Neurotrophic factors are traditionally viewed as secretory proteins that regulate neuronal survival and differentiation, but recent studies show that they also play an important role in synapse development and plasticity. My lab was among the first to demonstrate this novel function of trophic factors. We have made two important discoveries. One is that brain-derived neurotrophic factor (BDNF) acutely potentiates high frequency synaptic transmission, and promotes hippocampal LTP, a cellular model for learning and memory. The other is that neurotrophin-3 (NT3) facilitates the long-term maturation at developing neuromuscular junction (NMJ). We are continuing the studies on neurotrophic regulation of synapses. We have made a number of discoveries last year: 1) Molecular mechanisms underlying long-term regulation of hippocampal synapses by neurotrophic factors. While significant progress has been made in understanding the acute effects of neurotrophins at synapses, much less is known about the molecular mechanisms for the long-term synaptic effects. Using slice cultures, we demonstrated that BDNF induces two types of changes in the expression of synaptic proteins: a rapid, small increase in synaptophysin and synaptobrevin, and a slow but robust increase in synaptotagmin. The delayed increase in synaptotagmin was blocked by inhibition of cAMP pathway and protein synthesis, while the early increase in synaptophysin and synaptobrevin was not. These results suggest that unlike acute modulation, long-term regulation of hippocampal synapses by BDNF requires protein synthesis and cAMP--molecular mechanisms very similar to those used in activity-dependent long-term synaptic modulation. (JBC). 2) Signal transduction mechanisms for acute effects of neurotrophins. We showed last year that acute modulation of NT3 on transmitter release at the NMJ uses an unusual mechanism which involves calcium release from intracellular stores through inositol 1, 4, 5-trisphosphate (IP3) and/or ryanodine receptors, leading to an activation of calcium/calmodulin kinase II (CaMKII) (J. Cell Biol.). This year, we demonstrated that the acute effect of NT3 can be blocked by inhibition of PI3 kinase and IP3 receptors. However, neither stimulation of Ca2+ release from intracellular stores by photolysis of caged IP3 nor expression of a constitutively active phosphoinositide-3 kinase (PI3K*) in presynaptic motoneurons alone is sufficient to enhance transmission. Remarkably, photo-uncaging of IP3 in neurons expressing PI3K* elicits a marked synaptic potentiation, mimicking the NT3 effect. This study reveals a novel role of PI3 kinase in synaptic transmission, and suggests a general principle that combinational use of signaling pathways determines the specificity of neurotrophin actions. (Nature Neurosci.). 3) Acutely effects of GDNF on neuronal excitability and A-type K+ channels in midbrain dopaminergic neurons. GDNF has long been thought to be a potent neurotrophic factor for the survival of midbrain dopaminergic neurons, which are degenerated in Parkinson's disease. However, all the previous experiments were done on injured neurons. The physiological function of GDNF on normal neurons is not known. In this study, we discovered an unexpected, acute effect of GDNF on A-type potassium channels, leading to a potentiation of neuronal excitability, in the dopaminergic neurons in culture as well as in adult brain slices. Further, we found that GDNF regulates the K+ channels through a mechanism that involves activation of MAP kinase. Thus, this study has revealed, for the first time, an acute modulation of ion channels by GDNF. Our findings challenge the traditional view of GDNF as a long-term survival factor for midbrain dopaminergic neurons, and suggest that the normal function of GDNF is to regulate neuronal excitability, and consequently dopamine release. These results may have important implications in the treatment of Parkinson's disease. (Nature Neurosci.). 4) Long-term effects of GDNF on synaptic transmission and Ca2+ channels: Role of the Ca2+-binding protein frequenin. We have investigated the molecular mechanisms underlying the long-term effects of GDNF on synaptic transmission at the NMJ. Using Xenopus nerve-muscle co-cultures, we show that both spontaneous and evoked transmitter release at the neuromuscular synapses are enhanced after long-term treatment with GDNF. These effects are very similar to the synaptic potentiation elicited by presynaptic expression of frequenin, a neuron-specific Ca2+-binding protein. GDNF enhances the expression of frequenin in Xenopus motoneurons. Inhibition of frequenin expression or activity prevents the synaptic action of GDNF. GDNF also facilitates Ca2+ influx at the nerve terminals during evoked synaptic transmission by enhancing Ca2+ currents. The effect of GDNF on Ca2+ currents is blocked by inhibition of frequenin expression, occluded by over-expression of frequenin, and is selective to N-type Ca2+ channels. Thus, frequenin mediates GDNF-induced synaptic facilitation by potentiating N-type Ca2+ channels. These results have identified, for the first time, a molecular target that mediates the long-term, synaptic action of a neurotrophic factor. Our findings may also have general implications in the cell biology of neurotransmitter release.
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