ATP13A2 Gene Silencing in Drosophila Affects Autophagic Degradation of A53T Mutant α-Synuclein.

Mutations in ATP13A2 (PARK9), an autophagy-related protein, cause Kufor-Rakeb syndrome, an autosomal recessive, juvenile-onset form of parkinsonism. α-Synuclein (α-syn) is a presynaptic neuronal protein that forms toxic aggregates in Parkinson's disease (PD). We studied α-syn aggregation and autophagic flux in ATP13A2-knockdown Drosophila expressing either wild-type (WT) or mutant α-syn. Dopaminergic (DA) neuron loss was studied by confocal microscopy. Sleep and circadian activity were evaluated in young and old flies using a Drosophila activity monitor. Thirty-day-old ATP13A2-RNAi A53T-α-syn flies had increased Triton-insoluble α-syn levels, compared to control A53T-α-syn flies without ATP13A2-RNAi. Whole-brain staining revealed significantly fewer dopaminergic (DA) neurons in the PPL2 cluster of 30-day-old ATP13A2-RNAi flies expressing WT-, A30P-, and A53T-α-syn than in that of controls. In ATP13A2-RNAi A53T-α-syn flies, autophagic flux was decreased, as indicated by increased accumulation of Ref(2)P, the Drosophila p62 homologue. ATP13A2 silencing decreased total locomotor activity in young, and enhanced sleep features, similar to PD (decreasing bout length), in old flies expressing A53T-α-syn. ATP13A2 silencing also altered the circadian locomotor activity of A30P- and A53T-α-syn flies. Thus, ATP13A2 may play a role in the autophagic degradation of A53T-α-syn.

Silencing of Glucocerebrosidase Gene in Drosophila Enhances the Aggregation of Parkinson's Disease Associated α-Synuclein Mutant A53T and Affects Locomotor Activity.

Background: Mutations in glucocerebrosidase (GBA), a lysosomal enzyme are the most common genetic risk factor for developing Parkinson's disease (PD). We studied how reduced GCase activity affects α-synuclein (α-syn) and its mutants (A30P and A53T) aggregation, neurodegeneration, sleep and locomotor behavior in a fly model of PD. Methods: We developed drosophila with GBA gene knockdown (RNAi) (with reduced GCase activity) that simultaneously expresses either wildtype (WT) or mutants such as A30P or A53T α-syn. Western blot and confocal microscopy were performed to study the α-syn aggregation and neurodegeneration in these flies. We also studied the sleep and locomotor activity of those flies using Drosophila activity monitor (DAM) system. Results: Western blot analysis showed that GBA RNAi A53T α-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to α-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of α-syn remained unchanged. Confocal imaging of whole brain staining of 30 days old drosophila showed a statistically significant decrease in neuron numbers in PPL1 cluster in flies expressing α-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for α-syn aggregates showed an increased number of neurons staining for α-syn aggregates in A53T fly brain with GBA RNAi compared to control A53T flies, thus confirming our protein analysis finding that under decreased GBA enzyme activity, mutant A53T aggregates more than the control A53T without GBA silencing. Sleep analysis revealed decreased total activity in GBA silenced flies expressing mutant A53T compared to both A53T control flies and GBA RNAi flies without synuclein expression. Conclusion: In A53T flies with reduced GCase activity, there is increased α-syn aggregation and dopamine (DA) neuronal loss. This study demonstrates that reduced GCase activity both in the context of heterozygous GBA1 mutation associated with PD and in old age, contribute to increased aggregation of mutant α-syn A53T and exacerbates the phenotype in a fly model of PD.

Neuroprotective effect of nerolidol against neuroinflammation and oxidative stress induced by rotenone.

BACKGROUND: Parkinson disease (PD) is a movement disorder affecting 1 % of people over the age of 60. The etiology of the disease is unknown; however, accumulating evidence suggests that mitochondrial defects, oxidative stress, and neuroinflammation play important roles in developing the disease. Current medications for PD can only improve its symptoms, but are unable to halt its progressive nature. Although many therapeutic approaches are available, new drugs are urgently needed for the treatment of PD. Thus, the present study was undertaken to investigate the neuroprotective potential of nerolidol, a sesquiterpene alcohol, on a rotenone-induced experimental model of PD, where male Wistar rats intraperitoneally received rotenone (ROT) at a dose of 2.5 mg/kg of body weight once daily for 4 weeks. RESULTS: Nerolidol, which has antioxidant and anti-inflammatory properties, was injected intraperitoneally at 50 mg/kg of body weight, once daily for 4 weeks, and at 30 min prior to ROT administration. ROT administration significantly reduced the activities of antioxidant enzymes superoxide dismutase (SOD) and catalase (CAT), and the level of the antioxidant tripeptide glutathione (GSH). Moreover, ROT increased the levels of the lipid peroxidation product malondialdehyde (MDA), proinflammatory cytokines (IL-1ß, IL-6, and TNF-α), and inflammatory mediators (COX-2 and iNOS) in rat brain tissues. Immunostaining of brain tissue sections revealed a significant increase in the number of activated astrocytes (GFAP) and microglia (Iba-1), along with the concomitant loss of dopamine (DA) neurons in the substantia nigra pars compacta and dopaminergic nerve fibers in the striatum of ROT-treated rats. As expected, nerolidol supplementation to ROT-injected rats significantly increased the level of SOD, CAT, and GSH, and decreased the level of MDA. Nerolidol also inhibited the release of proinflammatory cytokines and inflammatory mediators. Finally, nerolidol treatment prevented ROT-induced glial cell activation and the loss of dopaminergic neurons and nerve fibers, and ultimately attenuated ROT-induced dopaminergic neurodegeneration. CONCLUSION: Our findings are the first to show that the neuroprotective effect of nerolidol is mediated through its anti-oxidant and anti-inflammatory activities, which strongly supports its therapeutic potential for the treatment of PD.

Glycyrrhizic acid Attenuates Neuroinflammation and Oxidative Stress in Rotenone Model of Parkinson's Disease.

Parkinson's disease (PD) is the second most common neurodegenerative disorder affecting humans. It is characterized by dopaminergic neurodegeneration, mitochondrial impairment, and oxidative stress, enhanced lipid peroxidation, and induction of pro-inflammatory cytokines. We evaluated the neuroprotective efficacy of glycyrrhizic acid (GA), an active component of licorice, against rotenone-induced-oxidative stress and neuroinflammation in a PD rat model. Since PD is progressive and chronic, we investigated the effect of chronic administration of GA for 4 weeks (50 mg/kg/day), 30 min prior to rotenone administration. Rotenone administration significantly reduced the activity of superoxide dismutase and catalase, and caused the depletion of reduced glutathione. A concomitant increase in the levels of the lipid peroxidation product malondialdehyde was observed. It also significantly enhanced the levels of pro-inflammatory cytokines in the midbrain and elevated the levels of inflammatory mediators such as cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Immunohistochemical analysis revealed significant increments in ionized calcium-binding adaptor molecule-1 (Iba-1) levels, and in glial fibrillary acidic protein (GFAP) levels, and loss of dopamine neurons in the substantia nigra pars compacta upon rotenone challenge. GA treatment significantly attenuated the dopamine neuron loss and decreased the Iba-1 and GFAP activation induced by the rotenone insult. GA also improved antioxidant enzyme activity, prevented glutathione depletion, inhibited lipid peroxidation, and attenuated induction of pro-inflammatory cytokines. Subsequently, GA attenuated the increased levels of the inflammatory mediators COX-2 and iNOS. In conclusion, GA protects against rotenone-induced-PD. The neuroprotective effects of GA are attributed to its potent antioxidative and anti-inflammatory properties.

Neuroprotective potential of ferulic acid in the rotenone model of Parkinson's disease.

Parkinson's disease (PD) is a chronic, progressive, and the second most common form of neurodegenerative disorders. In order to explore novel agents for the treatment of PD, in the current study, we have evaluated the neuroprotective efficacy of ferulic acid (FA) using rotenone (ROT)-induced rat model of PD. ROT was administered 2.5 mg/kg body weight to male Wistar rats for 4 weeks to induce the PD. Since PD is progressive and chronic in nature, the paradigm for evaluating FA was based on chronic administration for 4 weeks at the dose of 50 mg/kg, 30 minutes prior to ROT administration. ROT administration caused significant reduction in endogenous antioxidants such as superoxide dismutase, catalase, and glutathione. ROT challenge-induced lipid peroxidation evidenced by increased malondialdehyde following perturbation of antioxidant defense. Apart from oxidative stress, ROT also activated proinflammatory cytokines and enhanced inflammatory mediators such as cyclooxygenase-2 and inducible nitric oxide synthase. The immunofluorescence analysis revealed a significant increase in the number of activated microglia and astrocytes accompanied by a significant loss of dopamine (DA) neurons in the substantia nigra pars compacta area upon ROT injection. However, treatment with FA rescued DA neurons in substantia nigra pars compacta area and nerve terminals in the striatum from the ROT insult. FA treatment also restored antioxidant enzymes, prevented depletion of glutathione, and inhibited lipid peroxidation. Following treatment with FA, the inflammatory mediators such as cyclooxygenase-2 and inducible nitric oxide synthase and proinflammatory cytokines were also reduced. Further, the results were supported by a remarkable reduction of Iba-1 and GFAP hyperactivity clearly suggests attenuation of microglial and astrocytic activation. Results of our study suggest that FA has promising neuroprotective effect against degenerative changes in PD, and the protective effects are mediated through its antioxidant and anti-inflammatory properties.

Ginsenoside Rb1 inhibits fibrillation and toxicity of alpha-synuclein and disaggregates preformed fibrils.

Compelling evidence indicates that α-synuclein (α-syn) aggregation plays a central role in the pathogenesis of Parkinson's disease (PD) and other synucleinopathies. Identification of compounds that inhibit or reverse the aggregation process may thus represent a viable therapeutic strategy against PD and related disorders. Ginseng is a well-known medicinal plant that has been used in East Asia for more than two thousand years to treat several conditions. It is now understood that the pharmacological properties of ginseng can be attributed to its biologically active components, the ginsenosides, which in turn have been shown to have neuroprotective properties. We therefore sought to determine for the first time, the potential of the most frequently used and studied ginsenosides, namely Rg1, Rg3 and Rb1, as anti-amyloidogenic agents. The effect of Rg1, Rg3 and Rb1 on α-syn aggregation and toxicity was determined by an array of biophysical, biochemical and cell-culture-based techniques. Among the screened ginsenosides, only Rb1 was shown to be a potent inhibitor of α-syn fibrillation and toxicity. Additionally, Rb1 exhibited a strong ability to disaggregate preformed fibrils and to inhibit the seeded polymerization of α-syn. Interestingly, Rb1 was found to stabilize soluble non-toxic oligomers with no ß-sheet content, that were susceptible to proteinase K digestion, and the binding of Rb1 to those oligomers may represent a potential mechanism of action. Thus, Rb1 could represent the starting point for designing new molecules that could be utilized as drugs for the treatment of PD and related disorders.

Structure activity relationship of phenolic acid inhibitors of α-synuclein fibril formation and toxicity.

The aggregation of α-synuclein (α-syn) is considered the key pathogenic event in many neurological disorders such as Parkinson's disease (PD), dementia with Lewy bodies and multiple system atrophy, giving rise to a whole category of neurodegenerative diseases known as synucleinopathies. Although the molecular basis of α-syn toxicity has not been precisely elucidated, a great deal of effort has been put into identifying compounds that could inhibit or even reverse the aggregation process. Previous reports indicated that many phenolic compounds are potent inhibitors of α-syn aggregation. The aim of the present study was to assess the anti-aggregating effect of gallic acid (GA) (3,4,5-trihydroxybenzoic acid), a benzoic acid derivative that belongs to a group of phenolic compounds known as phenolic acids. By employing an array of biophysical and biochemical techniques and a cell-viability assay, GA was shown not only to inhibit α-syn fibrillation and toxicity but also to disaggregate preformed α-syn amyloid fibrils. Interestingly, GA was found to bind to soluble, non-toxic oligomers with no ß-sheet content, and to stabilize their structure. The binding of GA to the oligomers may represent a potential mechanism of action. Additionally, by using structure activity relationship data obtained from fourteen structurally similar benzoic acid derivatives, it was determined that the inhibition of α-syn fibrillation by GA is related to the number of hydroxyl moieties and their position on the phenyl ring. GA may represent the starting point for designing new molecules that could be used for the treatment of PD and related disorders.