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

Sowmithra,; Jagtap, Soham; Potdar, Chandrakanta; Yadav, Ravi; Pal, Pramod Kumar

doi:10.1016/j.scr.2020.101768

Cited by 7 publications

(7 citation statements)

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“…The iPSC lines used in this study are fully characterized and registered in hPSCreg. , Furthermore, and significantly, for this PD LRRK2 I1371V iPSC line, the PD diagnosis was not only performed using the Unified Parkinson’s Disease rating scale (UPDRS) score but also confirmed by [18F] fluoro-L-dopa [F-DOPA] positron emission tomography (F-DOPA PET). F-DOPA PET measures the uptake and trapping of dopamine precursors in nigrostriatal projections, and postmortem studies have shown that striatal F-DOPA measurements correlate with DA neuronal counts. , The iPSCs used were characterized for pluripotency as described before. , Midbrain DA neurons were generated from iPSCs through three stages of differentiation to generate neural progenitors (NPs), midbrain floor plate cells (FPCs), and eventually dopaminergic (DA) neurons (Figure A). The iPSCs were subjected to an NP induction medium while plated on a feeder-free geltrex ECM, as previously validated in human embryonic stem cells …”

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

“…Six clones from previously described iPSCs derived from LRRK2 I1371V PD patients (NIMHi001-A) and age- and gender-matched healthy subjects (NIMHAi006-A) screened for the PD-related genes through targeted-gene analysis were used. The iPSC colonies were maintained as described earlier. , The timeline and sequence of DA differentiation are provided in Figure A. The iPSC colonies were picked manually and plated on Geltrex (Gibco #A1413202)-coated six-well plates in a Stemflex medium (Gibco, #A3349401) at a density of 2.5 × 10 5 –3.0 × 10 5 cells/well.…”

Section: Methodsmentioning

confidence: 99%

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

Jagtap

Potdar

Yadav

et al. 2022

ACS Chem. Neurosci.

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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 S100β; 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.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

Jagtap

Potdar

Yadav

et al. 2022

ACS Chem. Neurosci.

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“…Five clones of previously reported iPSC lines for HC (NIMHAi006-A) and LRRK2-I1371V PD (NIMHi001-A) were used in this study [ 9 , 45 ]. Mouse embryonic fibroblast cells inactivated by mitomycin C on gelatin-coated dishes were used as the feeder layer for the iPSCs maintenance.…”

Section: Methodsmentioning

confidence: 99%

“…The G2019S mutation, which is present in the kinase domain, has been the most extensively researched, and it is the most common mutation in the Caucasian population. However, mutations in the GTPase domain (R1441C/G/H, Y1699C, I1371V) remain relatively less explored [ 3 , 4 ], and have higher incidence in the eastern hemisphere [ 5 , 6 , 7 , 8 , 9 ].…”

Section: Introductionmentioning

confidence: 99%

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

Banerjee

Raj

Potdar

et al. 2023

Cells

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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.

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Induced Pluripotent Stem Cell Derived Human Lung Organoids to Map and Treat the SARS-CoV2 Infections In Vitro

Bose

Kapoor

Nihad

2020

Advances in Experimental Medicine and Biology

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

Cited by 7 publications

References 5 publications

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

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

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

Induced Pluripotent Stem Cell Derived Human Lung Organoids to Map and Treat the SARS-CoV2 Infections In Vitro

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