Mechanisms Regulating Activity Dependent Synaptic Plasti

神经调节蛋白-1（NRG-1）被遗传学鉴定为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中的营养和分化因子的认识主要限于早期发育。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，并以活性依赖性方式在突触处加工和释放。在成人大脑中，我们发现ErbB受体与NMDAR共定位在神经元能突触后位点，并与PDZ结构域支架蛋白相互作用，这对于响应活动重塑突触非常重要。基于这些发现，我们提出NRG信号传导具有快速调节突触可塑性以响应活动的潜力。我们目前的工作支持这一假设。我们发现，虽然NRG-1对基础海马能突触传递没有影响，但它以活动依赖的方式逆转海马CA1区突触的长时程增强（LTP）。有趣的是，NRG-1逆转（去增强）LTP的潜力是时间依赖性的，它仅在引发LTP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，逆转LTP，也增加LTP水平在增强突触。使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受体电流来降低LTP。海马神经元转染受体融合的超椭圆绿色荧光蛋白（seGFP），一种形式的GFP，只有强烈的荧光时，表达在细胞表面，表明NRG-1刺激的内化表面GluR1的AMPA受体。NRG-1对LTP的这种新的调节对于调节神经元能突触的突触稳态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。 B。肌肉类型的活动依赖性调节 骨骼肌的大小和收缩特性通过锻炼来改变。在不同类型的运动中，不同的电脉冲模式可以调节肌肉质量，以及它们的慢收缩和快收缩特性。我们的长期目标是确定调节慢肌和快肌对活动的反应的信号通路。肌钙蛋白I慢（TnIs）和快（TcR）基因已作为我们的实验模型，因为这两个基因的表达是由慢或快模式的电脉冲，类似于由慢或快的运动神经元产生的调节。我们已经确定了缓慢的上游调控增强子（SURE）和快速内含子调控元件（FIRE）作为最小的DNA序列，分别调节TnIs和TnIs基因的纤维类型特异性转录。有趣的是，SURE元件是二分的：下游的一半调节肌肉特异性，上游的一半是纤维类型特异性所必需的。我们发现，一般转录因子3（GTF 3）结合到一个网站的上游一半的SURE。使用从随机序列库中选择转录因子DNA结合位点的方法，我们确定（G/A）GATT（A/G）是GTF 3共有位点，并且位于TnI SURE（和据报道由GTF 3调节的其他增强子）中。将GTF 3 DNA结合结构域定位于螺旋基序4，这是结合TnI SURE所必需且充分的。有趣的是，在患有威廉姆斯综合征（WS）的个体中，GTF 3在染色体7q11.2的~2.0 Mb微缺失中丢失。WS患者具有独特的身体、认知和行为异常，包括空间认知技能受损和肌病。我们的研究使用异位转染的GTF 3结构在成年肌肉和GTF 3基因敲除小鼠支持一个可能的作用，这个因素在调节肌肉收缩性能。 慢速和快速运动神经元的放电特性激活了在慢速或快速收缩肌肉中表达的基因的转录。负责感知和解码不同的动作电位模式，并将其转化为基因表达的特定变化的机制仍然未知。使用TnIs和TnIs增强子作为驱动绿色荧光蛋白（GFP）表达的报告构建体，我们发现用慢或快模式的电刺激刺激肌肉差异地调节这些增强子。缓慢，紧张性模式的去极化上调的SURE，而快速，阶段性模式增加消防转录。这些结果表明，TnI慢增强子和快增强子可以感知和响应不同的神经元活动模式。实验正在进行中，以确定特定的SURE和FIRE DNA调节元件，差异响应活动，以及介导这些影响的信号转导途径。

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.