The Role of Microglia in Models of ALS

描述(申请人提供)：肌萎缩侧索硬化症(ALS)是一种进展迅速的致命神经肌肉疾病；大约10%的ALS患者有家族病史。这些家族性肌萎缩侧索硬化症病例中有20%-25%是由Cu2+/Zn2+超氧化物歧化酶基因(MSOD1)突变引起的。在转基因小鼠中，mSOD1的过度表达也会在普遍表达时诱发疾病。然而，有证据表明，除了运动神经元，其他细胞可能需要产生ALS表型，即非细胞自主型。首先，mSOD1仅在神经元和星形胶质细胞中的表达不会导致疾病；然而，mSOD1的表达水平和时间与最初的mSOD1G93A小鼠不同。其次，来自嵌合小鼠的数据表明，被正常胶质细胞包围的表达mSOD1的神经元看起来没有受到损伤，而被表达mSOD的神经胶质细胞包围的正常神经元似乎异常。第三，病理存在于中枢神经系统的几种不同类型的细胞中，包括神经元、星形胶质细胞和小胶质细胞。为了确定mSOD1在ALS小胶质细胞和免疫细胞中表达的影响，我们选择了PU.1-/-小鼠。在出生时，这些小鼠缺乏巨噬细胞、中性粒细胞、T和B细胞，以及重要的小胶质细胞；这些小鼠只有在骨髓移植后才能存活，从而产生供者来源的CNS小胶质细胞。具体地说，我们的目标是：特定的目标1：确定mSOD1单独在小胶质细胞和免疫细胞中的表达是否可以诱发ALS样疾病和/或引发亚临床病理。我们正在将mSOD1-、正常人(H)SOD1和野生型来源的骨髓移植到PU.1-/-小鼠身上。PU.1-/-移植mSOD1骨髓的小鼠似乎没有表现出任何明显的运动神经元疾病的迹象；即使移植的小胶质细胞在形态上可能与对照组小鼠的小胶质细胞不同。我们将检查这些小鼠的亚临床病理。特定目的2：确定mSOD1在小鼠小胶质细胞和免疫细胞中的表达是否是启动和/或扩大mSOD1小鼠ALS样病所必需的。我们正在移植与PU.1-/-小鼠(mSOD1/PUI-/-)杂交的mSOD1小鼠。野生型骨髓移植的mSOD1/PU.1-/-小鼠表现为ALS样表型。有趣的是，这几只小鼠的病程可能比mSOD1/PU.1-/-小鼠骨髓移植的病程长。具体目的3：明确小胶质细胞介导运动神经元损伤的可能机制。我们正在培养被激活的野生型小胶质细胞损伤的原代运动神经元，mSOD1原代小胶质细胞似乎更加活跃，我们将评估它们损伤野生型和mSOD1运动神经元的能力。

DESCRIPTION (provided by applicant): Amyotrophic lateral sclerosis (ALS) is a rapidly progressive and fatal neuromuscular disease; approximately 10% of ALS patients have a family history of the disease. 20-25% of these familial ALS cases are caused by mutations in the Cu2+/Zn2+ superoxide dismutase gene (mSOD1). Overexpression of mSOD1 in transgenic mice also induces disease when ubiquitously expressed. However, evidence indicates that other cells, in addition to motor neurons, may be required to produce an ALS phenotype, i.e. non-cell autonomous. First, expression of mSOD1 in neurons-only and in astroglia-only did not produce disease; however, the level and timing of mSOD1 expression were not the same as the original mSOD1G93A mouse. Second, data from chimeric mice suggest that mSOD1-expressing neurons surrounded by normal glia appear uninjured, whereas normal neurons surrounded by mSOD-expressing glia appear abnormal. Third, pathology is present in several different cell types in the CNS, including neurons, astrocytes, and microglia. To determine the effects of mSOD1 expression in microglia and immune cells in ALS, we chose to use the PU.1-/- mouse. At birth, these mice lack macrophages, neutrophils, T and B cells, and importantly microglia; such mice survive only after bone marrow transplantation, resulting in donor-derived CNS microglia. Specifically, our aims are: Specific Aim 1: To determine if mSOD1 expression in microglia and immune cells alone can induce an ALS-like disease and/or initiate subclinical pathology. We are transplanting PU.1 -/- mice with mSOD1-, normal human (h)SOD1, and wildtype-derived bone marrow. PU.1 -/- mice transplanted with mSOD1 bone marrow did not appear to show any overt signs of motoneuron disease; even though the transplanted microglia may be morphologically different than microglia in control mice. We will examine these mice for subclinical pathology. Specific Aim 2: To determine if mSOD1 expression in microglia and immune cells is required for the initiation and/or amplification of an ALS-like disease in the mSOD1 mouse. We are transplanting the mSOD1 mice crossed with the PU.1 -/- mice (mSOD1/PU.I-/-). mSOD1/PU.1 -/- mice transplanted with wildtype bone marrow exhibit an ALS-like phenotype. Interestingly, the disease duration with these few mice may be longer than the duration of the mSOD1/PU.1 -/- mice transplanted with mSOD1 bone marrow. Specific Aim 3: To define potential mechanisms of microglia-mediated motor neuron injury. We are culturing primary motor neurons, which are injured by activated wildtype microglia, mSOD1 primary microglia appear more activated and we will assess their ability to injure wildtype and mSOD1 motor neurons.