The roles of postsynaptic density-95 protein (PSD-95) and its interactor, cypin, in the regulation of microtubule organization, dendrite morphology, and neuroprotection
Creators: name: Tseng, Chia-Yi
Publisher: name: No Publisher Supplied
Year: 2011

description: Dendritic patterning and development are critical for proper neuronal function and communication. A number of factors, including postsynaptic protein-95 (PSD-95) and its cytosolic interactor (cypin), coordinate dendrite branching through regulation of microtubule organization. Our laboratory has shown that PSD-95 decreases dendrite branching by disrupting microtubule organization and that cypin increases dendrite branching by directly regulating microtubule assembly. However, these previous studies assessing microtubule organization were performed in a cell-free system or a monkey kidney fibroblast cell line, COS-7, and they did not provide direct evidence for the roles of PSD-95 and cypin in microtubule organization in neurons. Here, by using electron microscopy, we show that overexpression of PSD-95 disrupts microtubule organization by increasing the number of microtubules crossing at dendrite branch points and increasing the spacing between microtubules. Furthermore, cypin promotes microtubule assembly by decreasing spacing between microtubules, suggesting an appearance of tight microtubule bundles.  Varicosity formation (focal swelling) in dendrites and loss of dendritic spines are the earliest characteristics of glutamate-induced excitotoxicity. Previous studies have shown that microtubule integrity relates to varicosity formation and dendrite recovery. Since cypin and PSD-95 are involved in the regulation of microtubule organization, we asked whether cypin and PSD-95 play a role in mediating changes in dendrite morphology in response to glutamate-induced excitotoxicity. To prevent excessive neuron death by overactivation glutamate receptors, a sublethal dose of N-methyl-D-aspartic acid (NMDA) was applied to primary hippocampal neurons. We found that microtubules are disrupted in response to this treatment. Overexpressing cypin and knocking down PSD-95 result in decreased varicosity size but increased percentage of neurons with varicosities and number of varicosities per unit of dendrite after sublethal NDMA exposure. Furthermore, neuroprotection from NMDA is seen under these conditions. We see opposite effects when PSD-95 is overexpressed and cypin is knocked down. Moreover, overexpressing cypin and knocking down PSD-95 lead to a reduction in the loss of dendritic protrusions after NMDA treatment. Here, we demonstrated that cypin and PSD-95 are involved in a pathway by which neurons alter their dendritic morphology in response to sublethal NMDA treatment.       Oxidative stress is one of the final pathways contributing to neuron death in glutamate-induced excitotoxicity. Previous studies show that cypin is involved in the production of uric acid (UA). Moreover, UA protects neurons from glutamate-induced excitotoxicity in the presence of astroglia in spinal cord cultures. By treating spinal cord neurons with 3-Morpholinosyndnomine (SIN-1) to mimic oxidative stress, we tested whether UA can reduce SIN-1-mediated neuronal death. We find that UA prevents SIN-1-induced neurotoxicity when added concurrently with and after SIN-1 treatment, and is the effects of UA are astroglia-independent. Taken together, the work in this thesis suggest that cypin and PSD-95 play a role in the regulation of microtubule organization, and hence, influence changes of dendrite morphology and neuronal survival in response to glutamate-induced and SIN-1-promoted excitotoxicity.