Biological And Biochemical Characterization Of Sigma Receptors

自从我在NIDA IRP的实验室在1982年发现sigma-1受体以来，许多临床前研究表明sigma-1受体及其相关配体与中风、健忘症、抑郁症、癌症、阿尔茨海默病、疼痛和可卡因成瘾有关。 在2021年10月1日至2022年9月30日的本财年中，我们发现了sigma-1受体的两种作用机制：(A)sigma-1受体在称为自噬的生存机制中发挥关键作用；(B)sigma-1受体在一种名为Wolfram综合征的临床未治疗疾病中起关键作用。 研究结果解释如下。 (A)巨自噬/自噬是细胞生存的基本过程，与许多疾病有关。自噬的一个关键步骤是通过KPNB1/Importin1将转录因子TFEB从胞浆运输到细胞核，通过核孔(NP)。在C9orf72亚型肌萎缩侧索硬化症-额颞叶变性(ALS-FTD)中，六核苷酸(G4C2)RNA扩展(HRE)破坏了TFEB的核质运输，损害了自噬。在这里，我们证明了一种分子伴侣，SIGMAR1/Sigma-1受体(Sigma非阿片细胞内受体1)，通过伴侣NP蛋白(即，核孔素)POM121招募KPNB1，促进TFEB向细胞核的转运。在NSC34细胞中，HRE通过干扰SIGMAR1和POM121之间的联系来减少TFEB的转运，导致TFEB、KPNB1和自噬标记LC3-II的核水平降低。过表达SIGMAR1或POM121，或用高选择性和强大的SIGMAR1激动剂普里多巴治疗，目前正处于ALS和亨廷顿病的2/3期临床试验中，可以挽救所有这些缺陷。我们的结果表明，核孔蛋白POM121不仅是一种结构性核孔蛋白，而且还建议使用SIGMAR1激动剂，如普里多巴定，用于自噬受损的疾病的治疗发展。 (B)Wolfram综合征是一种罕见的常染色体隐性遗传病，影响许多器官，会危及生命，目前尚无治疗方法。因此，本研究的目的是确定并提出一种新的相关治疗方法。其病理机制与内质网(ER)跨膜蛋白wolframin活性低下有关，这种跨膜蛋白参与内质网与线粒体之间的接触，称为线粒体相关ER膜(MAMs)。遗传突变通常会降低蛋白质的稳定性，改变其动态平衡，最终减少内质网到线粒体的钙转移，导致线粒体功能障碍和细胞死亡。在这里，我们证明激活Sigma-1受体(S1R)，参与钙转运的内质网驻留蛋白，可以抵消由于狼毒蛋白缺乏引起的MAM的功能改变。S1R激动剂Pre-084在体外恢复了钙离子传递和线粒体呼吸，并能够缓解在Wfs1abKO斑马鱼遗传动物模型中观察到的行为症状，即Wfs1abKO斑马鱼的多动和Wfs1Exon8小鼠的记忆障碍和焦虑。我们的发现为Wolfram综合征患者提供了一种新的治疗策略，通过使用配体操作的S1R伴侣有效地增强MAM功能。此外，这种策略可以扩展到其他涉及MAMs功能障碍的退行性疾病和线粒体疾病。

Since my laboratory at the NIDA IRP identified the sigma-1 receptor in 1982, many preclinical studies have shown that sigma-1 receptors and associated ligands are involved in stroke, amnesia, depression, cancer, Alzheimer disease, pain, and cocaine addiction. In this fiscal year from Oct 1/2021-Sept 30/2022, we discovered two mechanisms of action of the sigma-1 receptor: (A) The sigma-1 receptor plays a critical role in a survival mechanism called autophagy; and (B) The sigma-1 receptor is pivotal in a clinically unmet disease called the Wolfram's Syndrome. Findings are explained as follows. (A) Macroautophagy/autophagy is an essential process for cellular survival and is implicated in many diseases. A critical step in autophagy is the transport of the transcription factor TFEB from the cytosol into the nucleus, through the nuclear pore (NP) by KPNB1/importin1. In the C9orf72 subtype of amyotrophic lateral sclerosis-frontotemporal lobar degeneration (ALS-FTD), the hexanucleotide (G4C2)RNA expansion (HRE) disrupts the nucleocytoplasmic transport of TFEB, compromising autophagy. Here we show that a molecular chaperone, the SIGMAR1/Sigma-1 receptor (sigma non-opioid intracellular receptor 1), facilitates TFEB transport into the nucleus by chaperoning the NP protein (i.e., nucleoporin) POM121 which recruits KPNB1. In NSC34 cells, HRE reduces TFEB transport by interfering with the association between SIGMAR1 and POM121, resulting in reduced nuclear levels of TFEB, KPNB1, and the autophagy marker LC3-II. Overexpression of SIGMAR1 or POM121, or treatment with the highly selective and potent SIGMAR1 agonist pridopidine, currently in phase 2/3 clinical trials for ALS and Huntington disease, rescues all of these deficits. Our results implicate nucleoporin POM121 not merely as a structural nucleoporin, but also suggest the use of SIGMAR1 agonists, such as pridopidine, for therapeutic development of diseases in which autophagy is impaired. (B) The Wolfram syndrome is a rare autosomal recessive disease affecting many organs with life-threatening consequences and currently no treatment is available. Therefore, the aim of this study was to identify and propose a novel relevant therapy. The pathology is related to the deficient activity of wolframin, an endoplasmic reticulum (ER) transmembrane protein involved in contacts between ER and mitochondria termed mitochondria associated-ER membranes (MAMs). Inherited mutations usually reduce the proteins stability, altering its homeostasis and ultimately reducing ER to mitochondria Ca2+ transfer resulting in mitochondrial dysfunction and cell death. We here demonstrate that activation of the sigma-1 receptor (S1R), an endoplasmic reticulum resident protein involved in Ca2+ transfer, could counteract the functional alterations of MAMs due to wolframin deficiency. The S1R agonist PRE-084 restored Ca2+ transfer and mitochondrial respiration in vitro and was able to alleviate the behavioral symptoms observed in the genetic animal models of the disease, i.e. hyperlocomotion in Wfs1abKO zebrafish and memory deficits and anxiety in Wfs1Exon8 mice. Our findings provide a new therapeutic strategy for Wolfram syndrome patients, by efficiently boosting MAM function using the ligand operated S1R chaperone. Moreover, such strategy could be expanded to other degenerative and mitochondrial diseases involving MAMs dysfunction.