Diana Alejandra Quintero-Espinosa

Familial Alzheimer’s disease (FAD) is a complex multifactorial disorder clinically characterized by cognitive impairment and memory loss. Pathologically, FAD is characterized by intracellular accumulation of the protein fragment Aβ42 (iAβ), hyperphosphorylated microtubule-associated protein TAU (p-TAU), and extensive degeneration of basal forebrain cholinergic neurons of the nucleus basalis of Meynert (NbM) and the medial septal nucleus (MSN), mainly caused by mutations in the amyloid precursor protein (APP), presenilin 1 (PSEN1), and PSEN2 gene. Since the dopaminergic system may contribute to FAD symptoms, alterations in the nigro-hippocampal pathway may be associated with cognitive impairment in FAD. Interestingly, p-α-synuclein (p-α-Syn), Aβ, and p-TAU have been found to coexist in vulnerable regions of postmortem AD brains. However, the mechanism by which Aβ, p-TAU, and α-Syn coexist in DAergic neurons in AD brains has not been determined. We generated PSEN1 I416T dopaminergic-like neurons (DALNs) from I416T menstrual stromal cells (MenSCs) in NeuroForsk 2.0 medium for 7 days and then cultured them in minimal culture medium (MCm) for another 4 days. On day 11, DALNs were analyzed for molecular and pathological markers by flow cytometry and fluorescence microscopy. We found that mutant DALNs showed increased accumulation of iAβ as well as increased phosphorylation of TAU at S202/T205 compared to WT DALNs. Thus, mutant DALNs exhibited typical pathological hallmarks of Alzheimer’s disease. Furthermore, PSEN1 I416T DALNs showed concomitant signs of OS as evidenced by the appearance of oxidized sensor protein DJ-1 (i.e., DJ-1C106-SO₃) and apoptotic markers TP53, pS63-c-JUN, PUMA, and cleavage caspase 3 (CC3). Notably, these DALNs exhibited PD-associated proteins such as intracellular accumulation of α-Syn (detected as aggregates of pS129-α-Syn) and phosphorylation of LRRK2 kinase at residue S935. In addition, mutant DALNs showed a 17.16- and 6.17-fold decrease in DA-induced Ca²⁺ flux, compared to WT DALNs. These observations suggest that iAβ and p-TAU, together with p-α-Syn, and p-LRRK2 kinase, may damage DAergic neurons and thereby contribute to the exacerbation of neuropathologic processes in FAD. Remarkably, the LRRK2 inhibitor PF-06447475 (PF-475) significantly reversed PSEN1 I416T-induced neuropathological markers in DAergic neurons. PF-465 inhibitor reduced iAβ, oxDJ-1C106-SO₃, and p-TAU. In addition, this inhibitor reduced pS935-LRRK2, pS129-αSYN, pS63-c-JUN, and CC3. We conclude that the observed neuroprotective effects of PF-475 are due to direct inhibition of LRRK2 activity and that the LRRK2 protein is upstream of the molecular cascade of apoptosis and proteinopathy. Our results suggest that PF-475 is an effective neuroprotective agent against endogenous PSEN1 I416T-induced neurotoxicity in DALNs coexisting with Parkinson’s disease markers. Therefore, PF-475 may be of great therapeutic value in FAD.

Several efforts to develop new protocols to differentiate in in vitro human mesenchymal stromal cells (hMSCs) into dopamine (DA) neurons have been reported. We have formulated NeuroForsk 2.0 medium containing fibroblast growth factor type beta (FGFb), brain-derived neurotrophic factor (BDNF), melatonin, purmorphamine, and forskolin. We report for the first time that menstrual stromal cells (MenSCs) cultured in NeuroForsk 2.0 medium for 7 days transdifferentiated into DA-like neurons (DALNs) expressing specific DA lineage markers tyrosine hydroxylase-positive cells (TH+) and DA transporter-positive (DAT+) cells and were responsive to DA-induced transient Ca²⁺ influx. To test the usefulness of this medium, DALNs were exposed to rotenone (ROT), a naturally occurring organic neurotoxin used extensively to chemically induce an in vitro model of Parkinson’s disease (PD), which is a movement disorder characterized by the specific loss of DA neurons. We wanted to determine whether ROT induces apoptotic cell death and autophagy pathway under acute or chronic conditions in DALNs. Here, we report that acute ROT exposure induced several molecular changes in DALNS. ROT induced a loss of mitochondrial membrane potential (ΔΨ_m), high expression of parkin (PRKN), and high colocalization of dynamin-related protein 1 (DRP1) with the mitochondrial translocase of the outer membrane of mitochondria 20 (TOMM20) protein. Acute ROT also induced the appearance of DJ-1Cys¹⁰⁶-SO₃, as evidenced by the generation of H₂O₂ and oxidative stress (OS) damage. Remarkably, ROT triggered the phosphorylation of leucine-rich repeat kinase 2 (LRRK2) at residue Ser⁹³⁵ and phosphorylation of α-Syn at residue Ser¹²⁹, a pathological indicator. ROT induced the accumulation of lipidated microtubule-associated protein 1B-light chain 3 (LC3B), a highly specific marker of autophagosomes. Finally, ROT induced cleaved caspase 3 (CC3), a marker of activated caspase 3 (CASP3) in apoptotic DALNs compared to untreated DANLs. However, the chronic condition was better at inducing the accumulation of lysosomes than the acute condition. Importantly, the inhibitor of the LRRK2 kinase PF-06447475 (PF-475) almost completely blunted ROT-induced apoptosis and reduced ROT-induced accumulation of lysosomes in both acute and chronic conditions in DALNs. Our data suggest that LRRK2 kinase regulated both apoptotic cell death and autophagy in DALNs under OS. Given that defects in mitochondrial complex I activity are commonly observed in PD, ROT works well as a chemical model of PD in both acute and chronic conditions. Therefore, prevention and treatment therapy should be guided to relieve DALNs from mitochondrial damage and OS, two of the most important triggers in the apoptotic cell death of DALNs.

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.