Koichi J. Homma

Shrinkage of Dendritic Spines Associated with Long-Term Depression of Hippocampal Synapses

Activity-induced modification of neuronal connections is essential for the development of the nervous system and may also underlie learning and memory functions of mature brain. Previous studies have shown an increase in dendritic spine density and/or enlargement of spines after the induction of long-term potentiation (LTP). Using two-photon time-lapse imaging of dendritic spines in acute hippocampal slices from neonatal rats, we found that the induction of long-term depression (LTD) by low-frequency stimulation is accompanied by a marked shrinkage of spines, which can be reversed by subsequent high-frequency stimulation that induces LTP. The spine shrinkage requires activation of NMDA receptors and calcineurin, similar to that for LTD. However, spine shrinkage is mediated by cofilin, but not by protein phosphatase 1 (PP1), which is essential for LTD, suggesting that different downstream pathways are involved in spine shrinkage and LTD. This activity-induced spine shrinkage may contribute to activity-dependent elimination of synaptic connections.

Purification, Characterization, and cDNA Cloning of a Novel Growth Factor from the Conditioned Medium of NIH-Sape-4, an Embryonic Cell Line of Sarcophaga peregrina (Flesh Fly)

A growth factor from the conditioned medium of NIH-Sape-4, an embryonic cell line of the flesh fly, was purified to homogeneity. This growth factor, termed IDGF, stimulated the proliferation of NIH-Sape-4 cells in an autocrine manner; it was a homodimer of a protein with a molecular mass of 52 kDa, and its specific activity was comparable with those of mammalian growth factors. Immunoblotting experiments revealed that unfertilized mature eggs of the flesh fly contained this growth factor, a certain level of which was maintained throughout embryonic development. Analysis of cDNA for this growth factor showed that this factor is a novel protein consisting of 553 amino acid residues. No significant sequence similarity was found between this factor and other proteins except atrial gland granule-specific antigen of Aplysia californica.

Involvement of ACSL in local synthesis of neutral lipids in cytoplasmic lipid droplets in human hepatocyte HuH7

Lipid droplets (LDs) function as intracellular storage depots of neutral lipids. Recently, we identified long-chain acyl-coenzyme A synthetase 3 (ACSL3) as a major LD-associated protein in the human hepatocyte cell line HuH7. In this study, we investigated whether droplet-associated ACSL is involved in lipid metabolism in LDs. Addition of oleic acid (OA) to culture medium was shown to enhance the intracellular accumulation of LDs in the cells, which was accompanied by an increase of droplet ACSL3. When LD-enriched cells induced by OA were further incubated without OA for 3 days, ∼80% of LDs were retained in the cells. Conversely, cellular LD content was greatly decreased after the addition of an ACSL inhibitor, triacsin C. This was accompanied by a concomitant decrease of the droplet ACSL3. Incubation of isolated LD fractions with 14C-labeled OA or palmitic acid resulted in [14C]acyl-CoA generation in vitro, indicating the presence of ACSL activity in LDs. The droplet ACSL activity varied according to the quantity of LDs in their emergence and disappearance in cells. Incubation of the LD fraction with [14C]oleoyl-CoA resulted in radioactive triacylglycerol and cholesteryl esters. These results suggest that LD ACSL activity is involved in local synthesis of neutral lipids and LD formation.

A Truncated Tropo-Myosine-Related Kinase B Receptor, T1, Regulates Glial Cell Morphology via Rho GDP Dissociation Inhibitor 1

Through tropo-myosine-related kinase B (TrkB) receptors, brain-derived neurotrophic factor (BDNF) performs many biological functions such as neural survival, differentiation, and plasticity. T1, an isoform of TrkB receptors that lacks a tyrosine kinase, predominates in the adult mammalian CNS, yet its role remains controversial. In this study, to examine whether T1 transduces a signal and to determine its function, we first performed an affinity purification of T1-binding protein with the T1-specific C-terminal peptide and identified Rho GDP dissociation inhibitor 1 (GDI1), a GDP dissociation inhibitor of Rho small G-proteins, as a signaling protein directly associated with T1. The binding of BDNF to T1 caused Rho GDI1 to dissociate from the C-terminal tail of T1. Astrocytes cultured for 30 d expressed only endogenous T1 among the BDNF receptors. In 30 d cultured astrocytes, Rho GDI1, when dissociated in a BDNF-dependent manner, controlled the activities of the Rho GTPases, which resulted in rapid changes in astrocytic morphology. Furthermore, using 2 d cultured astrocytes that were transfected with T1, a T1 deletion mutant, or cyan fluorescent protein fusion protein of the T1-specific C-terminal sequence, we demonstrated that T1-Rho GDI1 signaling was indispensable for regulating the activities of Rho GTPases and for the subsequent morphological changes among astrocytes. Therefore, these findings indicate that the T1 signaling cascade can alter astrocytic morphology via regulation of Rho GTPase activity.

Truncated TrkB-T1 regulates the morphology of neocortical layer I astrocytes in adult rat brain slices

By altering their morphology, astrocytes, including those involved in the maintenance and plasticity of neurons and in clearance of transmitter, play important roles in synaptic transmission; however, the mechanism that regulates the morphological plasticity of astrocytes remains unclear. Recently, we reported that T1, a subtype of TrkB (a family of BDNF‐specific receptors), altered astrocytic morphology through the control of Rho GTPases in primary astrocyte cultures. In this study, we extended this observation to investigate acute neocortical slices from adult rats. T1 siRNA‐expression vectors were electroporated into astrocytes in neocortical layer I of living rats. In both normal slices and control vector‐electroporated slices, BDNF induced the elongation of the astrocytic processes and increased the branching of processes in slices after 1 h incubation. In contrast, in T1 siRNA‐electroporated slices, no such significant morphological changes were observed in the astrocytes. In addition, the number of synaptophysin+ sites in contact with GFAP+ processes increased in a BDNF–T1‐dependent manner without the increase in the total synaptophysin+ sites. Therefore, the present study provides evidence of the regulation of layer I astrocytic morphology by the BDNF–T1 signal in adult rat neocortical slices.

Chemotherapeutic activity of synthetic antimicrobial peptides: correlation between chemotherapeutic activity and neutrophil-activating activity

The chemotherapeutic activity of three synthetic antibacterial peptides was investigated. KLKLLLLLKLK‐NH2 and its d‐enantiomer showed significant chemotherapeutic activity in MRSA‐infected mice, whereas KLKLLLKLK‐NH2, which showed the highest antibacterial activity among them in vitro, was found to have almost no ability to prevent MRSA infection. These results suggest that the antibacterial activity of peptides assessed in vitro does not necessarily correlate with their chemotherapeutic activity. We found that KLKLLLLLKLK‐NH2 and its d‐enantiomer, but not KLKLLLKLK‐NH2, have the ability to activate human neutrophils to produce superoxide, suggesting that the prevention of MRSA infection by these peptides is not simply due to their direct bactericidal activity but to augmentation of the systemic defense mechanism mediated by neutrophils.

Thyroid hormone determines the start of the sensitive period of imprinting and primes later learning

Filial imprinting in precocial birds is the process of forming a social attachment during a sensitive or critical period, restricted to the first few days after hatching. Imprinting is considered to be part of early learning to aid the survival of juveniles by securing maternal care. Here we show that the thyroid hormone 3,5,3′-triiodothyronine (T3) determines the start of the sensitive period. Imprinting training in chicks causes rapid inflow of T3, converted from circulating plasma thyroxine by Dio2, type 2 iodothyronine deiodinase, in brain vascular endothelial cells. The T3 thus initiates and extends the sensitive period to last more than 1 week via non-genomic mechanisms and primes subsequent learning. Even in non-imprinted chicks whose sensitive period has ended, exogenous T3 enables imprinting. Our findings indicate that T3 determines the start of the sensitive period for imprinting and has a critical role in later learning.

3′ Phosphatase activity toward phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] by voltage-sensing phosphatase (VSP)

Voltage-sensing phosphatases (VSPs) consist of a voltage-sensor domain and a cytoplasmic region with remarkable sequence similarity to phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor phosphatase. VSPs dephosphorylate the 5′ position of the inositol ring of both phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] and phosphatidylinositol 4,5-bisphosphate [PI(4,5)P2] upon voltage depolarization. However, it is unclear whether VSPs also have 3′ phosphatase activity. To gain insights into this question, we performed in vitro assays of phosphatase activities of Ciona intestinalis VSP (Ci-VSP) and transmembrane phosphatase with tensin homology (TPTE) and PTEN homologous inositol lipid phosphatase (TPIP; one human ortholog of VSP) with radiolabeled PI(3,4,5)P3. TLC assay showed that the 3′ phosphate of PI(3,4,5)P3 was not dephosphorylated, whereas that of phosphatidylinositol 3,4-bisphosphate [PI(3,4)P2] was removed by VSPs. Monitoring of PI(3,4)P2 levels with the pleckstrin homology (PH) domain from tandem PH domain-containing protein (TAPP1) fused with GFP (PHTAPP1-GFP) by confocal microscopy in amphibian oocytes showed an increase of fluorescence intensity during depolarization to 0 mV, consistent with 5′ phosphatase activity of VSP toward PI(3,4,5)P3. However, depolarization to 60 mV showed a transient increase of GFP fluorescence followed by a decrease, indicating that, after PI(3,4,5)P3 is dephosphorylated at the 5′ position, PI(3,4)P2 is then dephosphorylated at the 3′ position. These results suggest that substrate specificity of the VSP changes with membrane potential.

Involvement of sapecin in embryonic cell proliferation of Sarcophaga peregrina (flesh fly).

Addition of antibodies against sapecin to the culture medium of NIH‐Sape‐4 cells derived from a Sarcophaga embryo greatly inhibited cell proliferation, whereas addition of sapecin stimulated cell proliferation. These results suggest that sapecin is involved in the proliferation of embryonic cells of Sarcophaga. Sapecin is known to have potent antibacterial activity, so it seems to have two different biological functions: i.e. protection against bacterial infection and stimulation of embryonic cell proliferation.

An 86 kDa diapause protein 1-like protein is a component of early-staged encapsulation-relating proteins in coleopteran insect, Tenebrio molitor larvae1

Recently, we reported two novel early‐staged encapsulation‐relating proteins (56 kDa and 48 kDa ERPs) isolated from the hemolymph of coleopteran insect, Tenebrio molitor larvae [Cho et al. (1999) Eur. J. Biochem. (in press)]. Here, a cDNA clone for another early‐staged encapsulation‐relating protein (86 kDa) was isolated. We found that the 86 kDa protein shows high homology with insect diapause protein 1. The 86 kDa protein was localized in the fat body and hemolymph, but not hemocyte lysate. A significant level of 86 kDa protein was detected in pre‐pupae stage, but it decreased rapidly at late larvae and pupae, and no protein was found in embryo, early larvae and adult stages. This diapause protein 1‐like protein is likely to be a component of early‐staged encapsulation‐relating proteins in the insect cellular defense reaction.