Human menstrual blood-derived mesenchymal stromal cells (MenSCs) have become not only an important source of stromal cells for cell therapy but also a cellular source for neurologic disorders in vitro modeling. By using culture protocols originally developed in our laboratory, we show that MenSCs can be converted into oating neurospheres (NSs) using the Fast-N-Spheres medium for 24-72h, and can be transdifferentiated into functional dopaminergic-like (DALNs, ~26% TH+/DAT+ ow cytometry) and cholinergic-like neurons (ChLNs, ~46% ChAT+/VAChT ow cytometry) which responded to dopamine-and acetylcholine-triggered neuronal Ca 2+ inward stimuli when cultured with the NeuroForsk and the Cholinergic-N-Run medium , respectively in a timely fashion (i.e., 4-7 days). Here, we also report a direct transdifferentiation method to induce MenSCs into functional astrocyte-like cells (ALCs) by incubation of MenSCs in commercial Gibco® Astrocyte Medium in 7-days. The MSCs-derived ALCs (~59% GFAP+/S100b+) were found to respond to glutamate-induced Ca 2+ inward stimuli. Altogether these results show that MenSCs are a reliable source to obtain functional neurogenic cells to further investigate the neurobiology of neurologic disorders.

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It is increasingly evident that LRRK2 kinase activity is involved in oxidative stress (OS)‐induced apoptosis—a type of regulated cell death and neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that a phenolic‐rich extract of avocado Persea americana var. Colinred peel (CRE, 0.01 mg/ml) restricts environmental neurotoxins paraquat (1 mM)/maneb (0.05 mM)‐induced apoptosis process through blocking reactive oxygen species (ROS) signaling and concomitant inhibition of phosphorylation of LRRK2 in nerve‐like cells (NLCs). Indeed, PQ + MB at 6 h exposure significantly increased ROS (57 ± 5%), oxidation of protein DJ‐1cys106SOH into DJ‐1Cys106SO3 ([~3.7 f(old)‐(i)ncrease]), augmented p‐(S935)‐LRRK2 kinase (~20‐f(old) (i)ncrease), induced nuclei condensation/fragmentation (28 ± 6%), increased the expression of PUMA (~6.2‐fi), and activated CASPASE‐3 (CASP‐3, ~4‐fi) proteins; but significantly decreased mitochondrial membrane potential (ΔΨm, ~48 ± 4%), all markers indicative of apoptosis compared to untreated cells. Remarkably, CRE significantly diminished both OS‐signals (i.e., DCF+ cells, DJ‐1Cys106SO3) as well as apoptosis markers (e.g., PUMA, CASP‐3, loss of ΔΨm, p‐LRRK2 kinase) in NLCs exposed to PQ + MB. Furthermore, CRE dramatically reestablishes the transient intracellular Ca2+ flow (~300%) triggered by dopamine (DA) in neuronal cells exposed to PQ + MB. We conclude that PQ + MB‐induced apoptosis in NLCs through OS‐mechanism, involving DJ‐1, PUMA, CASP‐3, LRRK2 kinase, mitochondria damage, DNA fragmentation, and alteration of DA‐receptors. Our findings imply that CRE protects NLCs directly via antioxidant mechanism and indirectly by blocking LRRK2 kinase against PQ + MB stress stimuli. These data suggest that CRE might be a potential natural antioxidant.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to dopaminergic neuronal vulnerability to oxidative stress (OS), mitochondrial impairment, and increased cell death in idiopathic and familial Parkinson’s disease (PD). However, how exactly this kinase participates in the OS-mitochondria-apoptosis connection is still unknown. We used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 LRRK2 knockout (KO) in the human embryonic kidney cell line 293 (HEK-293) to evaluate the cellular response to the mitochondrial inhibitor complex I rotenone (ROT), a well-known OS and cell death inducer. We report successful knockout of the LRRK2 gene in HEK-293 cells using CRISPR editing (ICE, approximately 60%) and flow cytometry (81%) analyses. We found that HEK-293 LRRK2 WT cells exposed to rotenone (ROT, 50 μM) resulted in a significant increase in intracellular reactive oxygen species (ROS, +7400%); oxidized DJ-1-Cys106-SO3 (+52%); phosphorylation of LRRK2 (+70%) and c-JUN (+171%); enhanced expression of tumor protein (TP53, +2000%), p53 upregulated modulator of apoptosis (PUMA, +1950%), and Parkin (PRKN, +22%); activation of caspase 3 (CASP3, +8000%), DNA fragmentation (+35%) and decreased mitochondrial membrane potential (ΔΨm, −58%) and PTEN induced putative kinase 1 (PINK1, −49%) when compared to untreated cells. The translocation of the cytoplasmic fission protein dynamin-related Protein 1 (DRP1) to mitochondria was also observed by colocalization with translocase of the outer membrane 20 (TOM20). Outstandingly, HEK-293 LRRK2 KO cells treated with ROT showed unaltered OS and apoptosis markers. We conclude that loss of LRRK2 causes HEK-293 to be resistant to ROT-induced OS, mitochondrial damage, and apoptosis in vitro. Our data support the hypothesis that LRRK2 acts as a proapoptotic kinase by regulating mitochondrial proteins (e.g., PRKN, PINK1, DRP1, and PUMA), transcription factors (e.g., c-JUN and TP53), and CASP3 in cells under stress conditions. Taken together, these observations suggest that LRRK2 is an important kinase in the pathogenesis of PD.

Human menstrual blood-derived mesenchymal stromal cells (MenSCs) have become not only an important source of stromal cells for cell therapy but also a cellular source for neurologic disorders in vitro modeling. By using culture protocols originally developed in our laboratory, we show that MenSCs can be converted into floating neurospheres (NSs) using the Fast-N-Spheres medium for 24-72h, and can be transdifferentiated into functional dopaminergic-like (DALNs, ~26% TH+/DAT+ flow cytometry) and cholinergic-like neurons (ChLNs, ~46% ChAT+/VAChT flow cytometry) which responded to dopamine- and acetylcholine- triggered neuronal Ca 2+ inward stimuli when cultured with the NeuroForsk and the Cholinergic-N-Run medium , respectively in a timely fashion (i.e., 4-7 days). Here, we also report a direct transdifferentiation method to induce MenSCs into functional astrocyte-like cells (ALCs) by incubation of MenSCs in commercial Gibco® Astrocyte Medium in 7-days. The MSCs-derived ALCs (~59% GFAP+/S100b+) were found to respond to glutamate-induced Ca 2+ inward stimuli. Altogether these results show that MenSCs are a reliable source to obtain functional neurogenic cells to further investigate the neurobiology of neurologic disorders.