Impaired Sonic Hedgehog Responsiveness of Induced Pluripotent Stem Cell-Derived Floor Plate Cells Carrying the LRRK2-I1371V Mutation Contributes to the Ontogenic Origin of Lower Dopaminergic Neuron Yield.

Lower population of dopaminergic (DA) neurons is known to increase susceptibility to Parkinson's disease (PD), and our earlier study showed a lower yield of DA neurons in Leucine-Rich Repeat Kinase Isoleucine 1371 Valine (LRRK2-I1371V) mutation-carrying PD patient-derived induced Pluripotent Stem Cells (iPSCs). Although the role of Sonic Hedgehog (SHH) in DA neurogenesis of floor plate cells (FPCs) is known, the effect of LRRK2 mutations on SHH responsiveness of FPCs impacting DA neuronal yield has not been studied. We investigated SHH responsiveness of FPCs derived from LRRK2-I1371V PD patient iPSCs with regard to the expression of SHH receptors Patched1 (Ptch1) and Smoothened (Smo), in conjunction with nuclear Gli1 (glioma-associated oncogene 1) expression, intracellular Ca2+ rise, and cytosolic cyclic adenosine monophosphate (cAMP) levels upon SHH induction. In addition, we examined the mechanistic link with LRRK2-I1371V gain-of-function by assessing membrane fluidity and Rab8A and Rab10 phosphorylation in SH-SY5Y cells and healthy control (HC) FPCs overexpressing LRRK2-I1371V as well as FPCs. Although total expression of Ptch1 and Smo was comparable, receptor expression on cell surface was significantly lower in LRRK2-I1371V FPCs than in HC FPCs, with distinctly lower nuclear expression of the downstream transcription factor Gli1. HC-FPCs transfected with LRRK2-I1371V exhibited a similarly reduced cell surface expression of Ptch1 and Smo. Intracellular Ca2+ response was significantly lower with corresponding elevated cAMP levels in LRRK2-I1371V FPCs compared with HC FPCs upon SHH stimulation. The LRRK2-I1371V mutant FPCs and LRRK2-I1371V-transfected SH-SY5Y and HC FPCs too exhibited higher autophosphorylation of phospho LRRK2 (pLRRK2) serine1292 and serine935, as well as substrate phosphorylation of Rab8A and Rab10. Concurrent increase in membrane fluidity, accompanied by a decrease in membrane cholesterol, and lower expression of lipid raft marker caveolin 1 were also observed in them. These findings suggest that impaired SHH responsiveness of LRRK2-I1371V PD FPCs indeed leads to lower yield of DA neurons during ontogeny. Reduced cell surface expression of SHH receptors is influenced by alteration in membrane fluidity owing to the increased substrate phosphorylation of Rab8A and reduced membrane protein trafficking due to pRab10, both results of the LRRK2-I1371V mutation.

Astrocytes Differentiated from LRRK2-I1371V Parkinson's-Disease-Induced Pluripotent Stem Cells Exhibit Similar Yield but Cell-Intrinsic Dysfunction in Glutamate Uptake and Metabolism, ATP Generation, and Nrf2-Mediated Glutathione Machinery.

Owing to the presence of multiple enzymatic domains, LRRK2 has been associated with a diverse set of cellular functions and signaling pathways. It also has several pathological mutant-variants, and their incidences show ethnicity biases and drug-response differences with expression in dopaminergic-neurons and astrocytes. Here, we aimed to assess the cell-intrinsic effect of the LRRK2-I1371V mutant variant, prevalent in East Asian populations, on astrocyte yield and biology, involving Nrf2-mediated glutathione machinery, glutamate uptake and metabolism, and ATP generation in astrocytes derived from LRRK2-I1371V PD patient iPSCs and independently confirmed in LRRK2-I1371V-overexpressed U87 cells. Astrocyte yield (GFAP-immunopositive) was comparable between LRRK2-I1371V and healthy control (HC) populations; however, the astrocytic capability to mitigate oxidative stress in terms of glutathione content was significantly reduced in the mutant astrocytes, along with a reduction in the gene expression of the enzymes involved in glutathione machinery and nuclear factor erythroid 2-related factor 2 (Nrf2) expression. Simultaneously, a significant decrease in glutamate uptake was observed in LRRK2-I1371V astrocytes, with lower gene expression of glutamate transporters SLC1A2 and SLC1A3. The reduction in the protein expression of SLC1A2 was also directly confirmed. Enzymes catalyzing the generation of γ glutamyl cysteine (precursor of glutathione) from glutamate and the metabolism of glutamate to enter the Krebs cycle (α-ketoglutaric acid) were impaired, with significantly lower ATP generation in LRRK2-I1371V astrocytes. De novo glutamine synthesis via the conversion of glutamate to glutamine was also affected, indicating glutamate metabolism disorder. Our data demonstrate for the first time that the mutation in the LRRK2-I1371V allele causes significant astrocytic dysfunction with respect to Nrf2-mediated antioxidant machinery, AT -generation, and glutamate metabolism, even with comparable astrocyte yields.

Dopaminergic Neurons Differentiated from LRRK2 I1371V-Induced Pluripotent Stem Cells Display a Lower Yield, α-Synuclein Pathology, and Functional Impairment.

Being a large multidomain protein, LRRK2 has several confirmed pathological mutant variants for PD, and the incidence of these variants shows ethnicity biases. I1371V, a mutation in the GTPase domain, has been reported in East-Asian populations, but there are no studies reported on dopaminergic (DA) neurons differentiated from this variant. The aim here was to assess the yield, function, and α-synuclein pathology of DA neurons differentiated from LRRK2 I1371V iPSCs. FACS analysis of neural progenitors (NPs) showed a comparable immunopositive population of cells for neural and glial progenitor markers nestin and S100ß; however, NPs from I1371V iPSCs showed lower clonogenic and proliferative capacities than healthy control NPs as determined by the neurosphere assay and Ki67 expression. Floor plate cells obtained from I1371V NPs primed with FGF8 showed distinctly lower immunopositivity for FOXA2 and CLIC5 than healthy control FPCs and similar DOC2B expression. On SHH addition, a similar mature neuronal population was obtained from both groups; however, the yield of TH-immunopositive cells was significantly lower in I1371V, with lower expression of mature DA neuronal markers En1, Nurr1, and DAT. Vesicular dopamine release and intracellular Ca2+ response with KCl stimulation were lower in I1371V DA neurons, along with a significantly reduced expression of resting vesicle marker VMAT2. A concurrently lower expression of PSD95/Syn-I immunopositive puncta was observed in I1371V differentiated cells. Further, higher phosphorylation of α-synuclein and aggregation of oligomeric α-synuclein in I1371V DA neurons were observed. Our data demonstrated conclusively for the first time that mutations in the I1371V allele of LRRK2 showed developmental deficit from the FPC stage and generated a lower yield/number of TH-immunopositive neurons with impairment in their function and synapse density along with increased α-synuclein pathology.

Reduction of phosphorylated α-synuclein through downregulation of casein kinase 2α alleviates dopaminergic-neuronal function.

Among the post-translational modifications of α-synuclein, phosphorylation has been reported to modulate the protein's nuclear localization, gene-expression and cytotoxicity. However, its effect on the functional performance of dopaminergic-neurons is not known. We aimed to evaluate the effect of siRNA-silencing of casein kinase (CK)2α in SH-SY5Y-cells overexpressing A53T α-synuclein, in alleviating phosphorylated α-synuclein serine129 (pSyn-129)-induced changes in intracellular Ca2+ ([Ca2+]i) response to physiological stimuli and vesicular-dopamine release. A53T transfection showed distinct increase in basal pSyn-129 expression with simultaneous nuclear localization, and CK2α siRNA decreased ROS-generation and pSyn-129 levels. A significant reduction was observed in KCl-induced ([Ca2+]i) response and vesicular-dopamine release in the A53T-transfected cells with a corresponding decrease in immunopositive-population of resting-vesicles (VMAT2). CK2α siRNA treatment showed recovery in [Ca2+]i rise with a corresponding upregulation of expression of voltage-gated Ca2+-channels (VGCC) CaV1.3 and CaV2.2 and RyR1 responsible for Ca2+ induced Ca2+ release from ER, VMAT2 expression and vesicular-dopamine release. Thus, using CK2α siRNA to reduce phosphorylation improved cellular-pathology in terms of ROS generation and pSyn-129 levels, as well as functional performance of DA-neuronal cells.

Mitochondrial Superoxide Dismutase Specifies Early Neural Commitment by Modulating Mitochondrial Dynamics.

Studies revealing molecular mechanisms underlying neural specification have majorly focused on the role played by different transcription factors, but less on non-nuclear components. Earlier, we reported mitochondrial superoxide dismutase (SOD2) to be essential for self-renewal and pluripotency of mouse embryonic stem cells (mESCs). In the present study, we found SOD2 to be specifically required for neural lineage, but not the meso- or endoderm specification. Temporally, SOD2 regulated early neural genes, but not the matured genes, by modulating mitochondrial dynamics-specifically by enhancing the mitochondrial fusion protein Mitofusin 2 (MFN2). Bio-complementation strategy further confirmed SOD2 to enhance mitochondrial fusion process independent of its antioxidant activity. Over-expression of SOD2 along with OCT4, but neither alone, transdifferentiated mouse fibroblasts to neural progenitor-like colonies, conclusively proving the neurogenic potential of SOD2. In conclusion, our findings accredit a novel role for SOD2 in early neural lineage specification.

Generation of induced pluripotent stem cells (NIMHi001-A) from a Parkinson's disease patient of East Indian ethnicity carrying LRRK2 I1371V variant.

Mutations in the Leucine Repeat Rich Kinase-2 (LRRK2) gene have been reported in familial Parkinson's disease (PD) cases. We have generated induced pluripotent stem cells (iPSCs) using Sendai-virus reprogramming-method from peripheral blood mononuclear cells of PD-patient of East-Indian ethnicity carrying the I1371V mutation in LRRK2 gene. PD diagnosis was performed using Unified Parkinson's Disease rating scale (UPDRS) score and confirmed by [18F]fluoro-l-dopa [F-DOPA] positron emission tomography (F-DOPA PET). The iPSC line was characterized for self-renewal and pluripotency. This cellular model will provide a valuable resource not only for drug-screening platform but also to understand the pathophysiology of this disease.