Targeting AMPA Receptor Splicing as a Therapeutic Approach for ALS

描述(申请人提供)：肌萎缩侧索硬化症(ALS)的特征是运动皮层、脑干和脊髓中运动神经元(MN)进行性和选择性的丧失，导致患者在确诊后2-5年内死亡。谷氨酸介导的中枢神经系统兴奋性毒性已被认为是ALS表现的主要因素。谷氨酸在肌萎缩侧索硬化症中的兴奋性毒性被认为是由于运动神经元处理过量钙内流的能力减弱所致，而钙内流是神经退行性变的触发因素。因此，降低中枢神经系统兴奋性钙离子内流的药物的开发被广泛认为是寻找ALS治疗方法的重要焦点。离子型AMPA受体是谷氨酸激活的离子通道，是钙离子内流和中枢神经系统兴奋性状态的关键调节因子。我们建议开发被称为反义寡聚体(AOS)的新型空间阻断核酸制剂，它将导致更多不敏感的AMPA受体的产生。AMPA受体是由4个亚基组成的五聚体，称为GluR1-4，以各种同聚体和异构体组合存在。所有GluR的两个选择性剪接变体称为“Flip”和“Flop”，通常在中枢神经系统中表达。AMPA“Flip”变种更能抵抗脱脂，而“Flip”变种则很容易脱敏。因此，当GluR1、GluR3和GluR4的触发器比率升高时，AMPA受体对脱敏具有抵抗力，神经元更具兴奋性，允许更多的钙内流。在这个方案中，我们将开发新的AO，其功能是特异性地降低GluR翻转异构体的表达，从而降低触发器/触发器比率。我们将在体外评估AO的疗效，包括评估其对AMPA电流的功能影响，并在正常小鼠脑室(Icv)注射后测试其疗效。我们认为，优化的AOS可能通过增加GluR亚单位特异性AMPA去分化而导致一种治疗ALS的新策略，从而产生神经保护作用。此外，有证据表明，在ALS中，钙离子渗透性AMPA受体的数量和触发器/触发器比率可能异常高，进一步支持了我们开发增加AMPA通道脱敏的化合物的理论基础。此外，我们的AOS可能适用于调节AMPA受体活性是当前药物发现焦点的其他疾病，包括癫痫、帕金森病、创伤性脑损伤、神经性疼痛和中风。 公共卫生相关性：我们建议在临床前研究中开发和验证称为空间位阻低聚物的化合物，最终可能导致一种新的治疗策略，通过使脊髓运动神经元的突触功能正常化来治疗人类肌萎缩侧索硬化症(ALS)。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is characterized by the progressive and selective loss of motor neurons (MNs) in the motor cortex, brainstem, and spinal cord, resulting in death of patients 2-5 years after diagnosis. Glutamate-mediated excitotoxicity in the CNS has been implicated as a major contributor to the manifestation of ALS. It is thought that glutamate-mediated excitotoxicity in ALS is due to diminished capacity of motor neurons to handle excessive Ca2+ influx, which serves as a trigger for neurodegeneration. Therefore, the development of drugs which lower Ca2+ influx associated with excitatory activity in CNS is widely considered as an important focal point in the search for therapeutics for ALS. Ionotropic AMPA receptors are glutamate-activated ion channels that are key regulators of Ca2+ influx and the excitatory state of the CNS. We propose to develop novel steric block nucleic acid agents called antisense oligomers (AOs) that will lead to the production of more desensitizable AMPA receptors. AMPA receptors are pentamers composed of 4 subunits termed GluR1-4, present in various homomeric and heteromeric combinations. Two alternatively spliced variants of all GluRs called "flip" and "flop" are normally expressed in the CNS. The AMPA "flip" variants are more resistant to desentization, whereas the "flop" variants are readily desensitized. Thus, when the flip/flop ratio of GluR1, GluR3 and GluR4 is elevated, the AMPA receptors are resistant to desensitization, and neurons are more excitable and permit greater influx of calcium. In this proposal, we will develop novel AOs that function to specifically lower GluR flip isoform expression, and thus decrease flip/flop ratio. We will evaluate AO efficacy in vitro, including assessment of their functional affects on AMPA currents and also test their efficacy in normal mice after intracerebroventricular (ICV) delivery. We propose that the optimized AOs could lead to a novel therapeutic strategy to treat ALS by increasing GluR subunit-specific AMPA desentization, resulting in neuroprotection. Also, there is evidence that the number of Ca2+-permeable AMPA receptors and the flip/flop ratio may be abnormally high in ALS, further supporting our rationale to develop compounds that increase AMPA channel desensitization. Also, our AOs may be applicable to other disorders where modulating AMPA receptor activity is a focus of current drug discovery, including epilepsy, Parkinsonism, traumatic brain injury, neuropathic pain, and stroke. PUBLIC HEALTH RELEVANCE: We propose to develop and validate compounds called steric block oligomers in pre-clincal studies that could ultimately lead to a novel therapeutic strategy to treat Amyotrophic lateral sclerosis (ALS) in humans, by normalizing synaptic function in spinal motor neurons.