News and Publications

Organization and Function of the Neuronal Cytoskeleton

During the past year, we made significant progress in research on neuronal development and regeneration. In 2 main projects, we focused on cytoskeletal proteins of nerve cells, key proteins that underlie the structure and morphologic flexibility required by neurons for transmitting, storing, and processing synaptic signals. We used biochemical, molecular biological, and microscopy-based approaches to understand the function of these molecules. Multiple-wavelength, time-lapse imaging of living neurons is an important tool that we use to uncover structure-function relationships for cytoskeletal proteins.

One project concerns microtubule-associated proteins (MAPs). These proteins are important in regulating the assembly and stability of microtubules. Two proteins we investigate, MAP2 and tau, have similarities and differences in their function. Surprisingly, our research last year revealed important distinctions between MAP2 and tau precisely within the regions that are most nearly identical in the 2 proteins. Abnormal regulation of tau plays a key role in Alzheimer's disease; so one of our goals is to understand the relationship between normal tau function and its role in pathologic changes. MAP2 has been implicated in the maturation of neuronal dendrites, but we speculate that MAP2 has an even earlier role in neuronal morphogenesis.

We recently showed that MAP2 regulates both actin and microtubule dynamics to promote neurite initiation in newly differentiated neurons. Previously, using mass spectrometry, we showed that phosphorylation sites at conserved, repeated motifs are key regulators of MAP2 function. Mutagenesis of these sites altered the association of MAP2 with the microtubule but not the association with the actin cytoskeleton. Using fluorescence-based time-lapse imaging and high-resolution confocal microscopy, we can track the behaviors of microtubules and actin filaments in the presence of normal and mutant forms of MAP2 in living neuronal cells. Our results indicate that MAP2 promotes neurite induction not only by stabilizing microtubules but also by coordinating their interaction with actin at the cell periphery.

A second project concerns the regulation of dendritic spines, specialized morphologic structures found at most forebrain synapses. Spines are the receptive contact points for synapses that release the neurotransmitter glutamate. Spines are vulnerable to injury in diseases such as stroke and epilepsy, in which excessive release of glutamate can induce neuronal injury and subsequent cell death (a condition called excitotoxicity). Furthermore, abnormal shapes and numbers of spines occur in patients with mental retardation and other cognitive disorders. Understanding how spines form, what regulates their stability, and how they recover from injury is therefore of therapeutic interest for several neurologic diseases.

Our most recent results suggested a neuroprotective role for spines, because preventing the collapse of dendritic spines attenuated neuronal cell death induced by a subsequent lethal stimulus. The spine cytoskeleton is composed mainly of actin filaments. We discovered that actin filaments in spines are broken down within minutes of an injury-inducing stimulus. However, this damage can be reversed within minutes under appropriate conditions, indicating for the first time that spines can regrow after they collapse. We are examining the physiologic consequences of spine loss and are searching for ways to prevent the loss and promote recovery.

In other studies, we are examining the molecular mechanisms that regulate the assembly and disassembly of actin filaments in spines. We identified several enzymatic pathways that participate in this event. Together these projects contribute to our understanding of molecular events in normal brain development and in regeneration of neuronal structure after injury and disease.

Publications

Dehmelt, L., Halpain, S. Actin and microtubules in neurite initiation: are MAPs the missing link? J. Neurobiol., in press.

Dehmelt, L., Smart, F.M., Ozer, R.S., Halpain, S. The role of MAP2 in the reorganization of microtubules and lamellipodia during neurite initiation. J. Neurosci., in press.

Halpain, S. Actin in a supporting role. Nat. Neurosci. 6:101, 2003.

Malmendahl, A., Halpain, S., Chazin, W.J. Nascent structural elements in the kinase anchoring domain of microtubule-associated protein 2. Biochem. Biophys. Res. Commun. 301:136, 2003.