| Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder in the world, and yet, the intrinsic causes of PD remain unclear. It is characterized by selective loss of dopamine (DA) neurons in substantia nigra compacta (SNc) and the deposition of proteinaceous inclusions called Lewy bodies (LBs), which primarily composed of a-synuclein. For several decades, the gold standard for symptomatic therapy of PD is based on dopamine replacement with levodopa (L-Dopa), the dopamine precusor. However, L-Dopa treatment is merely symptomatic and does not retard the progression of disease. Moreover, long-term treatment with L-Dopa often leads to the development of motor complications within 5 years of therapy combined with multiple non-motor manifestations. Direct stimulation of dopaminergic (DAergic) receptors with DA agonists has, therefore, become an alternative promising therapeutic method for PD.The monotherapy of DA agonists in early PD patients led to a lower overall incidence of motor complications compared to L-Dopa, but within 2-5 years from disease onset the majority of patients require the addition of L-Dopa to control the symptoms. Thus, DA agonists can be useful as monotherapy in the initial phase or combined with L-DOPA in advanced PD. Neuroprotective properties have been proposed for DA agonists, based on pre-clinical studies, but never confirmerd in patients. We have demonstrated previously that astrocytic D2 receptor (D2R) suppresses neuroinflammation and thus increases resistance of the SNc DA neurons to MPTP, but the underlying mechanism remained obscure. Further clarify the anti-inflammation role of D2R in astrocyte and address the potential mechanism of D2R agonist application in initial and advanced phase of PD, will provide a new perspective for the development of novel options for PD treatment.As the major component of LBs, α-Synuclein (α-Syn) is a highly conserved presynaptic protein of unknown function.Under disease conditions, α-syn misfolds, aggregates, and finally forms filamentous structures. Increasing evidences from animal models as well as data from genetic, biochemical and biophysical studies support the hypothesis that the processes of α-syn oligomerization and fibril growth have central roles in the pathogenesis of PD. Moreover, it is possible that the α-syn monomers might also pathologic by disrupting the activity of other molecules or signaling pathways. Nevertheless, the toxic forms of α-syn and the cellular pathways that are affected by α-syn remain unknown. Recent studies suggest that small amounts of α-syn are released from neuronal cells by unconventional exocytosis, and this extracellular α-syn can be transferred to neighboring glial cells, triggering numerous inflammation events. The evidence clearly shows that α-syn pathology seem to spread with the brain as the disease progresses, however, the impact role of α-syn on anti-neuroinflammation role of astrocytic dopamine D2 receptors and the different efficacy of D2R agonist in initial and advanced phase of PD, still remain unclear. Moreover, secreted α-syn also have a modulatory effect in adult neural stem cells/neural progenitor cells proliferation and differentiation, involving repair dysfunction in the progress of PD.However, the exact events underlying a-syn-induced impairment of adult neurogenesis in PD remain unknown. Dysregulation of adult neurogenesis is considered as a critical contributor to the pathogenesis of PD. Promoting adult neurogeneis thus has been regareded as a prospective strategy for self-recovery in degenerative diseases.Therefore, in the first part of our present work, we used WT/A53Ttg/tg mice primary astrocyte and human WT/A53T recombinant protein in order to investigate the role of D2R agonist on a-syn induced astrocytic inflammation, our results revealed that D2R agonist quinpiole inhibited LPS induced astrocytic inflammation which benefit to neuron survival while have no effect on a-syn induced inflammation. In the second part, we further found that quinpirole exerted anti-inflammation role through β3-Arr2-dependent mechanism. In addition, we demonstrated that a-syn have a negative role in β-Arr2-depedent signaling, thus canceled the anti-inflammation and neuroprotective role of quinpirole. Finally, using A53Ttg/tg mice, we found that NLRP3 inflammasome and TLR-4 activation-mediated neuroinflammaiton triggered by α-syn overexpression contributed to neurogenesis impairment in PD. Furthermore, miroRNA-7 is likely to advance a novel therapeutic strategy by enhacing neurogenesis in PD through post transcriptional inhibition of a-syn.Part Ⅰ The role of D2 receptors on α-syn-mediated astrocytic neuroinflammationAIM:To investigate the effect of D2R agonist on LPS/MPP+/a-syn-mediated neuroinflammtion.METHOD:After pre-treated A53Ttg/tg astrocyte with quinpirole 10 μM for 30 min, we used Real-time-PCR and Elisa to determine the mRNA levels and secretion of pro-cytokines such as IL-1β. Western blotting was used to analyze the expression of TLR4, p-IKKβ and p65 in A53Ttg/tg astrocyte. Midbrain and striatum tissues were separated from 12-month A53Ttg/tg mice. Western blotting was used to analyse the expression and existence form of a-syn. Primary midbrain astrocyte was cultured from new-born A53Ttg/tg mice. Western blotting and double-labeled immunocytofluoresence were used to o2bserve co-locolization of GFAP and a-syn and the expression and existence form of a-syn in astrocyte. Primary midbrain astrocyte was cultured from new-born (post-natal 1-3 days) wild-type (WT) mice. Astrocyte was pre-treated with quinpirole 10 μM for 30 min before LPS stimulation. We measured the mRNA level of pro-cytokines such as IL-1β 6 h after LPS stimulation using Real-time PCR. Secretion of IL-1β, TNF-a and IL-12 24 h after LPS stimulation was measured by Elisa. The expression of NF-κB singling-related protein TLR4, p-IKKβ and p65 were measured by western blotting. We measured the mRNA level and secretion of IL-1β, TNF-α and IL-12 48 h after MPP+ 50 μM stimulation using Real-time PCR and Elisa. WT primary astrocytes were pre-treated with quinpirole 10μM for 30 min before stimulation of exogenous human WT or A53T mutant monomer recombinant protein 10μg/ml for 24 h. We used Real-time PCR and Elisa to determine the mRNA levels and secretion of pro-cytokines such as IL-1β.Western blotting was used to analyze the expression of TLR4, p-IKKβ and p65 in WT astrocyte after α-syn stimulation. Real-time PCR and western blotting were used to evaluate the cytoplasmic and membrane mRNA level and expression of D2R upon α-syn overexpression. WT and A53Ttg/tg striatum synaptosome were extracted and exogenous human monomer recombinant protein was used to investigate the role of α-syn on D2R binding force through Radioligand receptor binding assays (RRA). After LPS stimulation for 24 h, we collected the astrocytic conditional medium, and mixed with neurobasal in portion of 1:2. Tyrosine hydroxylase (TH) immonohistochemistry was used to investigate the role of quinpirole on DA neuron survival and axon length. After a-syn stimulation for 24 h, we collected the astrocytic conditional medium, and mixed with neurobasal in portion of 1:2. Tyrosine hydroxylase (TH) immonohistochemistry was used to investigate the role of quinpirole on DA neuron survival and axon length.RESULTS:1) The mRNA levels and secretion of TNF-α, IL-1β, IL-6, IL-12 and IFN-y were higher, and the expression of TLR4, p-IKK|3 and p65 were increased in A53Ttg/tg mice astrocyte compared with control WT mice astrocyte. Quinpirole (10 μM) couldn’t inhibit the high level of astrocytic inflammation.2) a-Syn monomers were existed in midbrain and striatum from 12-month WT and A53Ttg/tg mice. a-Syn oligomers were existed in 12-month A53Ttg/tg mice midbrain and striatum but not WT mice.3) There was only a-syn monomers exist in cultured A53Ttg/tg mice astrocyte.4) LPS (100 ng/ml) stimulation greatly increased the mRNA level and secretion of TNF-a, IL-1β, IL-6, IL-12 and IFN-y, and increased the expression of TLR4, p-IKKβ and p65 in astrocyte. Quinpirole (10μM) significantly inhibited LPS induced astrocytic inflammation.5) MPP+(50 μM) stimulation increased the mRNA level and secretion of TNF-a, IL-1β and IL-12. Quinpirole (10 μM) significantly inhibited MPP+ induced astrocytic inflammation.6) Exogenous stimulation of human WT or A53T mutant a-syn monomer recombinant protein 10 μg/ml for 24 h significantly enhanced the mRNA levels and secretion of pro-inflammation cytokines such as IL-1β and increased the expression of TLR4, p-IKKβ and p65 in astrocyte. Quinpirole (10 μM) couldn’t inhibit the a-syn monomer induced astrocytic inflammation.7) a-Syn overexpression or exogenous stimulation signifantly increased the mRNA level and expression of astrocytic cytoplasmic and mebrane D2R.8) There was no difference between WT and A53Ttg/tg mice striatum synaptosome in D2R binding force.9) LPS (100 ng) stimulation significantly inhibited the number and axon length of TH+ neuron and quinpirole (10 μM) could resist LPS-induced neuron death effectively.10) WT or A53T mutant α-syn monomer recombinant protein (10 μg/ml) stimulation for 24 h significantly inhibited the number and axon length of TH+ neuon and quinpirole (10 μM) almost couldn’t resist α-syn-induced neuron death.CONCLUTION:1) α-Syn monomer induced significant inflammatory reaction in astrocyte.2) D2R agonist inhibited LPS induced astrocytic inflammation but not α-syn-induced inflammation.3) Activation of astrocytic D2R couldn’t alleviate α-syn-induced DA neuron death.Part Ⅱ a-Syn abolished the anti-inflammation role of astrocytic D2 receptors in a β-Arr2-dependent pathwayAIM:To investigate the regulation role and underlying mechanism of α-syn on the anti-inflammation role of quinipriole in astrocyte.METHOD:The role of quinpirole on aB-crystallin expression in astrocyte was oberserved by western blotting. cAMP direct immunoassay kit was used to investigate the role of quinpirole on astrocytic cAMP concentration. We used cAMP analogue (db-cAMP,100 μM) and AC activator (forskolin,10μM) to observe the role of quinpirole on IL-1β secretion using Elisa. The role of quinpirole on P-Arr2 expression in astrocyte was oberserved by western blotting. After down-regulating astrocytic P-Arr2 expression using siRNA, we measured the mRNA level and secretion of pro-cytokines such as IL-1β by Real-time PCR and Elisa; Western blotting was used to measure the expression of NF-κB singling-related protein TLR4, p-IKKβ and p65. The interaction between TAB1 and β3-Arr2/TAK1 was measured by co-Immunoprecipitation (Co-IP). The role of quinpirole on the level of p-TAK1 in normal state or after LPS stimulation was measured by western blotting. Western blotting and immunocytofluoresence were used to observe the role of α-syn on the expression of astrocytic aB-crystallin. Western blotting and Real-time PCR were used to observe the role of a-syn on the expression and mRNA level of astrocytic P-Arr2. After over expressed P-Arr2 in A53Ttg/tg astrocyte, we used Real-time PCR and Elisa to determine the mRNA levels and secretion of pro-cytokines such as IL-1β. Western blotting was used to analyze the expression of TLR4, p-IKKβ and p65 in A53Ttg/tg astrocyte. We observed the co-locolization of P-Arr2 and a-syn in HeLa cell after transfection with pDsRed2-Cl-SNCA and pcDNA3.1-GFP-β-arrestin 2 recombinant plasmids. Immunocytofluoresence were used to observe co-locolization of H3 and a-syn in A53Ttg/ts astrocyte. We constructed pGL3-β-arrestin 2 promoter luciferase reporter gene plasmid and transfected it with 3HA-WT/A53T-SNCA plasmids in Hek293T cell. Luciferase reporter gene assay was used to observe the role of a-syn on the luciferase activity of pGL3-β-arrestin 2. Chromatin-Immunoprecititation (Ch-IP) was used to directly determine the interaction between a-syn and β-Arr2. WT primary astrocytes were pre-treated with quinpirole 10 μM for 30 min before stimulation of exogenous human monomer a-syn recombinant protein 10 μg/ml for 24 h. Co Immunoprecititaion (Co-IP) was used to investigate the interaction between a-syn and β-Arr2 and the role of a-syn on the binding of P-Arr2 and TAB1. Western blotting was used to evaluate the role of quinpirole on a-syn-induced change of p-TAKl.RESULTS:1) Quinpirole had no effect on astrocytic aB-crystallin expression.2) Quinpirole inhibited the astrocytic cAMP concentration and db-cAMP (100 μM) and forskolin (10 μM) couldn’t influence the anti-inflammaiton role of quinpriole.3) Quinpirole enhanced the expression of β-Arr2 and down-regulation of P-Arr2 expression canceled the anti-inflammtion role of quinpriole.4) Quinpirole increased the binding of P-Arr2 and TAB1, and inhibited LPS-induced TAB1-TAK1 interaction, thus inhibited the p-TAKl level.5) There was no difference in expression of αB-crystallin in WT and A53Ttg/tg mice astrocyte and α-syn stimulation didn’t influence the expression of astrocytic αB-crystallin.6) α-Syn overexpression or exogenous stimulation signifantly inhibited the mRNA level and expression of astrocytic β-Arr2. Overexpression of β-Arr2 could restore the anti-inflammation role of quinpirole in A53Ttg/tg mice astrocyte effectively.7) α-Syn greatly inhibited the luciferase activity of β-Arr2 promoter. Ch-IP assay showed that there was an indirect interaction between α-syn and β-Arr2 promoter.8) α-Syn interacted with β-Arr2 and inhibited the interaction of β-Arr2 and TAB1, and finally increased the p-TAK1 level.CONCLUTION:1) Astrocytic D2R agonist stimulated GPCR-independent β-Arr2 signaling through promoting the interaction between β-Arr2 and TAB1, and sequentially inhibited LPS-induced production of TAB1-TAK1 complex, and thereby, inhibited astrocytic p-TAK1 level, thus possessed anti-inflammaiton properties.2) α-Syn monomer canceled the anti-inflammation role of astrocytic D2R through inhibition of astrocytic β-Arr2 mediated anti-inflammation signaling.Part Ⅲ The impact role and mechanism of α-synuclein induced neuroinflammation on adult neurogenesisAIM:To investigate the action and underlying mechanisms of α-syn-induced inflammation in the proliferation and differentiation of adult neural stem cells in SVZ.METHOD:BrdU immunohistochemistry assay was used to evaluate the SVZ proliferation in adult WT and A53Ttg/tg mice. Immnofluorescence labeling was used to observe the NeuN positive neuron, SOX2 positive stem cells and DCX positive early differentiated neurons, and the number of positive cells was calculated. BrdU immunohistochemistry was used to evaluate the SGZ proliferation in adult WT and A53Ttg/tg mice and immnofluorescence was used to observe the NeuN positive neuron and DCX positive early differentiated neurons, and the number of positive cells was calculated. We used Iba-1 immunohistochemistry to label the SVZ microglia in WT and A53Ttg/tg mice and Real-time PCR was used to analyze the mRNA level of cytokines including IL-1β, IL-4, IL-6, IL-10, IL-12 and TNF-a in both genotype mice SVZ. Western blotting was used to determine the expression of NF-κB signaling (TLR4, p-IKKβ and p65) and NLRP3 signaling (NLRP3, caspase-land IL-1β). The expression of NF-κB signaling (TLR4, p-IKKβ and p65) and NLRP3 signaling (NLRP3, caspase-land IL-1β) were observed in WT and a-syn knockout (a-syn-/-) mice SVZ through western blotting. Adult neural stem neural spheres in SVZ were cultured from WT and A53Ttg/tg mice and the number and diameter of the neural spheres were calculated. Immnofluorescence was used to observe the expression of a-syn and Nestin positive cells in both genotype adult neural stem neural,pheres. We used Real-time PCR to measure the mRNA level of NLRP3, NLRP1, NLRC4 and AIM2 in adult neural stem cells. BrdU and Tuj1 immnofluorescence labeling were used to observe the proliferation and differentiation of in vitro cultured neural stem cells of both genotypes. The expression of NF-κB signaling (TLR4, p-IKKp and p65) and NLRP3 signaling (NLRP3, caspase-1 and IL-1β) were observed in neural stem cells of WT and A53Ttg/tg mice by western blotting. WT neural stem cells were stimulated with exogenous A53T mutant a-syn monomer (10 μg/ml), and BrdU and Tuj1 immnofluorescence labeling were used to observe the proliferation and differentiation of cultured WT neural stem cells after application of CLI-095 (TLR4 inhibitor) and zYVAD (caspase-1 inhibitor). We prepared MPTP sub acute PD model using WT and caspase-1 knockout (Casp-1-/-) mice, and BrdU immunohistochemistry was used to evaluate the SVZ proliferation. WT and Casp-1-/- neural stem cells were cultured, and we calculated the number and diameter of the neural stem cells. The both genotypes of neural stem cells were treated with exogenous A53T mutant a-syn monomer (100 μg/ml), and BrdU and Tuj1 immnofluorescence labeling were used to observe the proliferation and differentiation. We prepared A53Ttg/ts;Casp-/- double crossed mice, and BrdU immunohistochemistry was used to evaluate the SVZ proliferation in normal state. Real-time PCR was applied to address the mRNA levels of microRNA-7 (miR-7) in WT and A53Ttg/tg mice SVZ and cultured neural stem cells. WT and A53Ttg/tg cultured SVZ neural stem cells were transfected with miR-7 (100 nM), and the expression of α-syn, TLR4, NLRP3 and IL-1(3 were determined by western blotting. BrdU and Tuj1 immnofluorescence labeling were used to observe the role of miR-7 transfection on proliferation and differentiation of A53Ttg/tg neural stem cells. After lateral ventricle injection of miR-7 mimics in A53Ttg/tg mice SVZ, we used western blotting to measure the expression of α-syn, NLRP3 and IL-1β. BrdU immunohistochemistry was used to evaluate the role of miR-7 injection on proliferation of neural stem cells, and Tuj 1 immnofluorescence labeling were used to observe the role of miR-7 injection on differentiation of A53Ttg/tg neural stem cellsRESULTS:1) In normal state, the number of BrdU+proliferation cells and DCX+early differentiated neuons in SVZ/SGZ of A53Ttg/tg mice were significantly lower than control WT mice.2) The Iba-1+ microglia in A53Ttg/tg mice SVZ were greatly activated and the mRNA levels of pro-inflammation cytokines including IL-1β, IL-6, IL-12 and TNF-a were increased remarkably, and the anti-inflammation cytokine IL-10 mRNA level was decreased. Western blotting results showed that the NF-κB signals and NLRP3 signaling were obviously activated.3) There was no difference in NLRP3 signaling in SVZ/SGZ between WT and α-syn-1- mice.4) The number and diameter of SVZ neural stem cells derived from A53Ttg/tg mice was considerably bellower than the WT ones.5) NLRP3, NLRP1, NLRC4 and AIM2 were all expressed on adult neural stem cells, and the expression of NLRP3 was the most abundant.6) The number of BrdU+ proliferative cells and Tuj1+ new-born neurons in A53Ttg/tg neural stem cells were lower than WT neural stem cells and the NF-κB signaling was significantly activated.7) CLI-095 and zYVAD alleviated the A53T a-syn monomer induced inhibition of the number of BrdU- proliferative cells and Tuj1+ new-born neurons, and the combination effect was stronger.8) Casp-1 knockout alleviated MPTP-induced inhibition of adult neurogenesis. The number of BrdU+ cells in SVZ of A53tg/tg;Casp-1-/- double crossed mice were more than A53Ttg/tg mice. Casp-1 knockout didn’t influence the number and diameter of cultured adult neural stem cells. Casp-1 knockout remarkably relieved A53T a-syn monomer induced inhibition of proliferation and differentiation of neural stem cells.9) The expression of miR-7 in SVZ of A53tg/tg mice was lower than WT mice. MiR-7 transfection inhibited the expression of a-syn, TLR4, NLRP3 and IL-1β in cultured A53Ttg/tg neural stem cells and enhanced the adult neurogenesis in A53Ttg/tg neural stem cells.10) Lateral ventricle injection of miR-7 significantly inhibited the expression of a-syn, NLRP3 and IL-1β in SVZ of A53Ttg/tg mice and enhanced the adult neurogenesis in SVZ of A53Ttg/tg mice.CONCLUTION:1) A53T a-syn overexpression developed an inhibition role on adult neurogeneis through NLRP3 inflammasome and TLR4 activation-mediated neuroinflammaiton and subsequently production of IL-1β.2) miR-7 effectively restored adult neurogenesis through post-transcriptional controlling a-syn expression.The major contributions of the present study lie in:1. α-Syn canceled the anti-inflammation and neuroprotection role of astrocytic D2 receptors. Astrocytic inflammation reaction can be triggered by a-syn monomer stimulation. Activation of astrocytic D2R inhibited LPS and MPP+-induced inflammation, but had no effect on a-syn-induced inflammation, which provided further confirmation of the neuroprotective role of D2R agonist in clinical cases.2. β-Arr2 biased signaling was evolved in the anti-inflammation role of astrocytic D2 receptors. D2R agonist inhibited LPS-induced neuroinflammation in a biased β-Arr2 signal-mediated but not G-protein-mediated manner. a-Syn canceled the anti-inflammation role of quinpirole in astrocyte through disrupting the assembling of β-Arr2 signal complex, providing us promising experimental evidence for the role of β-Arr2-biased agonist in astrocytic neuroinflammaiton and PD drug therapy.3. Neuroinflammation triggered by a-syn facilitated the adult neurogenesis impairment in PD. a-Syn induced activation of NLRP3 inflammasome and TLR4 activation-mediated neuroinflammtion contributed to the neurogenesis inhibition in PD pathogenesis. Promisingly, miR-7 is likely to reveal a novel therapeutic strategy by enhancing neurogenesis in PD and other a-synucleinopathies. |
PDF Full Text Request