The Role And Mechanism Of NOD2 In Parkinson’s Disease

BackgroundParkinson’s disease is one of the most common neurodegenerative diseases, characterized by a selective loss of dopaminergic (DA) neurons in the substantia nigra (SN) and a drastic decline in striatal dopamine concentrations. The mechanism of Parkinson’s disease is complex and so far it hasn’t been fully understood. There is increasing evidence showing that innate immunity is critically involved in the pathogenesis of Parkinson’s disease.Intracellular nucleotide-binding oligomerization domain (NOD) protein family, which is called NOD-like receptors (NLRs), is the research hot point of innate immunity. NOD2 is one of the first discovered NLRs and plays an important role in inflammatory homeostasis. It is reported that mutations in NOD2 gene are related to inflammatory diseases such as Crohn’s disease and Blau syndrome. However, the role and mechanism of NOD2 in Parkinson’s disease are still unknown.There is increasing evidence showing that oxidative stress induced by ROS is one of the main reasons resulting in PD injury. Superoxide O2-. produced by NADPH oxidase is an important resource of ROS. It has been reported that inhibiting the NOX2-dependent oxidative stress will improve the DA neuron injury in PD model. But in PD the relationship and the detailed mechanisms among NADPH oxidase and NOD2 is far from being fully characterized. Therefore, in this study, we will explore the role and mechanism of NoD2-mediated immuno-inflammation in the PD by using the wild type and NOD2 knockout mice. We will also investigate the mechanism of NADPH oxidase-derived ROS on NoD2 signaling pathway in PD. Our study will explain the role and mechanism of NOD2 in Parkinson’s disease for the first time and provide a significant target in the treatment of Parkinson’s disease.Methods1. To confirm the expression of NoD2 in PD mice induced by 6-OHDAWild type (WT) mice were placed in a stereotaxic device under 1.5% pentobarbital sodium anesthesia and given 6-OHDA or saline alone into two different sites of the striatum separately. The following experiments were conducted after 1 day,2 days,3 days,7 days,14 days and 21 days. 1.1 Behavioral analyses were evaluated by the apomorphine-induced rotation test.1.2 Western Blot was performed to detect the protein expression levels of NoD2 and tyrosine hydroxylase (TH) in substantia nigra and striatum.1.3 Immunofluorescent staining was used to detect NoD2 expression in the substantia nigra (SN) and striatum (STR) from WT mice on 14 days after 6-OHDA treatment.2. To confirm the role of NoD2 in PD.Wild type (WT) and NoD2 deficient (NoD2"’") mice were placed in a stereotaxic device under 1.5%pentobarbital sodium anesthesia and given 6-OHDA, muramyl dipeptide (MDP, an extrinsic ligand of NOD2) or saline alone into two different sites of the striatum separately. The following experiments were conducted after 7 days,14 days and 21 days.2.1 The protein levels of TH in substantia nigra(SN) and striatum(STR) were assessed by Western Blot and immunohistochemistry separately.2.2 The levels of DA and its metabolites DOPAC and HVA in mice striatum were measured by LC/MS.3. To explore the possible mechanism of PD induced by NoD2.Wild type (WT) and NoD2 deficient (NoD2’A) mice were placed in a stereotaxic device under 1.5%pentobarbital sodium anesthesia and given 6-OHDA, muramyl dipeptide (MDP, an extrinsic ligand of NoD2) or saline alone into two different sites of the striatum separately. The following experiments were conducted after 14 days.3.1 Immunofluorescent staining was performed to detect the microglial and astrocytic response.3.2 Cytometric Bead Array (CBA) was used to test the levels of pro-inflammatory mediators IL-6 and MCP-1.3.3 DCFH-DA fluorescence label method was used to detect the level of ROS in the striatum.3.4 The protein expression levels of gp91phox, cytoplasmic p47phox, membranous p47phox and total p47phox were measured by Western Blot.3.5 TUNEL staining was used to assess the apoptosis in substantia nigra.3.6 Western Blot was performed to detect the protein expression levels involved in apoptotic signaling pathways (BAX, Bcl-2, Cytochrome C, caspase-9, caspase-3).Results1. The expression of NoD2 in SN and STR was time-dependently increased in Parkinson’s disease mice induced by 6-OHDA.2. NOD2 stimulation reduced the protein level of TH time-dependently in SN and STR of WT mice.3. NoD2 deficiency improved the reduction of TH protein expression in SN and STR, and inhibited the reduction of DA and its metabolites in STR induced by 6-OHDA or MDP, which indicated NoD2 was involved in the injury of dopaminergic neurons.4. NoD2 deficiency attenuated the microglial and astrocytic activation and proliferation, reduced the production of pro-inflammatory cytokine IL-6 and chemokine MCP-1 induced by 6-OHDA or MDP, which indicated that N0D2 mediated inflammation was involved in PD.5.6-OHDA or MDP treatment increased the level of ROS in STR, and increased the expression of subunitie gp91phox and p47phox, and promoted p47phox translocation. NoD2 deficiency inhibited the activation of NoX2 induced by 6-OHDA or MDP, which indicated that NOX2-mediated oxidative stress contributed to NOD2-induced dopaminergic degeneration.6.6-OHDA or MDP treatment induced apoptosis in SN and increased the protein levels of BAX, Cytochrome C, caspase-9, caspase-3 and reduced the expression of Bcl-2. NoD2 deficiency attenuated the apoptosis induced by 6-OHDA or MDP, which revealed that apoptotic signaling pathway was involved in NOD2-induced dopaminergic degeneration.ConclusionsThe expression of NoD2 was increased in Parkinson’s disease mice induced by 6-OHDA.6-OHDA or MDP induced the activation of astrocytes and microglial cells, promoted the release of IL-6 and MCP-1, activated NoX2 and resulted in the dopaminergic degeneration and apoptosis. However, NoD2 deficiency reduced the injury of dopaminergic neurons induced by 6-OHDA or MDP, revealing that NoD2 was involved in the pathogenesis of Parkinson’s disease.