AI molecular property prediction for Parkinson's Disease reveals potential repurposing drug candidates based on the increase of the expression of PINK1.

BACKGROUND AND OBJECTIVE: Parkinson's Disease (PD), a common neurodegenerative disorder and one of the major current challenges in neuroscience and pharmacology, may potentially be tackled by the modern AI techniques employed in drug discovery based on molecular property prediction. The aim of our study was to explore the application of a machine learning setup for the identification of the best potential drug candidates among FDA approved drugs, based on their predicted PINK1 expression-enhancing activity. METHODS: Our study relies on supervised machine learning paradigm exploiting in vitro data and utilizing the scaffold splits methodology in order to assess model's capability to extract molecular patterns and generalize from them to new, unseen molecular representations. Models' predictions are combined in a meta-ensemble setup for finding new pharmacotherapies based on the predicted expression of PINK1. RESULTS: The proposed machine learning setup can be used for discovering new drugs for PD based on the predicted increase of expression of PINK1. Our study identified nitazoxanide as well as representatives of imidazolidines, trifluoromethylbenzenes, anilides, nitriles, stilbenes and steroid esters as the best potential drug candidates for PD with PINK1 expression-enhancing activity on or inside the cell's mitochondria. CONCLUSIONS: The applied methodology allows to reveal new potential drug candidates against PD. Next to novel indications, it allows also to confirm the utility of already known antiparkinson drugs, in the new context of PINK1 expression, and indicates the potential for simultaneous utilization of different mechanisms of action.

Radical Scavenging Is Not Involved in Thymoquinone-Induced Cell Protection in Neural Oxidative Stress Models.

Thymoquinone (TQ), an active compound from Nigella sativa seeds, is often described as a pharmacologically relevant compound with antioxidative properties, while the synthesis of TQ in the plant via oxidations makes it inapplicable for scavenging radicals. Therefore, the present study was designed to reassess the radical scavenging properties of TQ and explore a potential mode of action. The effects of TQ were studied in models with mitochondrial impairment and oxidative stress induced by rotenone in N18TG2 neuroblastoma cells and rotenone/MPP+ in primary mesencephalic cells. Tyrosine hydroxylase staining revealed that TQ significantly protected dopaminergic neurons and preserved their morphology under oxidative stress conditions. Quantification of the formation of superoxide radicals via electron paramagnetic resonance showed an initial increase in the level of superoxide radicals in the cell by TQ. Measurements in both cell culture systems revealed that the mitochondrial membrane potential was tendentially lowered, while ATP production was mostly unaffected. Additionally, the total ROS levels were unaltered. In mesencephalic cell culture under oxidative stress conditions, caspase-3 activity was decreased when TQ was administered. On the contrary, TQ itself tremendously increased the caspase-3 activity in the neuroblastoma cell line. Evaluation of the glutathione level revealed an increased level of total glutathione in both cell culture systems. Therefore, the enhanced resistance against oxidative stress in primary cell culture might be a consequence of a lowered caspase-3 activity combined with an increased pool of reduced glutathione. The described anti-cancer ability of TQ might be a result of the pro-apoptotic condition in neuroblastoma cells. Our study provides evidence that TQ has no direct scavenging effect on superoxide radicals.

Oxoglutarate dehydrogenase complex controls glutamate-mediated neuronal death.

Brain injury is accompanied by neuroinflammation, accumulation of extracellular glutamate and mitochondrial dysfunction, all of which cause neuronal death. The aim of this study was to investigate the impact of these mechanisms on neuronal death. Patients from the neurosurgical intensive care unit suffering aneurysmal subarachnoid hemorrhage (SAH) were recruited retrospectively from a respective database. In vitro experiments were performed in rat cortex homogenate, primary dissociated neuronal cultures, B35 and NG108-15 cell lines. We employed methods including high resolution respirometry, electron spin resonance, fluorescent microscopy, kinetic determination of enzymatic activities and immunocytochemistry. We found that elevated levels of extracellular glutamate and nitric oxide (NO) metabolites correlated with poor clinical outcome in patients with SAH. In experiments using neuronal cultures we showed that the 2-oxoglutarate dehydrogenase complex (OGDHC), a key enzyme of the glutamate-dependent segment of the tricarboxylic acid (TCA) cycle, is more susceptible to the inhibition by NO than mitochondrial respiration. Inhibition of OGDHC by NO or by succinyl phosphonate (SP), a highly specific OGDHC inhibitor, caused accumulation of extracellular glutamate and neuronal death. Extracellular nitrite did not substantially contribute to this NO action. Reactivation of OGDHC by its cofactor thiamine (TH) reduced extracellular glutamate levels, Ca2+ influx into neurons and cell death rate. Salutary effect of TH against glutamate toxicity was confirmed in three different cell lines. Our data suggest that the loss of control over extracellular glutamate, as described here, rather than commonly assumed impaired energy metabolism, is the critical pathological manifestation of insufficient OGDHC activity, leading to neuronal death.

The Reassessed Impact of Nicotine against Neurotoxicity in Mesencephalic Dopaminergic Cell Cultures and Neuroblastoma N18TG2 Cells.

Neuroprotective effects of nicotine are still under debate, so further studies on its effectiveness against Parkinson's disease are required. In our present study, we used primary dopaminergic cell cultures and N18TG2 neuroblastoma cells to investigate the effect of nicotine and its neuroprotective potential against rotenone toxicity. Nicotine protected dopaminergic (tyrosine hydroxylase immunoreactive) neurons against rotenone. This effect was not nAChR receptor-dependent. Moreover, the alkaloid at a concentration of 5 µM caused an increase in neurite length, and at a concentration of 500 µM, it caused an increase in neurite count in dopaminergic cells exposed to rotenone. Nicotine alone was not toxic in either cell culture model, while the highest tested concentration of nicotine (500 µM) caused growth inhibition of N18TG2 neuroblastoma cells. Nicotine alone increased the level of glutathione in both cell cultures and also in rotenone-treated neuroblastoma cells. The obtained results may be helpful to explain the potential neuroprotective action of nicotine on neural cell cultures.

Motor Cortex and Hippocampus Display Decreased Heme Oxygenase Activity 2 Weeks After Ventricular Fibrillation Cardiac Arrest in Rats.

Heme oxygenase (HO) and biliverdin reductase (BVR) activities are important for neuronal function and redox homeostasis. Resuscitation from cardiac arrest (CA) frequently results in neuronal injury and delayed neurodegeneration that typically affect vulnerable brain regions, primarily hippocampus (Hc) and motor cortex (mC), but occasionally also striatum and cerebellum. We questioned whether these delayed effects are associated with changes of the HO/BVR system. We therefore analyzed the activities of HO and BVR in the brain regions Hc, mC, striatum and cerebellum of rats subjected to ventricular fibrillation CA (6 min or 8 min) after 2 weeks following resuscitation, or sham operation. From all investigated regions, only Hc and mC showed significantly decreased HO activities, while BVR activity was not affected. In order to find an explanation for the changed HO activity, we analyzed protein abundance and mRNA expression levels of HO-1, the inducible, and HO-2, the constitutively expressed isoform, in the affected regions. In both regions we found a tendency for a decreased immunoreactivity of HO-2 using immunoblots and immunohistochemistry. Additionally, we investigated the histological appearance and the expression of markers indicative for activation of microglia [tumor necrosis factor receptor type I (TNFR1) mRNA and immunoreactivity for ionized calcium-binding adapter molecule 1 (Iba1])], and activation of astrocytes [immunoreactivity for glial fibrillary acidic protein (GFAP)] in Hc and mC. Morphological changes were detected only in Hc displaying loss of neurons in the cornu ammonis 1 (CA1) region, which was most pronounced in the 8 min CA group. In this region also markers indicating inflammation and activation of pro-death pathways (expression of HO-1 and TNFR1 mRNA, as well as Iba1 and GFAP immunoreactivity) were upregulated. Since HO products are relevant for maintaining neuronal function, our data suggest that neurodegenerative processes following CA may be associated with a decreased capacity to convert heme into HO products in particularly vulnerable brain regions.

Autophagy inhibitor 3-methyladenine could not modulate rotenone neurotoxicity in primary mesencephalic cell culture / El inhibidor de la autofagia 3-metiladenina no logra modular la neurotoxicidad de la rotenona en el cultivo primario de células mesencefálicas

Dysregulated autophagy, whether excessive or downregulated, has been thought to be associated with neurodegenerative disorders including Parkinson's disease. Accordingly, the present study was carried out to investigate whether 3-methyladenine, an autophagy inhibitor, can modulate the effects of rotenone on dopaminergic neurons in primary mesencephalic cell culture. Cultures were prepared from embryonic mouse mesencephala at gestation day 14. Four groups of cultures were treated on the 10th DIV for 48 h as follows: the first was kept as an untreated control, the second was treated with 3-methyladenine alone (1, 10, 100, 200 mM), the third was treated with 20 nM rotenone and the fourth was co-treated with 20 nM rotenone and 3-methyladenine (1, 10, 100, 200 mM). On the 12th DIV, cultured cells were stained immunohistochemically against tyrosine hydroxylase and culture media were used to measure the levels of lactate dehydrogenase. 3methyladenine had no effects on both the survival of dopaminergic neurons and the release of lactate dehydrogenase. Rotenone significantly decreased the number of dopaminergic neurons and increased the levels of lactate dehydrogenase in the culture media. When cultures concomitantly treated with 3-methyladenine and rotenone, 3-methyladenine had no effect against rotenone-induced dopaminergic cell damage and lactate dehydrogenase release into the culture medium. In conclusion, the autophagy inhibitor 3-methyladenine could not modulate rotenone-induced dopaminergic cell damage in primary mesencephalic cell culture.

Se estima que la autofagia desregulada, ya sea excesiva o con baja regulación, está asociada con trastornos neurodegenerativos, incluyendo la enfermedad de Parkinson. En consecuencia, el se realizó este estudio para investigar si la 3metiladenina, un inhibidor de la autofagia,puede modular los efectos de la rotenona en las neuronas dopaminérgicas en el cultivo primario de células mesencefálicas. Los cultivos se prepararon a partir de mesencéfalo de ratón embrionario el día 14 de gestación. Cuatro grupos de cultivos se trataron en el 10º DIV durante 48 h de la siguiente manera: el primer grupo se mantuvo como un control no tratado, el segundo se trató con 3-metiladenina sola (1, 10, 100, 200 mM), el tercer grupo se trató con rotenona 20 nM y el cuarto se trató conjuntamente con rotenona 20 nM y 3-metiladenina (1, 10, 100, 200 mM). En el 12º DIV; las células cultivadas fueron tratadas mediante tinción inmunohistoquímica en tirosina hidroxilasa y se usaron medios de cultivo para medir los niveles de lactato deshidrogenasa. La 3-metiladenina no tuvo efectos tanto en la supervivencia de las neuronas dopaminérgicas como en la liberación de lactato deshidrogenasa. La rotenona disminuyó significativamente el número de neuronas dopaminérgicas y se observó un aumento de los niveles de lactato deshidrogenasa en los medios de cultivo. Cuando los cultivos tratados concomitantemente con 3-metiladenina y rotenona, la 3metiladenina no tuvo efecto contra el daño celular dopaminérgico inducido por la rotenona y la liberación de lactato deshidrogenasa en el medio de cultivo. En conclusión, el inhibidor de la autofagia 3-metiladenina no moduló el daño celular dopaminérgico inducido por la rotenona en el cultivo celular mesencefálico primario.

Minocycline protects against acrylamide-induced neurotoxicity and testicular damage in Sprague-Dawley rats.

This study investigated the protective effects of minocycline against acrylamide (ACR)-induced neurotoxicity and testicular damage in Sprague-Dawley rats. Forty rats were divided into five groups (eight rats each). Group I received saline (0.5 mL/rat) daily for 10 days and served as the untreated control group. Group II received ACR (30 mg/kg body weight (b.w.)) daily for 10 days. Group III received ACR (30 mg/kg b.w.) daily for 10 days and subsequently minocycline (60 mg/kg b.w.) for five days. Group IV received ACR (30 mg/kg b.w.) daily for 10 days followed by saline for five days and served as the control group for the ACR-minocycline-treated group. Group V received minocycline (60 mg/kg b.w.) for five days. All treatments were administered orally. Rats in group I and V showed normal locomotor behavior and normal histology of the brain and testes. Administration of ACR (Group II and IV) resulted in weight loss and gait abnormalities. Furthermore, neuronal degeneration in the hippocampus and cerebellum and degeneration of the seminiferous tubular epithelium with formation of spermatid giant cells were observed. Ultrastructurally, ACR specifically damaged spermatogonia and spermatocytes. Acrylamide was also seen to cause a significant increase of malondialdehyde levels in the brain and testes. Treatment of ACR-administered rats with minocycline (Group III) significantly alleviated the loss of body weight and improved locomotor function. Minocycline also ameliorated neuronal degeneration and seminiferous tubular damage and decreased malondialdehyde concentrations. In conclusion, minocycline protects against neurotoxic effects of acrylamide and seminiferous tubular damage. Decreasing lipid peroxidation by minocycline might play a role in such protection.

Cannabidiol Protects Dopaminergic Neurons in Mesencephalic Cultures against the Complex I Inhibitor Rotenone Via Modulation of Heme Oxygenase Activity and Bilirubin.

Phytocannabinoids protect neurons against stressful conditions, possibly via the heme oxygenase (HO) system. In cultures of primary mesencephalic neurons and neuroblastoma cells, we determined the capability of cannabidiol (CBD) and tetrahydrocannabinol (THC) to counteract effects elicited by complex I-inhibitor rotenone by analyzing neuron viability, morphology, gene expression of IL6, CHOP, XBP1, HO-1 (stress response), and HO-2, and in vitro HO activity. Incubation with rotenone led to a moderate stress response but massive degeneration of dopaminergic neurons (DN) in primary mesencephalic cultures. Both phytocannabinoids inhibited in-vitro HO activity, with CBD being more potent. Inhibition of the enzyme reaction was not restricted to neuronal cells and occurred in a non-competitive manner. Although CBD itself decreased viability of the DNs (from 100% to 78%), in combination with rotenone, it moderately increased survival from 28.6% to 42.4%. When the heme degradation product bilirubin (BR) was added together with CBD, rotenone-mediated degeneration of DN was completely abolished, resulting in approximately the number of DN determined with CBD alone (77.5%). Using N18TG2 neuroblastoma cells, we explored the neuroprotective mechanism underlying the combined action of CBD and BR. CBD triggered the expression of HO-1 and other cell stress markers. Co-treatment with rotenone resulted in the super-induction of HO-1 and an increased in-vitro HO-activity. Co-application of BR completely mitigated the rotenone-induced stress response. Our findings indicate that CBD induces HO-1 and increases the cellular capacity to convert heme when stressful conditions are met. Our data further suggest that CBD via HO may confer full protection against (oxidative) stress when endogenous levels of BR are sufficiently high.

Neurotoxic effects of acrylamide on dopaminergic neurons in primary mesencephalic cell culture.

INTRODUCTION: Exposure to acrylamide is increasing worldwide as a result of its heavy use in industry and formation in carbohydrate-rich food cooked at high temperature. Despite its neurotoxicity, no studies have shown its toxic effects on dopaminergic neurons yet. Therefore, the current study was carried out to show whether acrylamide adversely affects primary cultured dopaminergic neurons. MATERIAL AND METHODS: Acrylamide (0.001, 0.01, 0.1, 1, 2 mM) was added to two different groups of primary mesencephalic cell cultures on the 9th day in vitro for 24 and 48 h, respectively. Moreover, a group of cultures was treated with lower concentrations of acrylamide (0.01, 0.05, 0.1, 0.5 mM) on the 6th day in vitro for 5 consecutive days to investigate its long-term effects on dopaminergic neurons. Following each treatment, culture media were obtained for measuring lactate dehydrogenase, and cultured cells were stained immunocytochemically against tyrosine hydroxylase and neuronal nuclear antigens. RESULTS: Treatment of cultures with acrylamide for 48 h significantly reduced the number of dopaminergic neurons, adversely altered the morphology of the surviving neurons and increased levels of lactate dehydrogenase in the culture media. Similar treatment of cultures with acrylamide also resulted in lower numbers of total neuronal cells as shown by a reduced expression of the neuronal nuclear antigen. Prolonged treatment of cultures with lower concentrations of acrylamide slightly reduced the survival of dopaminergic neurons but increased the release of lactate dehydrogenase into the culture media as well. CONCLUSIONS: The current study shows, for the first time, neurotoxicity of acrylamide on dopaminergic neurons in the primary mesencephalic cell culture.

Glutamine regulates mitochondrial uncoupling protein 2 to promote glutaminolysis in neuroblastoma cells.

Mitochondrial uncoupling protein 2 (UCP2) is highly abundant in rapidly proliferating cells that utilize aerobic glycolysis, such as stem cells, cancer cells, and cells of the immune system. However, the function of UCP2 has been a longstanding conundrum. Considering the strict regulation and unusually short life time of the protein, we propose that UCP2 acts as a "signaling protein" under nutrient shortage in cancer cells. We reveal that glutamine shortage induces the rapid and reversible downregulation of UCP2, decrease of the metabolic activity and proliferation of neuroblastoma cells, that are regulated by glutamine per se but not by glutamine metabolism. Our findings indicate a very rapid (within 1 h) metabolic adaptation that allows the cell to survive by either shifting its metabolism to the use of the alternative fuel glutamine or going into a reversible, more quiescent state. The results imply that UCP2 facilitates glutamine utilization as an energetic fuel source, thereby providing metabolic flexibility during glucose shortage. The targeting UCP2 by drugs to intervene with cancer cell metabolism may represent a new strategy for treatment of cancers resistant to other therapies.

Techniques to study phagocytosis and uptake of Leishmania tarentolae by J774 macrophages.

Leishmaniasis is a vector borne parasitic disease affecting millions of people worldwide and is spreading into further areas because of global warming. The development of new active substances against these single-cell eukaryotic parasites is of great importance. Leishmania tarentolae promastigotes (LtP) are non-pathogenic for mammals and serve as model organisms for pathogenic Leishmania in basic research. However, it is important to refine methods to study the process of the infection of mammalian macrophages by LtP and pathogenic Leishmania. Important stages of the infection are phagocytosis by macrophages and multiplication of Leishmania amastigotes in the phagolysosome of macrophages. In this study, advanced methods using electron spin resonance (ESR) spectroscopy and genetically manipulated LtP were used to monitor the infection of adherent J774 macrophages with LtP. An ESR method was established to detect the formation of superoxide radicals directly in adherent J774 cells and to investigate the effect of LtP on this activity. J774 cells responded with a burst of superoxide radicals in the presence of phorbol myristate acetate as positive control. In contrast, challenging J774 cells with LtP resulted in a much lower burst of superoxide radicals. To facilitate LtP detection in the phagolysosome of J774 macrophages, LtP expressing enhanced green fluorescent protein (EGFP-LtP) were constructed. After different infection times with EGFP-LtP, the J774 cells were visualized by phase contrast microscopy and the cell number was determined. The intramacrophage Leishmania tarentolae amastigotes (LtA) expressing EGFP were detected by fluorescence microscopy and then counted with ImageJ. These experiments showed that LtP are taken up by J774 cells and form intraphagolysosomal amastigotes. LtA under our conditions multiplied intracellularly and were able to persist about 48 h in J774 cells. These experiments showed that ESR spectroscopy of attached macrophages and the use of the EGFP-LtP are suitable methods to study the initial phase of Leishmania infection in vitro.

Rotenone: from modelling to implication in Parkinson's disease.

Rotenone ([2R-(2α,6aα,12aα)]-1,2,12,12a-tetrahydro-8,9-dimethoxy-2-(1-methylethenyl)-[1]benzopyran[3,4-b]furo [2,3-h][1]benzopyran-6(6aH)-one) is a naturally occurring compound derived from the roots and stems of Derris, Tephrosia, Lonchocarpus and Mundulea plant species. Since its discovery at the end of the 19th century, rotenone has been widely used as a pesticide for controlling insects, ticks and lice, and as a piscicide for management of nuisance fish in lakes and reservoirs. In 2000, Betarbet et al. reproduced most of the behavioural, biochemical and pathological features of Parkinson's disease (PD) in rotenone-treated rats. Since that time, rotenone has received much attention as it would be one of the environmental neurotoxins implicated in etiopathogenesis of PD. Moreover, it represents a common experimental model to investigate the underlying mechanisms leading to PD and evaluate the new potential therapies for the disease. In the current general review, we aimed to address recent advances in the hazards of the environmental applications of rotenone and discuss the updates on the rotenone model of PD and whether it is implicated in the etiopathogenesis of the disease.

Long-term neurotoxic effects of domoic acid on primary dopaminergic neurons.

Domoic acid, an excitatory neurotoxin produced by certain algae, reaches the food chain through accumulation in some sea organisms. To investigate its long-term neurotoxicity on dopaminergic neurons, prepared primary mesencephalic cell cultures were exposed to different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 8th day in vitro (DIV) for 4 days. On the 12th DIV, culture media were collected for measurement of lactate dehydrogenase and cultured cells were subjected to immunohistochemistry against tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and Hoechst 33342 dyes. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. Domoic acid significantly decreased the number of dopaminergic neurons and adversely affected their morphology, and slightly reduced the expression of neuronal nuclear antigen and glial fibrillary acidic protein. Co-treatment of cultures with domoic acid and the AMPA/KA or NMDA receptor antagonists NBQX and MK-801 rescued significant number of dopaminergic neurons. Domoic acid significantly decreased red:green fluorescence ratio of JC-1 and did not affect production of reactive oxygen species and apoptotic cell death. In conclusions, the present study reveals that long-term treatment of primary mesencephalic cell culture with domoic acid significantly destroyed dopaminergic neurons. This effect appears to be attributed to activation of AMPA/KA and NMDA receptors and mitochondrial damage.

Thiamine preserves mitochondrial function in a rat model of traumatic brain injury, preventing inactivation of the 2-oxoglutarate dehydrogenase complex.

BACKGROUND AND PURPOSE: Based on the fact that traumatic brain injury is associated with mitochondrial dysfunction we aimed at localization of mitochondrial defect and attempted to correct it by thiamine. EXPERIMENTAL APPROACH: Interventional controlled experimental animal study was used. Adult male Sprague-Dawley rats were subjected to lateral fluid percussion traumatic brain injury. Thiamine was administered 1 h prior to trauma; cortex was extracted for analysis 4 h and 3 d after trauma. KEY RESULTS: Increased expression of inducible nitric oxide synthase (iNOS) and tumor necrosis factor receptor 1 (TNF-R1) by 4 h was accompanied by a decrease in mitochondrial respiration with glutamate but neither with pyruvate nor succinate. Assays of TCA cycle flux-limiting 2-oxoglutarate dehydrogenase complex (OGDHC) and functionally linked enzymes (glutamate dehydrogenase, glutamine synthetase, pyruvate dehydrogenase, malate dehydrogenase and malic enzyme) indicated that only OGDHC activity was decreased. Application of the OGDHC coenzyme precursor thiamine rescued the activity of OGDHC and restored mitochondrial respiration. These effects were not mediated by changes in the expression of the OGDHC sub-units (E1k and E3), suggesting post-translational mechanism of thiamine effects. By the third day after TBI, thiamine treatment also decreased expression of TNF-R1. Specific markers of unfolded protein response did not change in response to thiamine. CONCLUSION AND IMPLICATIONS: Our data point to OGDHC as a major site of damage in mitochondria upon traumatic brain injury, which is associated with neuroinflammation and can be corrected by thiamine. Further studies are required to evaluate the pathological impact of these findings in clinical settings.

Neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture.

INTRODUCTION: Domoic acid is a potent marine neurotoxin produced by certain species of the diatom genus Pseudonitzschia. To our knowledge, there are no studies that have investigated neurotoxic effects of domoic acid on dopaminergic neurons. Accordingly, the present study was carried out to investigate the potential neurotoxic effects of domoic acid on dopaminergic neurons in primary mesencephalic cell culture. MATERIAL AND METHODS: Cultures prepared from embryonic mouse mesencephala (total of 250 embryos) were treated with different concentrations of domoic acid (0.1, 1, 10, 100 µM) on the 10th DIV for 48 h. On the 12th DIV, culture media were used for measurement of lactate dehydrogenase and cultured cells were subjected to immunostaining for tyrosine hydroxylase, neuronal nuclear antigen and glial fibrillary acidic protein, and fluorescence staining using H2DCFDA, JC-1 and DAPI stains. Moreover, roles of AMPA/KA and NMDA receptors in domoic acid neurotoxicity were also investigated. RESULTS: Domoic acid significantly decreased the number of dopaminergic neurons, decreased the expression of neuronal nuclear antigen and slightly affected astrocyte populations, and increased the release of lactate dehydrogenase into the culture media. AMPA/KA receptor antagonist NBQX but not NMDA receptor antagonist MK-801 significantly inhibited the neurotoxic effect of domoic acid on dopaminergic neurons. H₂DCFDA, JC-1 and DAPI fluorescence staining, respectively, revealed that DomA slightly raised ROS production, and significantly decreased mitochondrial membrane potential and increased apoptotic cell death of cultured cells. CONCLUSION: The current study presents for the first time the neurotoxic effects of domoic acid on dopaminergic neurons and this effect appears to be attributed to activation of AMPA/KA receptors on dopaminergic neurons.

Recent Advances on the Role of Neurogenesis in the Adult Brain: Therapeutic Potential in Parkinson's and Alzheimer's Diseases.

BACKGROUND: Generation of nascent functional neurons from neural stem cells in the adult brain has recently become largely accepted by the neuroscience community. In adult mammals including humans, the process of neurogenesis has been well documented in two brain regions; the subventricular zone of the lateral ventricles and the subgranular zone in the dentate gyrus of the hippocampus. METHOD: Some evidence has indicated neurogenesis in other regions of the adult mammalian brain such as the neocortex, cerebellum, striatum, amygdala and hypothalamus. These discoveries question a long standing dogma on nervous system regeneration and provide medical science with potential new strategies to harness the process of neurogenesis for treating neurological disabilities and neurodegenerative diseases. CONCLUSION: In this current review, we address the most recent advances on the role of neurogenesis in the adult brain and therapeutic potential in the two most common neurodegenerative disorders, Parkinson's and Alzheimer's diseases.

Tropism, intracerebral distribution, and transduction efficiency of HIV- and SIV-based lentiviral vectors after injection into the mouse brain: a qualitative and quantitative in vivo study.

Lentiviruses are suitable to transfer potential therapeutic genes into non-replicating cells such as neurons, but systematic in vivo studies on transduction of neural cells within the complete brain are missing. We analysed the distribution of transduced cells with respect to brain structure, virus tropism, numbers of transduced neurons per brain, and influence of the Vpx or Vpr accessory proteins after injection of vectors based on SIVsmmPBj, HIV-2, and HIV-1 lentiviruses into the right striatum of the mouse brain. Transduced cells were found ipsilaterally around the injection canal, in corpus striatum and along corpus callosum, irrespective of the vector type. All vectors except HIV-2SEW transduced also single cells in the olfactory bulb, hippocampus, and cerebellum. Vector HIV-2SEW was the most neuron specific. However, vectors PBjSEW and HIV-1SEW transduced more neurons per brain (means 41,299 and 32,309) than HIV-2SEW (16,102). In the presence of Vpx/Vpr proteins, HIV-2SEW(Vpx) and HIV-1SEW(Vpr) showed higher overall transduction efficiencies (30,696 and 27,947 neurons per brain) than PBjSEW(Vpx) (6636). The distances of transduced cells from the injection canal did not differ among the viruses but correlated positively with the numbers of transduced neurons. The presence of Vpx/Vpr did not increase the numbers of transduced neurons. Parental virus type and the vector equipment seem to influence cellular tropism and transduction efficiency. Thus, precision of injection and choice of virus pseudotype are not sufficient when targeted lentiviral vector transduction of a defined brain cell population is required.

Nutrient deprivation in neuroblastoma cells alters 4-hydroxynonenal-induced stress response.

4-hydroxy-2-nonenal (HNE), a toxic lipid peroxidation product, is associated with oxidative damage in cells and involved in various diseases including the initiation and progression of cancer. Cancer cells have a high, adaptable metabolism with a shift from oxidative phosphorylation to glycolysis and rely on high levels of glucose and glutamine as essential nutrients for cell growth. Here we investigated whether the toxic effects of HNE on the mitochondrial membrane potential (MMP) of cancer cells depends on their metabolic state by deprivation of glucose and/or glutamine. The addition of 16 µM HNE to N18TG2 neuroblastoma cells incubated in glucose medium led to a severe reduction of MMP, which was similar to the MMP of cells fed with both glucose and glutamine. In contrast, HNE addition to cells starved in glutamine medium increased their MMP slightly for a prolonged time period and this was accompanied by increased cellular survival. We found that ß-oxidation of HNE did not cause the increased MMP, since the aldehyde dehydrogenase was distinctly more active in cells with glucose medium. However, after blocking fatty acid ß-oxidation in cells starved in glutamine medium with etomoxir, which inhibits carnitine palmitoyltransferase 1, HNE addition induced a strong reduction of MMP similar to cells in glucose medium. Surprisingly, the effect of more toxic 4-oxo-2-nonenal was less pronounced. Our results suggest that in contrast to cells fed with glucose, glutamine-fed cancer cells are capable of ß-oxidizing fatty acids to maintain their MMP to combat the toxic effects of HNE.

THC (Δ9-Tetrahydrocannabinol) Exerts Neuroprotective Effect in Glutamate-affected Murine Primary Mesencephalic Cultures Through Restoring Mitochondrial Membrane Potential and Anti-apoptosis Involving CB1 Receptor-dependent Mechanism.

Aging-related neurodegenerative diseases, such as Parkinson's disease (PD) or related disorders, are an increasing societal and economic burden worldwide. Δ9-Tetrahydrocannabinol (THC) is discussed as a neuroprotective agent in several in vitro and in vivo models of brain injury. However, the mechanisms by which THC exhibits neuroprotective properties are not completely understood. In the present study, we investigated neuroprotective mechanisms of THC in glutamate-induced neurotoxicity in primary murine mesencephalic cultures, as a culture model for PD. Glutamate was administered for 48 h with or without concomitant THC treatment. Immunocytochemistry staining and resazurin assay were used to evaluate cell viability. Furthermore, superoxide levels, caspase-3 activity, and mitochondrial membrane potential were determined to explore the mode of action of this compound. THC protected dopaminergic neurons and other cell types of primary dissociated cultures from glutamate-induced neurotoxicity. Moreover, THC significantly counteracted the glutamate-induced mitochondrial membrane depolarization and apoptosis. SR141716A, a CB1 receptor antagonist, concentration-dependently blocked the protective effect of THC in primary mesencephalic cultures. In conclusion, THC exerts anti-apoptotic and restores mitochondrial membrane potential via a mechanism dependent on CB1 receptor. It strengthens the fact that THC has a benefit on degenerative cellular processes occurring, among others, in PD and other neurodegenerative diseases by slowing down the progression of neuronal cell death. Copyright © 2016 John Wiley & Sons, Ltd.

Comparable Neuroprotective Effects of Pergolide and Pramipexole on Ferrous Sulfate-Induced Dopaminergic Cell Death in Cell Culture.

BACKGROUND: Dopamine agonists are utilized clinically as an initial treatment in younger Parkinson's disease patients to delay the side effects associated with commencement of levodopa medication. These agonists also serveas adjunctive therapeutics with levodopa to lower the incidence of adverse motor symptoms in advanced stages of the disease. OBJECTIVES: To compare the neuroprotective effects of the dopamine agonists pergolide and pramipexole on ferrous sulfate-induced neurotoxicity in dopaminergic neurons from primary mesencephalic cell culture. METHODS: Pergolide (0.001-1 µM) and pramipexole (0.01-200 µM) were administered to 8 day primary murine mesencephalic cultures for 24 h. in the presence or absence of desferal, sulpiride or cycloheximide. Ferrous sulfate (450 µM) was then added for 24 hrs. Lactate dehydrogenase was assayed in the supernatant, glutathione concentrations measured in cell lysates and fixed cells were stained for tyrosine hydroxylase. RESULTS: Ferrous sulphate induced neurotoxity in cultures (p<0.0001) was abolished in the presence of the iron chelator desferal (p<0.008). Both pergolide (p<0.0001) and pramipexole (p<0.0001) significantly protected dopaminergic neurons against ferrous sulfate induced neurotoxicity and pramipexole helped preserve neurite morphology. Pramipexole treatment significantly reduced lactate dehydrogenase release (p<0.0001) as a measure of cellular injury. The dopamine receptor antagonist sulpiride (p<0.0001) and the protein synthesis inhibitor cycloheximide (p<0.0001) reduced the neuroprotective effects of pergolide indicating the involvement receptor stimulation and de novo protein synthesis in pergolide-mediated neuroprotection. Pramipexole also significantly reversed the decrease in cellular glutathione concentrations induced by ferrous sulfate (p<0.001). CONCLUSION: Both pergolide and pramipexole protect dopaminergic neurons against the neurotoxicity of ferrous sulfate. Pergolide specifically protects dopaminergic neurons through activation of dopamine receptors and de novo protein synthesis whereas pramipexole shows an overall effect through an antioxidant mechanism.