The Role And Mechanism Of Environmental Responsive Gene JWA On The Pathogenesis Of Parkinson’s Disease

Parkinson’s disease (PD) is the second most common neurodegenerative disease characterized by muscle rigidity, tremor, postural instability and paucity of voluntary movements, where only symptomatic treatment currently exists. The frequency of PD increases with age, almostly affecting1-4%of the population over60. The main neuropathological features of PD are the progressive degeneration of dopaminergic (DA) neurons and apparent gliosis with the presence of intracytoplasmic inclusions (Lewy bodies) in the mesencephalic substantia nigra compacta (SNc). Although several possible mechanisms of PD have been proposed, such as aging, oxidative stress and neuroinflammation, the explicit mechanism remains elusive. Clinically, L-DOPA is the first-class drug to treat PD, which only meliorates the symptoms but fails to protect DA neuron from degeneration.Therefore, new strategies or drugs to reverse or ameliorate the pathological process of DA neurons in PD patients would be of great clinical value.The significant activation of astrocytes is observed in PD and other neurodegenerative disorders. Astrocyte has been regarded as a critical participant in several neurodegenerative diseases, including PD. Astrocyte also plays crucial roles in adult central nervous system homeostasis, including synaptic glutamate uptake, extra-cellular potassium maintenance, and nutrient support for neurons.The JWA gene codes for a novel microtubule-binding protein, which regulates cancer cell migration via MAPK cascades and mediates differentiation of leukemic cell. JWA also responses to oxidative stress-induced base excision repair (BER) process via its regulation on the stability of XRCC1, a key regulator in BER process. These findings imply that JWA is essential to maintain redox homeostasis and may also regulate inflammatory responses. However, the role of JWA gene in the pathogenesis of PD has never been identified. In order to elucidate pathogenesis of PD, we tried to use astrocytic JWA null (JWAΔ2/Δ2/GFAP-Cre) mice and U251glioma cells to investigate the potential function of JWA on PD.Aims:To investigate the roles of astrocytic JWA expression in dopaminergic (DA) neuron degeneration and in the pathogenesis of Parkinson’s disease (PD).Methods:First, we produced adult male C57BL/6J mice with astrocytic JWA knockout (JWAΔ2/Δ2/GFAP-Cre) and littermate wild-type controls (JWALoxp/Loxp). Second, immunohistochemical studies and quantitative evaluation were used to observe the expression of Tyrosine hydroxylase (TH) and Glial fibrillary acidic Protein (GFAP) expression. The total numbers of GFAP, TH-positive cells were provided through Stereo-Investigator software. Nissl-stained neurons in mouse midbrain signified survival neurons. Third, six mice striatum tissues in each group were prepared for measurement of monoamine neurotransmitters by High performance liquid chromatography (HPLC). Forth, double-labeled immunofluorescence of ROS and TH in mouse SNc was carried out to evaluate the levels of ROS. Twelve hours after transfection of FLAG-CON or FLAG-JWA, U251cells were treated with MPP+(50μM) for24hours and the levels of ROS in U251cells were measured by flow cytometry. Fifth, we used commercial kits to determine the contents of intracellular glutathione (GSH). Sixth, Western blotting was used to analysis the expression of Ikkβ, IκB and p65both in vivo and in vitro models.Results:Conditional astrocytic JWA knockout resulted in significant activation of astrocytes (1.34x103±74.5vs.8.44×103±1.35×103, P<0.01) in mouse SNc, and accompanied by the loss of DA neurons (1.03×104±238vs.6.17×103±392, P< 0.001). We found that the dopamine level was significantly increased in the striatun of JWΔ2/Δ2/GFAP-Cre mice compared to the control mice (11.1±1.79nmol/g vs.22.5±2.23nmol/g, P<0.01); whereas the level of HVA (13.8±0.742nmol/g vs.10.6±0.870nmol/g, P<0.05) and the ratio of DOPAC/DA(3.28±0.414vs.2.02±0.285, P<0.05) were decreased. JWA deficiency obviously aggravated ROS accumulation in SNc compared to the wild type mice. Increasing JWA expression in U251glioma cells significantly inhibited ROS (3.60×104±572vs.2.87×104±397, P <0.001) whereas increased GSH production (298±0.667vs.365±7.09, P<0.001). The expression of NF-KB-p65in the nucleus of midbrain tissue was1.67-fold higher in JWAΔ2/Δ2/GFAP-Cre mice than that of JWALoxp/Loxp mice (P<0.01). In in vitro models, JWA overexpression increased NF-κB-p65to2.1-fold (P<0.01) in the cytoplasm and reduced to70%(P<0.001) of its levels in nucleus in U251cells in response to50μM MPP+treatment. The expression of IKKβ was1.7-fold (P<0.05) in JWAΔ2/Δ2/GFAP-Cre mice while IκB was0.5-fold (P<0.001) in the midbrain. The IκB was1.7-fold while the IKKβ and p-p65were approximately0.6-fold and0.5-fold in FLAG-JWA cells compared with the parental cells, respectively (P<0.001).Conclusion:Conditional astrocytic JWA knockout led to a significant increase of GFAP positive cells in SNc compared with JWALoxp/Loxp mice. Moreover, the number of neurons in SNc of astrocytic JWA conditional knockout mice was30%less than the control one under basic condition by the nissl’s staining. The astrocytic JWA deficiency mice showed a dramatic reduction of TH+neuronal number in SNc compared with those in JWALoxp/Loxp mice. JWA gene exerts neuroprotective roles against DA neuronal degeneration via modulating intracellular redox status and NF-κB signaling pathway.