Chronic Caffeine Treatment Protects Against ?-Synucleinopathy by Reestablishing Autophagy Activity in the Mouse Striatum.
ABSTRACT: Despite converging epidemiological evidence for the inverse relationship of regular caffeine consumption and risk of developing Parkinson's disease (PD) with animal studies demonstrating protective effect of caffeine in various neurotoxin models of PD, whether caffeine can protect against mutant ?-synuclein (?-Syn) A53T-induced neurotoxicity in intact animals has not been examined. Here, we determined the effect of chronic caffeine treatment using the ?-Syn fibril model of PD by intra-striatal injection of preformed A53T ?-Syn fibrils. We demonstrated that chronic caffeine treatment blunted a cascade of pathological events leading to ?-synucleinopathy, including pSer129?-Syn-rich aggregates, apoptotic neuronal cell death, microglia, and astroglia reactivation. Importantly, chronic caffeine treatment did not affect autophagy processes in the normal striatum, but selectively reversed ?-Syn-induced defects in macroautophagy (by enhancing microtubule-associated protein 1 light chain 3, and reducing the receptor protein sequestosome 1, SQSTM1/p62) and chaperone-mediated autophagy (CMA, by enhancing LAMP2A). These findings support that caffeine-a strongly protective environment factor as suggested by epidemiological evidence-may represent a novel pharmacological therapy for PD by targeting autophagy pathway.
Project description:Parkinson's disease (PD) is a common neurodegenerative disorder which is mostly sporadic but familial-linked PD (FPD) cases have also been found. The first reported gene mutation that linked to PD is ?-synuclein (?-syn). Studies have shown that mutations, increased expression or abnormal processing of ?-syn can contribute to PD, but it is believed that multiple mechanisms are involved. One of the contributing factors is post-translational modification (PTM), such as phosphorylation of ?-syn at serine 129 by G-protein-coupled receptor kinases (GRKs) and casein kinase 2? (CK2?). Another known important contributing factor to PD pathogenesis is oxidative and nitrosative stress. In this study, we found that GRK6 and CK2? can be S-nitrosylated by nitric oxide (NO) both in vitro and in vivo. S-nitrosylation of GRK6 and CK2? enhanced their kinase activity towards the phosphorylation of ?-syn at S129. In an A53T ?-syn transgenic mouse model of PD, we found that increased GRK6 and CK2? S-nitrosylation were observed in an age dependent manner and it was associated with an increased level of pSer129 ?-syn. Treatment of A53T ?-syn transgenic mice with N?-Nitro-L-arginine (L-NNA) significantly reduced the S-nitrosylation of GRK6 and CK2? in the brain. Finally, deletion of neuronal nitric oxide synthase (nNOS) in A53T ?-syn transgenic mice reduced the levels of pSer129 ?-syn and ?-syn in an age dependent manner. Our results provide a novel mechanism of how NO through S-nitrosylation of GRK6 and CK2? can enhance the phosphorylation of pSer129 ?-syn in an animal model of PD.
Project description:The misfolded ?-synuclein protein, phosphorylated at serine 129 (pSer129 ?-syn), is the hallmark of Parkinson disease (PD). Detected also in the enteric nervous system (ENS), it supports the recent theory that PD could start in the gut, rather than the brain. In a previous study, using a transgenic mouse model of human synucleinopathies expressing the A53T mutant ?-synuclein (TgM83), in which a neurodegenerative process associated with ?-synuclein occurs spontaneously in the brain, we have shown earlier onset of pSer129 ?-syn in the ENS. Here, we used this model to study the impact of paraquat (PQ) a neurotoxic herbicide incriminated in PD in agricultural workers) on the enteric pSer129 ?-syn expression in young mice. Orally delivered in the drinking water at 10?mg/kg/day for 6-8?weeks, the impact of PQ was measured in a time-dependent manner on weight, locomotor abilities, pSer129 ?-syn, and glial fibrillary acidic protein (GFAP) expression levels in the ENS. Remarkably, pSer129 ?-syn was detected in ENS earlier under PQ oral exposure and enteric GFAP expression was also increased. These findings bring additional support to the theory that neurotoxic agents such as PQ initiate idiopathic PD after oral delivery.
Project description:Parkinson’s disease (PD) is the second most common neurodegenerative disorder, characterized by dopaminergic neurodegeneration, motor impairment and non-motor symptoms. Epidemiological and experimental investigations into potential risk factors have firmly established that dietary factor caffeine, the most-widely consumed psychoactive substance, may exerts not only neuroprotective but a motor and non-motor (cognitive) benefits in PD. These multi-benefits of caffeine in PD are supported by convergence of epidemiological and animal evidence. At least six large prospective epidemiological studies have firmly established a relationship between increased caffeine consumption and decreased risk of developing PD. In addition, animal studies have also demonstrated that caffeine confers neuroprotection against dopaminergic neurodegeneration using PD models of mitochondrial toxins (MPTP, 6-OHDA, and rotenone) and expression of ?-synuclein (?-Syn). While caffeine has complex pharmacological profiles, studies with genetic knockout mice have clearly revealed that caffeine’s action is largely mediated by the brain adenosine A2A receptor (A2AR) and confer neuroprotection by modulating neuroinflammation and excitotoxicity and mitochondrial function. Interestingly, recent studies have highlighted emerging new mechanisms including caffeine modulation of ?-Syn degradation with enhanced autophagy and caffeine modulation of gut microbiota and gut-brain axis in PD models. Importantly, since the first clinical trial in 2003, United States FDA has finally approved clinical use of the A2AR antagonist istradefylline for the treatment of PD with OFF-time in Sept. 2019. To realize therapeutic potential of caffeine in PD, genetic study of caffeine and risk genes in human population may identify useful pharmacogenetic markers for predicting individual responses to caffeine in PD clinical trials and thus offer a unique opportunity for “personalized medicine” in PD.
Project description:Parkinson's disease (PD) is the second most common neurodegenerative disease. A key pathological feature of PD is Lewy bodies, of which the major protein component is ?-synuclein (?-syn). Human genetic studies have shown that mutations (A53T, A30P, E46K) and multiplication of the ?-syn gene are linked to familial PD. Mice overexpressing the human A53T mutant ?-syn gene develop severe movement disorders. However, the molecular mechanisms of ?-syn toxicity are not well understood. Recently, mitochondrial dysfunction has been linked with multiple neurodegenerative diseases including Parkinson's disease. Here we investigated whether mitochondrial motility, dynamics and respiratory function are affected in primary neurons from a mouse model expressing the human A53T mutation. We found that mitochondrial motility was selectively inhibited in A53T neurons while transport of other organelles was not affected. In addition, A53T expressing neurons showed impairment in mitochondrial membrane potential and mitochondrial respiratory function. Furthermore, we found that rapamycin, an autophagy inducer, rescued the decreased mitochondrial mobility. Taken together, these data demonstrate that A53T ?-syn impairs mitochondrial function and dynamics and the deficit of mitochondrial transport is reversible, providing further understanding of the disease pathogenesis and a potential therapeutic strategy for PD.
Project description:To investigate the putative interaction between chronic exposure to adenosine receptor antagonist caffeine and genetic influences on Parkinson's disease (PD), we determined whether deletion of the adenosine A(2A) receptor in knockout (KO) mice protects against dopaminergic neuron degeneration induced by a mutant human ?-synuclein (hm(2)-?SYN) transgene containing both A53T and A30P. The A(2A) KO completely prevented loss of dopamine and dopaminergic neurons caused by the mutant ?-synuclein transgene without altering levels of its expression. The adenosine A(2A) receptor appears required for neurotoxicity in a mutant ?-synuclein model of PD. Together with prior studies the present findings indirectly support the neuroprotective potential of caffeine and more specific A(2A) antagonists.
Project description:<h4>Background</h4>Parkinson's disease (PD) is characterized by degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNpc), accompanied by accumulation of α-synuclein, chronic neuroinflammation and autophagy dysfunction. Previous studies suggested that misfolded α-synuclein induces the inflammatory response and autophagy dysfunction in microglial cells. The NLRP3 inflammasome signaling pathway plays a crucial role in the neuroinflammatory process in the central nervous system. However, the relationship between autophagy deficiency and NLRP3 activation induced by α-synuclein accumulation is not well understood.<h4>Methods</h4>Through immunoblotting, immunocytochemistry, immunofluorescence, flow cytometry, ELISA and behavioral tests, we investigated the role of p38-TFEB-NLRP3 signaling pathways on neuroinflammation in the α-synuclein A53T PD models.<h4>Results</h4>Our results showed that increased protein levels of NLRP3, ASC, and caspase-1 in the α-synuclein A53T PD models. P38 is activated by overexpression of α-synuclein A53T mutant, which inhibited the master transcriptional activator of autophagy TFEB. And we found that NLRP3 was degraded by chaperone-mediated autophagy (CMA) in microglial cells. Furthermore, p38-TFEB pathways inhibited CMA-mediated NLRP3 degradation in Parkinson's disease. Inhibition of p38 had a protective effect on Parkinson's disease model via suppressing the activation of NLRP3 inflammasome pathway. Moreover, both p38 inhibitor SB203580 and NLRP3 inhibitor MCC950 not only prevented neurodegeneration in vivo, but also alleviated movement impairment in α-synuclein A53T-tg mice model of Parkinson's disease.<h4>Conclusion</h4>Our research reveals p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease, which could be a potential therapeutic strategy for PD. p38-TFEB pathways promote microglia activation through inhibiting CMA-mediated NLRP3 degradation in Parkinson's disease. In this model, p38 activates NLRP3 inflammasome via inhibiting TFEB in microglia. TFEB signaling negatively regulates NLRP3 inflammasome through increasing LAMP2A expression, which binds to NLRP3 and promotes its degradation via chaperone-mediated autophagy (CMA). NLRP3-mediated microglial activation promotes the death of dopaminergic neurons.
Project description:Parkinson's disease (PD) is a prevalent motor disease caused by the accumulation of mutated α-synuclein (α-Syn); however, its early stages are also characterized by non-motor symptoms, such as olfactory loss, cognitive decline, depression, and anxiety. The therapeutic effects of environmental enrichment (EE) on motor recovery have been reported, but its effects on non-motor symptoms remain unclear. Herein, we reveal the beneficial effects of EE on PD-related non-motor symptoms and changes in synaptic plasticity in the nucleus accumbens. To investigate its therapeutic effects in the early phase of PD, we randomly assigned eight-month-old mice overexpressing human A53T (hA53T) α-Syn to either the EE or standard condition groups for two months. Next, we performed behavioral tests and biochemical and histological analyses at 10 months of age. EE significantly alleviated locomotor hyperactivity and anxiety during the early stages of PD. It normalized the levels of tyrosine hydroxylase, phosphorylated and oligomeric α-Syn, and soluble <i>N</i>-ethylmaleimide-sensitive factor attachment protein receptor complex-forming proteins, including synaptosomal-associated protein, 25 kDa, syntaxin1, and vesicle-associated membrane protein 2 (VAMP2). Moreover, the interactions between VAMP2 and pSer129 α-Syn were markedly reduced following EE. The restoration of synaptic vesicle transportation status may underlie the neuroprotective effects of EE in hA53T α-Syn mice.
Project description:Many neuropathological and experimental studies suggest that the degeneration of dopaminergic terminals and axons precedes the demise of dopaminergic neurons in the substantia nigra, which finally results in the clinical symptoms of Parkinson disease (PD). The mechanisms underlying this early axonal degeneration are, however, still poorly understood. Here, we examined the effects of overexpression of human wildtype alpha-synuclein (?Syn-WT), a protein associated with PD, and its mutant variants ?Syn-A30P and -A53T on neurite morphology and functional parameters in rat primary midbrain neurons (PMN). Moreover, axonal degeneration after overexpression of ?Syn-WT and -A30P was analyzed by live imaging in the rat optic nerve in vivo. We found that overexpression of ?Syn-WT and of its mutants A30P and A53T impaired neurite outgrowth of PMN and affected neurite branching assessed by Sholl analysis in a variant-dependent manner. Surprisingly, the number of primary neurites per neuron was increased in neurons transfected with ?Syn. Axonal vesicle transport was examined by live imaging of PMN co-transfected with EGFP-labeled synaptophysin. Overexpression of all ?Syn variants significantly decreased the number of motile vesicles and decelerated vesicle transport compared with control. Macroautophagic flux in PMN was enhanced by ?Syn-WT and -A53T but not by ?Syn-A30P. Correspondingly, colocalization of ?Syn and the autophagy marker LC3 was reduced for ?Syn-A30P compared with the other ?Syn variants. The number of mitochondria colocalizing with LC3 as a marker for mitophagy did not differ among the groups. In the rat optic nerve, both ?Syn-WT and -A30P accelerated kinetics of acute axonal degeneration following crush lesion as analyzed by in vivo live imaging. We conclude that ?Syn overexpression impairs neurite outgrowth and augments axonal degeneration, whereas axonal vesicle transport and autophagy are severely altered.
Project description:Parkinson's disease (PD) is characterized by the loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies (LBs) in the surviving SNc neurons. LBs formation is caused by the accumulation of ?-synuclein (?-syn) or phosphorylated ?-syn at serine-129 (pSer129-?-syn), which is implicated in the pathological progression of PD. Salidroside (Sal), the main active ingredient of the root of Rhodiola rosea L., has been reported to have potent neuroprotective properties in our previous investigations. Here, we investigated the effects of Sal on 6-OHDA and overexpresssion of WT/A30P-?-syn-induced pathological ?-syn increase and the mechanism behind it in SH-SY5Y cells. We found Sal displays neuroprotective effects against 6-hydroxydopamine (6-OHDA)-induced cytotoxicity. Sal decreased the pSer129-?-syn level mainly by maintaining the normal function of ubiquitin-proteasome system (UPS). Furthermore, Sal promoted the clearance of ?-syn and protected the cell viability mainly through recovered the 20S proteasome activity in WT/A30P-?-syn-transfected cells. These data provide new mechanistic insights into the neuroprotective effects of Sal and Sal may be a promising therapy to slow neurodegeneration in PD. Highlights: Sal protects cells and decreases the pSer129-?-syn protein level in 6-OHDA-induced impairmental and dysfunctional SH-SY5Y cells. Sal promotes the clearance of ?-syn and protects the cell viability mainly through recovering the 20S proteasome activity in WT/A30P-?-syn plasmids transfected cells. Maintaining the normal function of the UPS may be one of the important mechanisms of Sal in neuroprotective effects.
Project description: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 ?-synuclein (?-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 ?-syn. Western blot and confocal microscopy were performed to study the ?-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 ?-syn flies (30 days old) had an increased level of Triton insoluble synuclein (that corresponds to ?-syn aggregates) compared to corresponding A53T flies without GBA RNAi (control), while mRNA expression of ?-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 ?-syn WT, A30P and A53T in the presence GBA RNAi compared to corresponding control. Staining with conformation specific antibody for ?-syn aggregates showed an increased number of neurons staining for ?-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 ?-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 ?-syn A53T and exacerbates the phenotype in a fly model of PD.