Abstract
Variations in the POLG1 gene encoding the catalytic subunit of the mitochondrial DNA polymerase gamma, have recently been associated with Parkinson’s disease (PD), especially in patients diagnosed with progressive external ophthalmoplegia (PEO). However, the majority of the studies reporting this association mainly focused on the genetic identification of the variation in POLG1 in PD patient primary cells, and determination of mitochondrial DNA copy number, providing little information about the cellular alterations existing in patient brain cells, in particular dopaminergic neurons. Therefore, through the use of induced pluripotent stem cells (iPSCs), we assessed cellular alterations in novel p.Q811R POLG1 (POLG1Q811R) variant midbrain dopaminergic neuron-containing spheroids (MDNS) from a female patient who developed early-onset PD, and compared them to cultures derived from a healthy control of the same gender. Both POLG1 variant and control MDNS contained functional midbrain regionalized TH/FOXA2-positive dopaminergic neurons, capable of releasing dopamine. Western blot analysis identified the presence of high molecular weight oligomeric alpha-synuclein in POLG1Q811R MDNS compared to control cultures. In order to assess POLG1Q811R-related cellular alterations within the MDNS, we applied mass-spectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1Q811R and control samples. Pro- and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1Q811R MDNS was suggested by the increase in PFKM and LDHA levels and confirmed using functional analysis of glycolytic rate and oxygen consumption levels. Our results validate the use of iPSCs to assess cellular alterations in relation to PD pathogenesis, in a unique PD patient carrying a novel p.Q811R variation in POLG1, and identify several altered pathways that may be relevant to PD pathogenesis.
Highlights
Parkinson’s Disease (PD) is the most prevalent neurodegenerative movement disorder associated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc)
Genetic analysis of the major PD genes disclosed the presence of a heterozygous genetic variant at exon 15 of the POLG1 gene (NM_002693.2): c.2432A > G (p.Gln811Arg)
Neurodegeneration of midbrain dopaminergic neurons is a key hallmark of PD
Summary
Parkinson’s Disease (PD) is the most prevalent neurodegenerative movement disorder associated with progressive dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc). A number of cellular processes, including mitochondrial function, have been implicated in the aetiology of PD [21, 25, 36]. First insights into the role of mitochondria in PD came from the observation that 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP) induces dopamine neuron death through inhibition of the complex I of the mitochondrial respiratory chain, resulting in parkinsonism [51]. A number of genes associated with familial forms of PD were identified to play a role in the maintenance of mitochondrial function, further suggesting its involvement in PD pathogenesis [1, 52]. Mitochondrial DNA (mtDNA) variations and depletion in PD have been extensively investigated, without definitive conclusions [48]. Higher levels of mtDNA deletions in SNpc of both PD and aged brains have been reported [9, 40], and reduced mtDNA copy number has been suggested as a biomarker of PD [19, 54]
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