The Modulatory Effects Of Uncoupling Protein 2 On Parkinson's Disease

Parkinson's disease (PD) is a chronic progressive neurodegenerative movement disorder, characterized by a profound and selective loss of dopaminergic neurons in the substantia nigra (SN), with presence of eosinophillic, intracytoplamic, proteinaceous inclusions termed as Lewy bodies (LB) and dystrophic Lewy neurites in surviving neurons. Clinical manifestations of this complex disease include motor impairments involving resting tremor, bradykinesia, postural instability, gait difficulty and rigidity. Current PD medications treat symptoms, mainly by L-DOPA administration; none halt or retard dopaminergic neuron degeneration. The main obstacle to developing neuroprotective therapies is a limited understanding of the key molecular events that provoke neurodegeneration.Although various hypotheses, including genetic factors, excitotoxicity, mitochondrial dysfunction, oxidative stress and inflammation have been proposed to be involved in the pathogenesis of PD, the exact mechanisms governing dopaminergic loss remain unclear. Mitochondrial oxidative stress has been implicated in a range of degenerative disease. Mitochondria dysfunction is a prominent feature in apoptosis and realease of pro-apoptotic proteins from the mitochondrial intermembrane space has been considered to be a critical event that occurs during apoptosis. Oxidative stress infers an imbalance between the formation of cellular oxidants and the antioxidative processes. Mitochondria are not only major source of reactive oxygen species (ROS) generation in aerobic cells, but they are also sensitive target for the damaging effects of oxygen radicals. Excessive ROS production impairs mitochondria membrane system, loss of mitochondrial membrane potential and activation mitochondrial permeabilty transiton pore, resulting in realease of pro-apoptotic proteins which subquently initiated mitochondrial apotosis pathway. Restoring mitochodrial function helps to provide neuroprotective effects on neurogentive disorders.Uncoupling proteins 2 (UCP2) is encoded by nuclear DNA and located in the inner membrane of the mitochondria. Its primary function is thought to be to translocate protons from the intermembrane space to the matrix of the mitochondria. In the individual mitochondrion, these proteins, through this process, may reduce the driving force of ATP synthase from catalyzing ATP synthesis, dissipate energy in the form of heat, diminish the production of superoxide anion, and decrease the likelihood of calcium entry to the mitochondrial matrix. The degeneration of substantia nigra compact (SNc) and ventral tegmental area (VTA) dopaminergic neurons is differential. One explaination is that UCP2 gene expression is different between SNc and VTA dopamine neurons, but the association requires further study.AIM: To investigate the role and the mechanism of UCP2 on MPTP-induced degeneration of dopaminergic neurons in MPTP/p PD model using UCP2 deficiency mice, and further study the underlying mechanism in vitro.METHODS: (1) Wild-type (UCP2+/+) and UCP2 knockout (UCP2-/-) mice treated with chronic MPTP intoxication protocol: 20 mg·kg-1 MPTP in saline was injected subcutaneously every 3.5 d over a period of 5 weeks, and were killed at 7 days after the last injection; (2) Immunohistochemistry was taken for tyrosine hydroxylase (TH), glial fibrillary acidic protein (GFAP), 5-bromodeoxyuridine (BrdU) and macrophage antigen complex-1 (MAC-1) expression. The total numbers of TH-positive neurons, GFAP-positive cells and MAC-1-positive cells in the substantia nigra pars compacts (SNc) and ventral tegmental area (VTA), and BrdU-positive cells in the subventricular zone (SVZ) and subgranular zone (SGZ) were obtained stereologically using the optical fractionator method; (3) The levels of FGF-2 (Fibroblast Growth Factor-2), GDNF (Glial cell line-Derived Neurotrophic Facor) and TNF-α(Tumor Necrosis Factor-α), IL-1β(Interleukin-1β) were determined by Realtime PCR, Western blotting and ELISA; (4) The levels of GRP78 (glucose-regulated protein 78), CHOP (C/EBP homologous protein) and NF-?B (Nuclear factor ?B) p65 were determined by Western blotting; (5) The levels of ROS (reactive oxygen species) in TH-positive neurons was determined by immuno fluorescence method; (6) Tyrosine Hydroxylase immunocytochemistry quantification of Tyrosine Hydroxylase immunoreactive (Thir) neuronal counts and processes was used to determine neuronal apoptosis; Assay of lactate dehydrogenase (LDH) released in neuronal media was used to determine neuronal neuronal injury; (7) RT-PCR and Western blotting were used to analysis the expression of UCP2 in astrocytes; Staining with Hoechst 33342 and assay of lactate dehydrogenase (LDH) were used to determine astroglia injury; Intracellular ROS was detected by molecular probe DCFH-DA.RESULTS: (1) UCP2 knockout not only aggravated MPTP-induced loss of SN dopaminergic neurons, but also caused the injury of dopaminergic neurons in the VTA. But UCP2 knockout dose not abolish the differential degeneration of dopaminergic neurons in the SN and VTA; (2) After MPTP treatment, UCP2 knockout enhanced the activation of astrocytes and microglia in SNc,VTA and inhibition of cell proliferation in SGZ and SVZ. UCP2 knockout promotes the generation of inflammation factors TNF-αand IL-1βformation,dose not effect the generation of nutritional factors FGF2 and GDNF; (3) UCP2 knockout enhanced MPTP-induced upregulation of GRP78, CHOP expression. UCP2 knockout also enhanced activation of NF-kB pathway by increasing p65 transported into nucleus; (4) UCP2 knockout significantly increased MPTP-induced upregulation of ROS generation in midbrain dopaminergic neurons; (5) UCP2 knockout enhanced MPP+-induced cytotoxicity in primary mesencephalic neurons and astrocytes; (6) Astrocytes expressed UCP2 protein, UCP2 knockout increased MPP+-induced upregulation of ROS generation in primary mesencephalic astrocytes. CONCLUSION:1. UCP2 knockout aggravates the loss of dopaminergic neurons , dose not abolish the differential degeneration of dopaminergic neurons in the SNc and VTA. The underlying mechanisms involve the enhanced activation and proliferation of astrocytes and microglia, inhibition of cell proliferation in SGZ and SVZ.2. UCP2 knockout increased the generation of ROS, enhanced ER stress and subsequent apoptosis pathway, consequently aggravating dopaminergic neurons damage.3. UCP2 knockout increased MPP+-induced upregulation of ROS generation in primary mesencephalic astrocytes, UCP2 knockout enhanced MPP+-induced cytotoxicity in primary mesencephalic neurons and astrocytes.The major contributions of the present study lie in:1. According to the reports, our study indicate that UCP2 is not involved in the differential degeneration of substantia nigra compact (SNc) and ventral tegmental area (VTA) dopaminergic neurons in PD models; UCP2 knockout increased the generation of ROS, enhanced ER stress and subsequent apoptosis pathway, consequently aggravating dopaminergic neurons damage.2. It was found that UCP2 also expressed in astrocytes. UCP2 knockout aggravated MPP+-induced cytotoxicity in primary mesencephalic neurons and astrocytes.