Pathological retrograde signaling in ALS

描述（由申请人提供）：脑源性神经营养因子（BDNF）激活受体TrkB，支持纯运动神经元在体外的存活，并在一些体内病变范例中拯救运动神经元。此外，在BDNF存在下生长的运动神经元将诱导易受毒性损伤的状态。例如，当运动神经元在包括BDNF在内的营养因子的混合物中生长时，TrkB拮抗剂（遗传或药理学）可以保护神经元免受与ALS相关的兴奋毒性和蛋白质毒性损伤。BDNF-TrkB信号传导的这种不良特性的机制尚不完全清楚，但已知依赖于从头蛋白合成。TrkB显示在轴突、树突和细胞体的质膜上。使用Campenot隔室培养系统，我们发现Trk在轴突-树突区域（而不是细胞体）的激活使运动神经元容易受到毒性损伤。由于轴突和树突在细胞和分子生物学水平上存在显著差异，因此确定易感信号来自哪个隔室是至关重要的。在具体目标#1中，我们将生成嵌合TrkB蛋白，其表达目标是轴突或树突结构域。接下来，我们将确定在哪个隔室中TrkB激活导致易损突变SOD毒性。轴-树突结构域的mrna至少部分不同于存在于细胞体中的mrna。微阵列分析为我们提供了候选分子，可能是BDNF作用的分子机制的基础。在具体目标#2中，我们将操纵强候选分子的表达，并确定它们是否会消除BDNF使运动神经元易受损伤的能力。这些高风险、高收益的实验将为运动神经元疾病的发病机制提供新的见解。

DESCRIPTION (provided by applicant): The activation of the receptor TrkB by brain derived neurotrophic factor (BDNF) supports the survival of pure motor neurons in vitro and rescues motor neurons in some in vivo lesion paradigms. In addition, growing motor neurons in the presence of BDNF will induce a state of vulnerability to toxic insults. For example, when motor neurons are grown in a cocktail of trophic factors including BDNF, TrkB antagonism (genetically or pharmacologically) protects against excitotoxic and proteotoxic insults relevant to ALS. The mechanism of this adverse property of BDNF-TrkB signaling is incompletely understood but is known to depend on de novo protein synthesis. TrkB is displayed on the plasma membrane of axons, dendrites and the cell soma. Using Campenot compartment culture system, we find that activation of Trk in the axo- dendritic domain (not the cell soma) renders motor neurons vulnerable to toxic insult. Since axons and dendrites differ dramatically at the cell and molecular biological level it is essential to determine from which compartment the vulnerability signal originates. In Specific aim #1, we will generate chimeric TrkB proteins that target its expression to either the axonal or the dendritic domain. Next we will determine in which compartment TrkB activation leads to vulernability mutant SOD toxicity. The repertoire of mRNAs in the axo-dendritic domain is at least partially distinct from those present in the cell body. A microarray analysis provides us with candidate molecules that might underlie the molecular mechanism of the effect of BDNF. In Specific aim #2, we will manipulate the expression strong candidate molecules and determine if they abrogate the capacity of BDNF to render motor neurons vulnerable to insult. These high-risk-high gain experiments will provide novel insight into the pathogenesis of motor neuron disease. PUBLIC HEALTH RELEVANCE: Amyotrophic Lateral Sclerosis (ALS) is a neurodegenerative disease in which motor neurons die and individuals become progressively weak. In rodent model systems, activation of a cell surface receptor called TrkB can make motor neurons vulnerable to insults relevant to ALS. In this proposal we will determine where on the surface of a motor neuron (e.g., axons versus dendrites) activation of TrkB renders them vulnerable to insult. Next we will study the importance of candidate molecules in axons or dendrites that undergo new synthesis upon activation of TrkB. These studies will bring mechanistic insight into why motor neurons die in ALS.