Translational Studies of Lipidomics-Associated Signaling Pathways in VCP Disease

描述（由申请人提供）：2000年Kimonis等人首次报道了与骨Paget病和额颞叶痴呆相关的包络体肌病（IBMPFD, OMIM 167320），随后于2004年将其归因于编码Valosin-Containing Protein （VCP）的基因突变。该病与进行性肢带肌无力和呼吸功能不全导致的早期死亡有关。肌肉病理表现为空泡、TDP-43和泛素阳性包涵体。鉴于VCP与其他常见神经退行性疾病的重叠，了解VCP疾病的细胞和分子病理生理机制具有很大的兴趣。我们的实验室已经生成了第一个具有最常见的VCP R155H突变的敲入小鼠模型，该突变具有人类VCP相关肌病的许多特征，包括进行性肌肉、骨骼、脊髓和脑病理。VCPR155H/R155H纯合子小鼠表现出快速进行性虚弱和加速病理，包括自噬体形成中断和线粒体功能障碍，通常在21天内死亡。然而，用富脂饲料（LED）喂养怀孕的VCPR155H/+杂合子母鼠，可逆转纯合子后代的致死表型。LED喂养小鼠显示出运动活动、肌肉病理和自噬/有丝自噬信号级联的改善。骨骼肌和肝脏的靶向脂质组学分析显示，组织中非酯化棕榈酸和神经酰胺水平升高（d18:1/16:0），后者与LC3-II标记的自噬体结合和招募受损线粒体进行溶酶体降解有关，也被称为“致命的线粒体自噬”。初步的体外研究表明，神经酰胺处理增加了成肌细胞中自噬标志物p62/SQSTM1和LC3-II的表达水平。我们推测，在喂食LED的纯合子小鼠中，降低神经酰胺中间体的水平可以改善VCP疾病的病理生理表现。在Aim 1中，我们将利用VCP小鼠模型和体外细胞系统作为一种创新的转化方法来探索VCP疾病中神经酰胺代谢改变的中间步骤。在Aim 2中，我们将研究LED补充剂对鞘脂诱导的致死性自噬、线粒体自噬、线粒体功能障碍和凋亡信号通路的修复机制。在Aim 3中，我们将通过药理药物评估还原性神经酰胺对疾病病理的影响。我们希望这些研究将为VCP及相关疾病的治疗提供潜在的新策略。

DESCRIPTION (provided by applicant): Inclusion Body Myopathy associated with Paget's disease of the bone, and Frontotemporal Dementia (IBMPFD, OMIM 167320), was first reported by Kimonis et al. in 2000 and was subsequently attributed to being caused by mutations in the gene encoding Valosin-Containing Protein (VCP) in 2004. The disease is associated with progressive limb girdle muscle weakness and early demise from respiratory insufficiency. Muscle pathology is significant for vacuoles, TDP-43 and ubiquitin-positive inclusions. There is great interest in understanding the cellular and molecular pathophysiological mechanism(s) underlying VCP disease in view of the overlap with other common neurodegenerative diseases. Our laboratory has generated the first knock-in mouse model with the most common VCP R155H mutation, which has many features of human VCP-associated myopathy including progressive muscle, bone, spinal cord and brain pathology. The VCPR155H/R155H homozygous mice exhibit rapid progressive weakness and accelerated pathology including disrupted autophagosome formation and mitochondrial dysfunction and typically die by 21 days. However, feeding pregnant VCPR155H/+ heterozygous dams with a lipid- enriched diet (LED) resulted in the reversal of the lethal phenotype in homozygous offspring. The LED fed mice showed improvement of motor activity, muscle pathology, and autophagy/mitophagy signaling cascades. A targeted lipidomic analysis of skeletal muscle and liver revealed elevations in tissue levels of non-esterified palmitic acid and ceramide (d18:1/16:0), the latter of which has been implicated in binding and recruiting LC3-II labeled autophagosomes to damaged mitochondria for lysosomal degradation, also termed 'lethal mitophagy'. Preliminary in vitro studies demonstrated that ceramide treatments increased expression levels of the autophagy markers p62/SQSTM1 and LC3-II in myoblasts. We hypothesize that decreasing the levels of the ceramide intermediates in the homozygote mice fed the LED ameliorates the pathophysiologic manifestations of VCP disease. In Aim 1, we will utilize the VCP mouse model and in vitro cell systems as an innovative translational approach to explore the intermediate steps in ceramide metabolism that are altered in VCP disease. In Aim 2, we will investigate mechanisms underlying LED supplementation rescue of the sphingolipid- induced lethal autophagy, mitophagy, mitochondrial dysfunction and apoptosis signaling pathways. In Aim 3, we will evaluate the effects of reducing ceramide on disease pathology by pharmacologic drugs. We hope that these studies will lead to potential new therapeutic strategies in VCP and related diseases.