INF2-mediated actin filament reorganization confers intrinsic resilience to neuronal ischemic injury.

During early ischemic brain injury, glutamate receptor hyperactivation mediates neuronal death via osmotic cell swelling. Here we show that ischemia and excess NMDA receptor activation cause actin to rapidly and extensively reorganize within the somatodendritic compartment. Normally, F-actin is concentrated within dendritic spines. However, <5 min after bath-applied NMDA, F-actin depolymerizes within spines and polymerizes into stable filaments within the dendrite shaft and soma. A similar actinification occurs after experimental ischemia in culture, and photothrombotic stroke in mouse. Following transient NMDA incubation, actinification spontaneously reverses. Na+, Cl-, water, and Ca2+ influx, and spine F-actin depolymerization are all necessary, but not individually sufficient, for actinification, but combined they induce activation of the F-actin polymerization factor inverted formin-2 (INF2). Silencing of INF2 renders neurons vulnerable to cell death and INF2 overexpression is protective. Ischemia-induced dendritic actin reorganization is therefore an intrinsic pro-survival response that protects neurons from death induced by cell edema.

Systematizing and cloning of genes involved in the cerebellar cortex circuit development.

The cerebellar cortical circuit of mammals develops via a series of magnificent cellular events in the postnatal stage of development to accomplish the formation of functional circuit architectures. The contribution of genetic factors is thought to be crucial to cerebellar development. Therefore, it is essential to analyze the underlying transcriptome during development to understand the genetic blueprint of the cerebellar cortical circuit. In this review, we introduce the profiling of large numbers of spatiotemporal gene expression data obtained by developmental time-series microarray analyses and in situ hybridization cellular mRNA mapping, and the creation of a neuroinformatics database called the Cerebellar Development Transcriptome Database. Using this database, we have identified thousands of genes that are classified into various functional categories and are expressed coincidently with related cellular developmental stages. We have also suggested the molecular mechanisms of cerebellar development by functional characterization of several identified genes (Cupidin, p130Cas, very-KIND, CAPS2) responsible for distinct cellular events of developing cerebellar granule cells. Taken together, the gene expression profiling during the cerebellar development demonstrates that the development of cerebellar cortical circuit is attributed to the complex but orchestrated transcriptome.

The physiological role of Homer2a and its novel short isoform, Homer2e, in NMDA receptor-mediated apoptosis in cerebellar granule cells.

Homer is a postsynaptic scaffold protein, which has long and short isoforms. The long form of Homer consists of an N-terminal target-binding domain and a C-terminal multimerization domain, linking multiple proteins within a complex. The short form of Homer only has the N-terminal domain and likely acts as a dominant negative regulator. Homer2a, one of the long form isoforms of the Homer family, expresses with a transient peak in the early postnatal stage of mouse cerebellar granule cells (CGCs); however, the functions of Homer2a in CGCs are not fully understood yet. In this study, we investigated the physiological roles of Homer2a in CGCs using recombinant adenovirus vectors. Overexpression of the Homer2a N-terminal domain construct, which was made structurally reminiscent with Homer1a, altered NMDAR1 localization, decreased NMDA currents, and promoted the survival of CGCs. These results suggest that the Homer2a N-terminal domain acts as a dominant negative protein to attenuate NMDAR-mediated excitotoxicity. Moreover, we identified a novel short form N-terminal domain-containing Homer2, named Homer2e, which was induced by apoptotic stimulation such as ischemic brain injury. Our study suggests that the long and short forms of Homer2 are involved in apoptosis of CGCs.

Interaction of Cupidin/Homer2 with two actin cytoskeletal regulators, Cdc42 small GTPase and Drebrin, in dendritic spines.

BACKGROUND: Homer is a postsynaptic scaffold protein that links various synaptic signaling proteins, including the type I metabotropic glutamate receptor subunits 1alpha and 5, the inositol 1,4,5-trisphosphate receptor, Shank and Cdc42 small GTPase. Overexpression of Homer induces changes in dendritic spine morphology in cultured hippocampal neurons. However, the molecular basis underpinning Homer-mediated spine morphogenesis remains unclear. In this study, we aimed to elucidate the structural and functional properties of the interaction between Cupidin/Homer2 and two actin-cytoskeletal regulators, Cdc42 small GTPase and Drebrin. RESULTS: Cupidin/Homer2 interacted with activated Cdc42 small GTPase via the Cdc42-binding domain that resides around amino acid residues 191-283, within the C-terminal coiled-coil domain. We generated a Cupidin deletion mutant lacking amino acids 191-230 (CPDDelta191-230), which showed decrease Cdc42-binding ability but maintained self-multimerization ability. Cupidin suppressed Cdc42-induced filopodia-like protrusion formation in HeLa cells, whereas CPDDelta191-230 failed to do so. In cultured hippocampal neurons, Cupidin was targeted to dendritic spines, whereas CPDDelta191-230 was distributed in dendritic shafts as well as spines. Overexpression of CPDDelta191-230 decreased the number of synapses and reduced the amplitudes of miniature excitatory postsynaptic currents in hippocampal neurons. Cupidin interacted with a dendritic spine F-actin-binding protein, Drebrin, which possesses two Homer ligand motifs, via the N-terminal EVH-1 domain. CPDDelta191-230 overexpression decreased Drebrin clustering in the dendritic spines of hippocampal neurons. CONCLUSION: These results indicate that Cupidin/Homer2 interacts with the dendritic spine actin regulators Cdc42 and Drebrin via its C-terminal and N-terminal domains, respectively, and that it may be involved in spine morphology and synaptic properties.

The Homer family proteins.

The Homer family of adaptor proteins consists of three members in mammals, and homologs are also known in other animals but not elsewhere. They are predominantly localized at the postsynaptic density in mammalian neurons and act as adaptor proteins for many postsynaptic density proteins. As a result of alternative splicing each member has several variants, which are classified primarily into the long and short forms. The long Homer forms are constitutively expressed and consist of two major domains: the amino-terminal target-binding domain, which includes an Enabled/vasodilator-stimulated phosphoprotein (Ena/VASP) homology 1 (EVH1) domain, and the carboxy-terminal self-assembly domain containing a coiled-coil structure and leucine zipper motif. Multimers of long Homer proteins, coupled through their carboxy-terminal domains, are thought to form protein clusters with other postsynaptic density proteins, which are bound through the amino-terminal domains. Such Homer-mediated clustering probably regulates or facilitates signal transduction or cross-talk between target proteins. The short Homer forms lack the carboxy-terminal domain; they are expressed in an activity-dependent manner as immediate-early gene products, possibly disrupting Homer clusters by competitive binding to target proteins. Homer proteins are also involved in diverse non-neural physiological functions.

HOMER2 binds MYO18B and enhances its activity to suppress anchorage independent growth.

MYO18B is a class XVIII myosin, cloned as a tumor suppressor gene candidate. To investigate the mechanisms of MYO18B-dependent tumor suppression, MYO18B-interacting proteins were searched for by a yeast two-hybrid screen. HOMER2, a Homer/Ves1 family protein, was identified as a binding partner of MYO18B. These proteins co-localized in the regions of membrane protrusion and stress fiber, which are known as ones with filamentous actin-rich structures. Expression of HOMER2 enhanced the ability of MYO18B to suppress anchorage-independent growth. These results indicate that HOMER2 and MYO18B cooperate together in tumor suppression.

SCG10, a microtubule destabilizing factor, stimulates the neurite outgrowth by modulating microtubule dynamics in rat hippocampal primary cultured neurons.

Microtubule dynamics, one of the key elements in neurite outgrowth, is regulated by various regulatory factors to determine the behavior of the neuronal growth cone and to form the specialized neuronal shape. SCG10 is a neuron-specific stathmin protein with a potent microtubule destabilizing factor and is enriched in the growth cones of the developing neurons. We investigated the functional role of SCG10 in neurite outgrowth using rat hippocampal primary cultured neurons. Genetic manipulation of SCG10 using a short-interfering RNA duplex markedly decreased the SCG10 expression level and significantly suppressed neurite outgrowth. This result was confirmed by immunodepletion experiments. On the other hand, the protein transduction of SCG10 using a polyarginine tag stimulated neurite outgrowth. Such manipulation of the SCG10 expression level affected microtubule morphology within the growth cones. A decrease in the SCG10 level converted the morphology to a more stable state, while an increase converted the morphology to a more dynamic state. However, an excess of SCG10 induced neurite retraction due to an excess of microtubule disassembly. These results suggest that SCG10 serves as an important regulatory factor of growth cone motility by enhancing microtubule dynamics, possibly through increasing the catastrophe frequency.