Search

B-cell acute lymphoblastic leukemia (B-ALL) is a hematologic disorder characterized by the abnormal proliferation and accumulation of immature B-lymphoblasts arrested at various stages of differentiation. Despite advances in treatment, a significant percentage of pediatric patients with precursor B-ALL still relapse. Therefore, alternative therapies are needed to improve the cure rates for pediatric patients. TPEN (N, N, N’, N’-tetrakis(2-pyridylmethyl)-ethylenediamine).is a pro-oxidant agent capable of selectively inducing apoptosis in leukemia cells. Consequently, it has been suggested that TPEN could be a potential agent for oxidative therapy. However, it is not yet known whether TPEN can selectively destroy leukemia cells in a more disease-like model, for example, the bloodstream and bone marrow (BM), in vitro. This investigation is an extension of a previous study that dealt with the effect of TPEN on ex vivo isolated/purified refractory B-ALL cells. Here, we evaluated the effect of TPEN on whole BM from nonleukemic patients (control) or pediatric patients diagnosed with de novo B-ALL or refractory B-ALL cells by analyzing the hematopoietic cell lineage marker CD34/CD19. Although TPEN was innocuous to nonleukemic BM (n=3), we found that TPEN significantly induced apoptosis in de novo (n = 5) and refractory B-ALL (n = 6) leukemic cell populations. Moreover, TPEN significantly increased the counts of cells positive for the oxidation of the stress sensor protein DJ-1, a sign of the formation of H2O2, and significantly increased the counts of cells positive for the pro-apoptotic proteins TP53, PUMA, and CASPASE-3 (CASP-3), indicative of apoptosis, in B-ALL cells. We demonstrate that TPEN selectively eliminates B-ALL cells independent of age, diagnosis status (de novo or refractory), sex, karyotype, or immunophenotype. Understanding TPEN-induced cell death in leukemia cells provides insight into more effective therapeutic oxidation-inducing anticancer agents.

Parkinson’s disease (PD) is a complex progressive neurodegenerative disorder involving impairment of bodily movement caused by the specific destruction of dopaminergic (DAergic) neurons. Mounting evidence suggests that PD might be triggered by an interplay between environmental neurotoxicants (e.g., paraquat, PQ), heavy metals (e.g., iron), and gene alterations (e.g., PARKIN gene). Unfortunately, there are no therapies currently available that protect, slow, delay, or prevent the progression of PD. Melatonin (Mel, N-acetyl-5-methoxy tryptamine) is a natural hormone with pleiotropic functions including receptor-independent pathways which might be useful in the treatment of PD. Therefore, as a chemical molecule, it has been shown that Mel prolonged the lifespan and locomotor activity, and reduced lipid peroxidation (LPO) in wild-type Canton-S flies exposed to PQ, suggesting antioxidant and neuroprotective properties. However, it is not yet known whether Mel can protect or prevent the genetic model parkin deficient in flies against oxidative stress (OS) stimuli. Here, we show that Mel (0.5, 1, 3 mM) significantly extends the life span and locomotor activity of TH > parkin-RNAi/ + Drosophila melanogaster flies (> 15 days) compared to untreated flies. Knock-down (K-D) parkin flies treated with PQ (1 mM) or PQ (1 mM)/iron (1 mM) significantly diminished the survival index and climbing abilities (e.g., 50% of flies were dead and locomotor impairment by days 4 and 3, respectively). Remarkably, Mel reverted the noxious effect of PQ or PQ/iron combination in K-D parkin. Indeed, Mel protects TH > parkin-RNAi/ + Drosophila melanogaster flies against PQ- or PQ/iron-induced diminish survival, locomotor impairment, and LPO (e.g., 50% of flies were death and locomotor impairment by days 6 and 9, respectively). Similarly, Mel prevented K-D parkin flies against both PQ and PQ/iron. Taken together, these findings suggest that Mel can be safely used as an antioxidant and neuroprotectant agent against OS-stimuli in selective individuals at risk to suffer early-onset Parkinsonism and PD.

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

Alzheimer’s disease (AD) is a progressive, degenerative disorder that mainly results in memory loss and a cognitive disorder. Although the cause of AD is still unknown, a minor percentage of AD cases are produced by genetic mutations in the presenilin-1 (PSEN1) gene. Differentiated neuronal cells derived from induced pluripotent stem cells (iPSCs) of patients can recapitulate key pathological features of AD in vitro; however, iPSCs studies focused on the p.E280 A mutation, which afflicts the largest family in the world with familial AD, have not been carried out yet. Although a link between the loss of the Y (LOY) chromosome in peripheral blood cells and risk for AD has been reported, LOY-associated phenotype has not been previously studied in PSEN1 E280 A carriers. Here, we report the reprogramming of fibroblast cells into iPSCs from a familial AD patient with the PSEN1 E280 A mutation, followed by neuronal differentiation into neural precursor cells (NPCs), and the differentiation of NPCs into differentiated neurons that lacked a Y chromosome. Although the PSEN1 E280 A iPSCs and NPCs were successfully obtained, after 8 days of differentiation, PSEN1 E280 A differentiated neurons massively died reflected by release and/ or activation of death markers, and failed to reach complete neural differentiation compared to PSEN 1 wild type cells.

To date, over 90 Parkinson’s disease (PD) risk variants have been reported from genome-wide association studies (GWAS). However, these GWAS efforts have been limited to individuals of European and East Asian ancestry. We performed the first GWAS of Latino PD patients from South America, comparing 807 cases against 690 controls followed by association testing of suggestive loci in a replication cohort of 1,234 cases and 439,522 controls. We demonstrated that SNCA plays a significant role in PD etiology in a Latino cohort and identified a suggestive locus near NRROS on chromosome 3 that appeared to be driven by Peruvian subjects. We also characterized the overlap of PD genetic architecture between Europeans and Latinos with a replication of significant variants identified by Nalls et al. in their 2019 GWAS1, finding 80% concordance in direction of effect. We then leveraged the population history of Latinos via admixture mapping, identifying a significant locus on chromosome 14 in a joint test of ancestries, driven by the Native American ancestral background, and a significant locus on chromosome 6 in our test of African ancestry, containing the genes STXBP6 and RPS6KA2, respectively. Ultimately, our work reflects the most comprehensive characterization of PD genetic architecture in Latinos to date.

In 2000, by using the binary galactose 4 (GAL4)/upstream activating sequence (UAS) system, Feany and Bender were the first to report a transgenic Drosophila melanogaster fly model expressing familial Parkinson's disease (PD)-linked mutants (Alanine53Treonine and Alanine30Proline) of human alpha-synuclein. Amazingly, these mutant Drosophila (as was not the case with normal Drosophila) replicated the essential features of human PD including adult-onset loss of dopaminergic neurons, filamentous intraneuronal inclusions containing alpha-synuclein, and locomotor dysfunction. We provide highlights in the history of Drosophila PD research with special attention paid to the knockdown transgenic Drosophila GAL4/UAS RNA interference system to characterize and understand the response of mutant parkin, dj-1β, and Lrrk flies to oxidative stress phenomena induced by PQ2+ intoxication, thereby exploring potential therapeutic treatments.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive disturbance as a consequence of the loss of cholinergic neurons in the brain, neuritic plaques and hyperphosphorylation of TAU protein. Although the underlying mechanisms leading to these events are unclear, mutations in presenilin 1 (PSEN1), e.g., E280A (PSEN1 E280A), are causative factors for autosomal dominant early-onset familial AD (FAD). Despite advances in the understanding of the physiopathology of AD, there are no efficient therapies to date. Limitations in culturing brain-derived live neurons might explain the limited effectiveness of AD research. Here, we show that mesenchymal stromal (stem) cells (MSCs) can be used to model FAD, providing novel opportunities to study cellular mechanisms and to establish therapeutic strategies. Indeed, we cultured MSCs with the FAD mutation PSEN1 E280A and wild-type (WT) PSEN1 from umbilical cords and characterized the transdifferentiation of these cells into cholinergic-like neurons (ChLNs). PSEN1 E280A ChLNs but not WT PSEN1 ChLNs exhibited increased intra- and extracellular Aβ42 peptide and TAU phosphorylation (at residues Ser202/Thr205), recapitulating the molecular pathogenesis of FAD caused by mutant PSEN1. Furthermore, PSEN1 E280A ChLNs presented oxidative stress (OS) as evidenced by the oxidation of DJ-1Cys106-SH into DJ-1Cys106-SO3 and the detection of DCF-positive cells and apoptosis markers such as activated pro-apoptosis proteins p53, c-JUN, PUMA and CASPASE-3 and the concomitant loss of the mitochondrial membrane potential and DNA fragmentation. Additionally, mutant ChLNs displayed Ca2+ flux dysregulation and deficient acetylcholinesterase (AChE) activity compared to control ChLNs. Interestingly, the inhibitor JNK SP600125 almost completely blocked TAU phosphorylation. Our findings demonstrate that FAD MSC-derived cholinergic neurons with the PSEN1 E280A mutation are a valid model of AD and provide important clues for the identification of targetable pathological molecules.

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.

Nasulysin-1, a new zinc-metalloproteinase from the snake venom of the hognose pit viper Porthidium nasutum, was purified to homogeneity using molecular exclusion chromatography and high performance liquid chromatography on a reverse phase column. The molecular mass of the purified enzyme was 25,900 kDa and pI 4.1, as determined by 1D and 2D polyacrylamide gel electrophoresis. Liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) analysis of the N-terminal amino acid sequence (1FSPRYIELVVVADHGMFKKYNSNLNTIR28; 1TASLANLEVWSK12; 1DLLPR6) of the purified nasulysin-1, shows close structural homology with other snake venom metalloproteinases isolated from different snake venoms. The purified nasulysin-1 showed specific apoptosis-inducing activity in Jurkat and K562 cells, a T-cell acute lymphocytic leukemia (ALL) and chronic myeloid leukemia (AML) cell model, respectively, without affecting the viability of human lymphocyte cells. After 48 h treatment, nasulysin-1 (20 μg/mL) induced loss of the mitochondrial membrane potential (ΔΨm), activated the apoptosis-inducing factor (AIF), activated the protease caspase-3, and induced chromatin condensation and DNA fragmentation, all hallmarks of apoptosis. These results strongly suggest that nasulysin-1 selectively induces apoptosis to eliminate leukemia cells. Thus, these data warrant further investigation into the use of the metalloproteinase protein, nasulysin-1 as a potential therapeutic agent for treating leukemia.

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%)/ H2O2 (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.

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.

Alzheimer’s disease (AD) is a neurodegenerative disorder characterized by progressive memory loss and cognitive disturbance as a consequence of the loss of cholinergic neurons in the brain, neuritic plaques and hyperphosphorylation of TAU protein. Although the underlying mechanisms leading to these events are unclear, mutations in presenilin 1 (PSEN1), e.g., E280A (PSEN1 E280A), are causative factors for autosomal dominant early-onset familial AD (FAD). Despite advances in the understanding of the physiopathology of AD, there are no efficient therapies to date. Limitations in culturing brain-derived live neurons might explain the limited effectiveness of AD research. Here, we show that mesenchymal stromal (stem) cells (MSCs) can be used to model FAD, providing novel opportunities to study cellular mechanisms and to establish therapeutic strategies. Indeed, we cultured MSCs with the FAD mutation PSEN1 E280A and wild-type (WT) PSEN1 from umbilical cords and characterized the transdifferentiation of these cells into cholinergic-like neurons (ChLNs). PSEN1 E280A ChLNs but not WT PSEN1 ChLNs exhibited increased intracellular soluble amyloid precursor protein (sAPPf) fragments and extracellular Aβ42 peptide and TAU phosphorylation (at residues Ser202/Thr205), recapitulating the molecular pathogenesis of FAD caused by mutant PSEN1. Furthermore, PSEN1 E280A ChLNs presented oxidative stress (OS) as evidenced by the oxidation of DJ-1Cys106-SH into DJ-1Cys106-SO3 and the detection of DCF-positive cells and apoptosis markers such as activated pro-apoptosis proteins p53, c-JUN, PUMA and CASPASE-3 and the concomitant loss of the mitochondrial membrane potential and DNA fragmentation. Additionally, mutant ChLNs displayed Ca2+ flux dysregulation and deficient acetylcholinesterase (AChE) activity compared to control ChLNs. Interestingly, the inhibitor JNK SP600125 almost completely blocked TAU phosphorylation. Our findings demonstrate that FAD MSC-derived cholinergic neurons with the PSEN1 E280A mutation provide important clues for the identification of targetable pathological molecules.

Barckground Alzheimer's disease (AD) is mainly caused by cellular loss and dysfunction of the basal forebrain cholinergic neurons and cholinergic axons in the cortex leading to slowly progressive decline in learning and memory performance. Unfortunately, no definitive treatment to halt neural cell loss exists to date. Therefore, it is necessary to obtain an unlimited source of cholinergic neurons for future pharmacological applications in AD. Human mesenchymal stromal cells (hMSCs) represent a unique source of cholinergic-like neurons (ChLNs). New method hWJ-MSCs were incubated with Cholinergic-N-Run medium for 4 and 7 days. Results hWJ-MSCs cultured with Cholinergic-N-Run medium differentiated into ChLNs in 4 days as evidenced by high levels of protein expression of the neuronal markers ChAT, VAChT, AChE, MAP2, β-Tubulin III, NeuN, TUC-4, NF-L and no expression of the immature marker SOX2, the dopaminergic marker TH, GABAergic marker GAD67 and glial marker GFAP. Comparison with existing method(s) The hWJ-MSCs form ChLNs (e.g., ∼26% IF+) within 20 days by using complex conditioned mediums that are expensive and time-consuming. We report for the first time, to our best knowledge, a direct method of hWJ-MSCs transdifferentiation into ChLNs (∼76% ChAT /VAChT assessed by immunofluorescence microscopy and flow cytometry) in an economic, efficient and timely fashion. Conclusions The fastest method to obtain ChLNs from hWJ-MSCs takes only four days using the one-step incubation medium Cholinergic-N-Run.

Background aims Stem cell transplantation is an excellent option for regenerative or replacement therapy. However, deleterious microenvironmental and endogenous factors (e.g., oxidative stress) compromise ongoing graft survival and longevity. Therefore, (transient or stable) genetically modified cells may be reasonably thought to resist oxidative stress–induced damage. Genetic engineering of mesenchymal stromal cells (MSCs) obtained from Wharton's jelly tissue may offer some therapeutic potential. PARKIN is a multifunctional ubiquitin ligase able to protect dopaminergic cells against stress-related signaling. We, therefore, evaluated the effect of the neurotoxicant 6-hydroxydopamine (6-OHDA) on regulated cell death signaling in MSCs and investigated whether overexpression of PARKIN in MSCs was capable of modulating the effect of 6-OHDA. Methods We transiently transfected Wharton's jelly–derived MSCs with an mCherry-PARKIN vector using the Lipofectamine LTX method. Naive MSCs and MSCs overexpressing PARKIN were exposed to increasing concentrations of 6-OHDA. We used light and fluorescence microscopy, flow cytometry, immunocytochemistry staining, in-cell Western and Western blot analysis. Results After 12–24 h of 6-OHDA exposure, we detected dichlorofluorescein (DCF)-positive cells (80%) indicative of reactive oxygen species (H2O2) production, reduced cell viability (40–50%), decreased mitochondrial membrane potential (ΔΨm, ~35–45%), DNA fragmentation (18–30%), and G1-arrested cell cycle in the MSCs. 6-OHDA exposure increased the expression of the transcription factor c-JUN, increased the expression of the mitochondria maintenance Phosphatase and tensin homologue-induced putative kinase 1 (PINK1) protein and increased the expression of pro-apoptotic PUMA, caspase-3 and apoptosis-inducing factor (AIF). 6-OHDA exposure also significantly augmented the oxidation of the oxidative stress sensor, DJ-1. Overexpression of PARKIN in MSCs not only significantly reduced the expression of cell death and oxidative stress markers but also significantly reduced DCF-positive cells (~50% reduction). Discussion 6-OHDA induced apoptosis in MSCs via generation of H2O2, activation of c-JUN and PUMA, mitochondrial depolarization and nuclei fragmentation. Our findings suggest that PARKIN protects MSCs against 6-OHDA toxicity by partly interacting with H2O2, reducing the expression of c-JUN, PUMA, AIF and caspase-3, and maintaining the mitochondrial ΔΨm.

Mendivil-Perez, Miguel et al., 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., This study was partially funded by the following grants: SAF2009-14037 from the Spanish Ministry of Economy and Competitivity (MINECO), CB/10/00238 from the Carlos III Health Institute, GREIB.PT_2010_04 from the CEIBiotic Program of the University of Granada, Spain, and CTS-101 from the Innovation, Science and Business Council, Junta de Andalucía, Spain. The study was carried out within the framework of the “Convenio Marco 206-2012” agreement between the University of Antioquia in Colombia and the University of Granada in Spain. MJ-Del-Rio and CV-P were supported by Colciencias grants #1115-657-740786 (contract 623-2014). MM-P and V-SM are associate researchers. MM-P is funded by the Colciencias, Enlaza-Mundos, and AUIP mobility programs. VS-M was funded by the AUIP mobility program.

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 ≫ 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.

Autosomal recessive Juvenile Parkinsonism (AR-JP) is a chronic, progressive neurodegenerative disorder caused by mutation in the PARKIN gene, and invariably associated with dopaminergic (DAergic) neuronal loss and brain iron accumulation. Since current medical therapy is symptomatic and lacks significant disease-modifying effects, other treatment approaches are urgently needed it. In the present work, we investigate the role of minocycline (MC) in paraquat (PQ)/iron-induced neurotoxicity in the Drosophila TH>parkin-RNAi/+ (w[*]; UAS-parkin-RNAi; TH-GAL4) fly and have shown the following: (i) MC increased life span and restored the locomotor activity of knockdown (KD) transgenic parkin flies in comparison with the control (vehicle) group; (ii) MC at low (0.1 and 0.3mM) and middle (0.5mM) concentrations protected, rescued and prevented KD parkin Drosophila against PQ toxicity. However, MC at high (1mM) concentration aggravated the toxic effect of PQ; (iii) MC protected and rescued DAergic neurons against the PQ toxic effect according to tyrosine hydroxylase (TH)>green-fluorescent protein (GFP) reporter protein microscopy and anti-TH Western blotting analysis; (iv) MC protected DAergic neurons against PQ/iron toxicity; (v) MC significantly abridged lipid peroxidation (LPO) in the protection, rescue and prevention treatment in TH>parkin-RNAi/+ flies against PQ or iron alone or combined (PQ/iron)-induced neuronal oxidative stress (OS). Our results suggest that MC exerts neuroprotection against PQ/iron-induced OS in DAergic neurons most probably by the scavenging activity of reactive oxygen species (ROS), and by chelating iron. Therefore, MC might be a potential therapeutic drug to delay, revert, or prevent AR-JP.

Context: Therapy for leukemia has a limited efficacy. There is a need to search for alternative anti-leukemia therapies. Persea americana Mill var. Hass (Lauraceae) is a tropical fruit (avocado) that might be used against cancer. Objective: To investigate whether P. americana induces death in Jurkat lymphoblastic leukemia cells. Materials and methods: Four ethanol extracts (0.1, 0.5, 1, 2 and 5 mg/mL) from avocado fruit (endocarp, whole seed, seed and leaves) were analyzed against Jurkat cells. Hydrogen peroxide generation by oxidation of 2',7'-dichlorodihydrofluorescein diacetate to the fluorescent compound 2',7'-dichlorfluorescein assay, acridine orange/ethidium bromide staining, flow cytometry analysis of annexin-V/7-amino-actinomycin, mitochondrial membrane potential and immunocytochemistry detection of transcription factor p53, caspase-3 and apoptosis-inducing factor (AIF) were evaluated. Results: Endocarp, seed, whole seed, and leaf (0.1 mg/mL) extracts induced significant apoptosis in Jurkat cells (p 0.001) in an oxidative stress-dependent fashion via mitochondrial membrane depolarization (52-87%), activation of transcription factor p53 (6.3-25.4%), protease caspase-3 (8.3-20%) and predominance of AIF reactivity (20.6-36%) in all extracts. Similar results were obtained with 0.5 mg/mL extracts. However, extract ≥1 mg/mL concentration induced necrosis (100%). Conclusions: P. americana extracts function as a pro-apoptotic compound. Leukemic cells are eliminated through an oxidative stress mechanism. This study contributes to the understanding of the molecular mechanism of the avocado and its therapeutic action on leukemia.

Previous studies have shown that polyphenols might be potent neuroprotective agents in Drosophila melanogaster wild type Canton-S acutely or chronically treated with paraquat (PQ), a selective toxin for elimination of dopaminergic (DAergic) neurons by oxidative stress (OS), as model of Parkinson's disease (PD). This study reports for the first time that knock-down (K-D) parkin Drosophila melanogaster (TH-GAL4; UAS-RNAi-parkin) chronically exposed to PQ (0.1-0.25 mM), FeSO(4) (Fe, 0.1mM), deferoxamine (DFO, 0.01 mM) alone or (0.1mM) PQ in combination with polyphenols propyl gallate (PG, 0.1mM) and epigallocathecin gallate (EGCG, 0.1, 0.5mM) showed significantly higher life span and locomotor activity than untreated K-D flies or treated with (1, 5, 20mM) PQ alone. Whilst gallic acid (GA, 0.1, 0.5mM) alone or in the presence of PQ provoked no effect on K-D flies, epicathecin (EC, 0.5mM) only showed a positive effect on prolonging K-D flies' life span. It is shown that PG (and EGCG) protected protocerebral posterolateral 1 (PPL1) DAergic neurons against PQ. Interestingly, the protective effect of low PQ concentrations, DFO and iron might be explained by a phenomenon known as "hormesis." However, pre-fed K-D flies with (0.1mM) PQ for 7 days and then exposed to (0.25 mM) for additional 8 days affect neither survival nor climbing of K-D Drosophila compared to flies treated with (0.25 mM) PQ alone. Remarkably, K-D flies treated with 0.1mM PQ (7 days) and then with (0.25 mM) PQ plus PG (8 days) behaved almost as flies treated with (0.25 mM) PQ. Taken these data suggest that antioxidant supplements that synergistically act with low pro-oxidant stimuli to prolong and increase locomotor activity become inefficient once a threshold of OS has been reached in K-D flies. Our present findings support the notion that genetically altered Drosophila melanogaster as suitable model to study genetic and environmental factors as causal and/or modulators in the development of autosomal recessive juvenile Parkinsonism (AR-JD)/PD. Most importantly, we have shown for the first time that low amounts of stressors induce a health-promoting extending effect in K-D parkin flies. Altogether our present results open new avenues for the screening, testing and development of novel antioxidant drugs against OS stimuli in neurodegenerative disorders.

Understanding the mechanism(s) by which dopaminergic (DAergic) neurons are eroded in Parkinson's disease (PD) is critical for effective therapeutic strategies. By using the binary tyrosine hydroxylase (TH)-Gal4/UAS-X RNAi Drosophila melanogaster system, we report that Dmp53, basket and drICE gene knockdown in dopaminergic neurons prolong life span (p < 0.05; log-rank test) and locomotor activity (p < 0.05; χ² test) in D. melanogaster lines chronically exposed to (1 mM) paraquat (PQ, oxidative stress (OS) generator) compared to untreated transgenic fly lines. Likewise, knockdown flies displayed higher climbing performance than control flies. Amazingly, gallic acid (GA) significantly protected DAergic neurons, ameliorated life span, and climbing abilities in knockdown fly lines treated with PQ compared to flies treated with PQ only. Therefore, silencing specific gene(s) involved in neuronal death might constitute an excellent tool to study the response of DAergic neurons to OS stimuli. We propose that a therapy with antioxidants and selectively "switching off" death genes in DAergic neurons could provide a means for pre-clinical PD individuals to significantly ameliorate their disease condition.

Leucine-rich repeat kinase 2 (LRRK2) has been linked to familial and sporadic Parkinson's disease. However, it is still unresolved whether LRRK2 in dopaminergic (DAergic) neurons may or may not aggravate the phenotype. We demonstrate that knocking down (KD) the Lrrk gene by RNAi in DAergic neurons untreated or treated with paraquat (PQ) neither affected the number of DAergic clusters, tyrosine hydroxylase (TH) protein levels, lifespan nor locomotor activity when compared to control (i.e. TH/+) flies. KD transgenic Lrrk flies dramatically increased locomotor activity in presence of TH enzyme inhibitor alpha-methyl-para-tyrosine (aMT), whereas no effect on lifespan was observed in both fly lines. Most importantly, KD Lrrk flies had reduced lipid peroxidation (LPO) index alone or in presence of PQ and the antioxidant minocycline (MC, 0.5mM). Taken together, these findings suggest that Lrrk appears unessential for the viability of DAergic neurons in D. melanogaster. Moreover, Lrrk might negatively regulate homeostatic levels of dopamine, thereby dramatically increasing locomotor activity, extending lifespan, and reducing oxidative stress (OS). Our data also indicate that reduced expression of Lrrk in the DAergic neurons of transgenic TH>Lrrk-RNAi/+ flies conferred PQ resistance and absence of neurodegeneration. The present findings support the notion that reduced/suppressed LRRK2 expression might delay or prevent motor symptoms and/or frank Parkinsonism in individuals at risk to suffer autosomal dominant Parkinsonism (AD-P) by blocking OS-induced neurodegenerative processes in the DAergic neurons.

The biometals iron (Fe), manganese (Mn) and copper (Cu) have been associated to Parkinson's disease (PD) and Parkinsonism. In this work, we report for the first time that acute (15 mM for up to 5 days) or chronic (0.5 mM for up to 15 days) Fe, Mn and Cu exposure significantly reduced life span and locomotor activity (i.e. climbing capabilities) in Drosophila melanogaster. It is shown that the concentration of those biometals dramatically increase in Drosophila's brain acutely or chronically fed with metal. We demonstrate that the metal accumulation in the fly's head is associated with the neurodegeneration of several dopaminergic neuronal clusters. Interestingly, it is found that the PPL2ab DAergic neuronal cluster was erode by the three metals in acute and chronic metal exposure and the PPL3 DAergic cluster was also erode by the three metals but in acute metal exposure only. Furthermore, we found that the chelator desferoxamine, ethylenediaminetetraacetic acid, and D: -penicillamine were able to protect but not rescue D. melanogaster against metal intoxication. Taken together these data suggest that iron, manganese and copper are capable to destroy DAergic neurons in the fly's brain, thereby impairing their movement capabilities. This work provides for the first time metal-induced Parkinson-like symptoms in D. melanogaster. Understanding therefore the effects of biometals in the Drosophila model may provide insights into the toxic effect of metal ions and more effective therapeutic approaches to Parkinsonism.

A significant association between HFE gene mutations and the HLA-A*03-B*07 and HLA-A*29-B*44 haplotypes has been reported in the Spanish population. It has been proposed that these mutations are probably connected with Celtic and North African ancestry, respectively. We aimed to find the possible ancestral association between HLA alleles and haplotypes associated with the HFE gene (C282Y and H63D) mutations in 214 subjects from Antioquia, Colombia. These were 18 individuals with presumed hereditary hemochromatosis (“HH”) and 196 controls. The HLA-B*07 allele was in linkage disequilibrium (LD) with C282Y, while HLA-A*23, A*29, HLA-B*44, and B*49 were in LD with H63D. Altogether, our results show that, although the H63D mutation is more common in the Antioquia population, it is not associated with any particular HLA haplotype, whereas the C282Y mutation is associated with HLA-A*03-B*07, this supporting a northern Spaniard ancestry.

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.

Wilson's disease is an autosomal recessive disorder of hepatic copper metabolism caused by mutations in a gene encoding a copper-transporting P-type ATPase. We report the clinical and molecular characterization of six members from three unrelated Colombian kindred. Completed sequence DNA analysis linked to the gene ATP-7B from patient wd-1 revealed a novel A to C transversion in exon 17 at position 3856 (A3856C) of the ATP-7B mRNA resulting in a threonine for proline substitution at position 1232 of the ATP-7B protein (T1232P). Additionally, two novel polymorphisms were detected (2785G:Gly875 in exon 11; and intron at +38 a > c:tgcgcccga in exon 19). All affected individuals were homozygous for the T1232P mutation and displayed neurologic and neuropsychiatric dominant onset. This work expands the knowledge about the number, type, and implication of mutations in WD.

Mutations in parkin are implicated in the pathogenesis of autosomal recessive juvenile parkinsonism (AR-JP) disease. We show that homozygote Cys212Tyr parkin mutation in AR-JP patients renders lymphocytes sensitive to dopamine, iron and hydrogen peroxide stimuli. Indeed, dopamine-induced apoptosis by four alternative mechanisms converging on caspase-3 activation and apoptotic morphology: (1) NF-kappaB-dependent pathway; mitochondrial dysfunction either by (2) H(2)O(2) or (3) hydroxyl exposure and (4) increase of unfolded-protein stress. We also demonstrate that 17beta-estradiol and testosterone prevent homozygote lymphocytes from oxidative stressors-evoked apoptosis. These results may contribute to understanding the relationship between genetic and environmental factors and iron in AR-JP.

The apolipoprotein E (APOE) gene plays a pivotal role in cholesterol metabolism. Since the discovery of the APOE*2 and APOE*4 as the major susceptibility alleles for several diseases including dyslipidemia, atherosclerosis, coronary heart disease, late-onset and early Alzheimer's disease, the APOE genotype might be considered as a potential predictive factor for both epidemiological research and diagnosis.The aim of this study is to report on the polymorphism of the APOE gene in the "Paisa" population from northwest Colombia (Antioquia) to obtain a population baseline of the existing variation in this locus.One thousand and one healthy voluntaries were genotyped for the APOE polymorphism using polymerase chain reaction-restriction fragment length polymorphism technique.The APOE*3/*3 genotype presented the highest frequency (66.33%) and the APOE*4/*4 had the lowest frequency (1.89%). Genotype frequencies comply with Hardy-Weinberg expectations. Allele frequencies obtained for APOE*2, APOE*3 and APOE*4 were 0.075 ± 0.005 (95% CI = 0.063-0.086), 0.814 ± 0.009 (0.797-0.831) and 0.111 ± 0.007 (0.098-0.125), respectively.Although globally the high-to-low APOE frequency follows the E*3E*4E*2 trend, the present APOE frequency data is in disagreement with some reports from South-American countries.

To establish the effect of low (11 mM) and high (55 mM) glucose concentrations (G11, G55) on Jurkat cells exposed to rotenone (ROT, a class 5 mitocan). We demonstrated that ROT induces apoptosis in Jurkat cells cultured in G11 by oxidative stress (OS) mechanism involving the generation of anion superoxide radical (O2∙-, 68%)/hydrogen peroxide (H2O2, 54%), activation of NF-κB (32%), p53 (25%), c-Jun (17%) transcription factors, and caspase-3 (28%), apoptosis-inducing factor (AIF, 36%) nuclei translocation, c-Jun N-terminal kinase (JNK) activation, and loss of mitochondria transmembrane potential (ΔΨm, 62%) leading to nuclei fragmentation (~10% and ~40% stage I-II fragmented nuclei, resp.). ROT induces massive cytoplasmic aggregates of DJ-1 (93%), and upregulation of Parkin compared to untreated cells, but no effect on PINK-1 protein was observed. Cell death marker detection and DJ-1 and Parkin expression were significantly reduced when cells were cultured in G55 plus ROT. Remarkably, metformin sensitized Jurkat cells against ROT in G55. Our results indicate that a high-glucose milieu promotes resistance against ROT/H2O2-induced apoptosis in Jurkat cells. Our data suggest that combined therapy by using mitochondria-targeted damaging compounds and regulation of glucose (e.g., metformin) can efficiently terminate leukemia cells via apoptosis in hyperglycemic conditions.

Recently, it has been demonstrated that there is no obvious correlation between DNA fragmentation (according to Terminal dUTP Nick-End Labeling technique) and the severity of amyloid-beta (Abeta) deposition and neurofibrillary tangle (NFT) formation in patients bearing mutations in presenilin 1[E280A]. Indeed, it was observed in 10 out of 48 brain sections TUNEL-positive labeling, while none showed classical apoptotic morphology. Based on these findings, we were interested to determine whether cortical cells from temporal and hippocampus post mortem brain sections die either by an apoptotic or necrotic process in FAD-brain sections labeled TUNEL positive compared with normal brain subjects labeled TUNEL-negative using electron microscopy (EM). We found that FAD-brain sections labeled TUNEL positive display the typical morphological characteristics of cell death by necrosis i.e. the nuclear chromatin form flocculent aggregates with poorly defined edges and electron lucent (it does not appears black on EM); the chromatin aggregates are irregularly scattered through the nucleus; mitochondria are swoolen with flocculent matrix densities. No apoptotic bodies were observed in any of the brain areas studied. These results may indicate that necrosis is the most generalized cell death process occurring in terminal PS1E280A brains and the DNA fragmentation of nuclei labeled by TUNEL technique may reflect DNA vulnerability. Thus, cell death by necrosis and the accompanying histopathological observations such as severe deposition of amyloid plaques and NFTs, severe gliosis, cortical depopulation, influx of lymphocytes indicative of a chronic inflammation may have an important impact on future therapeutic strategies in the treatment of PS1E280A patients.

Dopamine (DA) in combination with iron (Fe 2+ ) has been demonstrated to induce apoptosis in neuronal-like PC12 cells by an oxidative stress mechanism. To get a better insight of cell death and protective mechanisms in DA/Fe 2+ -induced toxicity, we investigated the effects of DA/Fe 2+ and the antioxidant action of 17β-estradiol (E2) in peripheral blood lymphocytes (PBL). We found that DA/Fe 2+ -induces apoptosis in PBL via a hydrogen peroxide (H 2 O 2 )-mediated oxidative mechanism, which in turn triggers a cascade of molecular events requiring RNA and de novo protein synthesis. We have also demonstrated that E2 prevents significantly DA/Fe 2+ -induced apoptosis in PBL by directly inhibiting the intracellular accumulation of peroxides generated by DA/Fe 2+ -reaction. This protective activity is independent of the presence or activation of the estrogen receptors (ERs). These data further support and validate our previous hypothesis that DA/Fe 2+ /H 2 O 2 could be a general mediator of oxidative stress through a common cell death mechanism in both neuronal and nonneuronal cells. These findings may be particularly relevant to the potential approaches to rescue and prolong the survival of neurons by estrogens in patients with Parkinson's disease (PD).

Recent studies have shown that Fez+ increases the oxidation of monoamines such as serotonin, dopamine and related toxins and that the formed oxidation products can undergo co-valent binding to free sulphydryl groups of proteins such as actin and "serotonin binding proteins" which are present in soluble brain extracts. Here we have tested the ability of ferrous iron to induce (3H)dopamine association to cytoplasmic proteins and we have established that a similar oxi- dation mechanism evidenced in vitro studies could be applied in cell culture. When PC12 cells were incubated with ferrous iron (ferrocene), the binding of (3H)dopamine to proteins was found to be two fold increased with respect to control. The iron is likely to accelerate the oxidation of dopamine to produce quinones which covalently bind to proteins and induce high-molecular protein aggregates. We evidenced that dopaminehron combination induced cell death in undifferentiated PC12 cells via an active cellular process evaluated in terms of morphological and biochemical changes indicative of apoptosis. We also demonstrated induction of lipid peroxidation when dopamine and ferrocene were present in high concentrations. Moreover, ascorbic acid diminished apoptosis but not the lipid peroxidation process. It might indicate that ferrocene and dopamine could produce oxidative stress of a different nature. These results show that the actions of dopamine and iron are essential in the induction of apoptosis and lipid peroxidation. However, there is no necessary causual link between lipid peroxidation and apoptosis. Our data also suggest that iron is capable of increasing the cytotox- icity of dopamine merely by increasing its rate of oxidation and without intervention of the monoamine oxidase B enzyme and, hence, both phenomenons may occur independently from each other in rat pheochromocytoma PC12. These obser- vations may have relevance to the understanding of the mechanism by which dopaminergic neurones are destroyed in some neurodegenerative disorders.

Reactivation of apoptosis appears as an ultimate goal to eliminate cancer cells. By using light and fluorescent microscopy, flow cytometry, immunohistochemistry staining, DNA fragmentation analysis, and pharmacological inhibition, we provide evidence that 2 pathways of cell death induced by glucose-starvation (GS)/oxidative stress (OS) run in parallel: apoptosis-inducing factor (AIF)-dependent and caspase-3 dependent mechanisms. However, the supremacy of 1 pathway over the other one relies on the availability of glucose, which is essential for the proper functioning of antioxidant cellular systems. It is shown that GS generates superoxide anion radical (O(2)(-))/hydrogen peroxide (H(2)O(2)), which are linked to the death ~45%-70% cells by AIF/mitochondria depolarization/chromatin condensation pathway and ~15%-30% death by nuclear factor-kappa B/p53/c-Jun/c-Jun N-terminal kinase /mitochondria depolarization/caspase-3 activation/DNA fragmentation mechanism at 24-48 h. Remarkably, chromatin condensation/nuclei fragmentation appeared to occur partially independent of the loss of the mitochondrial transmembrane potential (ΔΨ(m)) and plasma membrane damage. Interestingly, signaling inhibitors, antioxidants, or glucose protected cells if added immediately to culture devoid of glucose (P0.001), but only vitamin E and glucose significantly rescued cells at 3 h of GS compared to control. Taken together these data suggest that glucose deprivation might efficiently eliminate leukemia cells via apoptosis.

Acute lymphoblastic leukemia is still an incurable disease with resistance to therapy developing in the majority of patients. We investigated the effect of TPEN, an intracellular zinc chelator, in Jurkat and in ex vivo acute lymphoblastic leukemia (ALL) cells resistant to chemotherapy. Changes of nuclei morphology, reactive oxygen species generation, presence of hypodiploid cells, phosphatidylserine translocation, mitochondrial membrane depolarization, immunohistochemical identification of cell death signalling molecules, and pharmacological inhibition were assayed to detect the apoptotic cell death pathways. We found that TPEN induces apoptosis in both types of cells by a molecular oxidative stress pathway involving O(2)(•-) > H(2)O(2) >> NF-κB (JNK/c-Jun) >p53> loss ΔΨ(m)> caspase-3, AIF > chromatin condensation/DNA fragmentation. Interestingly, TPEN induced apoptosis independently of glucose; leukemic cells are therefore devoid of survival capacity by metabolic resistance to treatment. Most importantly, TPEN cytotoxic effect can eventually be regulated by the antioxidant N-acetyl-cysteine and zinc ions. Our data suggest that TPEN can be used as a potential therapeutic prooxidant agent against refractory leukemia. These data contribute to understanding the importance of oxidative stress in the treatment of ALL.

Previous studies have shown that polyphenols might be potent neuroprotective agents in Drosophila melanogaster, a valid model for PD, acutely treated with oxidative stress-stimulants. This study report for the first time that polyphenols exposure prolong life span (P < 0.05 by log-rang test) and restore locomotor activity (i.e., climbing capability, P < 0.05 by χ(2) test) of Drosophila melanogaster chronically exposed to paraquat compared to flies treated with paraquat alone in 1% glucose. We found that (10%) glucose partially prolongs life span and climbing in Drosophila exposed to iron, PQ or in combination, suggesting that both stimuli enhance a movement disorder in a concentration-dependent and temporal-related fashion. Moreover, chronic exposure of (1 mM) PQ/(0.5 mM) iron synergistically affect both survival and locomotor function independently of the temporal order of the exposure to the toxicants, but the survival is modulated in a concentration and temporal fashion by glucose. This investigation is the first to report that Ddc-GAL4 transgenic flies chronically fed with polyphenols increase life span (P < 0.05 by log-rang test) and enhance movement abilities (P < 0.05 by χ(2) test) compared to untreated Ddc-GAL4 or treated with paraquat in 1% glucose. Our present findings support the notion that Drosophila melanogaster might be a suitable model to study genetic, environmental and nutritional factors as causal and/or modulators in the development of PD. Most importantly, according to our model, we have demonstrated for the first time chronic polyphenols exposure as potential therapeutic compounds in the treatment of PD. These findings altogether open new avenues for the screening, testing and development of novel antioxidant drugs against oxidative stress stimuli.

Binding of [3H]serotonin and of [3H]dopamine to serotonin binding proteins (SBP) from soluble extracts of bovine frontal cortex is increased by Fe2+ but not by Fe3+. It was generally believed that Fe2+ first binds to sulfhydryl groups of SBP and that the monoamines form coordination bonds with the trapped iron. We report two series of findings that are incompatible with this mechanism. First, the binding of both radioligands is an irreversible process since it is not diminished when a large excess (1 mM) of serotonin or dopamine is added to a pre-equilibrated mixture of SBP, 0.1 mM Fe2+ and 0.2 microM radioligand. Once formed, binding is not impaired by chelating agents such as ethyleneglycoltetraacetic acid and desferal. Second, the Fe(2+)-stimulated binding is inhibited by reducing agents (sodium ascorbate, vitamin E, sodium metabisulfite) and by agents which deplete superoxide radicals (superoxide dismutase and hydrogen peroxide). Moreover, the effect of Fe2+ can be mimicked by oxidants (sodium periodate, potassium superoxide) and by the generation of superoxide radicals by the xanthine oxidase-catalysed oxidation of xanthine. To integrate these findings, we formulate the hypothesis that Fe2+ reacts with dissolved molecular oxygen to produce superoxide radicals, that these radicals oxidise [3H]serotonin and [3H]dopamine, and that the formed oxidation products bind covalently to cysteine residues of SBP. This alternative mechanism is also based on the ability of reagents which contain or modify sulfhydryl groups to decrease the binding and on the inability of hydroxyl radical scavengers (dimethyl sulfoxide, mannitol, ethanol and thiourea) to do so. Fe2+ is also able to irreversibly inactivate part of the binding sites on SBP (81% of the specific binding of [3H]serotonin, and 61% for [3H]dopamine). This Fe(2+)-mediated inactivation, as well as the covalent nature of the binding, preclude the interpretation of saturation and competition binding data in terms of reversible bimolecular interactions. Yet, such experiments indicate that, at the same concentration, [3H]dopamine binds to 2 to 3 times more sites than [3H]serotonin. Unlabelled dopamine acts also as a potent competitor at all the [3H]serotonin binding sites, whereas unlabelled serotonin only acts as a potent competitor at part (30%) of the [3H]dopamine binding sites. SBP were initially proposed to be involved in the storage, protection and/or transport of serotonin, and recently also of catecholamines. However, these potential functions of SBP can hardly be reconciled with the molecular mechanism of the binding. Moreover, it is conceivable that this binding actually represents an in vitro model for neurodegeneration.

Serotonin binding proteins (SBP) are present in the soluble fraction of bovine retina homogenates. These proteins can be precipitated with 30% ammonium sulphate and their binding and physicochemical characteristics are very similar to those of SBP in bovine and rat brain. Binding of [ 3 H]serotonin to bovine retina SBP requires Fe 2+ but not Fe 3+ . In the presence of an optimal concentration of Fe 2+ (0.1 mM), these proteins behave as a single class of non-cooperative sites for [ 3 H]serotonin ( B max = 242 ± 10 pmol/mg protein. K D = 0.22 ± 0.44 μM). Competition binding studies reveal that serotonin analogs possessing an hydroxyl group on the indole ring and catecholamine analogs possessing an intact catechol moiety are potent competitors ( K i from 0.12 to 0.3 μM). In both cases, the affinity is strongly decreased if aromatic hydroxyl groups are methoxylated. Catecholamine-SBP interactions can also be demonstrated directly by binding experiments with [ 3 H]dopamine. Binding of this catecholamine is greatly enhanced by Fe 2+ , to a lesser extent by Cu 2+ and Mn 2+ , but not by Fe 3+ . The Fe 2+ -dependent binding component is saturable ( B max = 505 ± 30 pmol/mg protein, K D = 0.34 ± 0.04 μM). The SBP from bovine retina show the same physicochemical properties as SBP from bovine and rat brain: they elute immediately after the void volume on a Sephacryl S100 HR (1.6 × 140 cm) gel filtration column (reflecting aggregation) and they migrate with apparent molecular weights of respectively 43 kDa and 57 kDa on native polyacrylamide gel electrophoresis. The serotonin-storing role of SBP in serotonergic neurones has already been well documented. Since the bovine retina is rich in dopamine and almost devoid of serotonin, the present data make us speculate that SBP could also be involved in the housekeeping of dopamine in retinal neurons.

Human peripheral blood lymphocytes have been useful as a putative model of oxidative stress-induced apoptosisfor Parkinson’s disease. The present work shows that rotenone, a mitochondrial complex I inhibitor, induced time- and con-centration-dependent apoptosis in lymphocytes which was mediated by anion superoxide radicals (O2•)) ⁄ hydrogen peroxide,depolarization of mitochondria, caspase-3 activation, concomitantly with the nuclear translocation of transcription factorssuch as NF-jB, p53, c-Jun and nuclei fragmentation. Since insulin-like growth factor-1 (IGF-1) interferes with a cell’s apopto-tic machinery when subjected to several stressful conditions, it is demonstrated here for the first time that IGF-1 effectivelyprotects lymphocytes against rotenone through PI-3K ⁄ Akt activation, down-regulation of p53 and maintenance of mitochon-drial membrane potential independently of ROS generation. These data might contribute to understanding the role played byIGF-1 against oxidative stress stimuli COL0010744

In order to establish causal or protective treatments for Parkinson's disease (PD), it is necessary to identify the cascade of deleterious events that lead to the dysfunction and death of dopaminergic neurons. Paraquat (PQ) is a pesticide used as xenobiotic compound to model PD. However, the mechanism(s) of PQ-induced cell death and the mechanism(s) of cytoprotection in a single cell model are still unknown. In this study, lymphocytes were treated with (0.1-1 mM) PQ. Apoptotic morphology was assessed with acridine orange/ethidium bromide staining. Further evaluation included (i) superoxide radicals, reflected by nitroblue tetrazolium reduction to formazan, (ii) the production of hydrogen peroxide, reflected by rhodamine-positive fluorescent cells, (iii) the generation of hydroxyl radicals, reflected by dimethylsulfoxide and melatonin ( radical)OH scavengers, (iv) activation and/or translocation of NF-kappaB, p53 and c-Jun transcription factors showed by immunocytochemical staining, and by ammonium pyrrolidinedithiocarbamate, pifithrin-alpha and SP600125 inhibition and (V) caspase-3 activation, reflected by caspase Ac-DEVD-cho inhibition. To elucidate the mechanism of cytoprotection, lymphocytes were treated with PQ in the presence of cannabinoids, insulin-like growth factor-1 and glucose. We provide evidence that PQ induces apoptosis in lymphocytes in a concentration- and time-dependent fashion by an oxidative stress mechanism involving O(2)( radical - ), H(2)O(2)/(( radical)OH) generation, simultaneous activation of NF-kappaB/p53/c-Jun transcription factors, mitochondrial depolarization and caspase-3 activation leading to morphological apoptosis. Moreover, dying lymphocytes are protected and rescued from PQ noxious stimuli by direct antioxidant effect by cannabinoids, receptor mediated signaling by IGF-1, and/or energetic protection by glucose. It is concluded that PQ-induced apoptosis in lymphocytes by a mechanism involving reactive oxygen species generation, mitochondrial dysfunction, transcriptional factors and caspase-3 activation. However, this cell death routine can be reversed by the action of cannabinoids, IGF-1 and glucose. These data may provide innovating therapeutic strategies to intervene environmentally or genetically susceptible PD population to oxidative stress.

Cannabinoids have been shown to function as protective agents via receptor-independent and/or receptor-dependent mechanisms against stressful conditions. However, the neuroprotective mechanism of cannabinoids is far from conclusive. Therefore, the genuine antioxidant impact of cannabinoids in vivo is still uncertain. In this study, we demonstrate for the first time that CP55,940, a nonselective CB(1)/CB(2) cannabinoid receptor agonist, significantly protects and rescues Drosophila melanogaster against paraquat (PQ) toxicity via a receptor-independent mechanism. Interestingly, CP55,940 restores the negative geotaxis activity (i.e., climbing capability) of the fly exposed to PQ. Moreover, Drosophila fed with (1-200 microM) SP600125, a specific inhibitor of the stress responsive Jun-N-terminal kinase (JNK) signaling, and 20 mM PQ increased survival percentage and movement function (i.e., climbing capability) when compared to flies only treated with PQ. Taken together our results suggest that exogenous antioxidant cannabinoids can protect against and rescue from locomotor dysfunction in wild type (Canton-S) Drosophila exposed to stress stimuli. Therefore, cannabinoids may offer promising avenues for the design of molecules to prevent, delay, or ameliorate the treatment of population at high risk of suffering Parkinson disease.

Dear Editor, Alzheimer’s disease (AD, OMIM#104300) is a progressive neurodegenerative disorder characterized by memory loss and cognitive and behavioral abilities impairment. At least three genes have consistently been associated with familial AD (FAD): Aβ precursor protein (APP, OMIM#104760), presenilin-1 (PSEN-1, OMIM#104311) and presenilin-2 (PSN2, OMIM#600759). Noticeably, a single base pair change from A to C (c.839A>C) at codon 280 (gAa-gCa) which results in a Glu-to-Ala substitution in PSEN-1 causes FAD in a large Colombian population in Antioquia region, northwest Colombia [1]. Given that this community is a genetic isolate population [2], this population is a unique resource for the study of modifier genes. Hereditary hemochromatosis (HH, OMIM#235200) is an autosomal recessive disorder of excessive iron uptake and deposition and damage to several organs of the body. HH is most often caused by at least two missense mutations in the gene HFE (chromosome 6p21.3): the c.C187G mutation of exon 2, which results in H63D change at protein translation; and the c.G845A mutation of exon 4, which results in C282Y. Interestingly, brains from AD patients have shown accumulation of iron [3]. Consequently, it is reasonable to think that HFE mutations may be pathological mediator of AD by contributing to brain iron deposition and/or earlier manifestation of dementia symptoms in AD patients. The aim of this study was to examine the allele frequencies of HFE mutations (H63D, C282Y) and their influence on the age of disease onset in a group of patients affected by FAD bearing the E280A mutation in PSEN-1 gene. This investigation was approved by the Ethical Committee of the University of Antioquia (Colombia), and it was supported by Colciencias grants #1115-041-8113 to CVP. A total of 105 individuals (67 women, 38 men) previously genotyped as E280A in PSEN-1 and 220 (117 women, 103 men, range age 18–65 years) non-demented controls, not relatives of FAD patients, were screened for H63D and C282Y in HFE gene by polymerase chain reaction method using the primers sequences reported elsewhere. Amplification mixtures were digested with Mbo I (exon 2) and Rsa I (exon 4) and subsequently analyzed by restriction fragment length polymorphism. Given that the age at onset FAD data (defined as the age at which the patient presented with memory impairment that interferes notably with daily activities) was only available from 75 individuals (52 women, 23 men; mean age of onset at 44.30±5.13; range 33–55 years), they were selected for associative studies between FAD PSN-1 and HH mutations. Tables 1 and 2 show the results of genotype and allelic analysis, respectively. Of notice, the H63D genotype frequencies of healthy normal individuals and FAD patients displayed almost identical distributions for the wild-type, heterozygous, and homozygous conditions [χ(2)=0.43, p=0.8061]. This genotype in both cohorts was at Hardy–Weinberg equilibrium. Similarly, there were no statistically significant differences in the allele frequencies between FAD patients and controls who were wild-type, heterozygous, and homozygous for the H63D alteration [χ(1)=0.39, p=0.5319]. In contrast, there were neither homozygote nor heterozygous for the C282Y mutation in the cohort of FAD patients, and one heterozygote was found among the control group. Given that neither the genotype nor the allelic frequencies of Ann Hematol (2008) 87:671–673 DOI 10.1007/s00277-008-0467-y

Hydrogen peroxide (H(2)O(2)) has been implicated as a key molecule in arresting embryonic development; however, its mechanism of action is not fully established. The aim of the present study was to determine the chronological generation of H(2)O(2) from oocyte to morula, and to examine the relationship of H(2)O(2) with loss of mitochondrial membrane potential, nuclear factor kappa-B (NF-kappaB), p53, caspase-3 activation, and cell death in bovine embryos in vitro. Accordingly, superoxide anion radicals were detected between 32 and 120 h after in vitro fertilization, but higher percentages of oxygen radicals were found in non-competent embryos (n=73, 22 to 34%) than in competent embryos (n=73, 0 to 1%; P

The role of insulin-like growth factor (IGF-1) as neural survival factor for the treatment of Alzheimer's disease has recently gained attention. The present study shows that IGF-1 protects lymphocytes from (10, 30 microM) Abeta[(25-35)] and (25, 50, 100 microM) H(2)O(2)-induced apoptosis through NF-kappaB activation and p53 down regulation involving the phosphoinositide 3-kinase (PI-3K)-dependent pathway as demonstrated by using either (25 microM) LY294002 (PI-3K inhibitor), (10 nM) ammonium pyrrolidinedithiocarbamate (PDTC; NF-kappaB inhibitor), 50 nM pifithrin-alpha (PFT; p53 inhibitor) or by using immunocytochemistry detection of NF-kappaB and p53 transcription factors activation. Importantly, IGF-1, PDTC and PFT were able to protect and rescue lymphocytes pre-exposed to 10 muM Abeta[(25-35)], even when the three compounds were added up-to 12 h post- Abeta[(25-35)] exposure. Altogether these results suggest that survival/rescue of lymphocytes from Abeta[(25-35)] toxicity is determined by p53 inactivation via IGF-1/ PI-3K pathway.

The Aβ deposition in the neuritic plaques is one of the major neuropathological hallmarks of the Alzheimer disease (AD). Studies in vitro have demonstrated that the Aβ 25–35 fragment, which contains the cytotoxic functional sequence of the amyloid peptide, induces neurotoxicity and cell death by apoptosis. Despite intense investigations, a complete picture of the precise molecular cascade leading to cell death in a single cellular model is still lacking. In this study, we provide evidence that Aβ 25–35 induce apoptosis either alone or in presence of iron in peripheral blood lymphocytes cells (PBL) in a concentration-dependent fashion by an oxidative stress mechanism involving: (1) the production of hydrogen peroxide (H 2 O 2 ), reflected by rhodamine-positive fluorescent cells, (2) activation and/or translocation of NF-κB, p53 and c-Jun transcription factors showed by immunocytochemical diaminobenzidine positive nuclei, (3) activation of NF-κB complex by electrophoretic mobility shift assay/immuno-blotting/and ammonium pyrrolidinedithiocarbamate (PDTC) inhibition, (4) caspase-3 activation, reflected by caspase Ac-DEVD-cho inhibition, (5) mRNA synthesis de novo according to actinomycin D cell death inhibition. These results are consistent with the notion that the Aβ 25–35 /H 2 O 2 generation precede the apoptotic process and that once H 2 O 2 is generated, it is able to trigger a specific cell death signalisation. Thus, taken together these results, we present a well-ordered cascade of the major molecular events leading PBL to apoptosis. These results may contribute to explain the importance of Aβ alone or in the presence of redox-available iron in association with Aβ plaques (and neurofibrillary tangles) in AD brains and the significant role played by H 2 O 2 as a second messenger of death signal in some degenerative diseases linked to oxidative stress stimuli.

The destruction of dopaminergic and serotonergic nerve cells by selective 6-hydroxydopamine (6-OHDA), 5,6-dihydroxytryptamine (5,6-DHT) and 5,7-dihydroxytryptamine (5,7-DHT), respectively, is a commonly used tool to investigate the mapping of neuronal pathways, elucidation of function and to mimic human neurodegenerative disease such as Parkinson’s and Alzheimer’s diseases. Despite intense investigations, a complete picture of the precise molecular cascade leading to cell death in a single cellular model is still lacking. In this study, we provide evidence that 6-OHDA, 5,6- and 5,7-DHT toxins-induced apoptosis in peripheral blood lymphocytes cells in a concentration-dependent fashion by a common oxidative mechanism involving: (1) the oxidation of toxins into quinones and production of the by-product hydrogen peroxide, reflected by desipramine—a monoamine uptake blocker—and antioxidants inhibition, (2) activation and/or translocation of nuclear factor-κB, p53 and c-Jun transcription factors, showed by immunocytochemical diaminobenzidine-positive stained nuclei, (3) caspase-3 activation, reflected by caspase Ac-DEVD-CHO inhibition, (4) mRNA and protein synthesis de novo according to cycloheximide and actinomycin D cell death inhibition. These results are consistent with the notion that uptake and intracellular autoxidation of those toxins precede the apoptotic process and that once H 2 O 2 is generated, it is able to trigger a specific cell death signalisation. Thus, taken together these results, we present an ordered cascade of the major molecular events leading peripheral blood lymphocytes to apoptosis. These results may contribute to explain the importance of H 2 O 2 as a second messenger of death signal in some degenerative diseases linked to oxidative stress stimuli.

The soluble serotonin-binding proteins (SBP) present in the adrenal medulla and in chromaffin cells, are very similar to those reported for the bovine brain and retina. Binding of [3H]serotonin and [3H]dopamine to these SBP is increased by Fe2+ but not by Fe3+. At an optimal concentration of Fe2+ (0.1 mM) these proteins behave as a single class of non-cooperative sites for [3H]serotonin (Bmax = 124 +/- 28 pmol/mg protein, KD = 0.51 +/- 0.13 microM) and [3H]dopamine (Bmax = 685 +/- 118 pmol/mg protein, KD = 0.46 +/- 0.06 microM). Binding of [3H]dopamine is also increased by Cu2+ and Mn2+, but to a lesser extent than by Fe2+. Catecholamines are good competitors for [3H]serotonin binding (Ki = 0.31 microM for dopamine, 0.6 microM for adrenaline and 0.9 microM for noradrenaline). The serotonin binding proteins from adrenal medulla elute in the void volume of a Sephacryl 100 HR gel filtration column, reflecting aggregation, and migrate mainly with an apparent molecular weight of 45 kDa in native polyacrylamide gel electrophoresis experiments. Subcellular localization studies and release experiments suggest that SBP are not present in chromaffin granules, but in the cytosol of purified chromaffin cells. The present data suggest that these proteins must have other functions than storing monoamines in synaptic vesicles.