Stroke is a leading cause of death and disability, yet there are few treatment options available to enhance stroke recovery. While it has long been known that astrocyte morphology changes in a graded fashion after stroke, little is known about the exact changes astrocytes undergo or the ways in which these responses may be manipulated to promote recovery. Reactive astrocytes have been considered as simple scar-forming cells in CNS injury. However, astrocytes have distinct effects on recovery depending on timing, location and astrocyte phenotype in some injury models, but this has not been studied in stroke. Here, I propose to identify phenotypic subsets of astrocytes and manipulate key astrocyte-secreted proteins within those subsets to determine molecular mechanisms of astrocyte responses to stroke, and their impact on tissue repair.
Aim 1 will examine the morphologic, phenotypic, and transcriptomic changes astrocytes undergo, in order to identify subpopulations of astrocytes with functional roles in tissue repair. To explore astrocyte morphology, one of several astrocyte-specific lentiviral vectors that drive expression of fluorophores will be injected in the peri-infarct cortex. These fluorescent proteins diffuse throughout the cell, clearly delinating the full architecture of the astrocyte and allowing a detaied analysis of changes in astrocyte length, polarity, complexity, and domain size. Phenotypic analysis will be conducted using specific astrocytic markers that reveal different aspects of astrocytic function that are related to tissue repair. Finally, the different transcriptomic change that different zones of astrocytes undergo will be determined using a line of mice in which tagged ribosomes are expressed in an astrocyte-specific manner combined with laser capture microscopy.
In Aim 2, astrocyte-secreted molecules that affect synapse formation and neural repair will be altered in subpopulation-specific fashions. Astrocyte-secreted proteins that promote neural repair are a newly identified and growing class of proteins about which little is known in stroke; their regional specificity is likely to play a crucial role in their action. Thereare three main known classes of protein that are secreted from astrocytes and affect synapse formation and neuronal repair: 1) those that help form synapses, 2) those that antagonize the first class, and 3) those that help synapses become electrically active. Preliminary results suggest that these classes are differentially regulated in different astrocytic subpopulations post stroke. I will first fully analyze the expression patterns of these molecules in different astrocytc subpopulations post-stroke. This information will inform interventional studies. As class 1 and 3 proteins are complementary in action, local delivery of exogenous proteins to those astrocytic subpopulations that do not express them post-stroke may improve recovery. Simultaneously, decreasing expression of class 2 proteins may allow the repair-mediating effects of class 1 and 3 proteins to be revealed. These modifications will be performed using localized delivery of astrocyte-specific delivery paradigms.
Stroke is a leading cause of death and disability worldwide, affecting almost 800,000 people a year in the United States alone. In this proposal, I will examine the effect stroke has on astrocytes, the most common cell in the brain, and manipulate the astrocyte response to stroke. Together, these experiments will reveal molecular systems affected in stroke that can be targeted to promote recovery.
| Nunez, Stefanie; Doroudchi, M Mehdi; Gleichman, Amy J et al. (2016) A Versatile Murine Model of Subcortical White Matter Stroke for the Study of Axonal Degeneration and White Matter Neurobiology. J Vis Exp : |
