Expression Patterns And Functions Of NDRG Family During Human And Mouse Brain Development

NDRG（N-Myc downstream-regulated gene） family is a set of new genes foundrecent years which consists of4family members: NDRG1~4. These members share57-65%amino acid homology. Through bioinformatics analysis, the NDRG2codingprotein has an ACP domain that can carry the acyl and a variety of nuclear transcriptionfactor binding sites. This is a common character of the NDRG family structure domain.This special structure may result in a common expression and some consistent functionamong the family members. The gene family is involved in the growth of cells.However,the exact mechanism is not very clear yet.Previous ariticals reported NDRG family has a different degree of expression inadult rat, mice and human brains. The expression of NDRG2in depression, stress andhypoxia are abnormal, indicating that NDRG2is highly correlated with brain diseases.Currently NDRG1,2are considered as brain differentiation related genes. NDRG4alsohas a higher expression in neural precursor cells, and is regulated by retinoic acid(RA) andother factors. There is no clear evidence of the expression of NDRG3during brain development. So, presumably, the wide expression of the family in the brain may beassociated with nervous system development. The present study has confirmed that thisgene family is closely related to the abnormal proliferation and differentiation of tumorcells. NDRG2is confirmed as a candidate tumor suppressor gene.The normal proliferation,differentiation and migration of neural progenitor cells iscritical for the development of embryonic brain. However, the mechanism is still not clear.Our previous study have found that NDRG2, a candidate tumor suppressor which is veryimportant for the proliferation and differentiation of cancer cells, is highly expressed inNPCs in mouse’s fetal brain tissue, but it’s not clear whether NDRG2has similarexpression pattern during human brain development. Besides, the four family members ofNDRG has highly amino acid homology and highly conservative. To explore theirfunction during brain development, it is necessary to co nfirm the distribution and cellularlocalization of them.ObjectsTo study the expression characteristics and the cell localization of NDRG2in humanfetal brain and the four NDRGs in mice after birth, then to lay the foundation for exploringthe NDRG family role in brain development.MethodsWe used experimental techniques such as immunohistochemical， immunefluorescence，RT-PCR，Western Blot，cell culture and electron microscopy to study theexpression and the cell localization of NDRG family in human fetal brain and mice invarious regions.(1)Using RT-PCR method to detect NDRG2mRNA expression in16to28weeks in different brain regions of fetal brain.(2) Using Western blot to detectNDRG2expression in28weeks in various regions.(3) Using Immune histochemicalmethod to detect NDRG2protain expression in human fetal brain.(4) Using NDRG2withGFAP or NeuN fluorescent double labeling to study its positioning in fetal brain cells.(5)Using Western Blot and immunofluorescence technology to detect the expression of fourNDRGs in the postnatal mouse brain and cellular localization,and to detect NDRG2 expression in neural precursor cells and their differentiated cells in vitro.(6) Using electronmicroscopy technology to detect the subcellular localization of NDRG2.Results(1)We detected the expression levels of NDRG2in different time of human fetalbrain by RT–PCR and Western Blot. We found that the NDRG2expression increased asgestational age matured,especially in the ventricular zone and subventricular zone(2)Using immunohistochemical method, we further confirmed that NDRG2mainlyexpressed in the ventricular zone and subventricular zone.Using double-labellingimmunofluorescence technology,We proved that NDRG2was expressed in astrocytes andrelatively mature neurons, mainly located in cytoplasm, some in the nucleus ofastrocytes.(3)Using Western Blot, we found the expression pattern of NDRG1-4membersat different time points in different brain regions of postnatal mice: NDRG2was expressedin hippocampus and subventricular zone in21day mice，the expression in120day micewas decreased significantly;NDRG1,3,4were also expressed in the subventicular zoneand hippocampus with no regular pattern.(4)Using immunofluorescence, we showed noexpression of NDRG1,3,4in neural precursor cells. NDRG2was mainly expressed in theastrocytes cytoplasm in mouse hippocampus.(5) In subcellular level, we found thatNDRG2was expressed in mitochondrial crest of astrocytes of neural precursor cells.Conclusion(1)NDRG2was widely expressed in human fetal brain functional area. Along withthe gestational age mature, the expression of NDRG2enhanced.The strongest expressionarea was in neural precursor cells,indicating that NDRG2was closely related to cellproliferation, differentiation and mature.(2) The expression patterns of NDRG familymembers were distinguished. NDRG2was mainly expressed in the neurogenesis areawhile NDRG1,3,4were involved in the differentiation of neural precursor cells.(3)NDRG2might regulate the proliferation and differentiation of precursor cells by affectingthe mitochondrial function.