| Parkinson’s disease (PD) is the second most common age-related neurodegenerative disorder in the world. It is a neurological disorder with evolving layers of complexity, and yet, the exact pathological mechanism remains unclear. The main pathological features of PD are the loss of dopaminergic neuron (DA neuron) and the deposition of Lewy bodies (LB) in substantia nigra pars compacta (SNc), accompanied by tremors and paralysis and other symptoms of parkinson-like movement. However, PD is now recognised as heterogeneous, with clinically significant non-motor features, such as depression. To date, levodopa replacement therapy is still in use to alleviate motor symptoms of PD, but it could not be terminated the process of PD pathology and even causes a series of side effects, worse the disease progression. Therefore, to clarify the pathogenesis of PD is particularly important for discovering therapeutic method for PD.Studies have shown that the pathological mechanisms of oxidative stress, mitochondrial dysfunction, misfolded protein accumulation, excitotoxicity, nerve regeneration disorder, neuroinflammation are synergistically involved in the DA neuronal damage in the process of PD. Neuroinflammation can occur in the early stage of PD, and the release cytokines from neurocytes could be regarded as biological markers of PD.In the first part of our present work, we have found that the release of interleukin-1β(IL-1β) from astrocytes exerts an important role in the pathogenesis of PD, but yet, the application of IL-1β receptor antagonist in animal models could not effectively improve the DA neuronal loss in PD. Thus, there is a great scientific significance for elucidating the regulation of astrocyte-mediated neuroinflammation and the exact pathological mechanism of DA neuronal injury, collectively, it will meaningful for the development of ideal neuroprotective agents for PD therapeutics.The initiation of the inflammatory response involves the recently discovered multiprotein complexes termed’inflammasomes’. Tschopp and colleagues initially described the inflammasome concept in the early 2000s when they revealed a critical link between tissue injury, innate immune processes and cysteinyl aspartate specific proteinase (caspase) dependent responses. An inflammasome is a cytosolic, multiprotein platform that enables the activation of pro-inflammatory caspases, chiefly caspase 1, through the induction of caspase-1 cleavage and release of IL-1β, IL-18 and other pro-inflammatory cytokine, eventually produce inflammation. For the reason that, inflammasomes play an important role in the innate immune system to resist pathological stimulated. It has been reported that the receptors of IL-1β and IL-18 were expressed in surface of neurocytes, and the different types of neurocytes regulate the function of immune and inflammation. Moreover, researchers are increasingly concerned about the inflammasome-mediated neuroinflammation in central nervous system (CNS). There are various kinds of inflammasome in CNS, and preliminary studies in our laboratory showed that NLRP3 inflammasome may be involved in the inflammatory process of PD, but yet, the regulation of inflammasome-mediated neuroinflammation has not been clearly elucidated in the pathomechanism of PD.Therefore, in the first part of this work, we have found the activation of inflammasomes in PD patients. Then we used wild type (WT) mice to induce acute and chronic 1-methyl,4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) PD mouse model, and extracted serum and primary bone marrow-derived macrophages (BMDM), even separated the tissues from brain, liver and spleen, in order to clarify the regulation of NLRP3 inflammasome in the process of PD. In addition, downregulation of NLRP3 protein in SNc by stereotactic interference could be suppressed MPTP-induced DA neuronal injury and glial cells activation, these data have been illustrated the activation of NLRP3 inflammasome is directly correlated with the process of PD. Secondly, we clarified the pathological mechanisms underlying ATP13A2 deficiency induced PD syndrome and further confirmed that NLRP3 inflammasome were involved in the regulation of PD process by using Atpl3a2 knockout (Atp13a2-/-) PD model mouse. The results have shown that ATP13A2 inhibited neuroinflammation via inhibiting astrocytic NLRP3 inflammasome activation, thereby delaying the injury of DA neurons and exerting a neuroprotective effect. Our study suggests that the astrocytic ATP13A2 or NLRP3 inflammasome is likely to effective targets for the regulation of neuroinflammation in the pathological processes of PD, and could be regarded as a novel strategy for the development of anti-inflammatory drugs. Since the progressive loss of DA neurons is mainly characteristic of PD, delay DA neuronal degeneration is a critical treatment strategy for PD. Finally, we observed the effects of caspase-1 deficiency or inhibitor (Z-YVAD) on MPTP/1-methyl-4-phenylpyridinium (MPP+)-induced DA neuronal apoptosis. We found that caspase-1 triggered PARP1/AIF signaling and contributed to DA neuronal apoptosis, beyond mflammasome. These findings provide a new pharmacological target for the development of neuroprotective drugs to suppress neuroinflammation and DA neuronal damage.Part I The role of NLRP3-Caspase-1 inflammasome in the process of Parkinson’s diseaseAIM:To investigate the role of NLRP3 inflammasome in the process of PD.METHOD:Secretion of caspase-1 and IL-1β from normal and PD patients’ serum detected by ELISA. Three to four months old male C57BL/6J mice were used to establish acute and chronic MPTP PD models, and the secretion of caspase-1 and IL-1β from serum were detected by ELISA. We measured the expression of procaspase-1, caspase-1, proIL-1β and IL-1β in the midbrain and NLRP1, NLRP2, NLRP3, NLRC4 and AIM2 in the midbrain, liver, and spleen by western blotting. After pre-treated primary bone marrow derived macrophages (BMDM) with lipopolysaccharide (LPS) 100 ng/ml for 6 h, then we treated BMDM with 5 mM ATP for 30 min or 10 μg/ml flagellin for 12 h or transfection with 10μg/ml MDP for 18 h or 2μg/ml dsDNA for 48 h, using ELISA and western blotting to determine secretion of IL-1β and the expression of procaspase-1, caspase-1, proIL-1β and IL-1β in BMDM. Downregulation the expression of NLRP3 in SNc by stereotactic injection of lentivirus wrapped miRNA-mus-Nlrp3 for 7 days, and we established acute MPTP model, detecting the expression of GFP protein by immunofluorescence, and the expression of NLRP3 by western blotting. Moreover, tyrosine hydroxylase (TH) immunohistochemistry was used to investigate the role of NLRP3 knockdown on DA neuronal survival, glial fibrillary acid protein (GFAP) and ionized calcium binding adapter molecule 1 (Iba-1)immunohistochemistry was used to detect the proliferation and activation of astrocytes and microglia.RESULTS:1) The secretion of caspase-1 and IL-1β were higher in serum of PD patients and MPTP/p PD model mice compared with normal or saline group. The expression of caspase-1 and IL-1β were increased in the midbrain of MPTP/p PD model mice.2) Compared with the corresponding saline group, the expression of NLRP1 and NLRP3 were significantly upregulated in the midbrain of acute and chronic MPTP mouse model.The expression of NLRP2, NLRP3 and NLRC4 were increased in the liver of acute MPTP model, and the expression of NLRP2 and NLRP3 were increased in the chronic MPTP model. The expression of NLRP3 and AIM2 were significantly upregulated in the spleen of acute and chronic MPTP mouse model.3) The expression of NLRP2 and NLRP3 were increased in the primary BMDM of acute and chronic MPTP model. After stimulation with different inflammasome activators, the expression of caspase-1 and IL-1β were increased by ATP and flagellin treatment, which the most significant change was shown in the NLRP3 inflammasome activator by ATP stimulation.4) The expression GFP was observed in SNc by stereotactic injection with mus-Nlrp3-GFP, which mus-Nlrp3-2764 could reduce about 63% NLRP3 protein expression.5) Downregulation the expression of NLRP3 significantly inhibited MPTP-induced the loss of TH neurons.6) Downregulation the expression of NLRP3 significantly inhibited MPTP-induced the proliferation and activation of glia in SNc.CONCLUTION:1) Inflammasome activated in the pathological conditions of PD.2) NLRP3 inflammasome plays a critical role in the regulation of neuroinflammation in PD.3) Downregulation of NLRP3 reduces MPTP-induced the loss of DA neurons and alleviates the proliferation and activation of glial cells in SNc, and exerts a neuroprotective effect.Part II The role and mechanism of astrocytic ATP13A2 deficiency in NLRP3-Caspase-1 inflammasome-mediated neuroinflammationAIM:To investigate the mechanism of ATP13A2 inhibits neuroinflammation in astrocytes.METHOD:The expression of ATP 13 A2 in the midbrain of chronic MPTP/p model was detected by western blotting. Real-time PCR and western blotting method to identify Atp13a2 knockout (Atpl3a2-/-) mice in the gene and protein level, respectively. Three to four months old male Atpl3a2+/+ and Atpl3a2-/- mice were used to establish chronic MPTP/p model of PD.The levels of monoamine neurotransmitters and amino acids in the striatum were measured by high performance liquid chromatography (HPLC). The lysosomal morphology was observed by transmission electron microscopy in the midbrain of both genotype mice. Immunofluorescence was used to observe LC3 spots accumulation and the western blotting method was used to detect the expression of LC3, p62 and autophagic substrate protein of a-synuclein in the midbrain. The rotarod test, locomotor activity test and pole test were performed to monitor the parkinsonian symptoms under baseline conditions and 7 days after the final injection of MPTP or saline. Immumohistochemical staining and western blotting was used to detect the expression of TH in the midbrain of both genotype mice. Immunofluorescence was used to observe the co-localization of TH neurons and a-synuclein, TH neurons and reactive oxygen species (ROS) in vivo. GFAP and Iba-1 immunohistochemical staining to label the astrocyte and microglia in Atpl3a2+/+ and Atpl3a2-/- mice under saline or MPTP/p treatment with quantifications by stereological software. The NF-κB signaling pathway related protein p65 and p-IKKP, and the inflammasome activation signaling associated protein NLRP3, procaspase-1, caspase-1, proIL-1β and IL-1β were detected by western blotting in the midbrain tissues of both genotype mice. Primary mesencephalic astrocytes were cultured from new-born Atp13a2+/+ and Atp13a2-/- mice, Real-time PCR and western blotting was used to detect the level of ATP13A2. After stimulation with 50μM MPP+ after 48 h, collected cell protein, western blotting was used to detect the influence of MPP+ on the expression of ATP13A2. H2DCF-DA fluorescent probe detected the influence of Atpl3a2 deficiecy on ROS generation mediated by MPP+stimulation in the primary astrocytes. The mRNA levels of TNF-a, IL-1β, IL-4, IL-6, IL-10 and neurotrophic factor GDNF, FGF2 were detected by Real-time PCR. After MPP+ stimulation for 48 h, we collected the astrocytic conditional medium from both genotype mice, and mixed with neurobasal in portion of 1:2. TH immunohistochemistry was used to observation the role of ATP 13A2 on DA neuronal survival and axon length. Administering two genotypes primary astrocytes with different inflammasome specific activator, the secretion of IL-1β was detected by ELISA. After MPP+ stimulation for 1.5,3,6,12, 24 h, the expression of p65 and p-IKKp were observed by western blotting. After MPP+ stimulation for 3,6,12,24,24 h, the expression of NLRP3, procaspase-1, caspase-1, proIL-1β and IL-1β were observed by western blotting. Small interfering RNA technology (siRNA-ATP13A2,100 nM) interference was used to downregulation the expression of ATP 13 A2 in WT primary astrocytes, supernatant IL-1β secretion and the expression of intracellular NLRP3, procaspase-1, caspase-1, proIL-1β and IL-1β were detected by western blotting. Western blotting and immunofluorescence were used to observe the change of cathepsin B (Cathepsin B) expression in both genotypes or the WT astrocytes of ATP 13 A2 interference with MPP+stimulation. Pre-incubated with cathepsin B activity inhibitor Z-FA-FMK 20 μM for 1 h, then stimulation with 50 μM MPP+ for 48 h, the expression of caspase-1 and IL-1β were detected by western blotting in the supernatant and cytoplasmic. Transfected with pcDNA3.1(-)-V5-ATP13A2 plasmid in both genotypes astrocytes, the release of TNF-a and IL-1β were detected by ELISA, and the expression of ATP13A2, cathepsin B, p-IKKβ, p65, NLRP3, caspase-1, proIL-1β and IL-1β were measured by western blotting.RESULTS:1) The expression ATP13A2 was reduced to 58% in of MPTP/p PD model mice compared to saline group.2) Atpl3a2 deficiency increased lysosomal membrane rupture and the damage of lysosomal structural integrity, continued induced autophagy dysfunction and α-synuclein accumulation in the midbrain brain tissue.3) Atpl3a2 deficiency significantly inhibited basal levels of striatal dopamine and its metabolites, and inhibited the release of aspartate and γ-aminobutyric acid in the striatum, while promoted the release of L-serine, there were no significant effect on the other amino acids release.4) Atpl3a2 deficiency induced motor dysfunction in mice.5) Atp13a2 deficiency induced the loss of TH neurons in SNc, and further aggravated MPTP/p-induced the accumulation of α-synuclein and generation of ROS within TH neurons.6) Atp13a2 deficiency increased the proliferation and activation of astrocytes and microglia in SNc.7) Atp13a2 deficiency aggravated MPTP/p-induced NLRP3 inflammasome activation in SNc.8) The protein of ATP13A2 was expressed in primary mesencephalic astrocytes. After stimulation with 50 μM MPP+ for 48 h, the expression of ATP 13 A2 was significantly decreased in WT astrocytes.9) Atp13a2 deficiency aggravated MPP+-induced ROS generation, and further increased MPP+-mediated the transcriptional levels of pro-inflammatory cytokines, inhibition of transcription levels of anti-inflammatory factors and neurotrophic factors, including IL-1β showed the most significant change on transcription level.10) The conditional medium from Atp13a2 deficiency astrocytes significantly inhibited the number and axon length of TH positive neurons.11) After stimulation with inflammasome specific activator in both genotypes of mouse primary astrocytes, Atp13a2 deficiency mainly induced the activation of NLRP3 inflammasome-mediated inflammation.12) Atp13a2 deficiency aggravated MPP+-induced the expression of p-IKKβ,p65, NLRP3, caspase-1, proIL-1β and IL-1β in astrocytes, and worsen the activation of NLRP3 inflammasome.13) ATP13A2-siRNA (100 nM) reduced the expression of astrocytic ATP13A2 and could increase the activation of MPP+-induced NLRP3 inflammasome activation.14) The deficiency or downregulation of ATP 13 A2 aggravated the MPP+-induced cathepsin B expression in astrocytes.15) Cathepsin B inhibitor Z-FA-FMK could effectively inhibit Atpl3a2 deficiency or MPP+-mediated NLRP3 inflammasome activation.16) Overexpresse of astrocytic ATP13A2 decreased Atp13a2 deficiency or MPP+-mediated cathepsin B expression and NLRP3 inflammasome activation and inhibited neuroinflammation.CONCLUTION:1) Atp13a2 deficiency increases the loss of DA neurons and exacerbates MPTP/p-induced the proliferation and activation of glia in SNc.2) ATP13A2 is expressed on astrocytes and participates in MPP+-mediated neurotoxin reactions.3) Knockout or knockdown astrocytic ATP13A2 exacerbates MPP+-induced NLRP3 inflammasome activation, and further promoting DA neuronal damage.4) Astrocytic ATP13A2 inhibits NLRP3 inflammasome activation by regulating the activity of the lysosomal cathepsin B.Part Ⅲ The impact role and mechanism of targeting caspase-1 on DA neuronal apoptosisAIM:To investigate the underlying mechanisms of caspase-1-induced DA neuronal injury.METHOD:Western blotting to detect the expression of caspase-1 in the midbrain of MPTP/p PD model mouse. Three to four months old male Caspase-1+/+ and Caspase-1-/- mice were used to establish chronic MPTP/p model of PD. Nissl staining to observe the morphology changes in neuron, and counts the number of Nissl positive neurons in both genotypes mice under saline or MPTP/p treatment. TH immunohistochemical staining to label the DA neuron in Caspase-1+/+ and Caspase-1-/- mice under saline or MPTP/p treatment with quantifications by stereological software. The rotarod test, locomotor activity test and pole test were performed to monitor the parkinsonian symptoms under baseline conditions and 7 days after the final injection of MPTP or saline. Western blotting to detect the expression of apoptosis-related protein B-cell lymphoma/leukemia-2 (Bcl-2), Bcl-2 associated X protein (Bax), cysteine-containing aspartate protease 7 (caspase-7), poly ADP-ribose polymerase-1 (PARP1) and apoptosis inducible factor (AIF) protein in midbrain tissues. Primary mesencephalic neurons were cultured from pregnant 13 to 16 days of Caspase-1+/+ and Caspase-1-/- mice, after 10μM MPP+ stimulation for 12 h, TH immunohistochemistry to observe and calculate the number of TH positive neurons and the length of neurite. Cultured human neuroblastoma SH-SY5Y neuronal cell lines, pre-incubation with caspase-1 inhibitor Z-YVAD 10\iM for 1 h and then stimulation with 200 μM MPP+ for 48 h, western blotting to detect the expression of caspase-1, Bcl-2 and Bax. Using Annexin-V and PI double staining combined with flow cytometry to detect cell apoptosis, cell viability was measured by MTT assay, Hoechst 33342 fluorescent staining was used to observe apoptotic cells. The role of Z-YVAD on caspase-7, PARP1 and AIF expression in SH-SY5Y cells were detected by western blotting. Real-time PCR and double digestion technology to construct 3HA-cleaved caspase-7 plasmids, and in combination with flow cytometry and western blotting to observe the effect of cleaved caspase-7 overexpression on caspase-1-mediated apoptosis.RESULTS:1)Compared with the saline group, MPTP/p induced the increase expression of caspase-1 in the midbrain, while no effect on the expression of procaspase-1.2) Under baseline, no significant effect on TH neurons between Caspase-1+/+ and Caspase-1-/- mice, caspase-1 deficiency significantly reduced the MPTP/p-induced the loss of neurons in SNc.3) Caspase-1 deficiency significantly improved MPTP/p-induced motor dysfunction.4) Caspase-1 deficiency improved the expression of MPTP/p-induced the downregulation of anti-apoptotic protein Bcl-2 and the upregulation of Bax.5) Caspase-1 deficiency inhibited the MPTP/p-induced caspase-7 clevage, and further suppressed the activation of PARP1/AIF signaling pathway, thus played a protective effect against apoptosis.6) Caspase-1 deficiency improved MPP+-induced the loss of TH positive neurons and shorten the length of axon.7) Caspase-1 inhibitor Z-YVAD (10βM) inhibited the expression of caspase-1, and decreased MPP+(200 μM)-induced the upregulation of Bax and the downregulation of Bcl-2 in SH-SY5Y cells.8) Z-YVAD inhibited MPP+-mediated neuronal apoptosis in SH-SY5Y cells.9) Overexpressing of cleaved caspase-7 significantly weakened the protection of Z-YVAD against neuronal apoptosis via increased PARP1 transported into the nuclear and the expression of cytoplasmic AIRCONCLUTION:1) MPTP/MPP+ induces the mature of caspase-1 involved in the regulation of PD pathological process.2) Caspase-1 deficiency inhibits MPTP/p-induced the loss of DA neurons and improves motor dysfunction.3) Z-YVAD targeted to modulate the activity of caspase-1 and inhibits the activated caspase-7 mediated PARP1/AIF signaling pathway activation, thus play protective effects against neuronal apoptosis.The major contributions of the present study lie in:1. NLRP3-Caspase-1 inflammasome activation is involved in the pathologic process of PD. Inflammasome activation is involved in PD patients and MPTP PD model mice, and the NLRP3 inflammsome exerts a critical role in the neurological damage of PD. Downregulation of NLRP3 in SNc by small interfering RNA interference technique, which significantly inhibits MPTP-induced the loss of DA neurons and the proliferation and activation glial cells, providing us academic foundation for targeting NLRP3 inflammasome activation in PD clinical drugs therapy.2. Astrocytic ATP13A2 inhibits NLRP3-Caspase-1 inflammasome activation mediated neuroinflammation. Atpl3a2 deficiency exacerbates the dysfunction of lysosome in astrocytes and induces the release of lysosomal cathepsin B,following NLRP3 inflammasome activation, thus increasing DA neuronal damage. Promisingly, ATP13A2 is likely to reveal a novel therapeutic strategy by inhibiting astrocyte-mediated neuroinflammation in PD and other lysosome storage diseases.3. Targeting caspase-1 alleviates the apoptosis of DA neurons in PD. Caspase-1 deficiency alleviates MPTP/p-induced DA neuronal apoptosis and motor dysfunction. Caspase-1 specific inhibitor Z-YVAD inhibits MPP+-induced caspase-7 clevage and PARP1/AIF signaling pathway mediated neuronal apoptosis in SH-SY5Y cells, providing us cleaved caspase-7 is essential for caspase-1-mediated neuronal apoptosis. These findings evoke a new target and strategy for the development of anti-apoptotic neuroprotective drug in PD. |
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