Pluripotent Stem Cell Derived Neurons as In Vitro Models for Studying Autosomal Recessive Parkinson's Disease (ARPD): PLA2G6 and Other Gene Loci

Abstract

Parkinson's disease (PD) is a neurodegenerative motor disorder which is largely sporadic; however, some familial forms have been identified. Genetic PD can be inherited by autosomal, dominant or recessive mutations. While the dominant mutations mirror the prototype of PD with adult-onset and L-dopa-responsive cases, autosomal recessive PD (ARPD) exhibit atypical phenotypes with additional clinical manifestations. Young-onset PD is also very common with mutations in recessive gene loci. The main genes associated with ARPD are Parkin, PINK1, DJ-1, ATP13A2, FBXO7 and PLA2G6. Calcium dyshomeostasis is a mainstay in all types of PD, be it genetic or sporadic. Intriguingly, calcium imbalances manifesting as altered Store-Operated Calcium Entry (SOCE) is suggested in PLA2G6-linked PARK 14 PD. The common pathways underlying ARPD pathology, including mitochondrial abnormalities and autophagic dysfunction, can be investigated ex vivo using induced pluripotent stem cell (iPSC) technology and are discussed here. PD pathophysiology is not faithfully replicated by animal models, and, therefore, nigral dopaminergic neurons generated from iPSC serve as improved human cellular models. With no cure to date and treatments aiming at symptomatic relief, these in vitro models derived through midbrain floor-plate induction provide a platform to understand the molecular and biochemical pathways underlying PD etiology in a patient-specific manner.

Keywords: Autophagic–lysosomal pathway; Calcium; Cellular reprogramming; Dopaminergic neurons; Lewy bodies; Mitophagy; PARK-14; Phospholipase A2; SOCE.

Figure 1. Structure of PLA2G6 (iPLA2β) protein.

The full length protein is shown with seven ankyrin repeats (pink circles), a proline-rich motif (blue box), a glycine-rich nucleotide binding motif (magenta), a lipase motif (orange with the active site highlighted), and a proposed C-terminal calcium-dependent calmodulin binding domain (purple). Numbers indicate amino acids.

Figure 2. A generalized schematic protocol for differentiation of human induced pluripotent stem cells (iPSCs) into midbrain dopaminergic (DA) neurons.

Human iPSCs are treated with small molecules, such as GSK3β inhibitors (GSK3i) and SMAD inhibitors along with Shh/ FGF8b to induce midbrain floor plate formation and subsequent midbrain DA specification. This is done either by means of direct differentiation from iPSCs or through embryoid bodies (EBs). The DA progenitors can be sorted with a midbrain cell surface marker like CORIN to achieve higher purity of DA neurons via elimination of unwanted contaminant cells. Mature midbrain DA neurons are generated from these progenitors by addition of mentioned factors at the end of 40-70 DIV (days in vitro) in total. * indicates factors that may be used in the final differentiation step, but not compulsory. ActA, activin A; AA, ascorbic acid; BDNF, brain derived neurotrophic factor; DAPT, γ-secretase inhibitor; dbcAMP, dibutyryl cyclic adenosine monophosphate; FGF8, fibroblast growth factor 8; GDNF, glial cell-derived neurotrophic factor; Shh, sonic hedgehog; TGFβ3, transforming growth factor beta-3.