UBIQUITIN TRANSGENIC MICE, AGING AND NEURODEGENERATION

泛素转基因小鼠、衰老和神经退行性变

• 批准号: 6779453
• 负责人: STEVEN Scott SCHREIBER
• 金额: $ 6.82万
• 依托单位: UNIVERSITY OF CALIFORNIA-IRVINE
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6779453
• 关键词: aging; apoptosis; central nervous system; disease /disorder model; gene expression; genetically modified animals; genotype; histology; immunocytochemistry; laboratory mouse; microinjections; model design /development; molecular cloning; mutant; neural degeneration; neuropathology; northern blottings; phenotype; protein degradation; terminal nick end labeling; tissue /cell culture; transfection; ubiquitin; western blottings

The topic of this proposal is applicable to Research Objectives 7 and 23 in PAR-03-056. The goal of this NIA pilot grant is to develop and characterize transgenic mouse lines that constitutively overexpress either wildtype or mutant ubiquitin in the aging central nervous system (CNS). We recently found that a reduction in the level of free ubiquitin following various neurotoxic insults leads to an accumulation of the pro-apoptotic protein, p53, and selective neurodegeneration. This novel cell death pathway is also associated with increased expression of a mutant form of ubiquitin, termed Ub+l, caused by a post-transcriptional frameshift in ubiquitin mRNA. By impairing ubiquitination and disrupting proteasome function Ub+l promotes additional protein accumulation and cell death. Both impaired ubiquitin-proteasome function and protein aggregates containing Ub+l have been demonstrated in Alzheimer's and other neurodegenerative diseases. However, the exact nature of the relationships between formation of Ub+l by "molecular misreading", abnormal ubiquitin-proteasome function and neuronal cell death remain poorly understood. We therefore propose to develop transgenic mouse lines that overexpress either free wild-type ubiquitin or mutant Ub+l in CNS neurons. Transgene expression will be directed by a Thyl promoter which is neuron-specific. An additional and highly beneficial feature of this expression system is that the Thyl promoter is inactive during embryonic and early postnatal life, thereby avoiding potentially adverse or confounding effects of transgene expression during early stages of development. Phenotypes will be characterized according to gross and microscopic neuropathology, apoptotic gene expression and the degree of cell death at different ages and in different brain regions. The immediate benefits provided by these mice will include establishing clinical relevance for the accumulation of Ub+l in age-related neurodegenerative diseases, and determining whether increasing the available pool of free ubiquitin is neuroprotective following adverse stimuli. Future applications will employ ubiquitin transgenic mice in high throughput screening studies to identify ubiquitin-modulating pharmacological agents for age-related neurodegenerative disorders such as Alzheimer's disease.

本提案的主题适用于PAR-03-056中的研究目标7和23。这项NIA试点资助的目标是开发和表征在衰老中枢神经系统（CNS）中组成型过表达野生型或突变型泛素的转基因小鼠品系。我们最近发现，各种神经毒性损伤后游离泛素水平的降低导致促凋亡蛋白p53的积累和选择性神经变性。这种新的细胞死亡途径还与泛素的突变形式（称为Ub+1）的表达增加有关，所述突变形式由泛素mRNA中的转录后移码引起。通过损害泛素化和破坏蛋白酶体功能，Ub+1促进额外的蛋白质积累和细胞死亡。已在阿尔茨海默病和其他神经退行性疾病中证实了受损的泛素-蛋白酶体功能和含有Ub+1的蛋白质聚集体。然而，在这方面， 通过“分子误读”形成Ub+1、异常的遍在蛋白-蛋白酶体功能和神经元细胞死亡之间的关系的确切性质仍然知之甚少。因此，我们建议开发在CNS神经元中过表达游离野生型泛素或突变体Ub+1的转基因小鼠系。转基因表达将由神经元特异性的Thyl启动子指导。该表达系统的另一个非常有益的特征是Thyl启动子在胚胎和出生后早期是无活性的，从而避免了转基因表达的潜在不利或混杂效应 在发育的早期阶段。将根据不同年龄和不同脑区的大体和显微神经病理学、凋亡基因表达和细胞死亡程度来表征表型。这些小鼠提供的直接好处将包括建立Ub+l在年龄相关神经退行性疾病中积累的临床相关性，并确定增加可用的游离泛蛋白库是否在不良刺激后具有神经保护作用。未来的应用将采用泛素转基因小鼠在高通量筛选研究，以确定泛素调节药物的年龄相关的神经退行性疾病，如阿尔茨海默氏病。