Regulation of cypin through snapin, a protein binding partner, and extrinsic signaling factors.

摘要

Proper dendrite development is important for normal neuronal communication. Disease states such as autism, Rett's syndrome, and Alzheimer's Disease can occur when there is aberrant dendrite branching. There are several known intrinsic factors involved in dendrite patterning: calcium activated proteins and the MEK/ERK pathway. In addition, a number of extrinsic factors can alter dendrite morphology, including neuronal activity and neurotrophins. Recently, our laboratory has shown that cypin is an intrinsic factor involved in dendrite patterning (Akum et al., 2004). Cypin overexpression in cultured hippocampal neurons increases dendrite branching while knocking down cypin decreases dendrite branching. In addition, cypin binds tubulin heterodimers and promotes microtubule assembly. Thus, cypin plays an important role in regulating dendrite morphology.; The focus of this dissertation is the regulation of cypin through its protein binding partner snapin (a SNAP-25 protein interactor), brain derived neurotrophic factor (BDNF) and neuronal activity, mimicked by treatment with potassium chloride. Here we show that snapin and cypin interact in vivo and that snapin competes tubulin heterodimer binding to cypin. This interaction also negatively affects cypin's ability to promote microtubule assembly. We also show that overexpression of snapin in cultured hippocampal neurons alters dendrite branching. These data show a novel function for snapin as a negative mediator of dendrite branching, possibly by regulating cypin-promoted microtubule assembly activity.; We find that BDNF increases cypin protein levels in hippocampal neurons. Interestingly, this effect seems to occur via a non-TrkB signaling pathway since K252a does not block the increase elicited by BDNF. BDNF also increases primary dendrite number through cypin via a TrkB-independent and mTOR-independent pathway. These data suggest that BDNF may signal to elevate cypin protein levels and increase dendrite branching via an alternate pathway from that of the TrkB-Pl3K/Akt-mTOR signaling pathway. Lastly, we show that cypin levels in cultured hippocampal neurons are also affected by potassium chloride. BDNF increases beta-catenin levels as well as PSD-95. Taken together, the work presented in this thesis suggests that cypin protein expression and function may be regulated through protein binding partners as well as BDNF and neuronal activity.