Colorado State University

Actin filaments (F-actin) constitute a dynamic component of the cytoskeleton of eukaryotic cells. Processes of cellular motility and cell division are dependent upon a pool of actin subunits capable of rapid assembly and disassembly in response to extracellular signals. The interaction of actin with the cell membrane and the spatial and temporal changes in actin organization that underlie cell movement are largely regulated by a number of actin binding proteins, among the most important being cofilin and its related family member, actin depolymerizing factor (ADF). ADF and cofilin are 18.5kDa proteins first isolated from vertebrate brain. All eukaryotic organisms, including plants and protists, express an ADF/cofilin-related protein. Genetic studies have shown that ADF/cofilins are essential for normal cell division and thus organismal maturation and survival, as well as for cell polarization and polarized cell migration such as occurs during development of multicellular organisms or during metastasis of cancer cells. In the nervous system, ADF/cofilins are essential for the initial formation of neurites, for their conversion into axons and dendrites, and for wiring the nervous system through pathfinding of neuronal growth cones. Memory and learning depend on proper regulation of ADF/cofilin in dendritic spines for both insertion of new receptors and for spine remodeling. Both in vitro and in vivo, at low concentration with respect to actin, ADF/cofilin serve to rapidly increase the dynamics of actin filaments but at high concentration they can bind to F-actin and stabilize filaments. Blocking ADF/cofilin activity completely inhibits cellular processes dependent upon actin reorganization.  ADF/cofilins also regulate myosin II binding to F-actin and thus influence cell contractile process though mechanisms other than actin depolymerization. In multicellular organisms, ADF/cofilins are regulated by phosphorylation of a single serine residue near the N-terminus of the protein. Since transmembrane signaling culminates in changes in the activity of protein kinases or phosphatases, ADF/cofilins are key proteins through which extracellular ligands bring about an alteration in cytoskeletal organization. In neuronal growth cones, this actin reorganization is the key to pathfinding by many extracellular guidance cues. ADF/cofilins also play key roles in neurodegenerative diseases.  They are rapidly activated (dephosphorylated) in response to energy depletion or oxidative stress. In many cell types, this hyperactivation results in the formation of cytoplasmic rods containing filaments of actin in a 1:1 complex with ADF/cofilins.  In neurons, rods form primarily within dendrites where they impair delivery of essential cargoes and block distal synapse activity. Rods are often abundant in human hippocampus from subjects suffering from dementia, such as in Alzheimer disease. Rods are induced experimentally in primary neuronal cultures by excitotoxic glutamate, amyloid-β peptides, proinflammatory cytokines such as TNFa, the HIV envelope protein, gp120, which is causative of AIDS dementia, and preformed fibrils of alpha-synuclein, a component in Lewy Bodies, a likely cause of dementia associated with Parkinson's disease.  Rods are also induced by energy depeletion and are a pathology associated with stroke. Rods contain oxidized cofilin dimers, suggesting that oxidative stress accompanies rod-inducing signals. The signaling pathway for rod formation from amyloid-β peptides, TNFa, gp120, and alpha-synuclein fibrils requires the expression of cellular prion protein, which is in a pathway leading to production of reactive oxygen species by membrane NADPH oxidase.  Sequestering of cofilin into rods may also affect the structure and dynamics of dendritic spines because cofilin is essential for the spine plasticity and remodeling that occurs during memory and learning. In mouse models of various neurodegenerative diseases or behavioral disorders, treatments that presumably modulate cofilin phosphoregulation, reverse the deficits of the disease/disorder. The final esearch in my laboratory addressed the role of ADF/cofilin in neurodegenerative diseases and brain disorders, including stroke. We developed a new method of brain slice culture that allowed us to follow the identical neurons over many weeks using fluorescence microscopy. We made and utilized viral vectors for expressing proteins in a cell-type specific manner and utilized computer imaging of confocal fluorescence images to quantify rods and analyze cell behavior and neuronal circuitry. We discovered that a small pentapeptide which is orally available and crosses the blood brain barrier could reverse rod pathology in both rodent and human neuronal cultures at a concentration of 1 pM, equal to two grains of salt in an olympic sized swimming pool. We hope that this ability to reverse rod pathology even in the presence of the rod inducer could translate to clinical treatments for multiple forms of dementia.