Preclinical modeling of chronic inhibition of the Parkinson's disease associated kinase LRRK2 reveals altered function of the endolysosomal system in vivo.

The most common mutation in the Leucine-rich repeat kinase 2 gene (LRRK2), G2019S, causes familial Parkinson's Disease (PD) and renders the encoded protein kinase hyperactive. While targeting LRRK2 activity is currently being tested in clinical trials as a therapeutic avenue for PD, to date, the molecular effects of chronic LRRK2 inhibition have not yet been examined in vivo. We evaluated the utility of newly available phospho-antibodies for Rab substrates and LRRK2 autophosphorylation to examine the pharmacodynamic response to treatment with the potent and specific LRRK2 inhibitor, MLi-2, in brain and peripheral tissue in G2019S LRRK2 knock-in mice. We report higher sensitivity of LRRK2 autophosphorylation to MLi-2 treatment and slower recovery in washout conditions compared to Rab GTPases phosphorylation, and we identify pS106 Rab12 as a robust readout of downstream LRRK2 activity across tissues. The downstream effects of long-term chronic LRRK2 inhibition in vivo were evaluated in G2019S LRRK2 knock-in mice by phospho- and total proteomic analyses following an in-diet administration of MLi-2 for 10 weeks. We observed significant alterations in endolysosomal and trafficking pathways in the kidney that were sensitive to MLi-2 treatment and were validated biochemically. Furthermore, a subtle but distinct biochemical signature affecting mitochondrial proteins was observed in brain tissue in the same animals that, again, was reverted by kinase inhibition. Proteomic analysis in the lung did not detect any major pathway of dysregulation that would be indicative of pulmonary impairment. This is the first study to examine the molecular underpinnings of chronic LRRK2 inhibition in a preclinical in vivo PD model and highlights cellular processes that may be influenced by therapeutic strategies aimed at restoring LRRK2 physiological activity in PD patients.

Effect of Zishenpingchan granule prepared from Chinese medicinal substances on the c-Jun N-terminal protein kinase pathway in mice with Parkinson's disease induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

OBJECTIVE: To investigate the regulatory mechanism of the c-Jun N-terminal protein kinase (JNK) signaling pathway in substantia nigra (SN) dopaminergic neurons inflammation and apoptosis, and the neuroprotective effect of Zishenpingchan granules in mice with Parkinson's disease (PD) induced by 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP). METHODS: PD model mice were established by intraperitoneally injecting MPTP. Sixty mice were divided into a model group, Traditional Chinese Medicine (TCM) group and control group. The mice of the TCM group were administered Zishenpingchan granules 7 days before PD induction. Seven days after PD induction, we examined locomotor activity, and performed the rotarod test and swimming test, to evaluate limb movement function. Furthermore, we used immunohistochemistry and western blotting to examine the expression of tyrosine hydroxylase (TH), cyclooxygenase-2 (Cox-2), caspase-3 and p-JNK. The terminal deoxynucleotidyl transferase mediated dUTP nick end labeling (TUNEL) method was used to examine neuron apoptosis in the SN. RESULTS: Compared with the control group, the mean score of locomotor activity, rotarod test and swimming test was significantly lower in the model group (P < 0.05); the TH-positive neuron expression was significantly decreased in the SN pars compacta (SNpc); the protein expression levels of Cox-2, caspase-3 and p-JNK was obviously increased; and the number of TUNEL-positive neurons in the SN was increased (P < 0.01). Compared with the model group, the mean score of neurobehavioral tests in the TCM group was obviously higher, the loss of TH-positive neurons ignificantly decreased, the protein expression levels of Cox-2, caspase-3 and p-JNK obviously decreased, and the number of TUNEL- positive neurons in the SN clearly decreased (P < 0.01). CONCLUSION: The JNK pathway plays an important role in the regulation of inflammation and apoptosis in nigral cells in PD mice. TCM can suppress the over-activation of the JNK pathway in the SN, and alleviate the inflammatory response in nigral cells and dopaminergic neuron apoptosis in PD mice.

A Small Molecule Activator of p300/CBP Histone Acetyltransferase Promotes Survival and Neurite Growth in a Cellular Model of Parkinson's Disease.

Parkinson's disease (PD) is a progressive neurodegenerative disease characterised by motor and non-motor symptoms, resulting from the degeneration of nigrostriatal dopaminergic neurons and peripheral autonomic neurons. Given the limited success of neurotrophic factors in clinical trials, there is a need to identify new small molecule drugs and drug targets to develop novel therapeutic strategies to protect all neurons that degenerate in PD. Epigenetic dysregulation has been implicated in neurodegenerative disorders, while targeting histone acetylation is a promising therapeutic avenue for PD. We and others have demonstrated that histone deacetylase inhibitors have neurotrophic effects in experimental models of PD. Activators of histone acetyltransferases (HAT) provide an alternative approach for the selective activation of gene expression, however little is known about the potential of HAT activators as drug therapies for PD. To explore this potential, the present study investigated the neurotrophic effects of CTPB (N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benzamide), which is a potent small molecule activator of the histone acetyltransferase p300/CBP, in the SH-SY5Y neuronal cell line. We report that CTPB promoted the survival and neurite growth of the SH-SY5Y cells, and also protected these cells from cell death induced by the neurotoxin 6-hydroxydopamine. This study is the first to investigate the phenotypic effects of the HAT activator CTPB, and to demonstrate that p300/CBP HAT activation has neurotrophic effects in a cellular model of PD.

Synthetic and Natural Monoamine Oxidase Inhibitors as Potential Lead Compounds for Effective Therapeutics.

Monoamine oxidases A and B (MAO-A and B) play a critical role in the metabolism of intracellular neurotransmitters of the central nervous system. For decades, MAO inhibitors have proven their clinical efficacy as potential drug targets for several neurological and neurodegenerative diseases. Use of first generation non selective MAO inhibitors as neuropsychiatric drugs elicited several side effects like hypertensive crisis and cheese reaction. Therefore their use is now limited due to non-selectivity towards MAO isoforms and inhibition of metabolizing enzymes like cytochrome P450. Development of selective and specific MAO inhibitors like moclobemide, toloxatone improves their efficacy as disease-modifying effects in monotherapy as well as adjunctive therapy. Recently a lot of research has been done to elucidate the pharmacological potential of medicinal plants and their isolated bioactive constituents having MAO inhibitory activity. Herbs containing MAO inhibitors are extensively used for the development of potent synthetic drugs and as safe and effective alternatives to the available synthetic drugs in the treatment of neurodegenerative diseases such as depression, Parkinson and Alzheimer's. In several diseases like Parkinson natural MAO inhibitors prevented the neuron denaturalization by their dual action via enhancing neurotransmission of dopamine as well as lowering the generation of free radicals and toxins. Currently development of selective MAO inhibitors is still under study to achieve more effective therapies by using Computer Aided Drug Designing, Ligand-based models and structure-activity hypothesis. These approaches also facilitate understanding the interaction of newly designed molecule with MAO enzymes and the rationalization of probable mechanisms of action.

Impact of physiological, pathological and environmental factors on the expression and activity of human cytochrome P450 2D6 and implications in precision medicine.

With only 1.3-4.3% in total hepatic CYP content, human CYP2D6 can metabolize more than 160 drugs. It is a highly polymorphic enzyme and subject to marked inhibition by a number of drugs, causing a large interindividual variability in drug clearance and drug response and drug-drug interactions. The expression and activity of CYP2D6 are regulated by a number of physiological, pathological and environmental factors at transcriptional, post-transcriptional, translational and epigenetic levels. DNA hypermethylation and histone modifications can repress the expression of CYP2D6. Hepatocyte nuclear factor-4α binds to a directly repeated element in the promoter of CYP2D6 and thus regulates the expression of CYP2D6. Small heterodimer partner represses hepatocyte nuclear factor-4α-mediated transactivation of CYP2D6. GW4064, a farnesoid X receptor agonist, decreases hepatic CYP2D6 expression and activity while increasing small heterodimer partner expression and its recruitment to the CYP2D6 promoter. The genotypes are key determinants of interindividual variability in CYP2D6 expression and activity. Recent genome-wide association studies have identified a large number of genes that can regulate CYP2D6. Pregnancy induces CYP2D6 via unknown mechanisms. Renal or liver diseases, smoking and alcohol use have minor to moderate effects only on CYP2D6 activity. Unlike CYP1 and 3 and other CYP2 members, CYP2D6 is resistant to typical inducers such as rifampin, phenobarbital and dexamethasone. Post-translational modifications such as phosphorylation of CYP2D6 Ser135 have been observed, but the functional impact is unknown. Further functional and validation studies are needed to clarify the role of nuclear receptors, epigenetic factors and other factors in the regulation of CYP2D6.

[Effect of Electroacupuncture on 26 S Proteasome and Nuclear Factor kappa B in Substantia Nigra of Rats with Rotenone-induced Parkinson's Disease].

OBJECTIVE: To explore the role of 26 S proteasome and nuclear factor kappa B (NFκB) in substantia nigra in the management of Parkinson's disease (PD) by acupuncture. METHODS: Forty-eight male SD rats were randomly divided into normal, sham operation, model and electroacupuncture (EA) groups (12 rats/group).. The PD model was established by 40-day consecutive subcutaneous injection of rotenone (1 mg/kg dissolved in dimethyl sulfoxide and normal saline) at the back shoulder. The rats in the sham operation group were treated by subcutaneous injection of dose of saline. "Fengfu" (GV 16) and "Taichong" (LR 3) acupoints were stimulated with EA at 2 Hz, 1 mA, 20 min of duration in each treatment, and daily for 28 consecutive days. The behavioral changes of rats in each group were measured and scored at 40th day and 68th day, respectively. Immunohistochemistry was used to determine the expression of tyrosine hydroxylase (TH) used to detect the expression of 26 S proteasome and NFκB and TH were measured by Western blot. RESULTS: In comparison with the normal and sham operation groups, the behavioral scores of rats in the model group were elevated, which were significantly decreased by EA intervention (P < 0.05). The expression of TH and 26 S proteasome decreased whereas the NFκB increased in the rats of model group (P < 0.05); and EA intervention reversed these changes (all P < 0.05). CONCLUSION: EA intervention can improve PD rats' behavioral changes, which is pobably related to its effects in reducing loss of TH-positive neurons, down-regulating NFκB protein expression, and up- regulating 26 S proteasome protein expression in the substantia nigra.

[Effects of electroacupuncture on proteasomes of substantia nigra in rats with Parkinson's disease].

OBJECTIVE: To explore the effects and action mechanism of electroacupuncture (EA) on Parkinson's disease (PD). METHODS: Forty-eight healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and an EA group, 12 rats in each one. Rats in the model group and EA group were treated with subcutaneous injection of rotenone (1mg/kg, dissolved in dimethyl sulfoxide and 0. 9 % normal saline) on neck and back for 40 days to establish rat model. Rats in the sham operation group were treated with injection of identical dose of dimethyl sulfoxide and 0. 9 %o normal saline at identical location which did not contain rotenone. After model establishment, rats in the EA group were treated with EA at "Fengfu" (GV 16) and "Taichong" (LR 3) with continuous wave (2 Hz, 1 mA), which was given 20 min per time, once a day for consecutive 28 days. Rats in the remaining groups were treated with fixation and immobilization without any other intervention. The rats behavioristics changes were observed and scored; immunohisto-chemistry was adopted to test the expression of tyrosine hydroxylase (TH); fluorescence spectrometry was used to detect the activities of 20 S ß1, ß2, ß5; western blot method was applied to measure the expression of 20S proteasome and its a subunit. RESULTS: Compared with the normal group and sham operation group, there was significant change of behavioristics in the model group, and TH positive neuron counting was obviously reduced; after treatment, the behavioristics score in the EA group was lower than that in the model group (P<0. 05), and TH positive neuron counting was significantly increased (P<0. 05). Compared with the normal group and sham operation group, the activities of 20 S ß1, ß2, ß5 in model group were significantly reduced (all P<0. 01), and those in the EA group were higher than those in the model group (P<0. 01). Compared with the normal group and sham operation group, the expression of 20S proteasome and its a subunit was reduced in the model group, and that in the EA group was higher than that in the model group (P<0. 05). CONCLUSION: EA could improve the loss of dopaminergic neurons induced by rotenone to prevent and treat PD, which is likely to be related with protecting the activity and expression of proteasomes in substantia nigra.

Carnosic acid protects against 6-hydroxydopamine-induced neurotoxicity in in vivo and in vitro model of Parkinson's disease: involvement of antioxidative enzymes induction.

The neuroprotective effects of carnosic acid (CA), a phenolic diterpene isolated from rosemary (Rosmarinus officinalis), have been widely investigated in recent years, however, its protection in in vivo still unclear. In this study, we investigated the behavioral activity and neuroprotective effects of CA in a rat model of Parkinson's disease (PD) induced by 6-hydroxydopamine (6-OHDA). Rats were treated with 20mg/kg body weight of CA for 3 weeks before 6-OHDA exposure. Results indicated that CA improved the locomotor activity and reduced the apomorphine-caused rotation in 6-OHDA-stimulated rats. Significant protection against lipid peroxidation and GSH reduction was observed in the 6-OHDA rats pretreated with CA. Pretreatment with CA increased the protein expression of γ-glutamate-cysteine ligase catalytic subunit, γ-glutamate-cysteine ligase modifier subunit, superoxide dismutase, and glutathione reductase compared with 6-OHDA-stimulated rats and SH-SY5Y cells. Immunoblots showed that the reduction of the Bcl-2/Bax ratio, the induction of caspase 3 cleavage, and the induction of poly(ADP-ribose) polymerase (PARP) cleavage by 6-OHDA was reversed in the presence of SB203580 (a p38 inhibitor) or SP600125 (a JNK inhibitor) in SH-SY5Y cells. Rats treated with CA reversed the 6-OHDA-mediated the activation of c-Jun NH2-terminal kinase and p38, the down-regulation of the Bcl-2/Bax ratio, the up-regulation of cleaved caspase 3/caspase 3 and cleaved PARP/PARP ratio, and the down-regulation of tyrosine hydroxylase protein. However, BAM7, an activator of Bax, attenuated the effect of CA on apoptosis in SH-SY5Y cells. These results suggest that CA protected against 6-OHDA-induced neurotoxicity is attributable to its anti-apoptotic and anti-oxidative action. The present findings may help to clarify the possible mechanisms of rosemary in the neuroprotection of PD.

Abberant protein synthesis in G2019S LRRK2 Drosophila Parkinson disease-related phenotypes.

LRRK2 mutations are a frequent cause of familial Parkinson disease (PD) and are also found in a number of sporadic PD cases. PD-linked G2019S and I2020T mutations in the kinase domain of LRRK2 result in elevated kinase activity, which is required for the toxicity of these pathogenic variants in cell and animal models of PD. We recently reported that LRRK2 interacts with and phosphorylates a number of mammalian ribosomal proteins, several of which exhibit increased phosphorylation via both G2019S and I2020T LRRK2. Blocking the phosphorylation of ribosomal protein s15 through expression of phospho-deficient T136A s15 prevents age-associated locomotor deficits and dopamine neuron loss caused by G2019S LRRK2 expression in Drosophila indicating that s15 is a pathogenic LRRK2 substrate. We previously described that G2019S LRRK2 causes an induction of bulk mRNA translation that is blocked by T136A s15 or the protein synthesis inhibitor anisomycin. Here, we report the protective effects of the eIF4E/eIF4G interaction inhibitor 4EGI-1, in preventing neurodegenerative phenotypes in G2019S LRRK2 flies, and discuss how our findings and those of other groups provide a framework to begin investigating the mechanistic impact of LRRK2 on translation.

A randomized clinical trial of high-dosage coenzyme Q10 in early Parkinson disease: no evidence of benefit.

IMPORTANCE: Coenzyme Q10 (CoQ10), an antioxidant that supports mitochondrial function, has been shown in preclinical Parkinson disease (PD) models to reduce the loss of dopamine neurons, and was safe and well tolerated in early-phase human studies. A previous phase II study suggested possible clinical benefit. OBJECTIVE: To examine whether CoQ10 could slow disease progression in early PD. DESIGN, SETTING, AND PARTICIPANTS: A phase III randomized, placebo-controlled, double-blind clinical trial at 67 North American sites consisting of participants 30 years of age or older who received a diagnosis of PD within 5 years and who had the following inclusion criteria: the presence of a rest tremor, bradykinesia, and rigidity; a modified Hoehn and Yahr stage of 2.5 or less; and no anticipated need for dopaminergic therapy within 3 months. Exclusion criteria included the use of any PD medication within 60 days, the use of any symptomatic PD medication for more than 90 days, atypical or drug-induced parkinsonism, a Unified Parkinson's Disease Rating Scale (UPDRS) rest tremor score of 3 or greater for any limb, a Mini-Mental State Examination score of 25 or less, a history of stroke, the use of certain supplements, and substantial recent exposure to CoQ10. Of 696 participants screened, 78 were found to be ineligible, and 18 declined participation. INTERVENTIONS: The remaining 600 participants were randomly assigned to receive placebo, 1200 mg/d of CoQ10, or 2400 mg/d of CoQ10; all participants received 1200 IU/d of vitamin E. MAIN OUTCOMES AND MEASURES: Participants were observed for 16 months or until a disability requiring dopaminergic treatment. The prospectively defined primary outcome measure was the change in total UPDRS score (Parts I-III) from baseline to final visit. The study was powered to detect a 3-point difference between an active treatment and placebo. RESULTS: The baseline characteristics of the participants were well balanced, the mean age was 62.5 years, 66% of participants were male, and the mean baseline total UPDRS score was 22.7. A total of 267 participants required treatment (94 received placebo, 87 received 1200 mg/d of CoQ10, and 86 received 2400 mg/d of CoQ10), and 65 participants (29 who received placebo, 19 who received 1200 mg/d of CoQ10, and 17 who received 2400 mg/d of CoQ10) withdrew prematurely. Treatments were well tolerated with no safety concerns. The study was terminated after a prespecified futility criterion was reached. At study termination, both active treatment groups showed slight adverse trends relative to placebo. Adjusted mean changes (worsening) in total UPDRS scores from baseline to final visit were 6.9 points (placebo), 7.5 points (1200 mg/d of CoQ10; P = .49 relative to placebo), and 8.0 points (2400 mg/d of CoQ10; P = .21 relative to placebo). CONCLUSIONS AND RELEVANCE: Coenzyme Q10 was safe and well tolerated in this population, but showed no evidence of clinical benefit. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00740714.

[Effect of electroacupuncture stimulation of "Fengfu" (GV 16) and "Taichong" (LR 3) on expression of COX-2 and tyrosine hydroxylase in Substantia Nigra in rats with Parkinson's disease].

OBJECTIVE: To observe the influence of electroacupuncture (EA) therapy on the expression of tyrosine hydroxylase (TH) and cyclooxygenase-2 (COX-2) proteins of Substantia Nigra cells(SNc) in the rotenone-induced Parkinson's disease(PD) rats, so as to explore the mechanism of EA underlying improvement of PD. METHODS: Forty rats were randomly divided into normal, sham-operation (sham), model and EA groups (n = 10/group). The PD model was established by successive subcutaneous injection of rotenone (highly selective lesions of nigrostriatal dopaminergic neurons) for 28 days. EA (2 Hz, 1 mA) was applied to bilateral "Fengfu" (GV 16) and "Taichong" (LR 3) for 20 min, once daily for 14 days. The expression levels of TH and COX-2 proteins in the Substantia Nigra of midbrain were detected with Western blotting. RESULTS: Compared with the normal group, the expression level of TH protein in the model group was significantly decreased (P < 0.01), and that of COX-2 protein in the model group was significantly increased (P < 0.01). After the EA treatment, the expression level of TH in the EA group was obviously upregulated (P < 0.01), and that of COX-2 protein in the EA group was considerably down-regulated (P < 0.01). No significant differences were found between the normal and sham groups in the expression levels of TH and COX-2 proteins (P > 0.05). CONCLUSION: EA therapy can decrease inflammation mediator COX-2 protein expression and upregulate TH protein expression in the Substantia Nigra of midbrain in PD rats, which may contribute to its effect in relieving PD in clinic.

[Effects of electroacupuncture on the expressions of neuroal nitric oxide synthase and astrocyte in dentate gyrus of rats with Parkinson's disease].

OBJECTIVE: To observe the changes in the expression of neuroal nitric oxide synthase (nNOS) and glial fibrillary acidic protein (GFAP) in dentate gyrus (DG) of rats with Parkinson's disease (PD) and effects of electroacupuncture (EA). METHODS: On the 7th day of stereotactic injection with 6-hydroxydopamine into right medial forebrain bundle, apomorphine-induced rotation was carried out to select the PD rats. The selected 12 rats were randomly divided into a model group (n = 6) and an EA group (n = 6). Moreover a normal group was established (n = 6). Then, the rats in EA group were treated with EA at "Hegu" (LI 4) and "Taichong" (LR 3), once a day for 21 days, and the other groups without any treatment. After EA treatment, the expressions of nNOS and GFAP in right DG were examined by immunohistochemistry method. RESULTS: The expression of nNOS in the right DG was weak in normal group, and the expression of nNOS in model group was significantly higher than that in normal group (P < 0.01), while in EA group, it was significantly lower than that in model (P < 0.01), with no difference between EA group and normal group (P > 0.05). The expression level of GFAP in model group was significantly higher than that in normal group (P < 0.01), while there was no difference in the number of GFAP positive cells between the above two groups (P > 0.05), and the number of GFAP positive cells in EA group was significantly increased compared with that in model group (P < 0.01), while with no significant difference in the expression level of GFAP between EA group and model group (P > 0.05). CONCLUSION: EA can reverse the increase of nNOS expression and promote the activation of astrocyte in DG on the injured side in rats with PD.

[Influence of electroacupuncture on p38-mitogen activated protein kinase in substantia nigra cells of rats with Parkinson disease model].

OBJECTIVE: To explore the role of inflammatory reaction mediated by p38-mitogen activated protein kinase (p38-MAPK) signal path on prevention and treatment of Parkinson disease (PD) model rats by electroacupuncture (EA). METHODS: Thirty-two healthy male SD rats were randomly divided into a normal group, a sham operation group, a model group and an EA group, eight rats in each one. The PD model was established in the model group and EA group by subcutaneous injection of rotenone in skin-back area (2 mg/kg, dissolved in sunflower oil, 2 mg/mL in density), while the injection of sunflower oil emulsion without rotenone at the same point and quantity as the model group was applied in the sham operation group. The normal group was not given any intervention. The EA treatment (continuous wave, 2 Hz in frequency, 1 mA in intensity, 20 min) was applied at "Fengfu" (GV 16) and "Taichong" (LR 3) in the EA group, once a day for continuously 14 days. No treatment was given in the other groups. The expression of tyrosine hydroxylase (TH), phosphorylated p38-MAPK, cyclooxygenase-2 (COX-2) in the substantia nigra were detected with immunohistochemical method. RESULTS: There was typical PD ethology change in the model group. Compared with the normal group and sham operation group, the expression of TH positive neuron in the substantia nigra in the model group was significantly decreased, while the expression of phosphorylated p38-MAPK and COX-2 were significantly increased (all P < 0.01). Compared with the model group, the expression of TH positive neuron in the EA group was apparently increased, while the expression of phosphorylated p38-MAPK and COX-2 were significantly decreased (all P < 0.01). CONCLUSION: The EA therapy could obviously reduce the expression of inflammation mediator COX-2, inhibit the phosphorylation of p38-MAPK, reduce the damage of dopaminergic neurons in the rats with PD, and this effect may be related with the impact of p38-MAPK signal path

Rescue of PINK1 protein null-specific mitochondrial complex IV deficits by ginsenoside Re activation of nitric oxide signaling.

PINK1, linked to familial Parkinson's disease, is known to affect mitochondrial function. Here we identified a novel regulatory role of PINK1 in the maintenance of complex IV activity and characterized a novel mechanism by which NO signaling restored complex IV deficiency in PINK1 null dopaminergic neuronal cells. In PINK1 null cells, levels of specific chaperones, including Hsp60, leucine-rich pentatricopeptide repeat-containing (LRPPRC), and Hsp90, were severely decreased. LRPPRC and Hsp90 were found to act upstream of Hsp60 to regulate complex IV activity. Specifically, knockdown of Hsp60 resulted in a decrease in complex IV activity, whereas antagonistic inhibition of Hsp90 by 17-(allylamino) geldanamycin decreased both Hsp60 and complex IV activity. In contrast, overexpression of the PINK1-interacting factor LRPPRC augmented complex IV activity by up-regulating Hsp60. A similar recovery of complex IV activity was also induced by coexpression of Hsp90 and Hsp60. Drug screening identified ginsenoside Re as a compound capable of reversing the deficit in complex IV activity in PINK1 null cells through specific increases of LRPPRC, Hsp90, and Hsp60 levels. The pharmacological effects of ginsenoside Re could be reversed by treatment of the pan-NOS inhibitor L-NG-Nitroarginine Methyl Ester (L-NAME) and could also be reproduced by low-level NO treatment. These results suggest that PINK1 regulates complex IV activity via interactions with upstream regulators of Hsp60, such as LRPPRC and Hsp90. Furthermore, they demonstrate that treatment with ginsenoside Re enhances functioning of the defective PINK1-Hsp90/LRPPRC-Hsp60-complex IV signaling axis in PINK1 null neurons by restoring NO levels, providing potential for new therapeutics targeting mitochondrial dysfunction in Parkinson's disease.

Heterogeneity of cholinergic denervation in Parkinson's disease without dementia.

Parkinson's disease (PD) is a multisystem neurodegenerative disorder. Heterogeneous clinical features may reflect heterogeneous changes in different brain regions. In contrast to the pronounced nigrostriatal denervation characteristic of PD, cholinergic changes are less marked. We investigated cholinergic innervation activity in PD subjects relative to normal subjects. Nondemented PD subjects (n=101, age 65.3±7.2 years) and normal subjects (n=29, age 66.8±10.9 years) underwent clinical assessment and [(11)C]methyl-4-piperidinyl propionate acetylcholinesterase and [(11)C]dihydrotetrabenazine monoaminergic positron emission tomography (PET) imaging. Cholinergic projection changes were heterogeneous for 65 out of 101 PD subjects who had neocortical and thalamic acetylcholinesterase activity within the normal range. The remainder had combined neocortical and thalamic (13/101), isolated neocortical (18/101), or isolated thalamic (5/101) acetylcholinesterase activity below the normal range. The low neocortical acetylcholinesterase activity subgroup had significantly lower global cognitive performance compared with the normal range subgroup (F=7.64, P=0.0069) with an independent effect for nigrostriatal denervation (F=7.60, P=0.0074). The low thalamic acetylcholinesterase activity subgroup did not differ from the normal thalamic acetylcholinesterase activity subgroup in cognitive performance or motor impairments except for a history of falls (P=0.0023). Cholinergic denervation is heterogeneous with reduced neocortical and/or thalamic acetylcholinesterase activity in 36% of nondemented PD subjects with corresponding clinical phenotypic variation. Results also show independent cognitive effects for both cholinergic and dopaminergic system changes in nondemented PD subjects.

Targeting Parkinson's - tyrosine hydroxylase and oxidative stress as points of interventions.

Parkinson's disease (PD) is characterized by the progressive loss of the dopaminergic neurons leading to decrease in striatal dopamine (DA) levels. In the present review, our focus was on recent advances in the treatment procedures of PD to achieve an increase in deficient tyrosine hydroxylase (TH) activity and/or expression. Stimulation of residual TH activity by the cofactors, 6R-L-erythro-tetrahydrobiopterin (BPH4) or NADH, or by brain transplant of natural TH-containing cells (fetal substantia nigra) or genetically engineered TH-containing cells, has been tried experimentally and clinically lately. As a promising approach to the gene therapy, intrastriatal expression of DAsynthesizing enzymes through transduction with separate adeno-associated virus (AAV) vectors/ marrow stromal cells (MSCs) or nonviral intravenous administration of rat transferrin receptor monoclonal antibody (TfRmAb)-targeted PEGylated immunoliposomes (PILs) has been found to be effective in animal models. Oxidative stress has been identified as one of the intermediary risk factors that could initiate and/or promote degeneration of DA neurons. TH itself is a prime target of oxidative/nitrosative injury. Certain superoxide dismutase and catalase mimetic prevented nitration of TH in cultured dopaminergic neurons. Therefore, development of therapeutic agents that can prevent formation of or specifically remove nitrating agents without interfering with normal neuronal function may protect protein from inactivation and provide means of limiting neuronal injury in PD. Non-pharmacological approaches such as diet therapy or use of active constituents of plants and phytomedicines have also emerged as a new - area of high interest. New treatment strategies for TH dysfunction rectification, a provision for neuroprotection in PD, seem to be on the horizon with many therapies under investigation.

Genetic mouse models for understanding LRRK2 biology, pathology and pre-clinical application.

Missense mutations in Leucine-Rich Repeat kinase 2 (LRRK2) are the most common cause of inherited Parkinson's disease (PD). Elucidation of LRRK2 biology and pathophysiology is central to the development of therapeutic intervention. Our group and others have developed a number of genetic mouse models of LRRK2 utilizing different genetic approaches. These models exhibit certain PD-related pathologies (e.g. impaired dopamine transmission and tauopathies) and abnormal motor functions, providing valuable insight into potential LRRK2-mediated pathogenesis of PD. However, not surprisingly they lack of substantial neuropathology and clinical syndromes of PD. Ongoing investigation of these models has begun to shed light on LRRK2 cellular functions and pathogenic pathways and is expected to assist the identification and validation of PD drug targets. This report summarizes the recent findings in our genetic LRRK2 models and discusses their utility in understanding much needed knowledge regarding early stage (pre-symptomatic) disease progression, drug target identification, and potential application in chemical screening focused on inhibitors of kinase activity of LRRK2.

Effect of Bushen Huoxue Decoction on the orphan receptor and tyrosine hydroxylase in the brain of rats with Parkinson's disease.

OBJECTIVE: To explore the effect of Bushen Huoxue Decoction (BHD) on the orphan receptor (Nurr1) and tyrosine hydroxylase (TH) in the brain of rats with Parkinson's disease (PD). METHODS: One hundred and twenty SD rats were divided into 100 in the model group and 20 in the normal control group, fifty-eight SD rats from the model group, established into PD model successfully by injuring their substantia nigra (SSN) with 6-hydroxydopamine, were divided equally into the model group and the test group, and they were treated with saline and BHD, respectively, for eight successive weeks. The change in the rats' behavior before and after treatment was observed by counting the cycles of rotation induced by apomorphine injection; the pathology of neurons, level of Nurr1 mRNA expression, and amount of TH positive cells in SSN were observed after treatment. RESULTS: The rats' behavior was improved in the tested group significantly, the rotation cycle after treatment being 84.0 ± 20.0 cycles/40 min, which was significantly lower than that in the model group (377.0 ± 62.3 cycles/40 min, P<0.01). Besides, the Nurr1 mRNA expression and TH positive cell in the test group were 0.97 ± 0.15 and 49.40 ± 14.72, respectively, which were significantly higher than those in the model group, 0.22 ± 0.03 and 5.45 ± 2.58, respectively (all P<0.01). CONCLUSION: BHD could treat PD by enhancing the Nurr1 mRNA expression, increasing the TH content in brain, and promoting the repairing of injured neuron in cerebral SSN.

HDAC inhibitor trichostatin A-inhibited survival of dopaminergic neuronal cells.

Histone deacetylase (HDAC) inhibitors have been shown associated with neurodegenerative diseases. However, their effects on survival of dopaminergic neurons remain uncertain. In the present study, the HDAC inhibitor trichostatin A (TSA) was tested in following dopaminergic neuronal cell lines: rat N27, mouse MN9D, and human SH-SY5Y cells. Results demonstrated that a single TSA treatment resulted in decreased cell survival and increased apoptosis in dopaminergic neuronal cells. Pre-treatment with TSA resulted in exacerbated neurotoxic damage to dopaminergic neurons induced by 1-methyl-4-phenylpyridinium and rotenone. These results suggest that HDAC inhibitors may influence Parkinson's disease pathogenesis by inhibiting survival and increasing vulnerability of dopaminergic neurons to neurotoxins. Our data also suggested the importance of prudent use of HDAC inhibitors in therapy.