Dendritic spines and their associated synapses become prematurely destabilized in psychiatric and neurodegenerative diseases. Proper control of the actin cytoskeleton is critical for the long-term structural stability of dendritic spines, bt currently little is known about the molecules and mechanisms that confer long-term structural stability on spines and the field remains understudied. We discovered that loss of integrin ?3?1 signaling through the Abl2/Arg nonreceptor tyrosine kinase causes widespread dendrite arbor loss and dendritic spine destabilization. Even though Arg inhibits the RhoA GTPase to stabilize dendrite arbors, this mechanism does not impact spine stability, raising the fundamental question of how Arg stabilizes spines. We provide evidence that Arg directly binds and stabilizes actin filaments and also regulates the binding and actions of the actin regulators cortactin and Arp2/3 complex on actin filaments. We also find that Arg-mediated recruitment of cortactin to dendritic spines is crucial for spine stability. Our proposal will test the highly innovative hypothesis that Arg interacts physically and functionally with actin filaments and actin regulatory proteins to directly regulate actin dynamics and thereby stabilize dendritic spines.
Our first aim will elucidate how Arg:cortactin interactions control actin dynamics. We find that Arg binding to actin filaments stabilizes them from depolymerization. Arg binding also recruits the actin-binding protein cortactin, which stabilizes actin filaments and increases actin branch formation by Arp2/3 complex. We will use total internal reflection microscopy to observe single filaments and to measure how Arg and cortactin affect actin filament stability, Arp2/3 complex-mediated branch formation, and cofilin-mediated actin filament severing. We will use mutants of these proteins that do not interact with each other or with actin filaments to identify which protein:protein interaction interfaces are critical for effects on actin dynamics. These studies will reveal how Ar and cortactin affect actin filament stability, branching, and turnover.
Our second aim will determine how Arg and cortactin modulate spine stability via effects on actin dynamics. We find that knockdown of Arg in neurons results in the loss of cortactin from spines and triggers their destabilization. We hypothesize this destabilization is due to the disruption of normal actin dynamics in spines. Knockdown of Arg or cortactin in established hippocampal neuron cultures compromises dendritic spine stability. These deficits can be quantitatively rescued by re-expression of shRNA-resistant versions of Arg or cortactin, respectively. Employing our collection of Arg and cortactin mutants, we will test how mutational disruption of key interaction interfaces in these proteins affects dendritic spine shape and stability. We will use fluorescence recovery after photobleaching (FRAP) of GFP-actin in spines to reveal how manipulations of Arg and cortactin function affect actin dynamics in spines and determine how this relates to the effects of these proteins on actin biochemistry and spine stability.
Connections between neurons become destabilized in psychiatric disorders, such as schizophrenia and major depression, and neurodegenerative disorders, such as Alzheimer's disease. Disruption of these connections results from a loss of key mechanisms that stabilize a neuron's elaborate structure. We have identified critical roles for the Arg and cortactin proteins in maintaining the structure of dendritic spines, small protrusions on neurons where connections are formed with other neurons. We will determine the mechanisms by which Arg and cortactin preserve synaptic connections and protect against disruption of neural circuits in the brain.