LRRK2 Kinase Inhibitor PF-06447475 Protects Drosophila melanogaster against Paraquat-Induced Locomotor Impairment, Life Span Reduction, and Oxidative Stress.

Parkinson's disease (PD) is a complex multifactorial progressive neurodegenerative disease characterized by locomotor alteration due to the specific deterioration of dopaminergic (DAergic) neurons in the substantia nigra pars compacta (SNpc). Mounting evidence shows that human LRRK2 (hLRRK2) kinase activity is involved in oxidative stress (OS)-induced neurodegeneration, suggesting LRRK2 inhibition as a potential therapeutic target. We report that the hLRRK2 inhibitor PF-06447475 (PF-475) prolonged lifespan, increased locomotor activity, maintained DAergic neuronal integrity, and reduced lipid peroxidation (LPO) in female Drosophila melanogaster flies chronically exposed to paraquat (PQ), a redox cycling compound, compared to flies treated with vehicle only. Since LRRK2 is an evolutionary conserved kinase, the present findings reinforce the idea that either reduction or inhibition of the LRRK2 kinase might decrease OS and locomotor alterations associated with PD. Our observations highlight the importance of uncovering the function of the hLRRK2 orthologue dLrrk2 in D. melanogaster as an excellent model for pharmacological screenings.

Combination of Tramiprosate, Curcumin, and SP600125 Reduces the Neuropathological Phenotype in Familial Alzheimer Disease PSEN1 I416T Cholinergic-like Neurons.

Familial Alzheimer's disease (FAD) is a complex and multifactorial neurodegenerative disorder for which no curative therapies are yet available. Indeed, no single medication or intervention has proven fully effective thus far. Therefore, the combination of multitarget agents has been appealing as a potential therapeutic approach against FAD. Here, we investigated the potential of combining tramiprosate (TM), curcumin (CU), and the JNK inhibitor SP600125 (SP) as a treatment for FAD. The study analyzed the individual and combined effects of these two natural agents and this pharmacological inhibitor on the accumulation of intracellular amyloid beta iAß; hyperphosphorylated protein TAU at Ser202/Thr205; mitochondrial membrane potential (ΔΨm); generation of reactive oxygen species (ROS); oxidized protein DJ-1; proapoptosis proteins p-c-JUN at Ser63/Ser73, TP53, and cleaved caspase 3 (CC3); and deficiency in acetylcholine (ACh)-induced transient Ca2+ influx response in cholinergic-like neurons (ChLNs) bearing the mutation I416T in presenilin 1 (PSEN1 I416T). We found that single doses of TM (50 µM), CU (10 µM), or SP (1 µM) were efficient at reducing some, but not all, pathological markers in PSEN 1 I416T ChLNs, whereas a combination of TM, CU, and SP at a high (50, 10, 1 µM) concentration was efficient in diminishing the iAß, p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 markers by -50%, -75%, -86%, and -100%, respectively, in PSEN1 I417T ChLNs. Although combinations at middle (10, 2, 0.2) and low (5, 1, 0.1) concentrations significantly diminished p-TAU Ser202/Thr205, DJ-1Cys106-SO3, and CC3 by -69% and -38%, -100% and -62%, -100% and -62%, respectively, these combinations did not alter the iAß compared to untreated mutant ChLNs. Moreover, a combination of reagents at H concentration was able to restore the dysfunctional ACh-induced Ca2+ influx response in PSEN 1 I416T. Our data suggest that the use of multitarget agents in combination with anti-amyloid (TM, CU), antioxidant (e.g., CU), and antiapoptotic (TM, CU, SP) actions might be beneficial for reducing iAß-induced ChLN damage in FAD.

X-Chromosome Association Study in Latin American Cohorts Identifies New Loci in Parkinson's Disease.

BACKGROUND: Sex differences in Parkinson's disease (PD) risk are well-known. However, the role of sex chromosomes in the development and progression of PD is still unclear. OBJECTIVE: The objective of this study was to perform the first X-chromosome-wide association study for PD risk in a Latin American cohort. METHODS: We used data from three admixed cohorts: (1) Latin American Research consortium on the Genetics of Parkinson's Disease (n = 1504) as discover cohort, and (2) Latino cohort from International Parkinson Disease Genomics Consortium (n = 155) and (3) Bambui Aging cohort (n = 1442) as replication cohorts. We also developed an X-chromosome framework specifically designed for admixed populations. RESULTS: We identified eight linkage disequilibrium regions associated with PD. We replicated one of these regions (top variant rs525496; discovery odds ratio [95% confidence interval]: 0.60 [0.478-0.77], P = 3.13 × 10-5 replication odds ratio: 0.60 [0.37-0.98], P = 0.04). rs5525496 is associated with multiple expression quantitative trait loci in brain and non-brain tissues, including RAB9B, H2BFM, TSMB15B, and GLRA4, but colocalization analysis suggests that rs5525496 may not mediate risk by expression of these genes. We also replicated a previous X-chromosome-wide association study finding (rs28602900), showing that this variant is associated with PD in non-European populations. CONCLUSIONS: Our results reinforce the importance of including X-chromosome and diverse populations in genetic studies. © 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

High Yield of Functional Dopamine-like Neurons Obtained in NeuroForsk 2.0 Medium to Study Acute and Chronic Rotenone Effects on Oxidative Stress, Autophagy, and Apoptosis.

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 (ΔΨm), 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.

LRRK2 Knockout Confers Resistance in HEK-293 Cells to Rotenone-Induced Oxidative Stress, Mitochondrial Damage, and Apoptosis.

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.

Rotenone Blocks the Glucocerebrosidase Enzyme and Induces the Accumulation of Lysosomes and Autophagolysosomes Independently of LRRK2 Kinase in HEK-293 Cells.

Parkinson's disease (PD) is a neurodegenerative disorder caused by the progressive loss of dopaminergic (DAergic) neurons in the substantia nigra and the intraneuronal presence of Lewy bodies (LBs), composed of aggregates of phosphorylated alpha-synuclein at residue Ser129 (p-Ser129α-Syn). Unfortunately, no curative treatment is available yet. To aggravate matters further, the etiopathogenesis of the disorder is still unresolved. However, the neurotoxin rotenone (ROT) has been implicated in PD. Therefore, it has been widely used to understand the molecular mechanism of neuronal cell death. In the present investigation, we show that ROT induces two convergent pathways in HEK-293 cells. First, ROT generates H2O2, which, in turn, either oxidizes the stress sensor protein DJ-Cys106-SH into DJ-1Cys106SO3 or induces the phosphorylation of the protein LRRK2 kinase at residue Ser395 (p-Ser395 LRRK2). Once active, the kinase phosphorylates α-Syn (at Ser129), induces the loss of mitochondrial membrane potential (ΔΨm), and triggers the production of cleaved caspase 3 (CC3), resulting in signs of apoptotic cell death. ROT also reduces glucocerebrosidase (GCase) activity concomitant with the accumulation of lysosomes and autophagolysosomes reflected by the increase in LC3-II (microtubule-associated protein 1A/1B-light chain 3-phosphatidylethanolamine conjugate II) markers in HEK-293 cells. Second, the exposure of HEK-293 LRRK2 knockout (KO) cells to ROT displays an almost-normal phenotype. Indeed, KO cells showed neither H2O2, DJ-1Cys106SO3, p-Ser395 LRRK2, p-Ser129α-Syn, nor CC3 but displayed high ΔΨm, reduced GCase activity, and the accumulation of lysosomes and autophagolysosomes. Similar observations are obtained when HEK-293 LRRK2 wild-type (WT) cells are exposed to the inhibitor GCase conduritol-ß-epoxide (CBE). Taken together, these observations imply that the combined development of LRRK2 inhibitors and compounds for recovering GCase activity might be promising therapeutic agents for PD.

PSEN1 E280A Cholinergic-like Neurons and Cerebral Spheroids Derived from Mesenchymal Stromal Cells and from Induced Pluripotent Stem Cells Are Neuropathologically Equivalent.

Alzheimer's disease (AD) is a chronic neurological condition characterized by the severe loss of cholinergic neurons. Currently, the incomplete understanding of the loss of neurons has prevented curative treatments for familial AD (FAD). Therefore, modeling FAD in vitro is essential for studying cholinergic vulnerability. Moreover, to expedite the discovery of disease-modifying therapies that delay the onset and slow the progression of AD, we depend on trustworthy disease models. Although highly informative, induced pluripotent stem cell (iPSCs)-derived cholinergic neurons (ChNs) are time-consuming, not cost-effective, and labor-intensive. Other sources for AD modeling are urgently needed. Wild-type and presenilin (PSEN)1 p.E280A fibroblast-derived iPSCs, menstrual blood-derived menstrual stromal cells (MenSCs), and umbilical cord-derived Wharton Jelly's mesenchymal stromal cells (WJ-MSCs) were cultured in Cholinergic-N-Run and Fast-N-Spheres V2 medium to obtain WT and PSEN 1 E280A cholinergic-like neurons (ChLNs, 2D) and cerebroid spheroids (CSs, 3D), respectively, and to evaluate whether ChLNs/CSs can reproduce FAD pathology. We found that irrespective of tissue source, ChLNs/CSs successfully recapitulated the AD phenotype. PSEN 1 E280A ChLNs/CSs show accumulation of iAPPß fragments, produce eAß42, present TAU phosphorylation, display OS markers (e.g., oxDJ-1, p-JUN), show loss of ΔΨm, exhibit cell death markers (e.g., TP53, PUMA, CASP3), and demonstrate dysfunctional Ca2+ influx response to ACh stimuli. However, PSEN 1 E280A 2D and 3D cells derived from MenSCs and WJ-MSCs can reproduce FAD neuropathology more efficiently and faster (11 days) than ChLNs derived from mutant iPSCs (35 days). Mechanistically, MenSCs and WJ-MSCs are equivalent cell types to iPSCs for reproducing FAD in vitro.

Characterizing the Genetic Architecture of Parkinson's Disease in Latinos.

OBJECTIVE: This work was undertaken in order to identify Parkinson's disease (PD) risk variants in a Latino cohort, to describe the overlap in the genetic architecture of PD in Latinos compared to European-ancestry subjects, and to increase the diversity in PD genome-wide association (GWAS) data. METHODS: We genotyped and imputed 1,497 PD cases and controls recruited from nine clinical sites across South America. We performed a GWAS using logistic mixed models; variants with a p-value <1 × 10-5 were tested in a replication cohort of 1,234 self-reported Latino PD cases and 439,522 Latino controls from 23andMe, Inc. We also performed an admixture mapping analysis where local ancestry blocks were tested for association with PD status. RESULTS: One locus, SNCA, achieved genome-wide significance (p-value <5 × 10-8 ); rs356182 achieved genome-wide significance in both the discovery and the replication cohorts (discovery, G allele: 1.58 OR, 95% CI 1.35-1.86, p-value 2.48 × 10-8 ; 23andMe, G allele: 1.26 OR, 95% CI 1.16-1.37, p-value 4.55 × 10-8 ). In our admixture mapping analysis, a locus on chromosome 14, containing the gene STXBP6, achieved significance in a joint test of ancestries and in the Native American single-ancestry test (p-value <5 × 10-5 ). A second locus on chromosome 6, containing the gene RPS6KA2, achieved significance in the African single-ancestry test (p-value <5 × 10-5 ). INTERPRETATION: This study demonstrated the importance of the SNCA locus for the etiology of PD in Latinos. By leveraging the demographic history of our cohort via admixture mapping, we identified two potential PD risk loci that merit further study. ANN NEUROL 2021;90:353-365.

Phenolic-rich extract of avocado Persea americana (var. Colinred) peel blunts paraquat/maneb-induced apoptosis through blocking phosphorylation of LRRK2 kinase in human nerve-like cells.

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.

Genome-Wide Analysis of Copy Number Variation in Latin American Parkinson's Disease Patients.

BACKGROUND: Parkinson's disease is the second most common neurodegenerative disorder and affects people from all ethnic backgrounds, yet little is known about the genetics of Parkinson's disease in non-European populations. In addition, the overall identification of copy number variants at a genome-wide level has been understudied in Parkinson's patients. The objective of this study was to understand the genome-wide burden of copy number variants in Latinos and its association with Parkinson's disease. METHODS: We used genome-wide genotyping data from 747 Parkinson's disease patients and 632 controls from the Latin American Research Consortium on the Genetics of Parkinson's disease. RESULTS: Genome-wide copy number burden analysis showed that patients were significantly enriched for copy number variants overlapping known Parkinson's disease genes compared with controls (odds ratio, 3.97; 95%CI, 1.69-10.5; P = 0.018). PRKN showed the strongest copy number burden, with 20 copy number variant carriers. These patients presented an earlier age of disease onset compared with patients with other copy number variants (median age at onset, 31 vs 57 years, respectively; P = 7.46 × 10-7 ). CONCLUSIONS: We found that although overall genome-wide copy number variant burden was not significantly different, Parkinson's disease patients were significantly enriched with copy number variants affecting known Parkinson's disease genes. We also identified that of 250 patients with early-onset disease, 5.6% carried a copy number variant on PRKN in our cohort. Our study is the first to analyze genome-wide copy number variant association in Latino Parkinson's disease patients and provides insights about this complex disease in this understudied population. © 2020 International Parkinson and Movement Disorder Society.

TPEN exerts selective anti-leukemic efficacy in ex vivo drug-resistant childhood acute leukemia.

Despite some advances in the treatment of acute lymphoblastic (ALL) and myeloid leukemia (AML) in recent years, there is still a prominent percentage of pediatric patients with a reduced overall prognosis. Therefore, other therapeutic approaches are needed to treat those patients. In the present study, we report that the metal chelator TPEN affected ΔΨm and DNA content in isolated CD34+ refractory cells from bone marrow ALL (n = 7; B-cell, n = 4; T-cell, n = 3) and AML (n = 3) pediatric patients. Furthermore, TPEN induced oxidation of hydrogen peroxide (H2O2) sensor protein DJ-1, induced up-regulation of BH3-only pro-apoptotic protein PUMA, transcription factor p53 and activated the executor protease CASPASE-3 as apoptosis markers, and reduced the reactivity of the cellular proliferating marker Ki-67 in all acute leukemic groups, and reduced the phosphorylation of c-ABL protein signal in an AML case. Remarkably, bone marrow cells from non-leukemic patients' cells (n = 2) displayed neither loss of ΔΨm nor loss of DNA content when exposed to TPEN. We conclude that TPEN selectively induces apoptosis in acute leukemic cells via reactive oxygen species (ROS) signaling mechanism. Understanding the pathways of TPEN-induced cell death may provide insight into more effective therapeutic ROS-inducing anticancer agents.

Neuroprotective Effects of Methanolic Extract of Avocado Persea americana (var. Colinred) Peel on Paraquat-Induced Locomotor Impairment, Lipid Peroxidation and Shortage of Life Span in Transgenic knockdown Parkin Drosophila melanogaster.

Parkinson's disease (PD) is a progressive neurodegenerative disorder associated with oxidative stress. Therefore, finding new antioxidant sources might be beneficial for its treatment. Avocado Persea americana is a fruit widely cultivated in tropical and subtropical climates worldwide. Although avocado by-products in the form of peel, seed coat and seeds are currently of no commercial use, they constitute a natural source of bioactive compounds. Methanolic (80%) extract obtained from lyophilized ground peels, seed coats, and seeds of the avocado Hass, Fuerte, Reed and Colinred varieties were analyzed for their total phenolic content (TPC) and their correlations with antioxidant capacity (AC) were assessed by ABTS, FRAP, and ORAC assays. For all varieties, the var. Colinred peel shows the highest TPC and AC. Further analysis showed that the var. Colinred peel presented major phenolic compounds B-type procyanidins and epicatechin according to HPLC-MS. The antioxidant effect of peel extract was evaluated upon in vivo oxidative stress (OS) model. We show for the first time that the peel extract can protect and/or prevent transgenic parkinDrosophila melanogaster fly against paraquat-induced OS, movement impairment and lipid peroxidation, as model of PD. Our findings offer an exceptional opportunity to test natural disease-modifying substances from avocado's by-products.

Vitamin E d-α-Tocopheryl Polyethylene Glycol Succinate (TPGS) Provokes Cell Death in Human Neuroblastoma SK-N-SH Cells via a Pro-Oxidant Signaling Mechanism.

Neuroblastoma (NB) is the most common neoplasm during infancy. Unfortunately, NB is still a lethal cancer. Therefore, innovative curative therapies are immediately required. In this study, we showed the prodeath activity of TPGS in human NB SK-N-SH cancer cells. NB cells were exposed to TPGS (10-80 µM). We report for the first time that TPGS induces cell death by apoptosis in NB cells via a pro-oxidant-mediated signaling pathway. Certainly, H2O2 directly oxidizes DJ-1 cysteine106-thiolate into DJ-1 cysteine106-sulfonate, indirectly activates the transcription factors NF-kappaB, p53, and c-JUN, induces the upregulation of mitochondria regulator proteins BAX/PUMA, and provokes the loss of mitochondrial membrane potential (ΔΨm) and the activation of caspase-3/AIF, leading to nuclear disintegration, demonstrated by cellular and biochemical techniques such as fluorescence microscopy, flow cytometry, and Western blot analysis. Since TPGS is a U.S. Food and Drug Administration (FDA)-approved pharmaceutical excipient, this molecule might be used in clinical trials for NB treatment.

Melatonin enhances neural stem cell differentiation and engraftment by increasing mitochondrial function.

Neural stem cells (NSCs) are regarded as a promising therapeutic approach to protecting and restoring damaged neurons in neurodegenerative diseases (NDs) such as Parkinson's disease and Alzheimer's disease (PD and AD, respectively). However, new research suggests that NSC differentiation is required to make this strategy effective. Several studies have demonstrated that melatonin increases mature neuronal markers, which reflects NSC differentiation into neurons. Nevertheless, the possible involvement of mitochondria in the effects of melatonin during NSC differentiation has not yet been fully established. We therefore tested the impact of melatonin on NSC proliferation and differentiation in an attempt to determine whether these actions depend on modulating mitochondrial activity. We measured proliferation and differentiation markers, mitochondrial structural and functional parameters as well as oxidative stress indicators and also evaluated cell transplant engraftment. This enabled us to show that melatonin (25 µM) induces NSC differentiation into oligodendrocytes and neurons. These effects depend on increased mitochondrial mass/DNA/complexes, mitochondrial respiration, and membrane potential as well as ATP synthesis in NSCs. It is also interesting to note that melatonin prevented oxidative stress caused by high levels of mitochondrial activity. Finally, we found that melatonin enriches NSC engraftment in the ND mouse model following transplantation. We concluded that a combined therapy involving transplantation of NSCs pretreated with pharmacological doses of melatonin could efficiently restore neuronal cell populations in PD and AD mouse models depending on mitochondrial activity promotion.

Metal chelator TPEN selectively induces apoptosis in K562 cells through reactive oxygen species signaling mechanism: implications for chronic myeloid leukemia.

Chronic myeloid leukemia (CML) is a hematologic disorder characterized by the constitutive expression of BCR-ABL tyrosine kinase. Although successful implementation of tyrosine kinase inhibitors for the treatment of CML remain a traditional choice for molecularly targeted therapy, some patients present primary or secondary resistance to such therapy. Therefore, alternative therapeutic strategies are required to treat resistant CML cells. Accordingly, new anti-proliferative and/or pro-apoptotic compounds would be needed for clinical treatment. In the present investigation, we demonstrate that TPEN (e.g. 3 µM), a lipid-soluble metal chelator, induces apoptosis in K562 cells via a molecular cascade involving H2O2 â‰« JNK, NF-κB > c-JUN, P73 > PUMA, BAX > loss of ΔΨm > CASPASE-3 > nuclei/DNA fragmentation. Fragmentation of the nuclei and DNA are indicative of cell death by apoptosis. Remarkably, the antioxidant N-acetyl-cysteine, and inhibitors of the transcription factors CASPASE 3 and (JNK) kinase, decreased oxidative stress (OS) and cell death in these cells. This is evidenced by fluorescence microscopy, flow cytometry and immunocytochemistry for OS markers (e.g. generation of H2O2 and DJ 1 oxidation) and nuclear expression of apoptotic markers (e.g. activated caspase 3 and JNK kinase). In addition, TPEN causes no detectable damage in human peripheral blood lymphocyte cells (hPBLCs). We conclude that TPEN selectively induces apoptosis in K562 cells via an OS-mechanism. Our findings may provide insight into more effective CML anticancer therapies.

Vitamin E synthetic derivate-TPGS-selectively induces apoptosis in jurkat t cells via oxidative stress signaling pathways: implications for acute lymphoblastic leukemia.

D-α-tocopheryl polyethylene glycol 1000 succinate (TPGS) is a water-soluble derivative of natural vitamin E commonly used as a drug delivery agent. Recently, TPGS alone has been reported to induce cell death in lung, breast and prostate cancer. However, the effect of TPGS on cancer cell viability remains unclear. Thus, this study was aimed to evaluate the cytotoxic effect of TPGS on human periphral blood lymphocytes (PBL) and on T cell acute lymphocytic leukemia (ALL) Jurkat clone E6-1 cells and its possible mechanism of action. PBL and Jurkat cells were treated with TPGS (10, 20, 40, 60, and 80 µM), and morphological changes in the cell nucleus, mitochondrial membrane potential (ΔΨm), and intracellular reactive oxygen species levels were determined by immune-fluorescence microscopy and flow cytometry. Cellular apoptosis markers were also evaluated by immunocytochemistry. In this study, TPGS induced apoptotic cell death in Jurkat cells, but not in PBL, in a dose-response manner with increasing nuclear DNA fragmentation, increasing cell cycle arrest, and decreasing ΔΨm. Additionally, TPGS increased dichlorofluorescein fluorescence intensity, indicative of H2O2 production, in a dose-independent fashion. TPGS increased DJ-1 Cys(106)-sulfonate, as a marker of intracellular stress and induced the activation of NF-κB, p53 and c-Jun transcription factors. Additionally, it increased the expression of apoptotic markers Bcl-2 related pro-apoptotic proteins Bax and PUMAand activated caspase-3. The antioxidant N-acetyl-L-cysteine and known pharmacological inhibitors protected the cells from the TPGS induced effects. In conclusion, TPGS selectively induces apoptosis in Jurkat cells through two independent but complementary H2O2-mediated signaling pathways. Our findings support the use of TPGS as a potential treatment for ALL.

Neuroprotective Effect of the LRRK2 Kinase Inhibitor PF-06447475 in Human Nerve-Like Differentiated Cells Exposed to Oxidative Stress Stimuli: Implications for Parkinson's Disease.

Leucine-rich repeat kinase 2 (LRRK2) has been implicated in oxidative stress (OS) and neurodegeneration in Parkinson's disease (PD). However, the pathophysiological mechanism of the LRRK2 kinase in neurons under stress stimuli is not yet understood. We demonstrate that rotenone (ROT), a mitochondria complex I inhibitor frequently used to generate in vitro and in vivo experimental models of PD, induces LRRK2 phosphorylation at serine 935 p-(S935) concomitant with cell death in nerve-like differentiated cells (NLCs). Indeed, ROT (50 µM) at 6 h exposure significantly increased reactive oxygen species (ROS) (~100 %), p-(S935)-LRRK2 kinase [~2 f(old)-(i)ncrease] level, induced nuclei condensation/fragmentation (16 %), increased the expression of NF-κB (5.6 f-i), p53 (5.3 f-i), c-Jun (5.4 f-i) transcription factors, activated caspase-3 (8.0 f-i) and AIF (6.8 f-i) proteins; but significantly decreased mitochondrial membrane potential (∆Ψm, ~21 %), indicative of apoptosis -a type of regulated cell death process- compared to untreated cells. Strikingly, the LRRK2 kinase inhibitor PF-06447475 (PF-475, 1 µM) protects NLCs against ROT induced noxious effect. The inhibitor not only blocked the p-(S935)-LRRK2 kinase phosphorylation but also completely abolished ROS, and significantly reversed all ROT-induced apoptosis signaling and OS associated markers to comparable control values. We conclude that wild-type LRRK2 may act as a pro-apoptotic factor under OS stimuli. Our findings suggest an association between OS and LRRK2 phosphorylation in the NLCs death process, as PD model. Therefore, the pharmacological inhibition of LRRK2 might help to understand the OS-mediated kinase activation in PD neurodegenerative disorder.

Fructose sensitizes Jurkat cells oxidative stress-induced apoptosis via caspase-dependent and caspase-independent mechanisms.

Whether fructose (FRU), as the sole energy source, confers a metabolic advantage on cancer cells against noxious stimuli is unknown. The aim of this study was to evaluate the effects of low (11 mM), moderate (25 mM), and high (55 mM) FRU concentrations alone or in combination with rotenone (ROT) or doxorubicin (DOX) in Jurkat cells, an acute lymphoblastic leukemia cell model. Glucose (GLU) was used as a control. Using different cell analysis techniques, we demonstrated that FRU was predominantly metabolized via oxidative phosphorylation (∼95%) (i.e., lactate production was reduced >120-fold), resulting in endogenous oxidative stress-induced conditions. The cells were characterized by generation of O2•- (43%)/ H2 O2 (40%) and activation of NF-κB (∼95-fold increase, fi), c-Jun-N terminal kinase (JNK), p53 (40-fi), and c-Jun (9-fi). In addition, we observed a loss of ΔΨm (10%), activation of caspase-3 (50-fi) and apoptosis-inducing factor (AIF, 2-fi), and condensation and fragmentation of the nuclei [20% by acridine orange/ethidium bromide/Hoechst (AO/EB/H) staining, 15% by flow cytometry] compared to those of GLU 11 at 24 h. Although DOX killed Jurkat cells independent of sugar content in the culture medium, leukemic cells in low, but not high, FRU were extremely sensitive to ROT. Taken together, our findings suggest that Jurkat cells are more susceptible to cell death if forced to shift from GLU metabolism (i.e., aerobic glycolysis) to FRU metabolism (i.e., oxidative phosphorylation) after treatment with mitochondria-targeting molecules. These observations may help elucidate the cell death mechanism of leukemic cells cultured in FRU.

Doxorubicin induces apoptosis in Jurkat cells by mitochondria-dependent and mitochondria-independent mechanisms under normoxic and hypoxic conditions.

In this study, we investigated the molecular mechanism of doxorubicin (dxr)-induced cytotoxicity on Jurkat cells - a model cell of human acute lymphoblastic leukemia - under normoxic (20% O2) and hypoxic (5% O2) conditions. Using in-cell western analysis, immunofluorescence, flow cytometry analysis, and biochemical inhibitors, we evaluated several oxidative stress (OS) and cell death markers. It was found that dxr (5-100 µmol/l) induced apoptosis by OS mechanisms involving DNA fragmentation (8-48%), loss of mitochondrial membrane potential (ΔΨm, 33-92%), and H2O2 production (15-42%) under normoxia. In addition, dxr (10 µmol/l) induced activation and/or nuclei translocation of NF-κB (6.6, 1.6-fold increase), p53 (4.3, 3.1 f), c-Jun (9.5, 5.0 f), apoptosis-inducing factor (AIF) (1.9, 3.9 f), caspase-3 (3.7, 1.9 f), overexpression of Parkin (2.1, 1.2 f)/PINK-1 (2.1 f) proteins, and reduced DJ-1 levels by half compared with untreated cells under normoxia, according to immunofluorescence and in-cell western analysis, respectively. In contrast, dxr (10 µmol/l) could not induce apoptosis in Jurkat cells under hypoxia. Effectively, dxr significantly reduced DNA fragmentation (6%), expression levels of cell death (e.g. p53, c-Jun, caspase-3, AIF), and OS (e.g. Parkin) markers, whereas it increased ΔΨm, hypoxia-inducible factor 1-α (HIF-1α, 3.1, 2.3 f), NF-κB (6.8, 2.0 f), and DJ-1 (1.3, 1.0 f) levels. This investigation suggests that dxr might efficiently eliminate acute lymphoblastic leukemia cells by OS-induced apoptosis under normoxic conditions through a minimal completeness of cell death signaling (i.e. mitochondria-caspase-3/AIF-dependent pathways) and through a direct DNA damage process. However, hypoxic conditions may reduce the effectiveness of dxr toxicity.

Alzheimer's Disease, Drosophila melanogaster and Polyphenols.

Alzheimer's disease (AD) is an insidious neurological disorder that affects memory, one of the human brain's main cognitive functions. Around 5.2 million Americans currently have AD, and the number threatens to climb to 7 million by 2020. Our native country, Colombia, is no exception with an estimated 260,000 individuals to be affected by AD in 2020. A large, genetically-isolated community in Antioquia, Colombia, with early-onset familial Alzheimer's disease due to a presenilin-1 mutation is ideally suited for the study of molecular mechanisms of AD, and hence accelerate the discovery of new or alternative treatment approaches. In this regard, polyphenols--also known as polyhydroxyphenols--have shown antioxidant activity, gene regulation, metal chelator and anti-amyloidogenic aggregation effects. However, further in vitro and in vivo investigations are warranted to validate their use in clinical trials. Drosophila melanogaster is increasingly being used as a valid in vivo model of AD. Here, we summarise data published within the past 16 years (1998-2014) on the molecular biology of AD and the use of polyphenols in the fly to understand the molecular actions and feasibility of these compounds in the treatment of AD.