Research Report 2010 - MDC

SHH. In line with these findings expression of Six3, adownstream target of SHH, is significantly down-regulatedin the forebrain of megalin -/- embryos and Shhexpression fails to be established properly in the ventralmidline of the forebrain during later stages of development.It is likely that the delay and decrease in the signalingof SHH during the initial patterning of the ventralmedial forebrain neural plate has detrimental consequencesfor CNS development in megalin mutants.A role for megalin in adult neurogenesis(Chandresh Gajera)In the adult brain, megalin is expressed in ependymalcells, a specialized epithelial cell layer lining the brainventricles. We found the strongest expression of thereceptor in the lateral wall of the lateral ventricles comparedto other ventricular regions. The lateral cell layerplays a crucial role in controlling the generation ofadult neurons from neuronal precursor cells in the subventricularzone (SVZ) of the lateral ventricles. The SVZis one of two regions of adult neurogenesis in themammalian forebrain. Neuronal precursors generatedin this region migrate to the olfactory bulb where theydifferentiate into mature neurons. Interestingly, SHHstimulates adult neurogenesis in the SVZ, and this effectrequires repression of BMP activity via chordin and noggin,similar to the situation seen in the embryonic neuraltube. Ependymal cells secrete noggin and thereby diminishthe inhibitory effect of BMP4 on SVZ neurogenesis.Recently, we obtained exciting new results from theanalysis of adult megalin-deficient mice that providedirect evidence for a role of the receptor in adult neurogenesis.Megalin deficiency does not affect the cellulararchitecture of the SVZ in mice. Nevertheless, mutantanimals show a decrease in cell proliferation in the lateralwall of the lateral ventricles (based on BrdU incorporationexperiments) affecting the neuronal precursorpopulation in the SVZ. The most prominent differencebetween megalin-deficient and control mice is foundby staining for GFAP (glial fibrillary acidic protein), amarker for neuronal precursor cells. Megalin -/- mice showa weaker and altered staining pattern for GFAP in theSVZ. Furthermore, megalin mutant mice show a decreasein nestin, a marker of undifferentiated neuronal precursors,in Dlx2, a marker of rapidly dividing precursors, andin PSA-NCAM, a marker of migrating neuroblasts.To address the mechanism whereby megalin influencesthe proliferation of neuronal precursors in the SVZ wetested the hypothesis that megalin may regulate thelevels of neurogenesis by inhibiting BMP4 activity inthe SVZ. We tested the expression of BMP2/4 andAbnormal SHH protein expression in megalin mutant embryosImmunostainings for Sonic hedgehog (SHH, red color) demonstrateexpression of the morphogen on coronal sections of wild type miceat E 8.25 in the prechordal plate (PcP) and in the overlying rostraldiencephalic ventral midline (RDVM) neuroepithelium (arrow,boundary indicated by the dotted line). Megalin protein is localizedon the apical surface of the neuroepithelium (green color) in wildtype embryos. In somite matched megalin deficient embryos SHHprotein could be detected in the prechordal plate but SHH expressionfails to be established in the overlying neuroepithelium during thisstage of development. The inset shows a schematic illustration ofthe developing CNS at E 8.0 in a sagittal view with the mesodermderived prechordal plate (PcP, purple) and the more caudal notocord(No) underlying the neuroepithelium of the diencephalon (Di) andthe floorplate (FP), respectively. Te: telencephalon, End: endodermdownstream mediators of this pathway. In these experiments,we discovered that there are increased levels ofBMP2/4 protein specifically in SVZ in megalin mutantsas compared to controls. We also detected a robust upregulationof phospho-Smad 1/5/8, and ID3, downstreammediators and targets of BMP signaling, respectively.Taken together, these results provide conclusiveevidence that megalin is a major regulator of adultneurogenesis in the SVZ, acting by suppressing theBMP4 signaling pathway. Remarkably, a similar function(to suppress BMP4 to activate SHH-dependentneurogenesis) is also performed by the receptor in theneural tube, supporting the concept that adult neurogenesisin the SVZ recapitulates features seen duringformation of neurons in the embryonic brain.Selected PublicationsWillnow, TE, Hammes, A, Eaton, S. (2007) Lipoproteins and their receptorsin embryonic development – more than cholesterol clearance.Development, 134, 3239-3249Hammes, A, Andreassen, TK, Spoelgen, R, Raila, J, Hubner, N, Schulz, H,Metzger, J, Schweigert, FJ, Luppa, PB, Nykjaer, A, Willnow, TE. (2005) Role ofendocytosis in cellular uptake of sex steroids. Cell 122(5): 751-762.Spoelgen, R, Hammes, A, Anzenberger, U, Zechner, D, Andersen, OM,Jerchow, B, Willnow, TE. (2005) LRP2/megalin is required for patterning ofthe ventral telencephalon. Development 132(2): 405-414.Cardiovascular and Metabolic Disease Research 11