REGULATED PROTEOLYSIS AND LONG-TERM MEMORY

突触可塑性的研究不仅对于了解正常的大脑功能，如学习和记忆，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容易处理的模型系统是海藻中感觉到运动神经元突触的长期突触前促进，海藻是学习和记忆的一种基本形式。长期的易化需要从神经递质5-羟色胺到核内的信号转导，以通过cAMP反应元件结合蛋白(CREB)激活基因转录。一直以来，信号通路抑制剂的作用是在缺乏足够刺激的情况下阻止记忆的形成。这些抑制物通过泛素-蛋白酶体途径降解。我们的总体目标是确定泛素-蛋白酶体途径如何发挥作用，以去除抑制长期促进作用的蛋白质，并了解这一途径中的重要调节检查点。早期的一项发现表明，cAMP依赖的蛋白激酶的调节亚单位被泛素途径降解，使该激酶持续活跃。我们的假设是，蛋白酶体酶核心上的调节复合体的调制有助于增加R亚基和其他底物的降解。我们最近的初步数据表明，蛋白酶体调节部分的两个亚基在形成长期易化过程中起着关键作用。我们也有直接证据表明，CREB抑制物CREB 1b可以通过泛素-蛋白酶体途径移除。CREB 1b的蛋白分解发生在CREB激活之前的一段时间窗口。我们的结果表明，以CREB lb为靶点的酶的活性是通过磷酸化来调节的。我们的第一个目标是证明两个蛋白酶体调节亚基的上调在诱导长期易化中起着重要作用。我们的第二个目标是分离和研究针对CREB 1b进行降解的酶，并证明它在诱导长期促进中发挥关键作用。泛素-蛋白酶体途径决定了CREB诱导基因的阈值，从而决定了形成长期记忆的阈值，这一概念可广泛应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，将使我们能够对突触可塑性的潜在机制获得新的见解。由于CREB激活的改变以及泛素-蛋白酶体途径在大脑的几个异常中被发现，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.