ABSTRACT
Huntington’s disease (HD) is a neurodegenerative disease caused by a CAG repeat expansion in the Huntingtin (HTT) gene. The resulting polyglutamine (polyQ) tract alters the function of the HTT protein. Although HTT is expressed in different tissues, the medium spiny projection neurons (MSNs) in the striatum are particularly vulnerable in HD. Thus, we sought to define the proteome of human HD patient-derived MSNs. We differentiated HD72 induced pluripotent stem cells and isogenic controls into MSNs and carried out quantitative proteomic analysis by two approaches. First, using data-dependent acquisitions with FAIMS (FAIMS-DDA) for label-free quantification on the Orbitrap Lumos mass spectrometer, we identified 6,323 proteins with at least two unique peptides (FDR ≤ 0.01). Of these, 901 proteins were significantly altered in the HD72-MSNs, compared to isogenic controls. Second, we quantitatively validated protein candidates by comprehensive data-independent acquisitions on a TripleTOF 6600 mass spectrometer quantifying 3,106 proteins with at least two unique peptides. Functional enrichment analysis identified pathways related to the extracellular matrix, including TGF-ý regulation of extracellular matrix, epithelial-mesenchymal transition, DNA replication, senescence, cardiovascular system, organism development, regulation of cell migration and locomotion, aminoglycan glycosaminoglycan proteoglycan, growth factor stimulus and fatty acid processes. Conversely, processes associated with the downregulated proteins included neurogenesis-axogenesis, the brain-derived neurotrophic factor-signaling pathway, Ephrin-A: EphA pathway, regulation of synaptic plasticity, triglyceride homeostasis cholesterol, plasmid lipoprotein particle immune response, interferon-γ signaling, immune system major histocompatibility complex, lipid metabolism and cellular response to stimulus. Moreover, proteins involved in the formation and maintenance of axons, dendrites, and synapses (e.g., Septin protein members) are dysregulated in HD72-MSNs. Importantly, lipid metabolism pathways were altered, and we found that lipid droplets accumulated in the HD72-MSNs, suggesting a deficit in lipophagy. Our proteomics analysis of HD72-MSNs identified relevant pathways that are altered in MSNs and confirm current and new therapeutic targets for HD.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Abbreviations: AD, Alzheimer’s disease; ApoE, apolipoprotein E; BDNF, brain-derived neurotrophic factor; CAG, cytosine adenine guanine; CNS, central nervous system; CREB, cAMP response element-binding protein; CV, compensation voltage; DDA, data-dependent acquisition; DE, differentially express; DIA, data-independent acquisition; ECM, extracellular matrix; EMT, epithelial-mesenchymal transition; FAIMS, high-field asymmetric waveform ion mobility spectrometry; FDR, false discovery rate; GABA, gamma aminobutyric acid; GO, Gene Ontology; GPX, glutathione peroxidase; HD, Huntington’s disease; HTT, Huntingtin; IGFBP7, insulin-like growth factor- binding protein 7; IFN-γ, interferon-gamma; IPA, Ingenuity Pathway Analysis; iPSC, induced pluripotent stem cell; LC-MS/MS, liquid chromatography-tandem mass spectrometry; LFQ, label- free quantitation; LGE, lateral ganglionic eminence; logFC, log fold change; MCM, minichromosome maintenance; MHC, major histocompatibility complex; mHTT, mutant Huntingtin; MS, mass spectrometry; MSN, medium spiny neurons; NSC, neural stem cells; PBS, phosphate buffered saline; PD, Parkinson’s disease; polyQ, polyglutamine; SASP, senescence- associated secretory phenotype; SMA, spinal muscular atrophy; STMN1, stathmin-1; TrkB, tyrosine kinase receptor B; WNT, wingless Int-1; XIC, extracted ion chromatogram.