Translational Studies of Lipidomics-Associated Signaling Pathways in VCP Disease

描述(由申请人提供)：与骨派杰氏病相关的包涵体肌病和额颞部痴呆(IBMPFD，OMIM 167320)，由Kimonis等人首次报道。在2000年，随后被归因于2004年编码含有Valosin的蛋白(VCP)的基因突变所致。这种疾病与进行性肢体腰带肌肉无力和呼吸功能不全导致的早期死亡有关。肌肉病理对空泡、TDP-43和泛素阳性包涵体有重要意义。鉴于VCP病与其他常见神经退行性疾病的重叠，了解VCP病的细胞和分子病理生理机制(S)具有重要意义。我们的实验室已经培育出第一个具有最常见的VCP R155H突变的敲入小鼠模型，该突变具有许多人类VCP相关肌病的特征，包括进行性肌肉、骨、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，包括自噬小体形成中断和线粒体功能障碍，通常在21天前死亡。然而，给怀孕的VCPR155H/+杂合子母鼠喂食富脂饮食(LED)会导致纯合子后代的致死表型逆转。喂食LED的小鼠表现出运动活动、肌肉病理和自噬/有丝分裂信号级联的改善。对骨骼肌和肝脏的定向脂肪组学分析显示，非酯化棕榈酸和神经酰胺(D18：1/16：0)的组织水平升高，后者参与结合和招募标记的自噬小体到受损的线粒体上进行溶酶体降解，也被称为致死性有丝分裂。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记p62/SQSTM1和LC3-II的表达水平。我们推测，降低喂饲LED的纯合子小鼠的神经酰胺中间产物水平可以改善VCP病的病理生理表现。在目标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翻译方法来探索VCP疾病中神经酰胺代谢的中间步骤。在目标2中，我们将研究补充LED拯救鞘磷脂诱导的致死性自噬、有丝分裂、线粒体功能障碍和凋亡信号通路的机制。在目的3中，我们将评估药物降低神经酰胺对疾病病理的影响。我们希望这些研究将为VCP和相关疾病带来潜在的新治疗策略。

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.