Abstract
Essential tremor (ET) is a common, progressive neurological disease characterized by an 8–12-Hz kinetic tremor. Despite its high prevalence, the patho-mechanisms of tremor in ET are not fully known. Through comprehensive studies in postmortem brains, we identified major morphological changes in the ET cerebellum that reflect cellular damage in Purkinje cells (PCs), suggesting that PC damage is central to ET pathogenesis. We previously performed a transcriptome analysis in ET cerebellar cortex, identifying candidate genes and several dysregulated pathways. To directly target PCs, we purified RNA from PCs isolated by laser capture microdissection and performed the first ever PC-specific RNA-sequencing analysis in ET versus controls. Frozen postmortem cerebellar cortex from 24 ETs and 16 controls underwent laser capture microdissection, obtaining ≥2000 PCs per sample. RNA transcriptome was analyzed via differential gene expression, principal component analysis (PCA), and gene set enrichment analyses (GSEA). We identified 36 differentially expressed genes, encompassing multiple cellular processes. Some ET (13/24) had greater dysregulation of these genes and segregated from most controls and remaining ETs in PCA. Characterization of genes/pathways enriched in this PCA and GSEA identified multiple pathway dysregulations in ET, including RNA processing/splicing, synapse organization/ion transport, and oxidative stress/inflammation. Furthermore, a different set of pathways characterized marked heterogeneity among ET patients. Our data indicate a range of possible mechanisms for the pathogenesis of ET. Significant heterogeneity among ET combined with dysregulation of multiple cellular processes supports the notion that ET is a family of disorders rather than one disease entity.
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Data Availability
Raw sequencing data and DEseq2 analysis for whole cerebellar cortex (GSE134878) [33] and laser-captured PCs (GSE197345) can be found at the NIH Gene Expression Omnibus (GEO).
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Acknowledgements
The authors would like to acknowledge their funding for this project from the NIH (R01 NS088257-01A1). There are no other financial disclosures or conflicts of interest to report for any author as it pertains to this research. Full list of financial support from the last 12-months for each author is provided. We would like to thank all patients that donated their brains for banking and the staff at the ETCBR at the New York Brain Bank, the NIH NeuroBioBank, and the Rush Alzheimer’s Disease Center Brain Bank. We thank Columbia University Genome Center and Dr. Erin Bush for their assistance and expertise on this project. The first author personally thanks William Zeizer for his instrumental knowledge of −80°C freezers.
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Contributions
Regina T. Martuscello: Designed and executed laser capture collection of PCs. Extracted RNA, quality control, prepared libraries, and sequenced samples. Analyzed data, prepared statistics, prepared figures, and wrote first draft manuscript.
Karthigayini Sivaprakasam: Analyzed raw RNA-sequencing data. Critiqued data and reviewed manuscript.
Whitney Hartstone: Assisted with laser capturing of PCs.
Sheng-Han Kuo: Critiqued data and reviewed manuscript.
Genevieve Konopka: Project design for RNA-sequencing analysis. Critiqued data and reviewed manuscript.
Elan D. Louis: Conceptualized project, critiqued data, and reviewed manuscript.
Phyllis L. Faust: Conceptualized project, project design, critiqued data, and manuscript.
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Dr. Martuscello, Ms. Hartstone and Dr. Sivaprakasam have no individual funding to report. Dr. Kuo has received research support from the National Institutes of Health: NINDS #R01 NS104423 (principal investigator), NINDS #R01 NS118179 (principal investigator), NINDS #R01 NS124854 (principal investigator). Dr. Konopka has received research support from the National Institutes of Health, NIMH #R01MH126481 (principal Investigator), NHGRI #R01HG011641 (principal Investigator), NINDS #UF1NS115821 (principal investigator), NICHD #R01HD099162 (co-investigator), NIMH #R01MH103517 (co-investigator); Simons Foundation for Autism Research #573689 (principal investigator); and The Welch Foundation #I-1997-20190330 (principal investigator). Dr. Louis has received research support from the National Institutes of Health: NINDS #R01 NS094607 (principal investigator), NINDS #R01 NS088257 (principal investigator), NINDS #R01 NS117745 (principal investigator), NINDS #R01 NS086736 (principal investigator), and NINDS #R01 NS124854 (co-investigator). Dr. Faust has received research support from the National Institutes of Health: NINDS #R01 NS086736 (co-investigator), NINDS #R01 NS088257 (principal investigator), NINDS #R01 NS117745 (principal investigator), NINDS #R01 NS124854 (principal investigator). No conflicts of interest to report from any author.
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Supplementary Information
Supplemental Table 1
Demographic, clinical and neuropathological information for all samples sequenced (PNG 7496 kb)
Supplemental Table 2
Top expressed genes in PCs isolated by laser capture microdissection. The top 20 genes expressed across all samples sequenced are shown. Gene localization in cerebellar cortex showing PC specific = only expressed in PCs; PC enriched = strongly expressed in PCs along with limited expression in other cerebellar cell types; PC expressed (high cerebellum expression) = expressed in PCs and high expression throughout the cerebellum. Localization in the cerebellum obtained from The Human Protein Atlas and The Allen Brain Atlas(PNG 3868 kb)
Supplemental Table 3
Differentially expressed pathways from ET versus Control analyzed by ssGSEA and compared via hierarchical cluster analysis. Pathways from heatmap (Fig. 4A) grouped with kindred gene ontology (GO) pathways shows various levels of dysregulation in control and ET cases. False discovery rate (FDR) is shown (PNG 5808 kb)
Supplemental Table 4
Differentially expressed pathways among ET samples analyzed by ssGSEA and compared via hierarchical cluster analysis. Pathways from heatmap (Fig. 5A) grouped with kindred pathways shows various levels of dysregulation and heterogeneity in ET cases. False discovery rate (FDR) is shown (PNG 6517 kb)
Supplemental Table 5
Cross validation analysis comparing PC enriched sequencing (LCM-seq) results to prior whole cerebellar cortex sequencing (whole lysate-seq) results (PNG 249 kb)
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Martuscello, R.T., Sivaprakasam, K., Hartstone, W. et al. Gene Expression Analysis of Laser-Captured Purkinje Cells in the Essential Tremor Cerebellum. Cerebellum 22, 1166–1181 (2023). https://doi.org/10.1007/s12311-022-01483-4
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DOI: https://doi.org/10.1007/s12311-022-01483-4