Outline


Jan

In the Department of Neurochemistry, neurochemical and neurobiological studies are carrying out to elucidate the function of various molecules which are involved in the differentiation, growth and regeneration of the nervous system. In particular, neurotrophic factors and neuron-glial interaction are the main research projects.


Microglia-derived neurotrophic factors

Accumulating lines of evidence indicated that glial cells play crucial roles in the development and maintenance of the nervous system. In our laboratory, to elucidate the microglial function in the brain, the neurotrophic factors which are secreted from microglia were investigated. We found that plasminogen and hepatocyte growth factor were produced in microglia which are neurotrophic for neurons. Furthermore, the production of plasminogen was revealed to be up-regulated in microglia following the axotomy of facial nerve.


Microglia-specific novel calcium binding protein

We successfully isolated a rat cDNA clone encoding a novel EF hand protein with a molecular weight of about 17 kDa and designated this gene Iba-1 (ionized calcium binding adapter molecule 1). The genomic copy of the Iba-1 gene was located within a segment of the major histocompatibility complex class III region between the Bat 2 and TNF genes. The Iba-1 gene was shown to be highly expressed in testis and spleen, but weakly expressed in brain, lung, and kidney. Among brain cells, the Iba-1 gene was specifically expressed in microglia. A screening of hemopoietic cell lines showed that the Iba-1 protein was clearly expressed in monoblastic cell lines but very weakly expressed in myeloid cell lines. Iba-1 protein is therefore suggested to act as an adapter molecule, mediating calcium signals that may function in a monocytic lineage including microglia.


Calcium dependent phospholipid binding protein, annexin V

We previously reported that annexin V promoted the survival of cultured rat neocortical neurons. In an effort to elucidate the mechanism underlying this neurotrophic activity of annexin V, we have attempted to identify the target or binding proteins of annexin V in neuronal cells. We screened an embryonic day 17 rat brain cDNA library by western blot using glutathione S-transferase-annexin V fusion protein as a probe and then isolated four clones showing binding to annexin V in a Ca2+- and phospholipid-dependent manner. Deduced amino acid sequences of two clones showed high homology with human X-linked Helicase2 (XH2) and DNA (cytosine-5) methyltransferase (DMTase) sequences, whereas the other two were not related to any known peptide sequence.


Physiological function of secreted form of amyloid precursor protein

The secreted form of amyloid precursor protein (APPs) including most of the extracellular domain of APP is released from the cell surface, suggesting physiological significance of APPs in vivo. We used the methylotrophic yeast Pichia pastoris as a host system for the production of recombinant APPs (rAPPs). Two rAPPss derived from isoforms of APP (APP695 and APP 770) were secreted into the culture medium from the yeast, which carried cDNA encoding the N-terminal portion of APP under the control of a P.pastoris alcohol oxidase promoter. Like APPss produced by the transfected COS-1 cells, the purified rAPPss from yeast were shown to be biologically active in terms of neurite outgrowth of embryonic rat neocortical explants. These rAPPss could be valuable tools for investigating the biological functions of APPss.

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