News and Publications

Organization and Function of the Neuronal Cytoskeleton

S. Halpain, J. Braga, L. Dehmelt, S. Graber, J. Mosbacher,* B. Roger, Y. Shiraishi

* Novartis Pharma AG, Basel, Switzerland

During the past year, we made significant progress in research relevant to 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 (Fig. 1).

One project concerns microtubule-associated proteins (MAPs). These proteins are important in regulating the assembly and stability of microtubules. We use both microscopy-based and molecular biological approaches to understand the structure and function of MAPs. Two proteins we investigate, MAP2 and tau, have similarities and differences in their function. 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 it has an even earlier role in neuronal morphogenesis.

We are testing the hypothesis 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 vs 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 support the hypothesis that MAP2 promotes neurite induction not only by stabilizing microtubules but also by coordinating their interaction with actin at the cell periphery.

Other findings implicate MAP2 as a downstream target of an important class of signaling molecules, the bone morphogenetic proteins. These proteins regulate the morphogenesis of many tissues, including bone, but they also stimulate growth and functional recovery of neuronal dendrites in animal models of stroke. We found that bone morphogenetic proteins cause rapid changes in neuronal morphology and that the changes require both gene transcription and protein kinase activity. Furthermore, phosphorylation of MAP2 is an early step in the signaling cascade.

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 suggest a neuroprotective role for spines, because collapse of the spine structure made neurons more susceptible to excitotoxicity. The spine cytoskeleton is composed mainly of actin filaments. We discovered that actin filaments in spines are rapidly broken down within minutes of an injury-inducing stimulus. However, this damage can be rapidly reversed within hours under appropriate conditions. We are examining the physiologic consequences of spine loss and are searching for ways to prevent loss and promote recovery.

In other studies, we are examining the molecular mechanisms that regulate the assembly and disassembly of actin filaments in spines. 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

Al-Bassam, J., Ozer, R.S., Safer, D., Halpain, S., Milligan, R.A. MAP2 and tau bind along the outer ridges of microtubule protofilaments. J. Cell Biol. 57:1187, 2002.

Halpain, S. Making new connections. Trends Cell Biol. 12:455, 2002.