REGULATED PROTEOLYSIS AND LONG-TERM MEMORY

突触可塑性的研究不仅对理解正常的大脑功能（如学习和记忆）具有重要意义，而且对于确定许多神经和精神疾病的潜在原因也是必要的。研究突触可塑性的一个可处理的模型系统是海马感觉-运动神经元突触的长期突触前促进，这是一种基本的学习和记忆形式。长期促进需要从神经递质5-HT到细胞核的信号转导，cAMP-响应元件结合蛋白（CREB）激活基因转录。在缺乏足够刺激的情况下，整个信号通路的抑制剂都在阻止记忆的形成。这些抑制剂通过泛素-蛋白酶体途径降解。我们的总体目标是确定泛素-蛋白酶体途径如何去除抑制长期促进的蛋白质，并了解该途径中重要的调节检查点。早期的一项发现表明，camp依赖性蛋白激酶的调控亚基被泛素途径降解，使该激酶持续活跃。我们的假设是，与蛋白酶体酶核相连的调节复合物的调节有助于增加R亚基和其他底物的降解。我们最近的初步数据表明，蛋白酶体调控部分的两个亚基在长期促进的形成中起关键作用。我们也有直接证据表明，通过泛素-蛋白酶体途径可以去除CREB 1b （CREB抑制因子）。CREB 1b的蛋白水解发生在CREB激活之前的一个时间窗口内。我们的研究结果表明，针对CREB lb蛋白水解去除的酶的活性受磷酸化调节。我们的第一个目标是表明两个蛋白酶体调节亚基的上调在诱导长期促进中起重要作用。我们的第二个目标是分离和研究针对CREB 1b降解的酶，并证明它在诱导长期促进中起关键作用。泛素-蛋白酶体途径决定CREB基因诱导的阈值，从而决定长期记忆形成的阈值，这一概念可以广泛适用于理解其他形式的神经元可塑性。我们的方法结合了分子、细胞和电生理技术，将使我们对突触可塑性的机制有新的认识。由于CREB激活的改变以及泛素-蛋白酶体途径在大脑的几种异常中被发现，这些见解将有助于确定神经元功能障碍的原因以及设计治疗干预措施。

Study of synaptic plasticity is not only important for understanding normal brain function such as learning and memory but also is necessary for identifying the underlying causes of many neurological and mental diseases. A tractable model system for investigating synaptic plasticity is long-term presynaptic facilitation of sensory-to-motor neuron synapses in Aplysia, an elementary form of learning and memory. Long-term facilitation requires signal transduction from the neurotransmitter 5-HT to the nucleus for activation of gene transcription by the cAMP- responsive element binding protein (CREB). All along the signaling pathway inhibitors operate to prevent memory formation in the absence of adequate stimuli. These inhibitors are degraded by the ubiquitin-proteasome pathway. Our overall goal is to determine how the ubiquitin-proteasome pathway functions to remove proteins that inhibit long-term facilitation and to understand the important regulatory checkpoints in this pathway. An earlier discovery showed that the regulatory subunit of the cAMP-dependent protein kinase is degraded by the ubiquitin pathway to make the kinase persistently active. Our hypothesis is that modulation of the regulatory complexes attached to the enzymatic core of the proteasome contributes to the increased degradation of R subunits and other substrates. Our recent preliminary data indicate that two subunits of the regulatory part of the proteasome play a critical role in the formation of long-term facilitation. We also have direct evidence for removal of CREB 1b, a CREB repressor, by the ubiquitin-proteasome pathway. The proteolysis of CREB 1b occurs during a time window preceding CREB activation. Our results indicate that the activity of the enzyme that targets CREB lb for proteolytic removal is regulated by phosphorylation. Our first aim is to show that the up-regulation of two proteasome regulatory subunits plays an important role in induction of long-term facilitation. Our second aim is to isolate and study the enzyme that targets CREB 1b for degradation and to demonstrate that it plays a critical role in inducing long-term facilitation. The concept that the ubiquitin-proteasome pathway operates to determine the threshold for gene induction by CREB and consequently threshold for formation of long-term memory could be widely applicable to understanding other forms of neuronal plasticity. Our approach that uses a combination of molecular, cellular and electrophysiological techniques would enable us to gain novel insights into the mechanisms underlying synaptic plasticity. Since alterations in CREB activation as well as the ubiquitin- proteasome pathway are seen in several abnormalities of the brain, these insights would be useful for identifying the causes of neuronal dysfunction as well as designing therapeutic interventions.