Understanding the age-dependent mitochondrial function in astrocytes after spinal cord injury via bi-directional manipulation of activity

项目摘要 在美国，每年有17000例新的脊髓损伤(SCI)病例，约30万人 患有慢性脊髓损伤。到目前为止，还没有FDA批准的治疗方法可以改善功能恢复。SCI日益增多 发生在老龄化人口中，伴随着年龄相关性的恢复率下降。然而，缺乏 将年龄作为关键生物学变量的研究已经成为转化临床前阶段的主要障碍 成功治疗老龄化的脊髓损伤人群的治疗。 与正常衰老相关的线粒体功能衰退表明，改善线粒体 脊髓损伤后功能恢复较好。然而，非选择性地增强线粒体活性 可根据年龄和受影响的细胞类型而有不同的影响，损害幼鼠的恢复，而 在年长的小鼠身上推广它。虽然这种年龄依赖效应的细胞和分子介体尚不清楚， 这表明，通过以线粒体功能下降为靶点，可能会达到更一致的效果 特定于细胞的方式。新的数据表明，星形胶质细胞中的线粒体活性随着年龄的增长而受损，而在 体外降低年轻星形胶质细胞中线粒体的活性会增加损伤的大小，反之，激活 线粒体缩小了病变的大小。这一点非常重要，因为急性星形胶质瘢痕在 脊髓损伤是有益的，可以减少炎症的扩散，保护备用的神经组织。体内初步研究 数据显示，随着年龄的增长，急性星形胶质细胞瘢痕形成减少，炎症标志物增加。 和病变大小，与功能恢复的减少有关。值得注意的是，飞行员数据显示了一个年龄- 星形胶质细胞增生症分子通路的依赖性变化，包括STAT3(信号)的减少 转录转导和激活因子)和PIAS3(激活的STAT3蛋白抑制物)的增加，两者都 对线粒体的活性有相反的作用。 因此，中心假设是星形胶质细胞线粒体功能的年龄相关性下降 随着年龄的增长，脊髓损伤后星形胶质细胞瘢痕形成受损，功能恢复减慢。这一假说将 在三个相关但独立的目标上进行测试： 这个项目的总体目标是证明促进星形胶质细胞中线粒体的活性可以减少 损伤的大小和促进老年动物脊髓损伤后的恢复。这些目标将通过减少 利用遗传学(Ndufs4flx；Ai14)和药理学在体外和体内星形胶质细胞的线粒体功能 策略(目标1)，通过在星形胶质细胞和药物中过表达PGC-1α来提高线粒体活性 促进线粒体功能选自一种新的高含量筛选试验(目标2)，并通过建立 STAT3和PIAS3在星形胶质细胞线粒体功能调节中的亚细胞作用 体外和体内功能的获得和丧失(转基因小鼠STAT3FLOX、PIAS3FLOX、SOCS3FLOX；过表达 核或线粒体STAT3和PIAS3)以及脊髓损伤后体内瞬时mRNA的表达(目标3)。

Project Summary In the United States, there are >17,000 new cases of spinal cord injury (SCI) every year, and ~300,000 people living with chronic SCI. To date, no FDA-approved treatment improves functional recovery. SCI is increasingly occurring in the aging populations, accompanied by an age-dependent decline in recovery. However, the lack of studies that incorporate age as a key biological variable has become a major obstacle in translating preclinical therapies into successfully treating the aging SCI population. The functional decline of mitochondria associated with normal aging suggests that improving mitochondrial function following SCI could result in better recovery. However, non-selectively enhancing mitochondria activity can have divergent effects depending on age and effected cell types, impairing recovery in young mice while promoting it in older mice. While the cellular and molecular mediators of this age-dependent effect are unknown, this suggests that more uniform effects may be achieved by targeting the decline in mitochondria function in a cell specific manner. New data suggest that mitochondrial activity in astrocytes is impaired with age, and that in vitro reducing mitochondrial activity in young astrocytes increases the injury size, and conversely, activation of mitochondria reduces the lesion size. This is of high importance because acute astroglial scar formation after SCI is beneficial, reducing the spread of inflammation and protecting spared neural tissue. Preliminary in vivo data suggests a reduction in acute astroglial scar formation with age and an increase in inflammation markers and lesion size, associated with a reduction in functional recovery. Remarkably, pilot data show an age- dependent changes in molecular pathways involved in astrogliosis including the reduction of STAT3 (signal transducer and activator of transcription) and increase of PIAS3 (Protein inhibitor of activated STAT3), both having opposite roles on mitochondrial activities. Thus, the central hypothesis is that the age-dependent decline in astrocytic mitochondrial functions impairs astroglial scar formation and reduces functional recovery after SCI with age. This hypothesis will be tested in three related, but independent, aims: The overall objective of this project is to demonstrate that promoting mitochondrial activity in astrocytes reduces lesion size and promotes recovery after SCI in aged animals. These objectives will be achieved by reducing mitochondrial function in astrocytes in vitro and in vivo, using genetic (Ndufs4flox;Ai14) and pharmacological strategies (Aim 1), by increasing mitochondrial activity using PGC-1α overexpression in astrocytes and drugs promoting mitochondrial functions chosen from a new High Content Screen assay (Aim 2), and by establishing the sub-cellular roles played by STAT3 and PIAS3 in modulating mitochondrial function in astrocytes using in vitro and in vivo gain and loss of function (transgenic mice STAT3flox, PIAS3flox, SOCS3flox; overexpression of nuclear or mitochondrial STAT3 and PIAS3) and in vivo transient mRNA expression after SCI (Aim 3).