Function of the Zinc Finger Protein ZPR1 in neurodegeneration

描述（由申请人提供）：拟研究的长期目标是了解锌指蛋白ZPR1在由存活运动神经元（SMN1）基因突变引起的脊髓性肌萎缩（SMA）发病机制中的生理作用。SMA以脊髓运动神经元变性为特征。神经元退化的细胞和分子机制尚不清楚，也没有治疗SMA的方法。SMN核积累缺陷是SMA的生化缺陷。在亚核体（包括宝石和Cajal体）中SMN的核积累需要ZPR1。SMA的严重程度与含核体的SMN呈负相关。SMA的严重程度可能受到修饰基因作用的影响。一个潜在的修饰基因是ZPR1，它在SMA患者中被下调。ZPR1的表达减少导致小鼠轴突缺损和脊髓运动神经元的丢失。本研究将确定ZPR1在神经元生长和分化中的功能，以及ZPR1作为SMA的保护调节剂的作用。我们将检验ZPR1表达增加可能改善SMA疾病严重程度的假设，以及ZPR1基因可能是SMA的保护性修饰因子。研究项目的具体目标是：目标1。探讨ZPR1在神经元中的生理功能。我们将通过体外培养的原代神经元，包括条件ZPR1 （F1/F1）敲除小鼠的脊髓运动神经元，以及通过感染表达Cre重组酶的腺病毒使ZPR1基因失活，来检测ZPR1缺失对神经元(A)的影响。(B)体内在内源性小鼠Hlxb9启动子控制下，将条件Zpr1 （F1/F1）小鼠与表达Cre重组酶的转基因Hb9-cre小鼠杂交。目标2。目的：探讨ZPR1过表达对疾病严重程度和SMA表型挽救的影响。(A)在小鼠ROSA26启动子的控制下产生过表达FLAG-ZPR1的转基因小鼠。我们将研究ZPR1过表达对小鼠神经元生长分化和生长发育的影响。(B)我们将通过将SMA模型小鼠与转基因Flag-Zpr1小鼠杂交来检测ZPR1过表达对SMA表型严重程度和挽救的影响。对ZPR1过表达的SMA小鼠进行表型检测。目标3。探讨ZPR1在SMN稳定和核积累中的作用。我们将研究SMN稳定的细胞和分子机制以及ZPR1和SMN复合物的核质运输。我们将研究与ZPR1相互作用的细胞质和核蛋白的作用，并可能介导ZPR1和SMN的核输出/输入。我们将研究磷酸化和脯氨酸顺反异构化的作用，它们可能调节ZPR1-SMN复合物的核质运输。我们将研究ZPR1缺乏对SMN复合物的稳定性和完整性、SMN的核积累和snRNP生物发生的影响。本研究结果将有助于进一步了解ZPR1在SMN核积累中的作用和SMA的发病机制。

DESCRIPTION (provided by applicant): The long-term goal of proposed research is to understand the physiological role of zinc finger protein ZPR1 in the pathogenesis of spinal muscular atrophy (SMA) caused by mutation of the survival motor neurons (SMN1) gene. SMA is characterized by degeneration of the spinal motor neurons. The cellular and molecular mechanisms of neuron degeneration are unclear and no treatment is available for SMA. The defect in nuclear accumulation of SMN is the biochemical defect in SMA. ZPR1 is required for nuclear accumulation of SMN in sub-nuclear bodies, including gems and Cajal bodies. The severity of SMA correlates negatively with SMN containing nuclear bodies. The severity of SMA may be influenced by the actions of modifier genes. One potential modifier gene is represented by ZPR1, which is down regulated in patients with SMA. The reduced expression of ZPR1 causes axonal defects and loss of spinal motor neurons in mice. The proposed research will determine the function of ZPR1 in the growth and differentiation of neurons and role of ZPR1 as a protective modifier of SMA. We will test the hypothesis that the increased expression of ZPR1 may ameliorate the severity of SMA disease and the ZPR1 gene may be a protective modifier of SMA. The Specific Aims of the research project are: Aim 1. To examine the physiological function of ZPR1 in neurons. We will examine the effect of ZPR1 deficiency in neurons (A) in vitro using cultured primary neurons, including spinal cord motor neurons from conditional Zpr1 (F1/F1) knockout mice and inactivation of the Zpr1 gene by infection with adenovirus expressing Cre recombinase. (B) In vivo by crossing conditional Zpr1 (F1/F1) mice with transgenic Hb9-cre mice expressing Cre recombinase under the control of endogenous mouse Hlxb9 promoter. Aim 2. To examine the effect of ZPR1 overexpression on the severity of disease and rescue of the SMA phenotype. (A) by generating a transgenic mouse overexpressing FLAG-ZPR1 under the control of a mouse ROSA26 promoter. We will examine the effect of ZPR1 overexpression on the growth and differentiation of neurons and growth and development of mice. (B) We will examine the effect of ZPR1 overexpression on severity and rescue of the SMA phenotype by crossing SMA model mice with transgenic Flag-Zpr1 mice. The phenotype of SMA mice with ZPR1 overexpression will be examined. Aim 3. To examine the role of ZPR1 in stabilization and nuclear accumulation of SMN. We will examine the cellular and molecular mechanisms of stabilization of SMN and nucleocytoplasmic trafficking of ZPR1 and SMN complexes. We will examine the role of cytoplasmic and nuclear proteins that interact with ZPR1 and may mediate nuclear export/import of ZPR1 and SMN. We will examine the role of phosphorylation and prolyl cis-trans isomerization that may regulate nucleocytoplasmic trafficking of ZPR1-SMN complexes. We will examine the effect of ZPR1 deficiency on the stability and integrity of SMN complex, nuclear accumulation of SMN and snRNP biogenesis. The findings of proposed study will provide insights into the function of ZPR1 in the nuclear accumulation of SMN and pathogenesis of SMA.