Research Project
7806407
DESCRIPTION (provided by applicant): Our reported data show that Thy-1-1V23 integrin interaction in neuron-astrocyte association stimulates astrocytes to form focal adhesions and stress fibers by integrin clustering and RhoA activation. Focal adhesions anchor stress fibers to the plasma membrane and are implicated in cell adhesion, migration, growth. Our submitted data indicate that Thy-1 requires both integrin and syndecan-4 to increase cell adhesion to the matrix via PKC1- and RhoA-dependent pathways. Interestingly, temporal RhoA activation is biphasic with a small peak of activity at 5min and a larger one at 20min. Since RhoA is reportedly activated downstream of PKC1, this collaborative grant seeks to determine whether PKC1 also follows a biphasic mode of activation downstream of integrins and syndecan-4, as well as to identify guanine nucleotide exchange factors (GEFs) required for Thy-1-induced RhoA activation. To test whether PKC1 is activated biphasically, molecules downstream of integrins or syndecans will be studied using null cells, dominant negative constructs or pharmacological inhibitors. To assess the contribution of each pathway, cell stimulation will be achieved using Thy-1 mutated in integrin/syndecan binding sites either individually or in combination. Additionally, spatio- temporal Rho activation upon Thy-1 stimulation will be followed in single cells transfected with Rho biosensors by FRET analysis. RhoGEFs will be identified in pull- down assays using GST-17ARhoA with high affinity for RhoGEFs. The use of mass spectrometry to analyze precipitated complexes will identify molecules involved in RhoA activation. Thus, molecular mechanisms governing Thy-1-induced morphological changes in astrocytes will be studied. Insights obtained should improve our understanding of astrogliosis, a process triggered upon brain injury that involves dramatic morphological changes in astrocytes, migration to form the glial scar and inhibition of neuronal regeneration in humans. This research will be done primarily in Chile, in collaboration with Dr. Keith Burridge from UNC, as an extension of NIH grant #RO1 GM29860-27. This collaboration was initiated using FIRCA funds (RO3 TW006024 awarded in 2002) where Dr. Burridge was the PI and Dr. Lisette Leyton the Foreign Investigator. PUBLIC HEALTH RELEVANCE: Astrocytes are ubiquitously present throughout the brain and associate intimately with neurons. Upon injury or in response to inflammation, astrocytes are activated, migrate, increase in size and participate in the formation of the glial scar. In doing so, damaged areas are segregated from unaffected tissue to avoid secondary lesions and a non-permissive environment for neuronal regeneration is created. Molecular mechanisms responsible for the lack of neuronal regeneration in the adult central nervous system remain controversial, possibly due to the participation of multiple inhibitory cell-cell, as well as cell-matrix interactions. Our interest focuses on a neuron-astrocyte interaction, described for the first time by our group 7 years ago, between Thy-1, a highly abundant neuronal surface molecule associated with inhibition of neurite outgrowth, and its reported astrocyte receptors, the 1V23 integrin and syndecan-4. With the studies proposed here, we will learn about molecular mechanisms that control Thy-1- induced morphological changes in astrocytes. The goal is to understand better astrocyte participation in the formation of the glial scar and inhibition of axonal growth. Much progress has been made in understanding the basic events underlying axon regeneration, but certainly not enough to achieve partial to complete recovery of such structures following central nervous system injury. The current proposal seeks a better understanding of the changes occurring in astrocytes upon interaction with Thy-1. A different line of ongoing research in our laboratory investigates the effect that 1V23 integrin interaction with Thy-1 might have in neurons. Our results indicate that this interaction triggers bidirectional signaling in both cells and leads to inhibition of neurite outgrowth in differentiating neurons, as well as axonal retraction of already existing processes. Thus, studies of this group are expected to yield a better understanding of molecular mechanisms controlling neurite outgrowth and astrocyte function. Such insights are of fundamental interest to brain physiology in general and may help in the development of more effective strategies to promote nerve regeneration.
