Investigation in the cannabigerol derivative VCE-003.2 as a disease-modifying agent in a mouse model of experimental synucleinopathy.

BACKGROUND: The cannabigerol derivative VCE-003.2, which has activity at the peroxisome proliferator-activated receptor-γ has afforded neuroprotection in experimental models of Parkinson's disease (PD) based on mitochondrial dysfunction (6-hydroxydopamine-lesioned mice) and neuroinflammation (LPS-lesioned mice). Now, we aim to explore VCE-003.2 neuroprotective properties in a PD model that also involves protein dysregulation, other key event in PD pathogenesis. METHODS: To this end, an adeno-associated viral vector serotype 9 coding for a mutated form of the α-synuclein gene (AAV9-SynA53T) was unilaterally delivered in the substantia nigra pars compacta (SNpc) of mice. This model leads to motor impairment and progressive loss of tyrosine hydroxylase-labelled neurons in the SNpc. RESULTS: Oral administration of VCE-003.2 at 20 mg/kg for 14 days improved the performance of mice injected with AAV9-SynA53T in various motor tests, correlating with the preservation of tyrosine hydroxylase-labelled neurons in the SNpc. VCE-003.2 also reduced reactive microgliosis and astrogliosis in the SNpc. Furthermore, we conducted a transcriptomic analysis in the striatum of mice injected with AAV9-SynA53T and treated with either VCE-003.2 or vehicle, as well as control animals. This analysis aimed to identify gene families specifically altered by the pathology and/or VCE-003.2 treatment. Our data revealed pathology-induced changes in genes related to mitochondrial function, lysosomal cell pathways, immune responses, and lipid metabolism. In contrast, VCE-003.2 treatment predominantly affected the immune response through interferon signaling. CONCLUSION: Our study broadens the neuroprotective potential of VCE-003.2, previously described against mitochondrial dysfunction, oxidative stress, glial reactivity and neuroinflammation in PD. We now demonstrate its efficacy against another key pathogenic event in PD as α-synuclein dysregulation. Furthermore, our investigation sheds light on the molecular mechanisms underlying VCE-003.2 revealing its role in regulating interferon signaling. These findings, together with a favorable ADMET profile, enhance the preclinical interest of VCE-003.2 towards its future clinical development in PD.

MORPHOLOGICAL AND BEHAVIORAL ANALYSIS OF THE PROTECTIVE EFFECTS OF BACTERIAL MELANIN IN A RAT MODEL OF PARKINSON'S DISEASE.

A comparative study of the morphological and functional state of the microvasculature of the substantia nigra pars compacta of the brain (SNc) and bone marrow of rats was carried out using the rotenone model of Parkinson's disease (PD) and with subsequent administration of bacterial melanin (BM). The detection of microvasculature was carried out according to the histoangiological method of Chilingaryan. Animal behavior was studied using a cylinder test. An analysis of morphometric data showed that, in comparison with control animals, experimental animals with rotenone dysfunction showed an increase in capillary diameters and a general reduction in the capillary link in SNc. Behavioral tests have shown that the animals with rotenone intoxication exhibit a form of behavior inherent in PD (freezing, immobility, apathy). Under the influence of BM, the diameter of the capillaries in the SNc approaches the norm, and the capillary link is restored. Due to the protective effect of BM in rats with rotenone intoxication, the trophism of the brain tissue increases as a result of the approach of the lumen of the vessels to the norm and the opening of new branches in the capillary network, an increase in the density of capillaries, which ensures the safety of nerve cells. Animal behavior indicators are close to normal. A comprehensive analysis of cytogenetic data of rat bone marrow was also carried out. In animals with PD, compared to controls, there is a significant increase in the amount of polyploid cells (PC) and a decrease in the level of mitotic index (MI), which usually manifests itself in inflammatory processes and is accompanied by inhibition of bone marrow hematopoiesis. Under the influence of BM, a tendency towards normalization of MI was noted and a significant decrease in the percentage of PC was obtained, which possibly indicates its beneficial effect. The data obtained suggest that BM can be used as a therapeutic agent in the treatment of PD.

Upregulated hexokinase 2 expression induces the apoptosis of dopaminergic neurons by promoting lactate production in Parkinson's disease.

Parkinson's disease (PD) is characterized by impaired mitochondrial function and decreased ATP levels. Aerobic glycolysis and lactate production have been shown to be upregulated in dopaminergic neurons to sustain ATP levels, but the effect of upregulated glycolysis on dopaminergic neurons remains unknown. Since lactate promotes apoptosis and α-synuclein accumulation in neurons, we hypothesized that the lactate produced upon upregulated glycolysis is involved in the apoptosis of dopaminergic neurons in PD. In this study, we examined the expression of hexokinase 2 (HK2) and lactate dehydrogenase (LDH), the key enzymes in glycolysis, and lactate levels in the substantia nigra pars compacta (SNpc) of a MPTP-induced mouse model of PD and in MPP+-treated SH-SY5Y cells. We found that the expression of HK2 and LDHA and the lactate levels were markedly increased in the SNpc of MPTP-treated mice and in MPP+-treated SH-SY5Y cells. Exogenous lactate treatment led to the apoptosis of SH-SY5Y cells. Intriguingly, lactate production and the apoptosis of dopaminergic neurons were suppressed by the application of 3-bromopyruvic acid (3-Brpa), a HK2 inhibitor, or siRNA both in vivo and in vitro. 3-Brpa treatment markedly improved the motor behaviour of MPTP-treated mice in pole test and rotarod test. Mechanistically, lactate increases the activity of adenosine monophosphate-activated protein kinase (AMPK) and suppresses the phosphorylation of serine/threonine kinase 1 (Akt) and mammalian target of rapamycin (mTOR). Together, our data suggest that upregulated HK2 and LDHA and increased lactate levels prompt the apoptosis of dopaminergic neurons in PD. Inhibition of HK2 expression attenuated the apoptosis of dopaminergic neurons by downregulating lactate production and AMPK/Akt/mTOR pathway in PD.

Neuroprotective Effects of the DPP4 Inhibitor Vildagliptin in In Vivo and In Vitro Models of Parkinson's Disease.

Parkinson's disease (PD) is characterized by loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc) of the midbrain. Restoration of nigrostriatal dopamine neurons has been proposed as a potential therapeutic strategy for PD. Because currently used PD therapeutics only help relieve motor symptoms and do not treat the cause of the disease, highly effective drugs are needed. Vildagliptin, a dipeptidyl peptidase 4 (DPP4) inhibitor, is an anti-diabetic drug with various pharmacological properties including neuroprotective effects. However, the detailed effects of vildagliptin against PD are not fully understood. We investigated the effects of vildagliptin on PD and its underlying molecular mechanisms using a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse model and a 1-methyl-4-phenylpyridium (MPP+)-induced cytotoxicity model. Vildagliptin (50 mg/kg) administration significantly attenuated MPTP-induced motor deficits as evidenced by rotarod, pole, and nest building tests. Immunohistochemistry and Western blot analysis revealed that vildagliptin increased tyrosine hydroxylase-positive cells in the SNpc and striatum, which was reduced by MPTP treatment. Furthermore, vildagliptin activated MPTP-decreased PI3k/Akt and mitigated MPTP-increased ERK and JNK signaling pathways in the striatum. Consistent with signaling transduction in the mouse striatum, vildagliptin reversed MPP+-induced dephosphorylation of PI3K/Akt and phosphorylation of ERK and JNK in SH-SY5Y cells. Moreover, vildagliptin attenuated MPP+-induced conversion of LC3B-II in SH-SY5Y cells, suggesting its role in autophagy inhibition. Taken together, these findings indicate that vildagliptin has protective effects against MPTP-induced motor dysfunction by inhibiting dopaminergic neuronal apoptosis, which is associated with regulation of PI3k/Akt, ERK, and JNK signaling transduction. Our findings suggest vildagliptin as a promising repurposing drug to treat PD.

Intragastric Administration of Casein Leads to Nigrostriatal Disease Progressed Accompanied with Persistent Nigrostriatal-Intestinal Inflammation Activited and Intestinal Microbiota-Metabolic Disorders Induced in MPTP Mouse Model of Parkinson's Disease.

Gut microbial dysbiosis and alteration of gut microbiota composition in Parkinson's disease (PD) have been increasingly reported, no recognized therapies are available to halt or slow progression of PD and more evidence is still needed to illustrate its causative impact on gut microbiota and PD and mechanisms for targeted mitigation. Epidemiological evidence supported an association between milk intake and a higher incidence of Parkinson's disease (PD), questions have been raised about prospective associations between dietary factors and the incidence of PD. Here, we investigated the significance of casein in the development of PD. The mice were given casein (6.75 g/kg i.g.) for 21 days after MPTP (25 mg/kg i.p. × 5 days) treatment, the motor function, dopaminergic neurons, inflammation, gut microbiota and fecal metabolites were observed. The experimental results revealed that the mice with casein gavage after MPTP treatment showed a persisted dyskinesia, the content of dopamine in striatum and the expression of TH in midbrain and ileum were decreased, the expression of Iba-1, CD4, IL-22 in midbrain and ileum increased continuously with persisted intestinal histopathology and intestinal barrier injury. Decreased intestinal bile secretion in addition with abnormal digestion and metabolism of carbohydrate, lipids and proteins were found, whereas these pathological status for the MPTP mice without casein intake had recovered after 24 days, no significant differences were observed with regard to only treated with casein. Our study demonstrates that intestinal pathologic injury, intestinal dysbacteriosis and metabolism changes promoted by casein in MPTP mice ultimately exacerbated the lesions to dopaminergic neurons.

The rostromedial tegmental nucleus is essential for non-rapid eye movement sleep.

The rostromedial tegmental nucleus (RMTg), also called the GABAergic tail of the ventral tegmental area, projects to the midbrain dopaminergic system, dorsal raphe nucleus, locus coeruleus, and other regions. Whether the RMTg is involved in sleep-wake regulation is unknown. In the present study, pharmacogenetic activation of rat RMTg neurons promoted non-rapid eye movement (NREM) sleep with increased slow-wave activity (SWA). Conversely, rats after neurotoxic lesions of 8 or 16 days showed decreased NREM sleep with reduced SWA at lights on. The reduced SWA persisted at least 25 days after lesions. Similarly, pharmacological and pharmacogenetic inactivation of rat RMTg neurons decreased NREM sleep. Electrophysiological experiments combined with optogenetics showed a direct inhibitory connection between the terminals of RMTg neurons and midbrain dopaminergic neurons. The bidirectional effects of the RMTg on the sleep-wake cycle were mimicked by the modulation of ventral tegmental area (VTA)/substantia nigra compacta (SNc) dopaminergic neuronal activity using a pharmacogenetic approach. Furthermore, during the 2-hour recovery period following 6-hour sleep deprivation, the amount of NREM sleep in both the lesion and control rats was significantly increased compared with baseline levels; however, only the control rats showed a significant increase in SWA compared with baseline levels. Collectively, our findings reveal an essential role of the RMTg in the promotion of NREM sleep and homeostatic regulation.

Anesthetic Effects on the Progression of Parkinson Disease in the Rat DJ-1 Model.

BACKGROUND: Parkinson disease is a chronic and progressive movement disorder characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNpc). The causes of Parkinson disease are not clear but may involve genetic susceptibilities and environmental factors. As in other neurodegenerative disorders, individuals predisposed to Parkinson disease may have an accelerated onset of symptoms following perioperative stress such as anesthesia, surgery, pain, and inflammation. We hypothesized that anesthesia alone accelerates the onset of Parkinson disease-like pathology and symptoms. METHODS: A presymptomatic Parkinson rat model (the protein, DJ-1, encoded by the Park7 gene [DJ-1], PARK7 knockout) was exposed to a surgical plane of isoflurane or 20% oxygen balanced with nitrogen for 2 hours on 3 occasions between 6 and 7 months of age. Acute and long-term motor and neuropathological effects were examined from 7 to 12 months of age in male DJ-1 rats, using the ladder rung, rotarod, and novel object recognition assays, as well as the immunohistochemical localization of tyrosine hydroxylase in dopaminergic neurons in the substantia nigra and ionized calcium-binding adaptor protein-1 (Iba-1) microglial activation in the substantia nigra and hippocampus. RESULTS: In the acute group, after the third anesthetic exposure at 7 months of age, the isoflurane group had a significant reduction in the density of dopaminergic neurons in the SNpc compared to controls. However, this reduction was not associated with increased microglial activation in the hippocampus or substantia nigra. With the ladder rung motor skills test, there was no effect of anesthetic exposure on the total number of foot faults or the ladder rung pattern in the acute group. The rotarod test also detected no differences before and after the third exposure in controls. For the long-term group, immunohistochemical analyses detected no differences in the density of dopaminergic neurons or microglial cells compared to unexposed DJ-1 rats from 8 to 12 months of age. The ladder rung test in the long-term group showed no differences in the total number of foot faults with time and exposure or between ladder rung patterns. The rotarod test detected no significant effect of exposure with time or between groups at any time point. The novel object recognition task in the long-term group revealed no differences in short- or long-term memory or in the number of rearings as a function of exposure. CONCLUSIONS: Multiple isoflurane exposures in this rat model of Parkinson disease transiently enhanced dopaminergic neurodegeneration in the SNpc that resolved over time and had no effects on progression in this Parkinson disease-like phenotype.

Noscapine Prevents Rotenone-Induced Neurotoxicity: Involvement of Oxidative Stress, Neuroinflammation and Autophagy Pathways.

Parkinson's disease is characterized by the loss of dopaminergic neurons in substantia nigra pars compacta (SNpc) and the resultant loss of dopamine in the striatum. Various studies have shown that oxidative stress and neuroinflammation plays a major role in PD progression. In addition, the autophagy lysosome pathway (ALP) plays an important role in the degradation of aggregated proteins, abnormal cytoplasmic organelles and proteins for intracellular homeostasis. Dysfunction of ALP results in the accumulation of α-synuclein and the loss of dopaminergic neurons in PD. Thus, modulating ALP is becoming an appealing therapeutic intervention. In our current study, we wanted to evaluate the neuroprotective potency of noscapine in a rotenone-induced PD rat model. Rats were administered rotenone injections (2.5 mg/kg, i.p.,) daily followed by noscapine (10 mg/kg, i.p.,) for four weeks. Noscapine, an iso-qinulinin alkaloid found naturally in the Papaveraceae family, has traditionally been used in the treatment of cancer, stroke and fibrosis. However, the neuroprotective potency of noscapine has not been analyzed. Our study showed that administration of noscapine decreased the upregulation of pro-inflammatory factors, oxidative stress, and α-synuclein expression with a significant increase in antioxidant enzymes. In addition, noscapine prevented rotenone-induced activation of microglia and astrocytes. These neuroprotective mechanisms resulted in a decrease in dopaminergic neuron loss in SNpc and neuronal fibers in the striatum. Further, noscapine administration enhanced the mTOR-mediated p70S6K pathway as well as inhibited apoptosis. In addition to these mechanisms, noscapine prevented a rotenone-mediated increase in lysosomal degradation, resulting in a decrease in α-synuclein aggregation. However, further studies are needed to further develop noscapine as a potential therapeutic candidate for PD treatment.

Adolescent Exposure to WIN 55212-2 Render the Nigrostriatal Dopaminergic Pathway Activated During Adulthood.

BACKGROUND: During adolescence, neuronal circuits exhibit plasticity in response to physiological changes and to adapt to environmental events. Nigrostriatal dopaminergic pathways are in constant flux during development. Evidence suggests a relationship between early use of cannabinoids and psychiatric disorders characterized by altered dopaminergic systems, such as schizophrenia and addiction. However, the impact of adolescent exposure to cannabinoids on nigrostriatal dopaminergic pathways in adulthood remains unclear. The aim of this research was to determine the effects of repeated activation of cannabinoid receptors during adolescence on dopaminergic activity of nigrostriatal pathways and the mechanisms underlying this impact during adulthood. METHODS: Male Sprague-Dawley rats were treated with 1.2 mg/kg WIN 55212-2 daily from postnatal day 40 to 65. Then no-net flux microdialysis of dopamine in the dorsolateral striatum, electrophysiological recording of dopaminergic neuronal activity, and microdialysis measures of gamma-aminobutyric acid (GABA) and glutamate in substantia nigra par compacta were carried out during adulthood (postnatal days 72-78). RESULTS: Repeated activation of cannabinoid receptors during adolescence increased the release of dopamine in dorsolateral striatum accompanied by increased population activity of dopamine neurons and decreased extracellular GABA levels in substantia nigra par compacta in adulthood. Furthermore, perfusion of bicuculline, a GABAa antagonist, into the ventral pallidum reversed the increased dopamine neuron population activity in substantia nigra par compacta induced by adolescent cannabinoid exposure. CONCLUSIONS: These results suggest that adolescent exposure to cannabinoid agonists produces disinhibition of nigrostriatal dopamine transmission during adulthood mediated by decreased GABAergic input from the ventral pallidum.

A history of ethanol drinking increases locomotor stimulation and blunts enhancement of dendritic dopamine transmission by methamphetamine.

Ethanol and psychostimulant use disorders exhibit comorbidity in humans and cross-sensitization in animal models, but the neurobiological underpinnings of this are not well understood. Ethanol acutely increases dopamine neuron excitability, and psychostimulants such as cocaine or methamphetamine increase extracellular dopamine through inhibition of uptake through the dopamine transporter (DAT) and/or vesicular monoamine transporter 2 (VMAT2). Psychostimulants also depress dopamine neuron activity by enhancing dendritic dopamine neurotransmission. Here, we show that mice with a previous history of ethanol drinking are more sensitive to the locomotor-stimulating effects of a high dose (5 mg/kg), but not lower doses (1 and 3 mg/kg) of methamphetamine or any tested dose of cocaine (3, 10, and 18 mg/kg), compared with water-drinking controls. We next investigated the impact of a history of ethanol drinking, in a separate group of mice, on methamphetamine- or cocaine-induced enhancement of dendritic dopamine transmission using whole-cell voltage clamp electrophysiology in mouse brain slices. Methamphetamine, applied at a concentration (10 µM) that affects both DAT and VMAT2, enhanced D2 receptor-mediated inhibitory postsynaptic currents (D2-IPSCs) in both groups, but this effect was blunted in mice with a history of ethanol drinking. As methamphetamine action at VMAT2 disrupts dopamine neurotransmission, these results may suggest enhanced action of methamphetamine at VMAT2. Furthermore, there were no differences in low-dose methamphetamine or cocaine-induced enhancement of D2-IPSCs, suggesting intact DAT function. Disruption of methamphetamine-induced enhancement of dendritic dopamine transmission would result in decreased inhibition of dopamine neurons, ultimately increasing downstream release and the behavioral effects of methamphetamine.

Neurotoxicity to dopamine neurons after the serial exposure to alcohol and methamphetamine: Protection by COX-2 antagonism.

A significant co-morbidity exists between alcohol and methamphetamine (Meth) in humans but the consequences and mechanisms underlying their co-morbid effects remain to be identified. A consequence associated with the abuse of either alcohol or Meth involves inflammation but little is known about the role of inflammation in a possible neurotoxicity arising from their co-exposure. Sprague Dawley rats were allowed 28 days of intermittent, voluntary access to 10% ethanol (EtOH) followed by a neurotoxic binge administration of Meth. EtOH drinking followed by Meth increased microglial cell counts and produced morphological changes in microglia of the substantia nigra pars compacta 2 h after Meth administration that were distinct from those produced by either EtOH or Meth alone. These effects preceded the activation of cleaved caspase-3 in dopamine cell bodies, as well as decreases in tyrosine hydroxylase (TH) immunoreactivity in the substantia nigra and dopamine transporter (DAT) immunoreactivity in the striatum measured at 7 days after Meth. Intervention with a selective COX-2 inhibitor during EtOH drinking prevented the changes in microglia, and attenuated the increase in cleaved caspase-3, and decreases in TH and DAT after Meth administration. Furthermore, motor dysfunction measured by a rotarod test was evident but only in rats that were exposed to both EtOH and Meth. The motor dysfunction was ameliorated by prior inhibition of COX-2 during EtOH drinking. The exaggerated neurochemical and behavioral deficits indicate that the comorbidity of EtOH and Meth induces a degeneration of the nigrostriatal pathway and support the role of inflammation produced by EtOH drinking that primes and mediates the neurotoxic consequences associated with the common co-morbidity of these drugs.

A Nigro-Vagal Pathway Controls Gastric Motility and Is Affected in a Rat Model of Parkinsonism.

BACKGROUND & AIMS: In most patients with Parkinson's disease, gastrointestinal (GI) dysfunctions, such as gastroparesis and constipation, are prodromal to the cardinal motor symptoms of the disease. Sporadic Parkinson's disease has been proposed to develop after ingestion of neurotoxicants that affect the brain-gut axis via the vagus nerve, and then travel to higher centers, compromising the substantia nigra pars compacta (SNpc) and, later, the cerebral cortex. We aimed to identify the pathway that connects the brainstem vagal nuclei and the SNpc, and to determine whether this pathway is compromised in a rat model of Parkinsonism. METHODS: To study this neural pathway in rats, we placed tracers in the dorsal vagal complex or SNpc; brainstem and midbrain were examined for tracer distribution and neuronal neurochemical phenotype. Rats were given injections of paraquat once weekly for 3 weeks to induce features of Parkinsonism, or vehicle (control). Gastric tone and motility were recorded after N-methyl-d-aspartate microinjection in the SNpc and/or optogenetic stimulation of nigro-vagal terminals in the dorsal vagal complex. RESULTS: Stimulation of the SNpc increased gastric tone and motility via activation of dopamine 1 receptors in the dorsal vagal complex. In the paraquat-induced model of Parkinsonism, this nigro-vagal pathway was compromised during the early stages of motor deficit development. CONCLUSIONS: We identified and characterized a nigro-vagal monosynaptic pathway in rats that controls gastric tone and motility. This pathway might be involved in the prodromal gastric dysmotility observed in patients with early-stage Parkinson's disease.

Effect of ghrelin on the motor deficit caused by the ablation of nigrostriatal dopaminergic cells or the inhibition of striatal dopamine receptors.

Ghrelin plays roles in a wide range of central functions by activating the growth hormone secretagogue receptor (GHSR). This receptor has recently been found in the substantia nigra (SN) to control dopamine (DA)-related physiological functions. The dysregulation of DA neurons in the SN pars compacta (SNc) and the consequent depletion of striatal DA are known to underlie the motor deficits observed in Parkinson's disease (PD). In the present study, we further investigated the role of the SN-ghrelin system in motor function under the stereotaxic injection of AAV-CMV-FLEX-diphtheria toxin A (DTA) into the SN of dopamine transporter (DAT)-Cre (DATSN::DTA) mice to expunge DA neurons of the SNc. First, we confirmed the dominant expression of GHSR1a, which is a functional GHSR, in tyrosine hydroxylase (TH)-positive DA neurons in the SNc of control mice. In DATSN::DTA mice, we clearly observed motor dysfunction using several behavioral tests. An immunohistochemical study revealed a dramatic loss of TH-positive DA neurons in the SNc and DAT-labeled axon terminals in the striatum, and an absence of mRNAs for TH and DAT in the SN of DATSN::DTA mice. The mRNA level of GHSR1a was drastically decreased in the SN of these mice. In normal mice, we also found the mRNA expression of GHSR1a within GABAergic neurons in the SN pars reticulata (SNr). Under these conditions, a single injection of ghrelin into the SN failed to improve the motor deficits caused by ablation of the nigrostriatal DA network using DATSN::DTA mice, whereas intra-SN injection of ghrelin suppressed the motor dysfunction caused by the administration of haloperidol, which is associated with the transient inhibition of DA transmission. These findings suggest that phasic activation of the SNc-ghrelin system could improve the dysregulation of nigrostriatal DA transmission related to the initial stage of PD, but not the motor deficits under the depletion of nigrostriatal DA. Although GHSRs are found in non-DA cells of the SNr, GHSRs on DA neurons in the SNc may play a crucial role in motor function.

Role of neuronal nitric oxide synthase in slowly progressive dopaminergic neurodegeneration in the Zitter rat.

Neuronal nitric oxide synthase (nNOS) is involved in nigrostriatal dopaminergic (DA) neurodegeneration. However, little is known about the distribution patterns and functions of nNOS in slowly progressive DA neurodegeneration. Here we describe the spatiotemporal change in nNOS expression over the course of neurodegeneration and the effect of short- or long-term treatment with the nNOS inhibitor, 7-nitroindazole (7-NI), in zitter (zi/zi) rats. In the substantia nigra pars compacta (SNc), nNOS expression was significantly increased with progression of neurodegeneration. nNOS-immunoreactive (ir) cells were in the vicinity of tyrosine hydroxylase-ir (TH-ir) DA neurons, and some of these cells were also positive for calbindin. nNOS in the caudate-putamen (CPu) showed little difference during progression of neurodegeneration. However, immunoelectron microscopic analysis revealed that abundant TH-ir fibers in the CPu were degenerated due to compression by vacuoles that contained swollen neuronal and glial elements. Additionally, lipid peroxidation as a marker of membrane oxidation was significantly increased in zi/zi rats. Short-term 7-NI treatment attenuated the increase in lipid peroxidation and inhibited the vacuolation in the CPu. Moreover, long-term 7-NI treatment significantly protected TH-ir neurons in the SNc, and TH-ir fibers and DA contents in the CPu. These results show that nNOS exacerbates slowly progressive DA neurodegeneration, and the neuroprotective effects of 7-NI may result from suppression of membrane oxidation that causes abnormal membrane structures in zi/zi rats.

Gastrodin microinjection suppresses 6-OHDA-induced motor impairments in parkinsonian rats: insights into oxidative balance and microglial activation in SNc.

PURPOSE OF THE RESEARCH: In this study, we appraised the effect of pre-treatment with intra-cerebro ventricular (i.c.v) microinjection of gastrodin (Gst) on catalepsy, motor imbalance, substantia nigra pars compacta (SNc) myeloperoxidase (MPO) activity, lipid peroxidation levels, nitric oxide (NO) production and total antioxidant capacity (TAC) in 6-hydroxydopamine (6-OHDA) rats model of PD. MATERIALS AND METHODS: Male Wistar rats were pre-treated with i.c.v microinjections of Gst (20, 40 and 80 µg/3 µl/rat) for five consecutive days. Then, catalepsy and motor balance were induced by unilateral infusion of 6-OHDA (8 µg/2 µl/rat) into the SNc. The anti-cataleptic and motor balance improving effect of Gst was assessed by the Bar test and Rotarod 3 weeks after neurotoxin injection, respectively. SNc MPO activity and lipid peroxidation levels, NO production and TAC were assessed at the end of behavioral experiments. RESULTS: Our data demonstrated that Gst pre-treatment significantly (p < 0.001) was prevented motor in-coordination and catalepsy in neurotoxin lesioned rats. The most motor improving effect was seen at 80 µg Gst (p < 0.001). Pre-treatment of parkinsonian rats with Gst meaningfully (p < 0.001) was suppressed MPO activity, lipid peroxidation and NO production. Furthermore, the TAC level in the SNc was increased (p < 0.001) in Gst-microinjected rats about to the normal non-parkinsonian animals. MAJOR CONCLUSIONS: In summary, pre-treatment with Gst abolished 6-OHDA-induced catalepsy and improved motor incoordination by decreasing: SNc MPO activity, lipid peroxidation levels and NO production, and restoring SNc levels of TAC to the levels of healthy rats.

Activation of AMPK/mTORC1-Mediated Autophagy by Metformin Reverses Clk1 Deficiency-Sensitized Dopaminergic Neuronal Death.

The autophagy-lysosome pathway (ALP) plays a critical role in the pathology of Parkinson's disease (PD). Clk1 (coq7) is a mitochondrial hydroxylase that is essential for coenzyme Q (ubiquinone) biosynthesis. We have reported previously that Clk1 regulates microglia activation via modulating microglia metabolic reprogramming, which contributes to dopaminergic neuronal survival. This study explores the direct effect of Clk1 on dopaminergic neuronal survival. We demonstrate that Clk1 deficiency inhibited dopaminergic neuronal autophagy in cultured MN9D dopaminergic neurons and in the substantia nigra pars compacta of Clk+/- mutant mice and consequently sensitized dopaminergic neuron damage and behavioral defects. These mechanistic studies indicate that Clk1 regulates the AMP-activated protein kinase (AMPK)/rapamycin complex 1 pathway, which in turn impairs the ALP and TFEB nuclear translocation. As a result, Clk1 deficiency promotes dopaminergic neuronal damage in vivo and in vitro, which ultimately contributes to sensitizing 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neuronal death and behavioral impairments in Clk1-deficient mice. Moreover, we found that activation of autophagy by the AMPK activator metformin increases dopaminergic neuronal survival in vitro and in the MPTP-induced PD model in Clk1 mutant mice. These results reveal that Clk1 plays a direct role in dopaminergic neuronal survival via regulating ALPs that may contribute to the pathologic development of PD. Modulation of Clk1 activity may represent a potential therapeutic target for PD.

Echinacoside's nigrostriatal dopaminergic protection against 6-OHDA-Induced endoplasmic reticulum stress through reducing the accumulation of Seipin.

Parkinson's disease (PD) is one of the most common neurodegenerative diseases. Recent epidemiological studies suggest that echinacoside (ECH), a phenylethanoid glycoside found in Cistanche deserticola, has a protective effect against the development of PD. However, the detailed mechanisms of how ECH suppresses neuronal death have not been fully elucidated. In this study, we confirmed that ECH protects nigrostriatal neurons against 6-hydroxydopamine (6-OHDA)-induced endoplasmic reticulum stress (ERS) in vivo and in vitro. ECH rescued cell viability in damaged cells and decreased 6-OHDA-induced reactive oxygen species accumulation in vitro. It also rescued tyrosine hydroxylase and dopamine transporter expression in the striatum, and decreased α-synuclein aggregation following 6-OHDA treatment in vivo. The validated mechanism of ECH activity was the reduction in the 6-OHDA-induced accumulation of seipin (Berardinelli-Seip congenital lipodystrophy 2). Seipin has been shown to be a key molecule related to motor neuron disease and was tightly associated with ERS in a series of in vivo studies. ECH attenuated seipinopathy by promoting seipin degradation via ubiquitination. ERS was relieved by ECH through the Grp94/Bip-ATF4-CHOP signal pathway.

Differential actions of AMP kinase on ATP-sensitive K+ currents in ventral tegmental area and substantia nigra zona compacta neurons.

ATP-sensitive K+ (K-ATP) channels play significant roles in regulating the excitability of dopamine neurons in the substantia nigra zona compacta (SNC). We showed previously that K-ATP channel function is up-regulated by AMP-activated protein kinase (AMPK). This study extended these studies to the neurons adjacent to the SNC in the ventral tegmental area (VTA). Using patch pipettes to record whole-cell currents in slices of rat midbrain, we found that the AMPK activator A769662 increased the amplitude of currents evoked by the K-ATP channel opener diazoxide in presumed dopamine-containing VTA neurons. However, current evoked by diazoxide with A769662 was significantly smaller in VTA neurons compared to SNC neurons. Moreover, a significantly lower proportion of VTA neurons responded to diazoxide with outward current. However, A769662 was able to increase the incidence of diazoxide-responsive neurons in the VTA. In contrast, A769662 did not potentiate diazoxide-evoked currents in presumed non-dopamine VTA neurons. These results show that AMPK activation augments K-ATP currents in presumed dopamine neurons in the VTA and SNC, although diazoxide-evoked currents remain less robust in the VTA. We conclude that K-ATP channels may play important physiological roles in VTA and SNC dopamine neurons.

Dopamine D4 receptor stimulation prevents nigrostriatal dopamine pathway activation by morphine: relevance for drug addiction.

Morphine is one of the most effective drugs used for pain management, but it is also highly addictive. Morphine elicits acute and long-term adaptive changes at cellular and molecular level in the brain, which play a critical role in the development of tolerance, dependence and addiction. Previous studies indicated that the dopamine D4 receptor (D4 R) activation counteracts morphine-induced adaptive changes of the µ opioid receptor (MOR) signaling in the striosomes of the caudate putamen (CPu), as well as the induction of several Fos family transcription factors. Thus, it has been suggested that D4 R could play an important role avoiding some of the addictive effects of morphine. Here, using different drugs administration paradigms, it is determined that the D4 R agonist PD168,077 prevents morphine-induced activation of the nigrostriatal dopamine pathway and morphological changes of substantia nigra pars compacta (SNc) dopamine neurons, leading to a restoration of dopamine levels and metabolism in the CPu. Results from receptor autoradiography indicate that D4 R activation modulates MOR function in the substantia nigra pars reticulata (SNr) and the striosomes of the CPu, suggesting that these regions are critically involved in the modulation of SNc dopamine neuronal function through a functional D4 R/MOR interaction. In addition, D4 R activation counteracts the rewarding effects of morphine, as well as the development of hyperlocomotion and physical dependence without any effect on its analgesic properties. These results provide a novel role of D4 R agonist as a pharmacological strategy to prevent the adverse effects of morphine in the treatment of pain.

Peptides semax and selank affect the behavior of rats with 6-OHDA induced PD-like parkinsonism.

Parkinson's disease (PD) is the second most common severe neurodegenerative disorder that is characterized by progressive degeneration of dopaminergic neurons (DA neurons) in the substantia nigra pars compacta (SNpc) region of the brain. In the present study, we investigated the effects of the synthetic regulatory peptides Semax (analog of an ACTH 4-10 fragment (ACTH4-10)) and Selank (analog of immunomodulatory taftsin) on behavior of rats with 6-hydroxidopamine (6-OHDA) induced PD-like parkinsonism. It was showed that both peptides did not affect motor activity of rats in elevated cross shaped maze and passive defensive behavior of the animals. At the same time, Selank decreased level of anxiety of rats with toxic damage of DA neurons in elevated cross shaped maze. Previously such effects of Selank were revealed in healthy rodents (rats and mice) with different models of psycho-emotional stress. Therefore, toxic damage of substantia nigra does not affect the response of the rat organism on this peptide.