Upregulation of a new microglial gene, mrf-1, in response to programmed neuronal cell death and degeneration.
Cerebellar granule neurons isolated from postnatal day 7 (P7) rats and grown in normal K+ medium begin to degenerate at approximately 4 d in vitro (DIV) and die. To search for genes upregulated in the process of neuronal cell death, differential hybridization was performed with subtracted cDNA probes and a cDNA library from 5 DIV. One of the genes isolated was microglial response factor-1 (mrf-1), which encoded a sequence of 177 amino acids with a single EF-hand calcium-binding motif. By Northern blots, the transcript was upregulated in cerebellar culture at 4 DIV, peaked at 6 DIV, and decreased at 7 DIV. Upregulation was also found when the apoptosis of granule cells was induced by replacing high K+ medium with normal K+ medium. However, when non-neuronal cells were thoroughly eliminated with aphidicolin, an antimitotic agent, the upregulation at 4-7 DIV did not occur. By immunocytochemistry, MRF-1 was detected at 5 DIV in OX-42-positive cells (microglia), and it exhibited an increase in response to granule cell death. MRF-1 levels in microglia purified from cerebral cortex also upregulated in the presence of 5 DIV granule cells. In the developing cerebellum in vivo, levels of mrf-1 mRNA transiently increased in the early postnatal stages, reaching a peak at P7 when cerebellar neurons and astrocytes undergo extensive apoptosis. In adult brain sections, MRF-1 was detected in the perikarya and processes of ramified/resting microglia, and peripheral motor nerve dissection prominently increased the expression in activated microglia surrounding injured central motoneurons. Therefore, mrf-1 appears to be one of the microglial genes that respond to neuronal cell death and degeneration.
Tanaka
S
S
Molecular Neurobiology Laboratory, Graduate Program in Biological Sciences, Hokkaido University, Sapporo 060-0810, Japan.
Suzuki
K
K
Watanabe
M
M
Matsuda
A
A
Tone
S
S
Koike
T
T
D000595:Amino Acid Sequence; D000818:Animals; D017209:Apoptosis / Q000502:physiology*; D019771:Axotomy; D001483:Base Sequence; D002135:Calcium-Binding Proteins; D002455:Cell Division / Q000502:physiology; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000235:genetics; D005786:Gene Expression Regulation / Q000502:physiology*; D007150:Immunohistochemistry; D008840:Microfilament Proteins; D017628:Microglia / Q000166:cytology / Q000502:physiology*; D008969:Molecular Sequence Data; D009410:Nerve Degeneration / Q000503:physiopathology*; D009419:Nerve Tissue Proteins / Q000235:genetics*; D009474:Neurons / Q000502:physiology*; D012333:RNA, Messenger / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D014018:Tissue Distribution
C100745:Aif1 protein, rat; D002135:Calcium-Binding Proteins; D018076:DNA, Complementary; D008840:Microfilament Proteins; D009419:Nerve Tissue Proteins; D012333:RNA, Messenger