| Summary: | 碩士 === 國立陽明大學 === 藥理學研究所 === 98 === Glutamate, an excitatory amino acid neurotransmitter is known to regulate neuronal activities and induce excitotoxicity in central nervous system (CNS) neurodegenerative diseases, including stroke and epilepsy. Kainic acid (KA), a selective agonist against a non-N-methyl-D-aspartate (NMDA)-type glutamate receptors, is commonly used in animal models for epilepsy by inducing seizures and a subsequent excitotoxic neuronal loss in the hippocampus. In addition to the physiological roles, including cellular homeostasis between biosynthesis and catabolism, autophagy, an intracellular self-eating mechanism, is also suggested as a type II programmed cell death. Indeed, clinical studies have demonstrated elevations in autophagy in patients with CNS neurodegenerative diseases, including traumatic brain injury and Parkinson’s disease. However, the role of autophagy in the etiology of CNS neurodegenerative diseases remains undetermined. In my thesis, two goals were pursued; one was to elucidate the role (neuroprotective or neurodestructive) of autophagy in KA-induced neurotoxicity and the other was the involvement of autophagy in melatonin-induced neuroprotection of KA-induced neurotoxicity.
C57BL6 mice were intraperitoneally administrated with KA (20 mg/kg). Western blot assay and DNA fragmentation were employed to study the involvement of α-synuclein aggregation, neuroinflammation as well as activation of endoplasmic reticulum (ER) and mitochondrial pathways in KA-induced apoptosis. Systemic KA elevated heme oxygenase-1 level and α-synuclein aggregation. At the same time, α-spectrin cleavage was demonstrated, suggesting the involvement of apoptosis and necrosis in KA-induced neurotoxicity. KA-induced increases in bcl-2 and aCaspase 3 as well as aCaspase 12 suggested the involvement of mitochondria and ER in KA-induced apoptosis, respectively. KA-induced neuroinflammation was evident by elevation in cyclooxygnease-2, glial fibrillary acidic protein and ED-1. A transient elevation in LC3-II level, which is a hallmark of autophagy, was observed 4-6 h after KA. Furthermore, elevations in lysosomal-associated membrane protein 2 (LAMP-2) and cathepsin B were demonstrated in KA-treated mice. Intrahippocampal infusion of siAtg7 for 5 days was required for the successful transfection. SiAtg7 significantly reduced KA-induced neuronal loss. Furthermore, KA-induced increases in LC3-II, LAMP-2 and cathepsin B as well as poly (ADP-ribose) polymerase were found in KA-treated mice. Local infusion of SiAtg7 decreased KA-induced elevations in ED-1, Glial fibrillary acidic protein and inducible nitric oxide synthase. Moreover, SiAtg7 reversed KA-induced reduction in mitochondrial DNA. These data indicate that autophagy was neurodestructive in KA-induced neurotoxicity.
A significant body of studies has reported that melatonin is neuroprotective against neurotoxicity by KA, excitatory amino acids and cortical ischemia. Several mechanisms have been suggested for melatonin-induced neuroprotection, including elevation in anti-oxidative defense enzymes and glutathione as well as inhibition of free radical formation and apoptosis. In my thesis, oral administration of melatonin (50 mg/kg) decreased KA-induced neuronal loss, α-synuclein aggregation, α-spectrin cleavage. Consistent with previous studies, melatonin reduced KA-induced aCaspases 3 and 12 level as well as DNA fragmentation. Moreover, systemic melatonin attenuated KA-induced increases in LC3-II elevation, LAMP-2 and cathepsin B. Taken together, my results suggest that autophagy may play a pro-death role in KA-induced neurotoxicity. Furthermore, melatonin exerts its neuroprotection through reducing autophagy in KA-induced neurotoxicity. Melatonin may be therapeutically useful for CNS neurodegenerative diseases.
|
|---|
