Testing efficacy of the nicotine protection of the substantia nigra pars compacta in a rat Parkinson disease model. Ultrastructure study.

Parkinson's disease (PD) is characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) with subsequent motor manifestations. This study aimed to assess the ameliorative effects of nicotine, in rotenone-induced PD rat model. Thirty adult male Albino Wistar rats were divided into three equal groups. Group I received an injection of normal saline. Group II received subcutaneous injection of rotenone at a dose of 1.5 mg/kg every other day. Group III received rotenone in the same previous dose and nicotine at a dose of 1.5 mg/kg daily. After 11 days of treatment, body weight (BW) and rat motor behavior were estimated. Specimens from the midbrain were processed for light and electron microscopy. The expression of tyrosine hydroxylase (TH), α-synuclein, and GFAP was examined. Serum levels of total antioxidant capacity (TAC) and malondialdehyde (MDA), and striatal levels of dopamine (DA) were analyzed. Group III revealed a significant improvement in BW and motor activity. Nicotine upregulated the expression of TH, downregulated the expression of α-synuclein and GFAP. The levels of MDA and TAC were improved but were still far from those of the control. Striatal DA levels increased. Nicotine activated the neurons and glial cells. The vascular endothelium, however, did not elicit improvement. Although nicotine ameliorated the loss of the dopaminergic neurons and motor deficit, it did not show improvement of vascular endothelium. It is still necessary to examine nicotin's ability to maintain the dopaminergic neurons in a good functioning state.

Lactate and Pyruvate Activate Autophagy and Mitophagy that Protect Cells in Toxic Model of Parkinson's Disease.

Intracellular quality control regulated by autophagy process is important for maintenance of cellular homeostasis. Deregulation of autophagy and more specifically mitophagy leads to accumulation of the misfolded proteins and damaged mitochondria that in turn leads to the cell loss. Alteration of autophagy and mitophagy has shown to be involved in the number of disorders including neurodegenerative diseases. Autophagy and mitophagy could be activated by short-time acidification of the cytosol; however, most of the compounds which can induce it are toxic. Here, we tested several organic compounds which are involved in cellular metabolism on their ability to change intracellular pH and induce mitophagy/autophagy. We have found that lactate and pyruvate are able to reduce intracellular pH in non-toxic concentrations. Short-term (2 h) and long-term (24 h) incubation of the cells with lactate and pyruvateinduced mitophagy and autophagy. Incubation of the SH-SY5Y cells or primary neurons and astrocytes with lactate or pyruvate also activated mitophagy and autophagy after MPP + treatment that led to recovery of mitochondrial function and protection of these cells against apoptotic and necrotic death. Thus, pyruvate- or lactate-induced acidification of cytosol activates cell protective mitophagy and autophagy.

Metabolomics Fingerprint Induced by the Intranigral Inoculation of Exogenous Human Alpha-Synuclein Oligomers in a Rat Model of Parkinson's Disease.

Parkinson's disease (PD) is considered a synucleinopathy because of the intraneuronal accumulation of aggregated α-synuclein (αSyn). Recent evidence points to soluble αSyn-oligomers (αSynO) as the main cytotoxic species responsible for cell death. Given the pivotal role of αSyn in PD, αSyn-based models are crucial for the investigation of toxic mechanisms and the identification of new therapeutic targets in PD. By using a metabolomics approach, we evaluated the metabolic profile of brain and serum samples of rats infused unilaterally with preformed human αSynOs (HαSynOs), or vehicle, into the substantia nigra pars compacta (SNpc). Three months postinfusion, the striatum was dissected for striatal dopamine (DA) measurements via High Pressure Liquid Chromatography (HPLC) analysis and mesencephalon and serum samples were collected for the evaluation of metabolite content via gas chromatography mass spectrometry analysis. Multivariate, univariate and correlation statistics were applied. A 40% decrease of DA content was measured in the HαSynO-infused striatum as compared to the contralateral and the vehicle-infused striata. Decreased levels of dehydroascorbic acid, myo-inositol, and glycine, and increased levels of threonine, were found in the mesencephalon, while increased contents of fructose and mannose, and a decrease in glycine and urea, were found in the serum of HαSynO-infused rats. The significant correlation between DA and metabolite content indicated that metabolic variations reflected the nigrostriatal degeneration. Collectively, the metabolomic fingerprint of HαSynO-infused rats points to an increase of oxidative stress markers, in line with PD neuropathology, and provides hints for potential biomarkers of PD.

Advances in modelling alpha-synuclein-induced Parkinson's diseases in rodents: Virus-based models versus inoculation of exogenous preformed toxic species.

Aggregates of alpha-synuclein (αSyn) have been described in Parkinson's disease (PD) patients, and recent evidence has suggested that the most toxic αSyn species in PD are small soluble aggregates including oligomers, prefibrils, protofibrils. The physiological function of αSyn is still highly debated, with a possible role in synaptic vesicle trafficking and release at the presynaptic compartment, and in the regulation of gene expression in the nucleus. Emerging evidence indicate that most of αSyn functions are related with the crucial ability to bind biological membranes, which is associated with structural conversion from a disordered monomer to an α-helical enriched structure. Conformational properties of αSyn can be modulated by a number of factors including post-translational modifications, gene duplication and triplication-driven overexpression, single point mutations, environmental changes, which affect membrane binding and the protein propensity to aggregate in toxic species. The recognized toxic role of αSyn in PD has laid the rational for purposing of αSyn-based, neuropathologically relevant preclinical models of PD. Different approaches have led to the establishment of transgenic models, viral vector-based models, and more recently models based on the intracerebral inoculation of exogenous αSyn preformed fibrils/oligomers. Here, we overview and compare viral vector-based models of αSyn overexpression and models obtained by direct intracerebral infusion of in vitro preformed αSyn species. The advantages and pitfalls associated with these different approaches are discussed.

Vulnerability of glia and vessels of rat substantia nigra in rotenone Parkinson model.

BACKGROUND: Astrocytes have been implicated as potentially exerting both neurotoxic and neuroprotective activities in Parkinson's disease (PD). Whether glial cells negatively impact the neuron integrity remains to be determined. We aimed to assess the vulnerability of glia and vessels in rat substantia nigra in a rotenone PD model. MATERIAL AND METHODS: Twenty adult male albino rats were divided into two equal groups: vehicle-control group (received dimethylsulfoxide + polyethylene glycol (PEG)-300, 1:1 v/v) and rotenone-treated group (received six doses of rotenone, 1.5 mg/kg/48 h s.c.). Using histological, ultrastructural, biochemical, and morphometric techniques, astrocytes, microglia, vessels, and total antioxidant capacity have been assessed. RESULTS: The rotenone-treated group revealed an increase in the number of astrocytes compared to the control, conformational changes of the immature form, disruption of the outer mitochondrial membrane, and no increase in glial filaments. Dark microglia appeared in close vicinity of blood capillaries. The blood capillaries displayed an increase in number compared to the control, degenerated apoptotic endothelium, and pericytes and an increase in string vessels. The total antioxidant level significantly increased in rotenone-treated group (p < 0.001) compared to the control group. CONCLUSION: Our results demonstrated that oxidative stress and mitochondrial dysfunction involved nigral cellular elements other than dopaminergic neurons. These included astrocytes, microglia, vascular endothelial cells, and pericytes, which might result in promoting damage to the neurons.

Gut-brain and brain-gut axis in Parkinson's disease models: Effects of a uridine and fish oil diet.

Recent investigations have focused on the potential role of gastrointestinal (GI) abnormalities in the pathogenesis of Parkinson's disease (PD). The 'dual-hit' hypothesis of PD speculates that a putative pathogen enters the brain via two routes: the olfactory system and the GI system. Here, we investigated (1) whether local exposures of the neurotoxin rotenone in the gut or the brain of mice could induce PD-like neurological and GI phenotypes as well as a characteristic neuropathology in accordance with this 'dual-hit hypothesis' and (2) the effects of a diet containing uridine and fish oil providing docosahexaenoic acid (DHA), in both models. Mice were given rotenone either orally or by an injection in the striatum. Dietary interventions were started 1 week before rotenone exposures. We found that (1) both oral and intrastriatal administration of rotenone induced similar PD-like motor deficits, dopaminergic cell loss, delayed intestinal transit, inflammation, and alpha-synuclein accumulation in the colon; (2) the uridine and DHA containing diet prevented rotenone-induced motor and GI dysfunctions in both models. The models suggest possible bidirectional communication between the gut and the brain for the genesis of PD-like phenotype and pathology. The dietary intervention may provide benefits in the prevention of motor and non-motor symptoms in PD.

Promising Effects of Neurorestorative Diets on Motor, Cognitive, and Gastrointestinal Dysfunction after Symptom Development in a Mouse Model of Parkinson's Disease.

Parkinson's disease (PD) is characterized by the progressive degeneration of dopaminergic nigrostriatal neurons, with reductions in the function and amount of dopaminergic synapses. Therefore, synapse loss and membrane-related pathology provide relevant targets for interventions in PD. We previously showed the beneficial preventive effects of a dietary intervention containing uridine and DHA, two precursors for membrane synthesis, in the intrastriatal rotenone model for PD. Here, we examined the therapeutic potential of the same dietary intervention on motor, cognitive, and gastrointestinal symptoms. In addition, we tested the effects of an extended nutritional formula based on the same precursors plus other nutrients that increase membrane phospholipid synthesis as well as prebiotic fibers. C57BL/6J mice received a unilateral rotenone injection in the striatum. Dietary interventions started 28 days after surgery, when motor-symptoms had developed. Readout parameters included behavioral tasks measuring motor function and spatial memory as well as intestinal function and histological examination of brain and gut to assess PD-like pathology. Our results show that rotenone-induced motor and non-motor problems were partially alleviated by the therapeutic dietary interventions providing uridine and DHA. The extended nutritional intervention containing both precursors and other nutrients that increase phospholipid synthesis as well as prebiotic fibers was more effective in normalizing rotenone-induced motor and non-motor abnormalities. The latter diet also restored striatal DAT levels, indicating its neurorestorative properties. This is the first study demonstrating beneficial effects of specific dietary interventions, given after full development of symptoms, on a broad spectrum of motor and non-motor symptoms in a mouse model for PD.

Mitomycin-treated undifferentiated embryonic stem cells as a safe and effective therapeutic strategy in a mouse model of Parkinson's disease.

Parkinson's disease (PD) is an incurable progressive neurodegenerative disorder. Clinical presentation of PD stems largely from the loss of dopaminergic neurons in the nigrostriatal dopaminergic pathway, motivating experimental strategies of replacement based on cell therapy. Transplantation of dopaminergic neurons derived from embryonic stem cells significantly improves motor functions in rodent and non-human primate models of PD. However, protocols to generate dopaminergic neurons from embryonic stem cells generally meet with low efficacy and high risk of teratoma formation upon transplantation. To address these issues, we have pre-treated undifferentiated mouse embryonic stem cells (mESCs) with the DNA alkylating agent mitomycin C (MMC) before transplantation. MMC treatment of cultures prevented tumorigenesis in a 12 week follow-up after mESCs were injected in nude mice. In 6-OH-dopamine-lesioned mice, intrastriatal injection of MMC-treated mESCs markedly improved motor function without tumor formation for as long as 15 months. Furthermore, we show that halting mitotic activity of undifferentiated mESCs induces a four-fold increase in dopamine release following in vitro differentiation. Our findings indicate that treating mESCs with MMC prior to intrastriatal transplant is an effective to strategy that could be further investigated as a novel alternative for treatment of PD.

Intraventricular injection of 6-hydroxydopamine results in an increased number of tyrosine hydroxylase immune-positive cells in the rat cortex.

Previously we have demonstrated that intraventricular injection of 6-hydroxydopamine (6-OHDA) results in increased proliferation and de-differentiation of rat cortical astrocytes into progenitor-like cells 4 days after lesion (Wachter et al., 2010). To find out if these cells express tyrosine hydroxylase (TH), the rate-limiting enzyme in the catecholamine synthesis pathway, we performed immunohistochemistry in the rat cortex following intraventricular injection of 6-OHDA. Four days after injection we demonstrated a strong emergence of TH-positive (TH(+)) somata in the cortices of 6-OHDA-lesioned animals. The number of TH(+) cells in the cortex of 6-OHDA-lesioned animals was 15 times higher than in sham-operated animals, where virtually no TH(+) somata occurred. Combining TH immunohistochemistry with classical Nissl stain yielded complete congruency, and ∼45% of the TH(+) cells co-expressed calretinin, which indicates an interneuron affiliation. There was no co-staining of TH with other interneuron markers or with glial markers such as glial fibrillary acidic protein (GFAP) or the neural stem/progenitor marker Nestin, nor could we find co-localization with the proliferation marker Ki67. However, we found a co-localization of TH with glial progenitor cell markers (Sox2 and S100ß) and with polysialylated-neural cell adhesion molecule (PSA-NCAM), which has been shown to be expressed in immature, but not recently generated cortical neurons. Taken together, this study seems to confirm our previous findings with respect to a 6-OHDA-induced expression of neuronal precursor markers in cells of the rat cortex, although the TH(+) cells found in this study are not identical with the potentially de-differentiated astrocytes described recently (Wachter et al., 2010). The detection of cortical cells expressing the catecholaminergic key enzyme TH might indicate a possible compensatory role of these cells in a dopamine-(DA)-depleted system. Future studies are needed to determine whether the TH(+) cells are capable of DA synthesis to confirm this hypothesis.