Function of the Zinc Finger Protein ZPR1 in neurodegeneration

描述(申请人提供)：拟议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症(SMA)发病机制中的生理作用。SMA的特点是脊髓运动神经元变性。神经元变性的细胞和分子机制尚不清楚，SMA尚无治疗方法。SMN的核聚集缺陷是SMA的生化缺陷。SMN在包括宝石和卡哈尔小体在内的亚核体中的核积累需要ZPR1。SMA的严重程度与含有核小体的SMN呈负相关。SMA的严重程度可能受修饰基因作用的影响。一个潜在的修饰基因是ZPR1，它在SMA患者中表达下调。ZPR1的表达减少会导致小鼠脊髓运动神经元的损伤和轴突的损伤。这项研究将确定ZPR1在神经元生长和分化中的作用，以及ZPR1作为SMA的保护性修饰物的作用。我们将验证这一假设，即ZPR1的表达增加可能改善SMA疾病的严重程度，并且ZPR1基因可能是SMA的保护性修饰。本研究的具体目的是：目的1.研究ZPR1在神经元中的生理功能。我们将使用培养的原代神经元，包括条件Zpr1(F1/F1)基因敲除小鼠的脊髓运动神经元，以及感染表达Cre重组酶的腺病毒使Zpr1基因失活，来研究ZPR1缺陷对神经元(A)的影响。(B)在小鼠内源性Hlxb9启动子的控制下，将条件性Zpr1(F1/F1)小鼠与表达Cre重组酶的转基因HB9-cre小鼠进行体内杂交。目的2.检测ZPR1过表达对SMA表型挽救和病情严重程度的影响。(A)通过在小鼠rosa26启动子的控制下产生过表达FLAG-ZPR1的转基因小鼠。我们将检测ZPR1过表达对小鼠神经元生长和分化以及生长发育的影响。(B)我们将通过将SMA模型小鼠与转基因Flag-Zpr1小鼠杂交，研究ZPR1过表达对SMA表型的严重程度和挽救的影响。将检测ZPR1过表达的SMA小鼠的表型。目的3.研究ZPR1在SMN的稳定和核积聚中的作用。我们将研究SMN的稳定以及ZPR1和SMN复合体的核质转运的细胞和分子机制。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的核进出口。我们将研究磷酸化和Pro顺反异构化的作用，这可能调节ZPR1-SMN复合体的核质运输。我们将研究ZPR1缺乏对SMN复合体的稳定性和完整性、SMN的核积累和SNRNP生物发生的影响。本研究结果将为进一步研究ZPR1在SMN核积聚和SMA发病机制中的作用提供理论依据。 与公共卫生相关：脊髓性肌萎缩症(SMA)，一种由存活运动神经元(SMN)蛋白缺乏引起的神经肌肉疾病。SMA是美国婴儿死亡的主要原因之一，估计有750万美国人是疾病携带者。对于这种儿童早期的毁灭性遗传病，目前还没有治疗方法。众所周知，SMA疾病的严重程度受修饰基因的作用影响。这项拟议的研究将确定锌指蛋白ZPR1在改善SMA疾病严重程度方面的作用。将ZPR1确定为SMA的保护性修饰物将有助于开发治疗SMA的新策略。

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. PUBLIC HEALTH RELEVANCE: Spinal muscular atrophy (SMA), a neuromuscular disease caused by deficiency of the survival motor neuron (SMN) protein. SMA is one of the leading causes of infant mortality in the United States with an estimated 7.5 million Americans as carriers of disease. No treatment is available for this devastating genetic disease of early childhood. The severity of SMA disease is known to be influenced by the actions of modifier genes. The proposed study will determine the role of zinc finger protein ZPR1 in ameliorating the severity of SMA disease. Identification of ZPR1 as protective modifier of SMA will allow development of novel strategies for the treatment of SMA.