Mechanisms Regulating Activity Dependent Synaptic Plasti

神经调节蛋白-1 （NRG-1）是一种精神分裂症易感基因，但其在成人大脑中的功能尚不清楚。我们目前对NRG-1作为外周和中枢神经系统的营养和分化因子的认识主要局限于早期发育。尽管NRG-1及其ErbB酪氨酸激酶受体（ErbB 1-4）在成年啮齿动物和人脑中表达，但对其功能知之甚少。NRG-1在海马中表达，并以活动依赖的方式在突触中加工和释放。在成人大脑中，我们发现ErbB受体在谷氨酸能突触后位点与NMDARs共定位，并与pdz结构域支架蛋白相互作用，这对于突触在活动响应中重塑是重要的。基于这些发现，我们提出NRG信号在响应活动时具有快速调节突触可塑性的潜力。我们目前的工作支持这一假设。我们发现，虽然NRG-1对基础谷氨酸突触传递没有影响，但它以活动依赖的方式逆转海马CA1突触的长期增强（LTP）。有趣的是，NRG-1逆转（去电位）LTP的潜力是时间依赖性的，它仅在LTP引发后的前30分钟内起作用。我们发现ErbB抑制剂阻断NRG-1，以及刺激依赖性的LTP逆转，并增加LTP在增强突触的水平。利用膜片钳和细胞生物学技术，我们证明了NRG-1通过选择性地减少AMPA而不是NMDA受体电流来减弱LTP。转染超黄道绿色荧光蛋白（seGFP）受体的海马神经元的实时成像表明，NRG-1刺激表面含有glur1的AMPA受体的内化。超黄道绿色荧光蛋白是一种仅在细胞表面表达时才发出强烈荧光的GFP形式。NRG-1对LTP的这种新调控对于谷氨酸突触的突触稳态调节以及理解精神分裂症等复杂疾病的分子机制具有重要意义。

Neuregulin-1 (NRG-1) was genetically identified as a schizophrenia susceptibility gene, but its function in the adult brain is unknown. Our present knowledge of NRG-1 as a trophic and differentiation factor in the peripheral and central nervous system is mostly restricted to early development. Although NRG-1 and its ErbB tyrosine kinase receptors (ErbB 1-4) are expressed in the adult rodent and human brain, little is known about their functions. NRG-1 is expressed in the hippocampus, and is processed and released at synapses in an activity-dependent manner. In adult brain, we showed that ErbB receptors colocalize with NMDARs at glutamatergic postsynaptic sites and interact with PDZ-domain scaffolding proteins, which are important for remodeling synapses in response to activity. Based on these findings, we proposed that NRG signaling had the potential to rapidly modulate synaptic plasticity in response to activity. Our present work supports this hypothesis. We found that while NRG-1 has no effect on basal glutamatergic synaptic transmission, it reverses long-term potentiation (LTP) at hippocampal CA1 synapses in an activity-dependent fashion. Interestingly, the potential of NRG-1 to reverse (depotentiate) LTP is time-dependent, it only works within the first 30 min after eliciting LTP. We found that ErbB inhibitors block NRG-1, as well as stimulus-dependent, reversal of LTP, and also increase LTP levels at potentiated synapses. Using patch clamp and cell biological techniques, we demonstrated that NRG-1 depotentiates LTP by selectively reducing AMPA, but not NMDA, receptor currents. Live imaging of hippocampal neurons transfected with receptors fused to superecliptic green fluorescent protein (seGFP), a form of GFP that only fluoresces strongly when expressed on the cell surface, indicate that NRG-1 stimulates the internalization of surface GluR1-containing AMPA receptors. This novel regulation of LTP by NRG-1 has important implications for the modulation of synaptic homeostasis at glutamatergic synapses, and for understanding molecular mechanisms that underlie complex disorders like schizophrenia. B. ACTIVITY-DEPENDENT REGULATION OF MUSCLE TYPES Skeletal muscle size and contractile properties are modified by exercise. The different patterns of electrical impulses elicited during distinct types of exercise regulate muscle mass, and their slow- and fast-twitch contractile properties. Our long-term objective is to identify the signaling pathways that regulate the properties of slow- and fast-twitch muscles in response to activity. The troponin I slow (TnIs) and fast (TnIf) genes have served as our experimental model because expression of both genes is regulated by either slow or fast patterns of electrical impulses, similar to those produced by slow- or fast-firing motor neurons. We have identified the slow upstream regulatory enhancer (SURE) and the fast intronic regulatory element (FIRE) as minimal DNA sequences that regulate the fiber-type-specific transcription of the TnIs and TnIf genes, respectively. Interestingly, the SURE element is bipartite: the downstream half regulates muscle specificity and the upstream half is necessary for fiber-type specificity. We found that the General Transcription Factor 3 (GTF3) binds to a site in the upstream half of SURE. Using a method to select transcription factor DNA binding sites from random pools of sequences, we determined that (G/A)GATT(A/G) is the GTF3 consensus site and is located in the TnI SURE (and other enhancers reported to be regulated by GTF3). The GTF3 DNA binding domain was mapped to helix motif 4, which is necessary and sufficient to bind the TnI SURE. Interestingly, GTF3 is lost in a ~2.0 Mb micro-deletion of chromosome 7q11.2 in individuals with Williams Syndrome (WS). Persons with WS have distinctive physical, cognitive and behavior abnormalities that include impaired spatial cognitive skills and myopathies. Our studies using ectopically transfected GTF3 constructs in adult muscles and GTF3 knock-out mice support a possible role for this factor in regulating muscle contractile properties. Slow and fast motor neuron firing properties activate the transcription of genes expressed in either slow- or fast-twitch muscles. The mechanisms responsible for sensing and decoding distinct patterns of action potentials, and converting them into specific changes in gene expression, remain unknown. Using the TnIs and TnIf enhancers as reporter constructs driving expression of the green fluorescent protein (GFP), we found that stimulation of muscles with either slow or fast patterns of electrical stimuli differentially regulate these enhancers. Slow, tonic patterns of depolarization upregulate the SURE, while fast, phasic patterns increase FIRE transcription. These results indicate that the TnI slow and fast enhancers can sense, and respond to, distinct patterns of neuronal activity. Experiments are in progress to identify the specific SURE and FIRE DNA regulatory elements that differentially respond to activity, and the signal transduction pathways that mediate these effects.