Studies of Hereditary Neurological Disease: Disease Mechanisms

最近，我们的研究集中在三种神经肌肉疾病：由于雄激素受体（AR）中的多聚谷氨酰胺扩增引起的脊髓延髓肌萎缩症（SBMA），由于蛋白SMN缺乏引起的常染色体隐性脊髓性肌萎缩症（SMA），以及由于senataxin突变引起的肌萎缩侧索硬化症4型（ALS 4）。具体的研究成果包括： (1)SBMA属于多聚谷氨酰胺（polyQ）疾病家族，其由蛋白质介导的毒性功能获得机制引起。polyQ疾病蛋白的神经毒性可以通过在特定位点的磷酸化来修饰，从而为疾病特异性治疗的开发提供了理论基础。我们试图鉴定调节polyQ-AR磷酸化的信号通路以用于治疗开发。我们报告，细胞周期蛋白依赖性激酶2（CDK 2）磷酸化polyQ-AR特异性在Ser 96。CDK 2对polyQ-AR的磷酸化增加了蛋白质的稳定性和毒性，并受到腺苷酸环化酶（AC）/蛋白激酶A（PKA）信号通路的负调控。为了将这些发现转化为治疗，我们开发了一种垂体腺苷酸环化酶激活多肽（PACAP）的类似物，一种AC/PKA通路的有效激活剂。慢性鼻内给药PACAP类似物敲入SBMA小鼠减少Ser 96磷酸化，促进polyQ-AR降解，并改善疾病结果。这些结果提供了基于使用PACAP类似物的非侵入性治疗是SBMA的治疗选择的原则证据。 (2)SBMA和其他polyQ疾病与由于突变而获得异常错误折叠构象的蛋白质相关。为了防止错误折叠的蛋白质毒性，细胞激活由分子伴侣和降解途径（蛋白酶体和自噬）组成的蛋白质质量控制（PQC）系统。PQC系统的低效激活导致错误折叠的蛋白质积累，最终导致神经元细胞死亡，而聚集蛋白的有效的大自噬/自噬介导的降解是有益的。后者依赖于由动力蛋白和特异性分子伴侣（如HSPB 8-BAG 3-HSPA 8复合物）介导的主动逆行转运。使用表达SBMA和ALS中涉及的聚集倾向蛋白的细胞模型，我们证明了抑制动力蛋白介导的逆行转运，这会损害错误折叠物种的自噬靶向，不会增加它们的聚集。相反，动力蛋白抑制与突变体ARpolyQ、SOD 1、截短的TARDBP/TDP-43和扩增的polyGP C9 ORF 72产物的减少的积累和增加的清除相关。增强的错误折叠蛋白质清除率是由蛋白酶体介导的，而不是由自噬介导的，并且与HSPA 8辅伴侣BAG 1的上调相关。与此同时，BAG 1的过表达增加了蛋白酶体介导的这些错误折叠蛋白的清除。我们的数据表明，当错误折叠的蛋白质不能被有效地运输到细胞的核周区域时，在那里它们要么被自噬降解，要么被储存到攻击体中，细胞激活一种补偿机制，该机制依赖于BAG 1的诱导，以动力蛋白独立的方式将HSPA 8结合的货物靶向蛋白酶体。 (3)SMA是一种常染色体隐性遗传性神经肌肉疾病，是婴儿死亡的主要遗传原因之一。SMA动物模型研究表明，增加SMN蛋白水平可改善疾病表型。我们的研究小组先前确定并优化了一系列新的小分子，具有良好的效力和毒性特征以及合理的药代动力学，能够增加SMA患者源性细胞中的SMN蛋白水平。我们现在已经表明，ML 372，这一系列的代表，几乎加倍的残留SMN蛋白的半衰期从SMN 2基因座表达通过阻断其泛素化和随后的蛋白酶体降解。ML 372增加SMA小鼠肌肉、脊髓和脑组织中的SMN蛋白水平。重要的是，ML 372治疗改善了翻正反射并延长了重度SMA小鼠模型的生存期。这些结果表明，通过选择性抑制其泛素化来减缓SMN降解可以改善SMA模型小鼠的运动表型和寿命。 (4)星形胶质细胞是中枢神经系统的主要支持细胞，负责谷氨酸清除、代谢支持、对损伤的反应和信号传递的调节。星形胶质细胞与SMA和其他神经退行性疾病有关。SMN蛋白水平的星形胶质细胞特异性拯救已显示可减轻小鼠的疾病表现。然而，星形胶质细胞中SMN缺乏可能导致SMA的机制尚不清楚，星形胶质细胞活性缺乏的方面也不清楚。因此，这是值得确定SMN缺陷的星形胶质细胞，损害正常功能的缺陷。我们发现，SMA星形胶质细胞培养来自小鼠脊髓的两种性别是缺乏支持WT和SMN缺陷的运动神经元来自男性，女性和混合性别的来源，这种缺陷可能会减轻分泌因子。特别是，SMN缺陷型星形胶质细胞与对照组相比单核细胞化学活性蛋白1（MCP 1）分泌水平降低，MCP 1恢复刺激培养的运动神经元的神经突生长。因此，纠正MCP 1缺陷可能是SMA的一种新的治疗方法。

Recently our research has focused on three neuromuscular diseases: spinal and bulbar muscular atrophy (SBMA) due to polyglutamine expansion in the androgen receptor (AR), autosomal recessive spinal muscular atrophy (SMA) due to deficiency of the protein SMN, and amyotrophic lateral sclerosis type 4 (ALS4) due to mutation in senataxin. Specific research accomplishments include the following: (1) SBMA belongs to a family of polyglutamine (polyQ) diseases, which are caused by protein-mediated toxic gain-of-function mechanisms. The neurotoxicity of the polyQ diseases proteins can be modified by phosphorylation at specific sites, thereby providing a rationale for the development of disease-specific treatments. We sought to identify signaling pathways that modulate polyQ-AR phosphorylation for therapy development. We report that cyclin-dependent kinase 2 (CDK2) phosphorylates polyQ-AR specifically at Ser96. Phosphorylation of polyQ-AR by CDK2 increases protein stabilization and toxicity and is negatively regulated by the adenylyl cyclase (AC)/protein kinase A (PKA) signaling pathway. To translate these findings into therapy, we developed an analog of pituitary adenylyl cyclase activating polypeptide (PACAP), a potent activator of the AC/PKA pathway. Chronic intranasal administration of the PACAP analog to knock-in SBMA mice reduced Ser96 phosphorylation, promoted polyQ-AR degradation, and ameliorated disease outcome. These results provide proof of principle that noninvasive therapy based on the use of PACAP analogs is a therapeutic option for SBMA. (2) SBMA and other polyQ diseases are associated with proteins that because of the mutation acquire aberrant misfolded conformations. To prevent misfolded protein toxicity, cells activate a protein quality control (PQC) system composed of chaperones and degradative pathways (proteasome and autophagy). Inefficient activation of the PQC system results in misfolded protein accumulation that ultimately leads to neuronal cell death, while efficient macroautophagy/autophagy-mediated degradation of aggregating proteins is beneficial. The latter relies on an active retrograde transport, mediated by dynein and specific chaperones, such as the HSPB8-BAG3-HSPA8 complex. Using cellular models expressing aggregate-prone proteins involved in SBMA and ALS, we demonstrated that inhibition of dynein-mediated retrograde transport, which impairs the targeting to autophagy of misfolded species, does not increase their aggregation. Rather, dynein inhibition correlates with a reduced accumulation and an increased clearance of mutant ARpolyQ, SOD1, truncated TARDBP/TDP-43 and expanded polyGP C9ORF72 products. The enhanced misfolded protein clearance is mediated by the proteasome rather than by autophagy, and correlates with the upregulation of the HSPA8 cochaperone BAG1. In line, overexpression of BAG1 increases the proteasome-mediated clearance of these misfolded proteins. Our data suggest that when the misfolded proteins cannot be efficiently transported toward the perinuclear region of the cells, where they are either degraded by autophagy or stored into the aggresome, the cells activate a compensatory mechanism that relies on the induction of BAG1 to target the HSPA8-bound cargo to the proteasome in a dynein-independent manner. (3) SMA is an autosomal recessive neuromuscular disease and one of the leading inherited causes of infant mortality. Studies in animal models of SMA have shown that increasing SMN protein levels ameliorates the disease phenotype. Our group previously identified and optimized a new series of small molecules, with good potency and toxicity profiles and reasonable pharmacokinetics, that were able to increase SMN protein levels in SMA patient-derived cells. We have now shown that ML372, a representative of this series, almost doubles the half-life of residual SMN protein expressed from the SMN2 locus by blocking its ubiquitination and subsequent degradation by the proteasome. ML372 increased SMN protein levels in muscle, spinal cord, and brain tissue of SMA mice. Importantly, ML372 treatment improved the righting reflex and extended survival of a severe mouse model of SMA. These results demonstrate that slowing SMN degradation by selectively inhibiting its ubiquitination can improve the motor phenotype and lifespan of SMA model mice. (4) Astrocytes are the primary support cells of the CNS and are responsible for glutamate clearance, metabolic support, response to injury, and regulation of signal transmission. Astrocytes have been implicated in SMA as in in other neurodegenerative disorders. Astrocyte-specific rescue of SMN protein levels has been shown to mitigate disease manifestations in mice. However, the mechanism by which SMN deficiency in astrocytes may contribute to SMA is unclear and what aspect of astrocyte activity is lacking is unknown. Therefore, it is worthwhile to identify defects in SMN-deficient astrocytes that compromise normal function. We showed that SMA astrocyte cultures derived from mouse spinal cord of both sexes are deficient in supporting both WT and SMN-deficient motor neurons derived from male, female, and mixed-sex sources and that this deficiency may be mitigated with secreted factors. In particular, SMN-deficient astrocytes have decreased levels of monocyte chemoactive protein 1 (MCP1) secretion compared with controls and MCP1 restoration stimulates outgrowth of neurites from cultured motor neurons. Correction of MCP1 deficiency may thus be a new therapeutic approach to SMA.