Calcitriol restrains microglial M1 polarization and alleviates dopaminergic degeneration in hemiparkinsonian mice by boosting regulatory T-cell expansion.

OBJECTIVE: Vitamin D deficiency is a risk factor for Parkinson's disease (PD) and vitamin D supplementation robustly alleviates neurodegeneration in PD models. However, the mechanisms underlying this effect require further clarification. Current evidence suggests that harnessing regulatory T cells (Treg) may mitigate neuronal degeneration. In this study, we investigated the therapeutic effects of vitamin D receptor activation by calcitriol on PD, specifically focusing on its role in Treg. METHODS: Hemiparkinsonian mice model was established through the injection of 6-OHDA into the striatum. Mice were pretreated with calcitriol before 6-OHDA injection. The motor performance, dopaminergic neuronal survival, contents of dopamine, and dopamine metabolites were evaluated. The pro-inflammatory cytokines levels, T-cell infiltration, mRNA expression of indicated microglial M1/M2 phenotypic markers, and microglial marker in the midbrain were detected. Populations of Treg in the splenic tissues were assessed using a flow cytometry assay. PC61 monoclonal antibody was applied to deplete Treg in vivo. RESULTS: We show that calcitriol supplementation notably improved motor performance and reduced dopaminergic degeneration in the 6-OHDA-induced PD model. Mechanistically, calcitriol promoted anti-inflammatory/neuroprotective Treg and inhibited pro-inflammatory/neurodestructive effector T-cell generation in this model. This process significantly inhibited T-cell infiltration in the midbrain, restrained microglial activation, microglial M1 polarization, and decreased pro-inflammatory cytokines release. This more favorable inflammatory microenvironment rescued dopaminergic degeneration. To further verify that the anti-inflammatory effects of calcitriol are associated with Treg expansion, we applied an antibody-mediated Treg depletion assay. As predicted, the anti-inflammatory effects of calcitriol in the PD model were diminished following Treg depletion. CONCLUSION: These findings suggest that calcitriol's anti-inflammatory and neuroprotective effects in PD are associated with its potential to boost Treg expansion.

Therapeutic Effects of Selenium on Alpha-Synuclein Accumulation in Substantia Nigra Pars Compacta in a Rat Model of Parkinson's Disease: Behavioral and Biochemical Outcomes.

Parkinson's disease (PD) is the second most common progressive neurodegenerative disorder characterized by the accumulation of accumulated alpha-synuclein (α-Syn) in substantia nigra. Research has shown that selenium (Se) can protect neural cells through the actions of selenoproteins, including selenoprotein P (SelP) and selenoprotein S (SelS), which participate in endoplasmic reticulum-associated protein degradation (ERAD). In this study, we investigated the potential protective role of Se in a pre-clinical PD rat model.We aimed to evaluate the therapeutic effects of Se administration in the 6-hydroxydopamine (6-OHDA) induced unilateral rat PD model. Male Wistar rats were utilised for unilateral PD animal model which were subjected to stereotaxic surgery and injected with 20 µg 6-OHDA/5 µl 0.2% ascorbate saline. After confirming the model, the rats were intraperitoneally injected with 0.1, 0.2, and 0.3 mg/kg of sodium selenite for 7 days. We then performed behavioral tests, including apomorphine-induced rotation, hanging, and rotarod tests. Following sacrifice, we analysed the substantia nigra area of the brain and serum for protein quantification, element analysis, and gene expression analysis.Our results indicate that the administration of 0.3 mg/kg of Se improved the motor deficiency in hanging, rotarod, and apomorphine-induced rotational tests. While there was no significant improvement in the expression of α-Syn, Se increased the expression of selenoproteins. Additionally, levels of selenoproteins, Se, and α-Syn both brain and serum were re-established by the treatment, suggesting the role of Se on the α-Syn accumulation. Furthermore, Se improved PD-induced biochemical deficits by increasing the levels of SelS and SelP (p<0.005).In conclusion, our findings suggest that Se may have a protective role in PD. 0.3 mg/kg dosage of Se increased the expression of selenoproteins, reduced the accumulation of α-Syn in the brain, and improved PD-induced motor deficits. These results suggest that Se may be a potential therapeutic option for PD treatment.

Heated Leaf Extract of Coriandrum sativum L. Protects Nigral Dopaminergic Degeneration in Rats.

Coriandrum sativum L. (coriander), which is an annual herb of the Apiaceae family, has been traditionally used as a remedy. Here we tested whether heated extract of coriander leaf protects nigral dopaminergic neurodegeneration after exposure to 6-hydroxydopamine (6-OHDA). After injection of 6-OHDA into the rat substantia nigra pars compacta (SNpc), dopaminergic degeneration, which was determined by tyrosine hydroxylase immunostaining, was rescued by co-injection of CaEDTA, an extracellular Zn2+ chelator, suggesting that extracellular Zn2+ influx is involved in neurodegeneration. Both intracellular Zn2+ dysregulation determined by ZnAF-2 fluorescence and dopaminergic degeneration in the SNpc induced by 6-OHDA were rescued by co-injection of 0.25% coriander extract, which also reduced reactive oxygen species (ROS) production in the SNpc determined by aminophenyl fluorescein fluorescence. The present study suggests that coriander leaf extract protects nigral dopaminergic neurodegeneration induced by intracellular Zn2+ dysregulation. It is likely that the nutraceutical property of coriander leaf extract contributes to the protection via reducing ROS production.

Potential anti-Parkinsonian's effect of S-(+)-linalool from Cinnamomum osmophloeum ct. linalool leaves are associated with mitochondrial regulation via gas-1, nuo-1, and mev-1 in Caenorhabditis elegans.

Parkinson's disease (PD) is one of the prevalent neurodegenerative diseases, and developing new treatments from natural products is of particular interest. Essential oils from Cinnamomum osmophloeum ct. linalool leaves contain high levels (~95%) of S-(+)-linalool. The neuroprotective effects of linalool have been previously described, yet the underlying molecular mechanisms remain largely unknown. This study aimed to investigate the potential anti-Parkinsonian's effect of S-(+)-linalool on mitochondrial regulation and decipher the underlying molecular mechanisms in Caenorhabditis elegans PD model. Essential oils at 20 mg/L and 20 mg/L S-(+)-linalool each significantly attenuated the damaging effects of 6-hydroxydopamine (6-OHDA) on dopaminergic (DA) neurons and decreased the mitochondrial unfolded protein response (UPRmt ) to antimycin. RNAi knockdown of mitochondrial complex I (gas-1, nuo-1), and complex II (mev-1) genes prevented the improvement of mitochondrial activity by S-(+)-linalool. The protective effects of S-(+)-linalool on 6-OHDA-induced behavior changes were absent in a DA-specific strain of C. elegans produced by gas-1, nuo-1, and mev-1 RNAi knockdown. These results suggest the potential anti-Parkinsonian's effect of S-(+)-linalool is associated with mitochondrial activity and regulated by gas-1, nuo-1, and mev-1 in C. elegans. Our findings suggest that S-(+)-linalool might be a promising candidate for therapeutic application to inhibit the progression of PD.

The modulatory effect of 100 Hz electroacupuncture on striatal synaptic plasticity in unilateral lesioned 6-OHDA rats.

Recent studied have reported that impaired striatal synaptic plasticity played a crucial role in Parkinson's disease (PD). Previous studies have suggested that electroacupuncture (EA) alleviated the motor deficits in PD patients and animal models. However, the mechanisms underlying this protection need to be further elucidated. In this study, we found that EA-induced improvement of motor deficits in the 6-hydroxydopamine (6-OHDA) rat model doesn't act through dopaminergic system. EA rescued the decreased striatal long-term potentiation (LTP) in 6-OHDA rats. In addition, the declined expression of N-methyl-D-aspartic acid receptor subunit 2B (NR2B) in the striatum was remarkably up-regulated by EA. The EA-induced improvement of LTP can be eliminated by NR2B-selective inhibitor. It is indicated that EA-induced recovery of striatal LTP was correlated with the up-regulation of NR2B subunit. EA was also found to rescue the decreased dendritic arborization and the spine density in the striatum of 6-OHDA rats. Meanwhile, EA suppressed striatal glutamate content and vesicular glutamate transporter 1 which is expressed in cortico-striatal glutamatergic projections. The decrease of striatal glutamate content induced by decortication, EA treatment or a combination of both reversed the loss of striatal spine density in 6-OHDA rats. It is indicated that EA-induced reduction of cortico-striatal glutamate transmission contributes to the recovery of striatal spine density. In conclusion, the therapeutic effect of EA on the motor deficits of 6-OHDA rats was mediated by rescuing cortico-striatal glutamate transmission and striatal synaptic plasticity.

Neuroprotective effect of bromelain in 6-hydroxydopamine induced in vitro model of Parkinson's disease.

BACKGROUND: This study was designed to investigate the neuroprotective effects of bromelain, which is known to have anti-oxidant and anti-inflammatory properties, against the neurotoxicity (induced by 6-OHDA) in SH-SY5Y cells. METHODS AND RESULTS: To establish Parkinson's Disease (PD) model in cell culture conditions, SH-SY5Y cells were exposed to 200 µM 6-OHDA for 1 day. Prior to 6-OHDA treatment, SH-SY5Y cells had been pre-treated with bromelain (25 µg/mL, 50 µg/mL, 75 µg/mL and 100 µg/mL). After 1 day, cell viability was determined with the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) and lactate dehydrogenase (LDH) assays. Oxidative stress was assessed with total antioxidant capacity (TAC), total oxidant status (TOS), glutathione reductase (GR) and malondialdehyde (MDA) analyses. The effect of the bromelain in SH-SY5Ycells was also examined by 4',6-diamidino-2-phenylindole (DAPI) staining. We found that 6-OHDA increased LDH leakage, and cellular apoptosis in SH-SY5Y cells. 6-OHDA aggravated oxidative stress by increasing TOS, MDA and GR and eventually promoted apoptosis in SH-SY5Y cells, while pretreatment with bromelain attenuated these toxic effects of 6-OHDA. CONCLUSIONS: These findings indicated that bromelain, with its neuroprotective features can be useful for neuroprotection in PD.

Effect of Roucongrong (Herba Cistanches Deserticolae) decoction on the substantia nigra through Wnt/ß-catenin signaling pathway in rats with Parkinson's disease induced by 6-hydroxydopamine hydrochloride.

OBJECTIVE: To investigate the effect of Roucongrong (Herba Cistanches Deserticolae) decoction on the substantia nigra in rats with Parkinson's disease (PD) induced by 6-hydroxydopamine hydrochloride (6-OHDA). To further determine whether the Wnt/ß-catenin signaling pathway is involved in the action. METHODS: A rat model of PD was established by intracranial injection of 6-OHDA. Subsequently, three concentrations of Roucongrong (Herba Cistanches Deserticolae) decoction were prepared and administered to rats by gavage therapy for 14 d. Behavioral changes were measured in PD rats. In vivo tyrosine hydroxylase (TH) levels in the substantia nigra were examined by immunohistochemistry. Additionally, gene and protein expression levels of members of the Wnt/ß-catenin signaling pathway were examined by Western blotting and polymerase chain reaction. Lastly, a Wnt/ß-catenin inhibitor was used to investigate the mechanism of action in 1-methyl-4-phenylpyridinium (MPP + )- treated MES23.5 cells in vitro. RESULTS: Roucongrong (Herba Cistanches Deserticolae) decoction improved performance in the stride and gait adjustment tests in PD rats. It also increased TH in the substantia nigra and altered the expression of genes and proteins in the Wnt/ß-catenin signaling pathway. Wnt/ß-catenin inhibitor reduced the effect of Roucongrong (Herba Cistanches Deserticolae) decoction in MPP +-treated MES23.5 cells. CONCLUSION: Roucongrong (Herba Cistanches Deserticolae) decoction may promote neuronal survival in PD in vivo and in vitro by increasing TH content in the substantia nigra and by activating the Wnt/ß-catenin signaling pathway.

Marijuana improved motor impairments and changes in synaptic plasticity-related molecules in the striatum in 6-OHDA-treated rats.

Using marijuana has become popular and is allowed for medical purposes in some countries. The effect of marijuana on Parkinson's disease is controversial and Medical marijuana may benefit for motor and non-motor symptoms of patients with Parkinson's disease. No research has been conducted to fully prove the benefits, risks, and uses of marijuana as a treatment for patients with Parkinson's disease. In the present study, several different approaches, including behavioral measures and the western blot method for protein level assay, were used to investigate whether exposure to marijuana affects the motor and synaptic plasticity impairment induced by 6-OHDA. Marijuana consumption significantly decreased apomorphine-induced contralateral rotation, beam travel time, beam freeze time, and catalepsy time, but significantly increased latency to fall in the rotarod test, balance time, and protein level of PSD-95 and dopamine receptor D1 in the 6-OHDA + marijuana group. These results suggest that marijuana may be helpful for motor disorders and synaptic changes in patients with Parkinson's disease.

Oat polyphenol avenanthramide-2c confers protection from oxidative stress by regulating the Nrf2-ARE signaling pathway in PC12 cells.

Accumulating evidence has demonstrated that cellular antioxidant systems play essential roles in retarding oxidative stress-related diseases, such as Parkinson's disease. Because nuclear factor erythroid 2-related factor 2 (Nrf2) is a chief regulator of cellular antioxidant systems, small molecules with Nrf2-activating ability may be promising neuroprotective agents. Avenanthramide-2c (Aven-2c), avenanthramide-2f (Aven-2f) and avenanthramide-2p (Aven-2p) are the most abundant avenanthramides in oats, and they have been documented to possess multiple pharmacological benefits. In this work, we synthesized these three compounds and evaluated their cytoprotective effect against oxidative stress-induced PC12 cell injuries. Aven-2c displayed the best protective potency among them. Aven-2c conferred protection on PC12 cells by scavenging free radicals and activating the Nrf2-ARE signaling pathway. Pretreatment of PC12 cells with Aven-2c efficiently enhanced Nrf2 nuclear accumulation and evoked the expression of a set of cytoprotective molecules. The mechanistic study also supports that Nrf2 activation is the molecular basis for the cellular action of Aven-2c. Collectively, this study demonstrates that Aven-2c is a potent Nrf2 agonist, shedding light on the potential usage of Aven-2c in the treatment of neuroprotective diseases.

Theacrine, a purine alkaloid from kucha, protects against Parkinson's disease through SIRT3 activation.

BACKGROUND: Oxidative damage of dopaminergic neurons is the fundamental causes of Parkinson's disease (PD) that has no standard cure at present. Theacrine, a purine alkaloid from Chinese tea Kucha, has been speculated to benefit the neurodegeneration in PD, through similar actions to its chemical analogue caffeine, albeit excluding side effects. Theacrine has nowadays gained a lot of interest for its multiple benefits, while the investigations are weak and insufficient. HYPOTHESIS/PURPOSE: It is well-known that tea has a wide range of functions, especially in the prevention and treatment of neurodegenerative diseases. Theacrine is an active monomer compound in Camellia assamica var. kucha Hung T. Chang & H.S.Wang (Kucha), which appears to be effective and safe in PD therapy. The aim of this study is to examine its actions in diverse PD models and explore the mechanisms. STUDY DESIGN: For determination of theacrine's effects, we employed diverse oxidative damage-associated PD models, including 6-OHDA-treated rats, MPTP-treated mice/zebrafish and MPP+-treated SH-SY5Y cells, and using caffeine, selegiline and depranyl as positve control. For investigation and verification of the mechanisms, we utilized approaches testing mitochondrial function-related parameters and enzyme activity as well as applied gene knockdown and overexpression. METHODS: We employed behavioral tests including spontaneous activity, pole, swimming, rotarod and gait, immunohistochemistry, HPLC, flow cytometry, immunohistochemistry, Western blot, gene knockdown by siRNA and overexpression by plasmid in this study. RESULTS: Theacrine is demonstrated to retrieve the loss of dopaminergic neurons and the damages of behavioral performance in multiple animal models of PD (6-OHDA-treated rats and in MPTP-treated mice and zebrafish). The followed data of MPP+-treated SH-SY5Y cells indicate that theacrine relieves apoptosis resulted from oxidative damage and mitochondrial dysfunction. Further investigations illustrate that theacrine activates SIRT3 directly. It is of advantage to prevent apoptosis through SIRT3-mediated SOD2 deacetylation that reduces ROS accumulation and restores mitochondrial function. This concept is elaborated by 3TYP that inhibits SIRT3 enzyme activity and knockdown/overexpression of SIRT3 gene, demonstrating a crucial role of SIRT3 in theacrine-benefited dopaminergic neurons. CONCLUSION: Theacrine prevents apoptosis of dopaminergic neurons through directly activating SIRT3 which deacetylating SOD2 and restoring mitochondrial functions.

Dopaminergic Modulation of Orofacial Mechanical Hypersensitivity Induced by Infraorbital Nerve Injury.

While the descending dopaminergic control system is not fully understood, it is reported that the hypothalamic A11 nucleus is its principle source. To better understand the impact of this system, particularly the A11 nucleus, on neuropathic pain, we created a chronic constriction injury model of the infraorbital nerve (ION-CCI) in rats. ION-CCI rats received intraperitoneal administrations of quinpirole (a dopamine D2 receptor agonist). ION-CCI rats received microinjections of quinpirole, muscimol [a gamma-aminobutyric acid type A (GABAA) receptor agonist], or neurotoxin 6-hydroxydopamine (6-OHDA) into the A11 nucleus. A von Frey filament was used as a mechanical stimulus on the maxillary whisker pad skin; behavioral and immunohistochemical responses to the stimulation were assessed. After intraperitoneal administration of quinpirole and microinjection of quinpirole or muscimol, ION-CCI rats showed an increase in head-withdrawal thresholds and a decrease in the number of phosphorylated extracellular signal-regulated kinase (pERK) immunoreactive (pERK-IR) cells in the superficial layers of the trigeminal spinal subnucleus caudalis (Vc). Following 6-OHDA microinjection, ION-CCI rats showed a decrease in head-withdrawal thresholds and an increase in the number of pERK-IR cells in the Vc. Our findings suggest the descending dopaminergic control system is involved in the modulation of trigeminal neuropathic pain.

Ginsenoside Re Mitigates 6-Hydroxydopamine-Induced Oxidative Stress through Upregulation of GPX4.

Ginsenosides are active components found abundantly in ginseng which has been used as a medicinal herb to modify disease status for thousands of years. However, the pharmacological activity of ginsenoside Re in the neuronal system remains to be elucidated. Neuroprotective activity of ginsenoside Re was investigated in SH-SY5Y cells exposed to 6-hydroxydopamine (6-OHDA) to induce cellular injury. Ginsenoside Re significantly inhibited 6-OHDA-triggered cellular damage as judged by analysis of tetrazolium dye reduction and lactose dehydrogenase release. In addition, ginsenoside Re induced the expression of the antioxidant protein glutathione peroxidase 4 (GPX4) but not catalase, glutathione peroxidase 1, glutathione reductase, or superoxide dismutase-1. Furthermore, upregulation of GPX4 by ginsenoside Re was mediated by phosphoinositide 3-kinase and extracellular signal-regulated kinase but not by p38 mitogen-activated protein kinase or c-Jun N-terminal kinase. Ginsenoside Re also suppressed 6-OHDA-triggered cellular accumulation of reactive oxygen species and peroxidation of membrane lipids. The GPX4 inhibitor (1S,3R)-RSL3 reversed ginsenoside Re-mediated inhibition of cellular damage in SH-SY5Y cells exposed to 6-OHDA, indicating that the neuronal activity of ginsenoside Re is due to upregulation of GPX4. These findings suggest that ginsenoside Re-dependent upregulation of GPX4 reduces oxidative stress and thereby alleviates 6-OHDA-induced neuronal damage.

Complementation of dopaminergic signaling by Pitx3-GDNF synergy induces dopamine secretion by multipotent Ntera2 cells.

Human teratocarcinoma cell line Ntera2 (NT2) expresses dopamine signals and has shown its safe profile for clinical applications. Attempts to restore complete dopaminergic (DAergic) phenotype enabling these cells to secrete dopamine have not been fully successful so far. We applied a blend of gene transfer techniques and a defined medium to convert NT2 cells to fully DAergic. The cells were primarily engineered to overexpress the Pitx3 gene product and then cultured in a growth medium supplemented with knockout serum and retinoic acid to form embroid bodies (EBs). Trypsinization of EB colonies produced single cells ready for differentiation. Neuronal/DAergic induction was promoted by applying conditioned medium taken from engineered human astrocytomas over-secreting glial cell-derived neurotrophic factor (GDNF). Immunocytochemistry, reverse-transcription and real-time polymerase chain reaction analyses confirmed significantly induced expression of molecules involved in dopamine signaling and metabolism including tyrosine hydroxylase, Nurr1, dopamine transporter, and aromatic acid decarboxylase. High-performance liquid chromatography analysis indicated release of dopamine only from a class of fully differentiated cells expressing Pitx3 and exposed to GDNF. In addition, Pitx3 and GDNF additively promoted in vitro neuroprotection against Parkinsonian toxin. One month after transplantation to the striatum of 6-OHDA-leasioned rats, differentiated NT2 cells survived and induced significant increase in striatal volume. Besides, cell implantation improved motor coordination in Parkinson's disease (PD) rat models. Our findings highlight the importance of Pitx3-GDNF interplay in dopamine signaling and indicate that our strategy might be useful for the restoration of DAergic fate of NT2 cells to make them clinically applicable toward cell replacement therapy of PD.

Protective effects of DA-9805 on dopaminergic neurons against 6-hydroxydopamine-induced neurotoxicity in the models of Parkinson's disease.

With the elderly population rapidly growing, the prevalence of Parkinson's disease (PD) is quickly increasing because neurodegenerative disorders are usually late-onset. Herbal medicines and formula are adjuvant therapies of conventional PD agents, which result in serious side effects with long-term use. This study evaluated the neuroprotective effects of DA-9805, a standardized herbal formula that consists of an ethanolic extract of Moutan Cortex Radix, Angelica Dahuricae Radix, and Bupleuri Radix against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity in vitro and in vivo. In PC12 cells, DA-9805 at concentrations of 1 and 10 µg/mL ameliorated cell viability, which was reduced by 6-OHDA. In addition, DA-9805 activated the extracellular-regulated kinase-nuclear transcription factor-erythroid 2-related factor 2 pathway, subsequently stimulating antioxidative enzymes such as NAD(P)H:quinone oxidoreductase 1 and catalase and suppressing apoptosis. Furthermore, DA-9805 prevented 6-OHDA-induced movement impairment, as well as a decrease of dopaminergic neurons and dopamine transmission in rodents. Taken together, these results suggest that the mixed herbal formula DA-9805 may be a pharmaceutical agent for preventing or improving PD.

Synthesis of andrographolide analogues and their neuroprotection and neurite outgrowth-promoting activities.

Alzheimer's disease (AD) is the most prevalent neurodegenerative disease and remains incurable. Currently, neuronal injury and synapse loss have been considered to be main features in the pathophysiology of AD. Thus, modulation of neuronal survival and neurite outgrowth may represent an efficient strategy for the treatment of AD. Based on the isolates from the traditional medicine Andrographis paniculata, a series of andrographolide analogues were prepared and evaluated for the neuroprotection and neurotrophic activity. Two compounds (3 and 12) could effectively inhibit LPS-induced NO production and iNOS expression as well as proinflammatory cytokines TNF-α and IL-6. Moreover, pretreatment with 3 and 12 could protect neurons against microglia-mediated neurotoxicity. Further, H2O2- and 6-OHDA induced neurotoxicity in PC12 cells were also attenuated by the novel 12. Our next study indicated that compounds 1, 4 and 10 promoted NGF-induced neurite outgrowth in PC12 cells, with 10 the most potent. To clarify the underlying mechanisms of active compounds (3, 10 and 12), system pharmacology was employed. The results revealed that muscarinic acetylcholine receptors (mAChRs) may be the main targets of 12 against AD, while thyroid hormone signaling pathway was involved in the mechanisms of 10. These study point to the therapeutic potential of andrographolide analogues against AD.

Reduction of HIP2 expression causes motor function impairment and increased vulnerability to dopaminergic degeneration in Parkinson's disease models.

Huntingtin interaction protein 2 (HIP2) is an E2 ubiquitin-conjugating enzyme associated with neurodegenerative diseases, and HIP2 mRNA has been implicated as a potential blood biomarker for Parkinson's disease (PD). However, it is unclear whether the alteration of HIP2 expression may contribute to the development of PD, and whether the change of HIP2 in blood could reflect its expression in the brain or motor functions in PD patients. In this study, we established a mouse line with HIP2 haploinsufficiency. The reduction of the HIP2 expression led to spontaneous motor function impairment and dopaminergic neuronal loss. Furthermore, HIP2 haploinsufficiency increased the susceptibility of mice to 6-hydroxydopamine (6-OHDA) and caused severe loss of dopaminergic neurons. Interestingly, in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model for PD, we observed concurrent, highly correlated decrease of HIP2 expression in the brain and in the blood. Using blood samples from more than 300 patients, we validated the decreased HIP2 mRNA in PD patients, including de novo patients. Finally, in a 1-year, 20-patient study, we observed reversed blood HIP2 mRNA levels accompanying improved motor and overall daily functions in 75% of the PD patients with instructed Tai Chi training. Therefore, our in vivo studies have indicated HIP2 insufficiency as a contributing factor for PD, and functionally validated blood HIP2 as a useful and reversible biomarker for PD.

Evaluation of phytomedicinal potential of perillyl alcohol in an in vitro Parkinson's Disease model.

Preclinical Research & Development Parkinson's disease (PD) is the second most common neurodegenerative disorder that affects approximately 10 million people worldwide. The risk of developing PD and similar neurodegenerative disorders increases with age and an estimated 4% people are diagnosed with the disease before reaching the age of 50. Oxidative stress, cytotoxicity, and mitochondrial dysfunction are common features exhibited in the development of PD. The 6-hyroxydopamine (6-OHDA) model of PD is one of the most well characterized and studied models of the disease. 6-OHDA, a neurotoxin, can induce most characteristic features of the disease, including mitochondrial dysfunction in-vivo and in-vitro. SH-SY5Y is a neuroblastoma cell line of human origin that has been used for dose response studies on PD in the past. Based on previous data, we have used SH-SY5Y cells as an in-vitro model of PD to analyse the phytomedicinal potential of perillyl alcohol (PA), a monoterpenoid obtained from essential oils of various plants such as sage, peppermint and lavender. We have found that pretreatment with PA (10 µM and 20 µM) mitigated 6-OHDA (150 µM) induced cytotoxicity in a dose-dependent manner. We observed marked restoration of cell viability and mitochondrial membrane potential (MMP) as well as reduced reactive oxygen species generation, Cytochrome c immunofluorescence and DNA fragmentation after treatment with PA. On the basis of on our data, we have come to the conclusion that PA demonstrates sufficient neuroprotective activity to provide new avenues in therapy of PD and its apparent target being restoration of MMP can lead to better understanding of the disease.

Lateral but not Medial Hypothalamic AMPK Activation Occurs at the Hypoglycemic Nadir in Insulin-injected Male Rats: Impact of Caudal Dorsomedial Hindbrain Catecholamine Signaling.

The hypothalamic energy sensor adenosine 5'-monophosphate-activated protein kinase (AMPK), an important regulator of counter-regulatory responses to hypoglycemia, responds to pharmacological manipulation of hindbrain AMPK activity. Dorsomedial hindbrain A2 noradrenergic neurons express hypoglycemia-sensitive metabolo-sensory biomarkers, including AMPK. Here, adult male rats were pretreated by intra-caudal fourth ventricular administration of the selective neurotoxin 6-hydroxydopamine (6-OHDA) to determine if catecholamine signaling from the aforesaid site governs hypothalamic AMPK activation during insulin-induced hypoglycemia (IIH). Micropunched arcuate (ARH), ventromedial (VMH), paraventricular (PVH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissues were obtained at the neutral protamine Hagedorn insulin-induced hypoglycemic nadir, coincident with A2 AMPK activation, for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant metabolic neuropeptides. ARH, VMH, LHA, and DMH norepinephrine levels were altered according to insulin dose; 6-OHDA-mediated reversal of these responses was site-specific. IIH elevated LHA and reduced VMH pAMPK protein, profiles that were respectively unchanged or increased by 6-OHDA. PVH and ARH pAMPK was resistant to IIH, but augmented in ARH of neurotoxin- plus insulin-treated rats. ARH neuropeptide Y (NPY) and pro-opiomelanocortin (POMC) proteins were correspondingly increased or refractory to IIH; 6-OHDA pretreatment normalized NPY and elevated POMC expression after insulin injection. Results demonstrate site-specific bi-directional adjustments in hypothalamic AMPK reactivity to hypoglycemia. Intensification of ARH/VMH pAMPK by 6-OHDA implies dorsomedial hindbrain improvement of energy balance in those sites during IIH. Neurotoxin-mediated augmentation versus suppression of basal catabolic (ARH POMC/VMH steroidogenic factor-1) or IIH-associated anabolic (ARH NPY) neuropeptide profiles, respectively, may involve local AMPK-dependent against independent mechanisms.

Involvement of adenosine triphosphate-sensitive potassium channels in the neuroprotective activity of hydrogen sulfide in the 6-hydroxydopamine-induced animal model of Parkinson's disease.

Studies have shown that hydrogen sulfide (H2S) exerts a neuroprotective effect and may have a therapeutic value for treating neurodegenerative diseases including Parkinson's disease. However, little is known about the mechanisms underlying the neuroprotective activity of H2S in vivo. Here, we evaluated the effect of glibenclamide, an ATP-sensitive potassium channel blocker, on the neuroprotective activity of H2S in the 6-hydroxydopamine (6-OHDA) animal model of Parkinson's disease. 6-OHDA was administered by stereotaxic surgery into the medial forebrain bundle. Sodium hydrosulfate (NaHS, 3 and 5.6 mg/kg), as a donor of H2S, alone or in combination with glibenclamide (5 mg/kg), was daily injected for 7 days starting 1-2 h before the stereotaxic surgery. After an apomorphine-induced rotational test, the number of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta was determined by immunofluorescence. The striatal dopamine level and oxidative stress markers were also measured in brain homogenates. Pretreatment with NaHS significantly attenuated 6-OHDA-induced motor asymmetry in the rotational test. Histological and biochemical evaluations demonstrated that NaHS, especially at high dose, increased the survival of tyrosine hydroxylase-positive neurons in the substantia nigra pars compacta and reduced the decreasing effect of 6-OHDA on striatal dopamine levels. However, co-administration of glibenclamide reversed the antiparkinsonian and neuroprotective effects of NaHS. However, glibenclamide did not change the reducing effect of NaHS on 6-OHDA-induced overproduction of malondialdehyde. Our data show that ATP-sensitive potassium channels are involved in the antiparkinsonian and neuroprotective effects of H2S in the 6-OHDA animal model of Parkinson's disease.

Dopamine is Required for Activity-Dependent Amplification of Arc mRNA in Developing Postnatal Frontal Cortex.

The activity-regulated gene Arc/Arg3.1 encodes a postsynaptic protein crucially involved in glutamatergic synaptic plasticity. Genetic mutations in Arc pathway and altered Arc expression in human frontal cortex have been associated with schizophrenia. Although Arc expression has been reported to vary with age, what mechanisms regulate Arc mRNA levels in frontal cortex during postnatal development remains unclear. Using quantitative mRNA analysis of mouse frontal cortical tissues, we mapped the developmental profiles of Arc expression and found that its mRNA levels are sharply amplified near the end of the second postnatal week, when mouse pups open their eyes for the first time after birth. Surprisingly, electrical stimulation of the frontal cortex before eye-opening is not sufficient to drive the amplification of Arc mRNA. Instead, this amplification needs both electrical stimulation and dopamine D1-type receptor (D1R) activation. Furthermore, visual stimuli-driven amplification of Arc mRNA is also dependent on D1R activation and dopamine neurons located in the ventral midbrain. These results indicate that dopamine is required to drive activity-dependent amplification of Arc mRNA in the developing postnatal frontal cortex and suggest that joint electrical and dopaminergic activation is essential to establish the normal expression pattern of a schizophrenia-associated gene during frontal cortical development.