Cellular alterations identified in pluripotent stem cell-derived midbrain spheroids generated from a female patient with progressive external ophthalmoplegia and parkinsonism who carries a novel variation (p.Q811R) in the POLG1 gene

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 (POLG1 Q811R) 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 POLG1 Q811R MDNS compared to control cultures. In order to assess POLG1 Q811R-related cellular alterations within the MDNS, we applied massspectrometry based quantitative proteomic analysis. In total, 6749 proteins were identified, with 61 significantly differentially expressed between POLG1 Q811R and control samples. Pro-and anti-inflammatory signaling and pathways involved in energy metabolism were altered. Notably, increased glycolysis in POLG1 Q811R 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.

Patient-Derived Midbrain Organoids to Explore the Molecular Basis of Parkinson's Disease

“…More recent evolutions of these protocols have confirmed the initial observations, as well as aimed at increasing the quality and reproducibility of hMO (67,(72)(73)(74)(75)(76) and better estimating their yield of mDA neurons. For instance, using a high content image analysis approach, Smits et al (73) showed that TH+ cells composed 62% of all cells after 1 month of differentiation, while Ahfeldt et al (74) found using a knock-in TH:tdtomato line, that TH+ cells composed ∼38% of total cells at a similar timepoint.…”

“…Interestingly, a recent study focusing on a novel variation in the POLG1 gene (Q811R), previously linked to progressive external ophthalmoplegia and parkinsonism ( 75 ), found significant increases in hMO TH+ cells after 100 days of culture compared to those from a gender-matched control. This study also reported an increased production of NM in response to DA treatment, which may have neurotoxic effects in the long run.…”

Insights into Lewy body disease from rare neurometabolic disorders

“…This study did not show consistently higher levels of Alzheimer-type pathology compared to that observed in normal ageing, and Lewy body pathology appeared to conform to Braak’s scheme for the distribution of Lewy bodies in PD (Braak et al 2003 ). Consistent with the proposition that POLG mutations may increase vulnerability to Lewy body pathology, stem cells derived from a patient with a POLG mutation had increased levels of high molecular weight α-synuclein and higher levels of α-synuclein phosphorylated at serine 129 (Chumarina et al 2019 ).…”

Modelling Mitochondrial Disease in Human Pluripotent Stem Cells: What Have We Learned?

“…Transcriptomics and other “omics” approaches, particularly in combination (i.e., multi-omics), have the potential to provide a more complete picture of where and how processes are being regulated in a disease state [ 189 ]. These studies could aid pathway identification in differentiated cell types to inform downstream drug screens and elucidate disease specific cellular mechanisms, with some mitochondrial disease hPSC models reporting these data already ( Table A3 and Table A4 ) [ 48 , 65 , 98 , 99 , 104 , 119 , 190 , 191 ]. Despite the hurdles to screening, feasibility has been shown in some model systems and these approaches could be adapted to other terminally differentiated cell types [ 98 , 131 ].…”