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

Abstract

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.

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

“…SH-SY5Y cell lines overexpressing these variants of LRRK2 mutations show differential susceptibility between the kinase and GTPase domains [16]. In contrast to the findings in LRRK2 G2019S PD iPSCs-derived DA neurons [17], an impairment in the development of DA neurons with respect to yield was observed in the iPSCs of LRRK2-I1371V PD patients [18]. These studies highlight the need for studying the function and commitment of other cell types carrying the GTPase domain mutation as well.…”

The landscape of rare genetic variation associated with inflammatory bowel disease and Parkinson’s disease comorbidity

“…Furthermore, we identified 11 additional candidate LRRK2 variants that may contribute to IBD-PD comorbidity. Previous studies have already reported L119P, S1228T, R1628P, M1646T, and Y2189C as PD risk variants [ 66 – 69 ], while I1371V has been shown to cause increased phosphorylation and aggregation of α-synuclein in neurons [ 70 ]. Additionally, P1542S is a common variant (gnomAD MAF = 0.03) and has been listed as a CD-associated polymorphism in HGMD [ 38 ].…”

Structural insights into the GTP-driven monomerization and activation of a bacterial LRRK2 homolog using allosteric nanobodies

“…Indeed, we previously showed that Nb Roco1 increases the GTPase activity of the slow CtRoco-L487A mutant (Leemans et al, 2020 ). L487 is located in the Roc Switch 1 loop and the mutation is orthologous to the I1371V PD mutation in LRRK2 (Jagtap et al, 2022 ;Paisán-Ruíz et al, 2005 ). Therefore, we first performed single turnover GTPase experiments by mixing 5 µM CtRoco-L487A with 5 µM GTP in presence of an excess of both Nb Roco1 and Nb Roco2 .…”