch S.M. Li X.J. Nuclear and neuropil aggregates in Huntington’s disease: relationship to neuropathology.J. Neurosci. 1999; 19 (10087066): 2522-253410.1523/JNEUROSCI.19-07-02522.1999Crossref PubMed Google Scholar), showed that GFP-positive MSNs contained intranuclear diffuse Htt-positive signals in 4-week-old R6/2;Scn4b-Venus mice (Fig. 1C and Fig. S2). These diffuse Htt signals are called “nuclear accumulations” (NAs), which are distinct from the neuronal intranuclear inclusions (NIIs) observed in the degenerating neurons of HD mouse models and HD patients (4Nucifora Jr., F.C. Sasaki M. Peters M.F. Huang H. Cooper J.K. Yamada M. Takahashi H. Tsuji S. Troncoso J. Dawson V.L. Dawson T.M. Ross C.A. Interference by huntingtin and atrophin-1 with cbp-mediated transcription leading to cellular toxicity.Science. 2001; 291 (11264541): 2423-242810.1126/science.1056784Crossref PubMed Scopus (906) Google Scholar, 23Meade C.A. Deng Y.P. Fusco F.R. Del Mar N. Hersch S. Goldowitz D. Reiner A. Cellular localization and development of neuronal intranuclear inclusions in striatal and cortical neurons in R6/2 transgenic mice.J. Comp. Neurol. 2002; 449 (12115678): 241-26910.1002/cne.10295Crossref PubMed Scopus (86) Google Scholar). Indeed, at 4 weeks, EM48- and ubiquitin-positive aggregates are mainly NAs or small-size NIIs in the striatum of R6/2, whereas the NIIs became obvious in association with a decrease in NAs at 8 weeks (Fig. S3). The percentage of NA-positive MSNs was more than 99% (n = 417 NA+Venus+ out of 418 Venus+) at 4 weeks. We thus decided to use the MSNs of 4-week-old R6/2;Scn4b-Venus and its control (WT;Scn4b-Venus) mice for array profiling for the following reasons: 1) mRNA levels of Scn4b, a representative DEG in HD, were already altered at this stage; 2) protein levels of Venus in MSNs of R6/2 mice were similar to those of control mice; and 3) secondary effects of transcriptional disruption would be small at this early stage. To purify MSNs, we dissected striatal regions from the brains of 4-week-old R6/2;Scn4b-Venus and its control mice and dissociated them with papain. After staining with propidium iodide (PI) to detect dead cells, the dissociated cells were sorted with FACS, and finally 5,000–10,000 Venus-positive MSNs were obtained (Fig. 2 (A–C) and Fig. S4). RNA quality of the sorted MSNs was assessed using Agilent Bioanalyzer to confirm no clear degradation (RIN > 7.0) (Fig. S5). Amplified and labeled MSN cDNAs were generated from the total RNA and hybridized to an Agilent SurePrint G3 Mouse GE 8x60K Microarray (FACS-array) (Fig. 2A). To compare DEPs between purified MSNs and whole striatum, we performed microarray analysis using whole striatal samples from 4-week-old R6/2 and control mice (STR-array) (Fig. 2A). No degradation of total RNA of the striatal samples was confirmed by Agilent Bioanalyzer (RIN > 8.0) (Fig. S6). To validate purification of FACS-sorted MSNs, we performed qPCR using cell type–specific markers. The expression levels of cell type–specific markers for oligodendrocyte (Mbp, Mag, Mog, Sox10, and Gjc2), astrocyte (Gfap, Gjb6, Egfr3, Aqp4, and Slc1a2), microglia (Cx3cr1, Itgam, Tmem119, and Fcrl), and striatal interneurons (Calb2, Chat, Sst, Pvalb, Npy, and Htr3a) (24Silberberg G. Bolam J.P. Local and afferent synaptic pathways in the striatal microcircuitry.Curr. Opin. Neurobiol. 2015; 33 (26051382): 182-18710.1016/j.conb.2015.05.002Crossref PubMed Scopus (74) Google Scholar) were very low in the FACS-purified MSNs compared with whole striatal samples (Fig. 2D). Only the expression of Th, a marker of the striatal interneuron subtype, was detected in purified MSNs (Fig. 2D). The result suggests that a subset of Th-expressing interneurons could remain in FACS-purified MSN fractions or detect local expression of TH mRNA in the terminals of nigrostriatal projection fibers (25Gervasi N.M. Scott S.S. Aschrafi A. Gale J. Vohra S.N. MacGibeny M.A. Kar A.N. Gioio A.E. Kaplan B.B. The local expression and trafficking of tyrosine hydroxylase mRNA in the axons of sympathetic neurons.RNA. 2016; 22 (27095027): 883-89510.1261/rna.053272.115Crossref PubMed Scopus (19) Google Scholar). In contrast, the expression of MSN subtype markers, such as Drd1a and Tac1 (striatonigral MSN markers) and Drd2 and Penk (striatopallidal MSN markers), was detected in the FACS-purified MSNs (Fig. 2D). Comparative analysis between FACS-purified MSNs gene expression data (WT;Scn4b-Venus, raw signal > 3,000, 3,014 probes) (Table S1) and Heiman’s MSN-enriched gene expression data (3,897 genes) (26Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. Suárez-Fariñas M. Schwarz C. Stephan D.A. Surmeier D.J. Greengard P. Heintz N. A translational profiling approach for the molecular characterization of CNS cell types.Cell. 2008; 135 (19013281): 738-74810.1016/j.cell.2008.10.028Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar) revealed that 745 probes (24.7%) in our data overlapped with their MSN-enriched gene expression data (Fig. 2E). Furthermore, well-known striatal-enriched genes, Ppp1r1b, Ptpn5, Arpp19, Arpp21, Gnal, Rasd2, Rgs9, Adcy5, Gng7, Rasgrp2, Pde1b, Pde10a, Gpr88, Rarb, Strn4, Foxp1, and Zfp503 (26Heiman M. Schaefer A. Gong S. Peterson J.D. Day M. Ramsey K.E. Suárez-Fariñas M. Schwarz C. Stephan D.A. Surmeier D.J. Greengard P. Heintz N. A translational profiling approach for the molecular characterization of CNS cell types.Cell. 2008; 135 (19013281): 738-74810.1016/j.cell.2008.10.028Abstract Full Text Full Text PDF PubMed Scopus (735) Google Scholar), were contained in our FACS-purified MSN gene expression data (Table S1). These results indicate that we obtained an MSN-enriched fraction containing both striatonigral and striatopallidal MSN subtypes by FACS. Microarray data for FACS-purified MSNs and whole striatal samples are available in the GEO database under accession numbers GSE113928, GSE113929, and GSE113930. First, we performed qPCR validation for FACS- and STR-array data. The qPCR results of known dysregulated genes (14Seredenina T. Luthi-Carter R. What have we learned from gene expression profiles in Huntington’s disease?.Neurobiol. Dis. 2012; 45 (21820514): 83-9810.1016/j.nbd.2011.07.001Crossref PubMed Scopus (103) Google Scholar) showed consistent results with log2 -fold change (FC) values of the microarray data (Fig. 3, A and B). This result indicates that both FACS- and STR-array data certainly include mHTT-associated differentially expressed genes. Next, to identify differentially expressed probes of FACS-array (FACS-array DEPs), microarray data were statistically analyzed using GeneSpring software. We identified 1,014 probes showing different signals in R6/2 MSNs (absolute (abs) FC > 1.5, p < 0.05, raw signal > 100) (Table S2). To find DEPs showing enrichment in FACS-array compared with STR-array, we examined the ratio of absolute FC of FACS-array DEPs to corresponding probes in STR-array (FACS-FC/STR-FC). The FACS-array DEPs showing FACS-FC/STR-FC > 1.5 were selected as FACS-enriched DEPs. The FACS-enriched DEPs referred to more detectable DEPs by FACS-array than STR-array due to their higher FC. On the other hand, the FACS-array DEPs showing similar FC and the same directional change as STR-array (FACS-FC/STR-FC < 1.5) were selected as FACS-nonenriched DEPs (Fig. S7A). Because FACS-“enriched” DEPs should include “dramatically changed” DEPs compared with STR-array, we included the DEPs showing opposite expression change compared with corresponding STR-probes (up and down or down and up). Those probes were also considered “dramatically changed” DEPs and included in the FACS-enriched DEP set (Fig. S7A). To obtain an overview of the array data, we plotted the log2 FCs of the FACS-array DEPs against those of corresponding probes in STR-array (Fig. 3C and Fig. S8A). DEPs located on the positive direction area of the x axis (from 0 to 5) are up-regulated genes in R6/2 MSNs, and DEPs located on the negative direction area of the x axis (from 0 to −5) are down-regulated genes in R6/2 MSNs. Likewise, DEPs located on the positive direction area of the y axis (from 0 to 5) are up-regulated genes in R6/2 striatum, and DEPs located on the negative direction area of the y axis (from 0 to −5) are down-regulated genes in R6/2 striatum (Fig. 3, C and D). In addition, DEPs located on the opposite direction area (x axis, 0–5; y axis, 0 to −5; x axis, 0 to −5; y axis, 0–5) are the DEPs showing opposite expression regulation between FACS-array and STR-array. We identified 677 FACS-enriched DEPs (pink dots in Fig. 3C) and 337 FACS-nonenriched DEPs (blue dots in Fig. 3C) (Table S2). Notably, known dysregulated genes such as Scn4b (FC = −2.22, p = 0.0068) and Rgs4 (FC = −1.63, p = 0.0076) were mostly found in FACS-nonenriched DEPs (red dots in Fig. 3C). We next identified DEPs strongly detected by STR-array (STR-enriched DEPs) in the same way as the FACS-array DEPs. To extract STR-enriched DEPs, we examined the ratio of absolute FC of STR-array DEPs to corresponding probes in FACS-array (STR-FC/FACS-FC). The STR-array DEPs showing STR-FC/FACS-FC > 1.5 were selected as STR-enriched DEPs, and the STR-array DEPs showing similar FC to FACS-array (FACS-FC/STR-FC < 1.5) were selected as STR-nonenriched DEPs (Fig. S7A). The DEPs showing opposite expression change with corresponding FACS-probes (up and down or down and up) were included as STR-enriched DEPs in the same way as the FACS-enriched DEPs (Fig. S7A). We identified 170 STR-enriched DEPs (purple dots in Fig. 3D) and 326 STR-nonenriched DEPs (green dots in Fig. 3D) (Table S3). Log2 FCs of the STR-array DEPs against those of corresponding probes in FACS-array (Fig. 3D and Fig. S8B) showed that known dysregulated genes, Scn4b (FC = −2.33, p = 0.0003) and Rgs4 (FC = −2.24, p = 0.0031), were contained in STR-nonenriched DEPs (red dots in Fig. 3D). Taken together, we created four probe set from FACS- and STR-array DEPs according to enrichment of the two different arrays: FACS-enriched DEPs, FACS-nonenriched DEPs, STR-enriched DEPs, and STR-nonenriched DEPs. Because known dysregulated genes were enriched in the FACS-nonenriched DEPs, FACS-enriched DEPs could contain DEPs that are masked in commonly used profiling analyses. To explore the biological implications for each probe set, we carried out GO enrichment analysis using DAVID 6.8 (RRID:SCR_001881), which is a web-accessible program to investigate functional associations among differentially expressed genes (27Huang da W. Sherman B.T. Lempicki R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources.Nat. Protoc. 2009; 4 (19131956): 44-5710.1038/nprot.2008.211Crossref PubMed Scopus (22690) Google Scholar). The top 5 annotation clusters in each probe set produced by a functional annotation clustering tool are presented in Fig. 4 (genes of all clusters are listed in Table S4). Notably, FACS-enriched DEPs were associated with “regulation of transcription,” “DNA damage,” and “DNA repair,” whereas the other three probe sets were associated with neuronal functions and properties such as “ion transport,” “calcium signaling pathway,” and “synapse.” These results suggest that FACS-enriched DEPs may be more relevant to basic cellular function rather than neuronal function. Next, we performed further selection using more stringent criteria, because some of the probes with low signal value (less than 500 in microarray data) were difficult to verify by qPCR (data not shown). To select more verifiable DEPs, we eliminated the probes with raw signal < 500 from the normalized FACS-array data." @default.
• W3033574098 created "2020-06-12" @default.
• W3033574098 creator A5005919258 @default.
• W3033574098 creator A5009883805 @default.
• W3033574098 creator A5014705038 @default.
• W3033574098 creator A5018691239 @default.
• W3033574098 creator A5021667234 @default.
• W3033574098 creator A5042510713 @default.
• W3033574098 creator A5068795703 @default.
• W3033574098 creator A5073378704 @default.
• W3033574098 creator A5080074650 @default.
• W3033574098 creator A5085222181 @default.
• W3033574098 date "2020-07-01" @default.
• W3033574098 modified "2023-10-14" @default.
• W3033574098 title "FACS-array–based cell purification yields a specific transcriptome of striatal medium spiny neurons in a murine Huntington disease model" @default.
• W3033574098 cites W1859650365 @default.
• W3033574098 cites W1962249623 @default.
• W3033574098 cites W1973721788 @default.
• W3033574098 cites W1980792888 @default.
• W3033574098 cites W2006764478 @default.
• W3033574098 cites W2011796077 @default.
• W3033574098 cites W2015534979 @default.
• W3033574098 cites W2019165280 @default.
• W3033574098 cites W2026802661 @default.
• W3033574098 cites W2028010727 @default.
• W3033574098 cites W2031215175 @default.
• W3033574098 cites W2032864658 @default.
• W3033574098 cites W2037256348 @default.
• W3033574098 cites W2040268901 @default.
• W3033574098 cites W2049861653 @default.
• W3033574098 cites W2059801721 @default.
• W3033574098 cites W2060130891 @default.
• W3033574098 cites W2061119206 @default.
• W3033574098 cites W2062682951 @default.
• W3033574098 cites W2064274168 @default.
• W3033574098 cites W2067709327 @default.
• W3033574098 cites W2073489133 @default.
• W3033574098 cites W2078807269 @default.
• W3033574098 cites W2080053685 @default.
• W3033574098 cites W2081634890 @default.
• W3033574098 cites W2091387807 @default.
• W3033574098 cites W2099685145 @default.
• W3033574098 cites W2105821580 @default.
• W3033574098 cites W2108102646 @default.
• W3033574098 cites W2109410863 @default.
• W3033574098 cites W2114311651 @default.
• W3033574098 cites W2115995998 @default.
• W3033574098 cites W2121642311 @default.
• W3033574098 cites W2122440289 @default.
• W3033574098 cites W2123491442 @default.
• W3033574098 cites W2124066825 @default.
• W3033574098 cites W2125309982 @default.
• W3033574098 cites W2127176239 @default.
• W3033574098 cites W2129630363 @default.
• W3033574098 cites W2133589330 @default.
• W3033574098 cites W2133775329 @default.
• W3033574098 cites W2136367337 @default.
• W3033574098 cites W2148908926 @default.
• W3033574098 cites W2150626351 @default.
• W3033574098 cites W2156257249 @default.
• W3033574098 cites W2158217645 @default.
• W3033574098 cites W2160260282 @default.
• W3033574098 cites W2160748841 @default.
• W3033574098 cites W2169735361 @default.
• W3033574098 cites W2171808363 @default.
• W3033574098 cites W2274495055 @default.
• W3033574098 cites W2341971745 @default.
• W3033574098 cites W2517341189 @default.
• W3033574098 cites W2765265311 @default.
• W3033574098 cites W2917951125 @default.
• W3033574098 cites W584280763 @default.
• W3033574098 doi "https://doi.org/10.1074/jbc.ra120.012983" @default.
• W3033574098 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/7380199" @default.
• W3033574098 hasPubMedId "https://pubmed.ncbi.nlm.nih.gov/32499373" @default.
• W3033574098 hasPublicationYear "2020" @default.
• W3033574098 type Work @default.
• W3033574098 sameAs 3033574098 @default.
• W3033574098 citedByCount "7" @default.
• W3033574098 countsByYear W30335740982020 @default.
• W3033574098 countsByYear W30335740982021 @default.
• W3033574098 countsByYear W30335740982022 @default.
• W3033574098 countsByYear W30335740982023 @default.
• W3033574098 crossrefType "journal-article" @default.
• W3033574098 hasAuthorship W3033574098A5005919258 @default.
• W3033574098 hasAuthorship W3033574098A5009883805 @default.
• W3033574098 hasAuthorship W3033574098A5014705038 @default.
• W3033574098 hasAuthorship W3033574098A5018691239 @default.
• W3033574098 hasAuthorship W3033574098A5021667234 @default.
• W3033574098 hasAuthorship W3033574098A5042510713 @default.
• W3033574098 hasAuthorship W3033574098A5068795703 @default.
• W3033574098 hasAuthorship W3033574098A5073378704 @default.
• W3033574098 hasAuthorship W3033574098A5080074650 @default.
• W3033574098 hasAuthorship W3033574098A5085222181 @default.
• W3033574098 hasBestOaLocation W30335740981 @default.
• W3033574098 hasConcept C104317684 @default.
• W3033574098 hasConcept C142724271 @default.
• W3033574098 hasConcept C1491633281 @default.
• W3033574098 hasConcept C150194340 @default.

• next