Neuroprotective mechanism of HO-1 and autophagy in neurodegenerative diseases

• Main Authors: Shih-Ya Hung, 洪詩雅
• Other Authors: Wei-Mei Fu
• Format: Others
• Language: en_US
• Published: 2009
• Online Access: http://ndltd.ncl.edu.tw/handle/63639168289536566939

博士 === 國立臺灣大學 === 藥理學研究所 === 97 === Heme oxygenase-1 (HO-1) is up-regulated in response to oxidative stress and catalyzes the degradation of pro-oxidant heme to carbon monoxide (CO), Fe2+ and bilirubin. Intense HO-1 immunostaining in the brain of Parkinsonism is demonstrated, indicating that HO-1 may be involved in the pathogenesis of Parkinsonism. We here locally injected adenovirus containing human HO-1 gene (Ad-HO-1) into rat substantia nigra concomitantly with 1-methyl-4-phenylpyridinium (MPP+). Seven days after injection of MPP+ and Ad-HO-1, the brain was isolated for immunostaining, measurement of dopamine content and inflammatory cytokines. It was found that over-expression of HO-1 significantly increased the survival rate of dopaminergic neuron; reduced the production of TNF-α and IL-1β in substantia nigra; antagonized the reduction of striatal dopamine content in MPP+-lesioned side and also up-regulated BDNF and GDNF expression in substantia nigra. Apomorphine-induced rotation following MPP+-treatment was also inhibited by Ad-HO-1. On the other hand, inhibition of HO enzymatic activity by ZnPPIX facilitated the MPP+-induced rotatory behavior and enhanced the reduction of dopamine content. HO-1 over-expression also exerted protection of dopaminergic neurons against MPP+-induced neurotoxicity in midbrain neuron-glia co-cultures. Over-expression of HO-1 increased the expression of BDNF and GDNF in astrocytes and BDNF in neurons. Our results indicate that HO-1 induction exerts neuroprotection both in vitro and in vivo. Endogenous induction of HO-1 is involved in the neuroprotection in Parkinsonism. We then examined the action of downstream products of HO-1, bilirubin and CO. Bilirubin is a potent antioxidant and neuroprotectant. Neurotrophic factors of BDNF and GDNF also play important roles in survival and morphological differentiation of dopaminergic neurons. We have previously found that HO-1 induction by adenovirus containing human HO-1 gene (Ad-HO-1) in substantia nigra of rat increases BDNF and GDNF expression. As mentioned above, HO-1 in the enhancement of neurotrophic factor expression. Treatment of anti-BDNF/GDNF antibody significantly enhanced dopaminergic neuronal death and Ad-HO-1 co-treatment was able to antagonize the apoptosis effect. Injection Ad-HO-1 into substantia nigra of adult rat for 24 h, the confocal imaging shows that HO-1 induction appeared in dopaminergic neuron, astrocyte and microglia. HO-1 induced-BDNF/GDNF mRNA expression in substantia nigra was 26/21 folds of contralateral Ad-injected side. The downstream product of bilirubin also increased GDNF expression in glia-enriched cultures through ERK and PI3K-Akt pathways, which further enhanced NF-kB (p65) nuclear translocation. In addition, bilirubin also enhanced BDNF expression through similar pathway in cortical neuron-enriched cultures. We also examined the effect of another HO product of CO by using CO donor. [Ru(CO)3Cl2]2 induced neurotrophic factor expression via sGC-PKG pathway in both neuron and glia. Our results indicate that the downstream products of HO-1, bilirubin and CO, modulate BDNF and GDNF expression in neuron and astrocyte. Autophagy is a degradation pathway for the turnover of dysfunctional organelles or aggregated proteins in cells. Extracellular accumulation of β-amyloid peptide has been reported to be a major cause of Alzheimer''s disease (AD) and large numbers of autophagic vacuoles accumulate in the brain of AD patient. However, how autophagic process is involved in Aβ-induced neurotoxicity and how Aβ peptide is transported into neuron and metabolized is still unknown. In order to study the role of autophagic process in Aβ-induced neurotoxicity, EGFP-LC3 was over-expressed in SH-SY5Y cells (SH-SY5Y/pEGFP-LC3). It was found that treatment with Aβ25-35, Aβ1-42 or serum-starvation induced strong autophagy response in SH-SY5Y/pEGFP-LC3. Confocal double-staining image showed that exogenous application of Aβ1-42 in medium caused the co-localization of Aβ1-42 with LC3 in neuronal cells. Concomitant treatment of Aβ with a selective α7nAChR antagonist, α-bungarotoxin (α-BTX), enhanced Aβ-induced neurotoxicity in SH-SY5Y cells. On the other hand, nicotine (nAChR agonist) enhanced the autophagic process and also inhibited cell death following Aβ application. In addition, nicotine but not α-BTX increased primary hippocampal neuronal survival following Aβ treatment. Furthermore, using Atg7 siRNA to inhibit autophagosome formation in an early step or α7nAChR siRNA to knockdown α7nAChR significantly enhanced Aβ-induced neurotoxicity. Confocal double-staining image shows that nicotine treatment in the presence of Aβ enhanced the co-localization of α7nAChR with autophagosomes. These results suggest that α7nAChR may act as a carrier to bind with eAβ and internalize into cytoplasm and further inhibit Aβ-induced neurotoxicity via autophagic degradation pathway. Our results suggest that autophagy process plays a neuroprotective role against Aβ-induced neurotoxicity. Defect in autophagic regulation or Aβ-α7nAChR transport system may impair the clearance of Aβ and enhance the neuronal death.