News and Publications

Organization and Function of the Neuronal Cytoskeleton

S. Halpain, L. Dehmelt, S. Graber, R. Lim, R. Ozer, F. Smart

During the past year, we made significant progress in research relevant to neuronal regeneration and neural development. Two projects focus on cytoskeletal proteins of nerve cells, key proteins that underlie the structure and morphologic flexibility that neurons require for storing and processing synaptic signals.

One project concerns the regulation of dendritic spines, specialized structures at many brain synapses. Neurons in the forebrain contain thousands of spinelike protrusions along the dendrites of the cells. These protrusions are the major receptive contact points at synapses that release the neurotransmitter glutamate. The function of the protrusions is not entirely clear; however, abnormal numbers and morphology of dendritic spines are associated with mental retardation, indicating that these structures are important in cognitive function. Spines are highly vulnerable to injury in diseases such as stroke and epilepsy. Understanding how spines form, what regulates their stability, and how they recover from injury is of great interest in devising therapies for several neurologic diseases.

The cytoskeleton of spines is composed mainly of actin filaments. We discovered that the filaments are rapidly broken down within minutes of an injury-inducing stimulus. This damage to spines can be rapidly reversed within hours under appropriate conditions. In other studies, we are examining the mechanisms involved in spine collapse and spine recovery. Two important factors we identified are intracellular levels of calcium and the calcium-dependent enzyme calcineurin. In addition, in collaboration with colleagues in the Department of Neuropharmacology, we showed that actin filaments play essential roles in physiologic plasticity at synapses.

Our second project focuses on a class of proteins called microtubule-associated proteins (MAPs). These proteins are important in regulating the assembly and stability of microtubules. Two proteins we study, MAP2 and tau, share a similar microtubule-binding domain but differ in the rest of the molecule. We are using both microscopy-based and molecular biological approaches to understand the structure-function relationship for these molecules.

Tau is a molecule involved in Alzheimer's disease and related neurodegenerative conditions of cognitive and motor impairment. Tau forms into abnormal structures called neurofibrillary tangles in such diseases, but MAP2 does not. The molecular basis for the tangles inside diseased neurons remains unclear. We are applying our knowledge of MAP function and regulation toward solving this problem.

MAP2 binds both microtubules and actin filaments, and we propose that it is an important regulator of cytoskeletal dynamics in neuronal dendrites. In previous work, we showed that the phosphorylation state of MAP2 is dynamically regulated by the neurotransmitter glutamate, the major excitatory neurotransmitter in the brain in mammals. Using mass spectroscopy, we identified specific phosphorylation sites as key regulators of MAP2 function. Mutagenesis of these sites altered the association of MAP2 with the microtubule versus association with the actin cytoskeleton. We also uncovered a potential new function for MAP2 as a scaffolding protein, suggesting that MAP2 not only organizes cytoskeletal proteins but also binds signaling molecules in a phosphorylation-dependent manner.

In other studies, we are exploring the signal transduction pathways that control MAP2 phosphorylation and localization in vivo. We are transfecting cultured neurons with wild-type and mutant forms of MAP2 and tau in order to probe the function of these proteins during the development and stabilization of dendrites and axons. Such information will be important in understanding both the molecular events in normal neurologic development and those in regeneration of neuronal structure and synaptic connections after injury and disease.

PUBLICATIONS

Lim, R.W.L., Halpain, S. Regulated association of microtubule-associated protein 2 (MAP2) with Src and Grb2: Evidence for MAP2 as a scaffolding protein. J. Biol. Chem. 275:20578, 2000.

Ozer, R., Halpain, S. Phosphorylation-dependent localization of microtubule-associated protein MAP2c to the actin cytoskeleton. Mol. Biol. Cell 11:3573, 2000.

Smart, F.M., Halpain, S. Regulation of dendritic spine stability. Hippocampus 10:542, 2000.

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