Abstract
SummaryMitochondrial protein (MP) assemblies undergo alterations during neurogenesis, a complex process vital in brain homeostasis and disease. Yet which MP assemblies remodel during differentiation remains unclear. Here, using mass spectrometry-based co-fractionation profiles and phosphoproteomics, we generated mitochondrial interaction maps of human pluripotent embryonal carcinoma stem cells and differentiated neuronal-like cells, which presented as two discrete cell populations by single-cell RNA sequencing. The resulting networks, encompassing 6,442 high-quality associations among 600 MPs, revealed widespread changes in mitochondrial interactions and site-specific phosphorylation during neuronal differentiation. By leveraging the networks, we show the orphan C20orf24 as a respirasome assembly factor whose disruption markedly reduces respiratory chain activity in patients deficient in complex IV. We also find that a heme-containing neurotrophic factor, neuron-derived neurotrophic factor [NENF], couples with Parkinson disease-related proteins to promote neurotrophic activity. Our results provide insights into the dynamic reorganization of mitochondrial networks during neuronal differentiation and highlights mechanisms for MPs in respirasome, neuronal function, and mitochondrial diseases.
Highlights
Mitochondria are dynamic organelles crucial for a number of essential cellular functions in neurons, including oxidative phosphorylation (OXPHOS), neuronal differentiation, and synapse formation (Nunnari and Suomalainen, 2012)
biochemical fractionation (BF)/MS Co-elution Profiles from mt Extracts of NTera2 ECSCs and retinoic acid (RA)-Induced differentiated neuronal-like cells (DNLCs) To establish a map of native human mt macromolecular assemblies involved in neurogenesis, we applied our BF/MS strategy (Havugimana et al, 2012) to mt extracts isolated from chemically cross-linked cultures of NTera2 ECSCs and DNLCs (Figure 1A)
We found that 10% (1,889) of the total transcripts (19,587) in DNLCs were significantly (q % 0.01; Table S1) altered with a more than 2-fold change in expression when compared with ECSCs, resulting in 1,176 downregulated and 713 upregulated genes
Summary
Mitochondria (mt) are dynamic organelles crucial for a number of essential cellular functions in neurons, including oxidative phosphorylation (OXPHOS), neuronal differentiation, and synapse formation (Nunnari and Suomalainen, 2012). Mt are crucial for neurogenesis, a dynamic process in which neural stem cells differentiate into neurons via a neurogenic gene expression program (Khacho et al, 2018). The decline in neurogenesis leads to cognitive impairment associated with various degenerative disorders, and impaired mt may contribute to such deterioration (Fernandez et al, 2019; Khacho et al, 2018); the underlying mechanisms triggering these changes are poorly understood. Global changes in gene expression (Busskamp et al, 2014) and proteome dynamics (Frese et al, 2017) have been observed across various stages of neuronal development in multiple cell types. The physiological functions of mt proteins (MPs), as well as the organization of the full repertoire of cell-context-dependent, native human mtPPIs and resulting multiprotein complexes (MPCs) before and after neuronal differentiation are far from complete
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