Scientific Report 2003-2004 - Cleveland Clinic Lerner Research ...

THE MACKLINLABORATORYPROJECT STAFFTatyana Gudz, Ph.D.Mika Yoshida, M.D.RESEARCH ASSOCIATEAna Flores, Ph.D.POSTDOCTORAL FELLOWYuko Fujita, Ph.D.TECHNICAL ASSISTANTSChristine AgaibiEid DarwishCindy KangasKapila NavaratneElizabeth ShickCOLLABORATORSMartha J. Miller, M.D., Ph.D. 1Robert H. Miller, Ph.D. 2Stephen A. Stohlman, Ph.D. 3Bruce L. Trapp, Ph.D. 41Dept. of Pediatrics, CaseWestern Reserve Univ.,Cleveland, OH2Dept. of Neurosciences, CaseWestern Reserve Univ.,Cleveland, OH3Dept. of Molec. Biol./Immunol., Univ. of SouthernCalif., Los Angeles, CA4Dept. of Neurosciences, CCFThe Department of NeurosciencesGenes, Molecular Signaling Regulationof Normal Brain DevelopmentThe long-range goal of my research programis understanding the molecular signals thatregulate normal brain development. Themain research effort is directed to oligodendrocytedifferentiation and myelin biogenesis. We focus ona gene that is primarily expressed in myelinatingoligodendrocytes, the myelin proteolipid protein(PLP), and the closely related DM20 protein,which are the most abundant proteins of the CNSmyelin membrane. Point mutations in this gene arelethal, and the affected animals die by 3-4 weeksof age. Mutations in other oligodendrocyte genesthat generate important proteins for the myelinmembrane do not kill the animals at young ages.We are particularly interested in why mutations inthe PLP protein should be so devastating.We have used the PLP gene promoter togenerate transgenic animals overexpressingenhanced GFP (EGFP) in oligodendrocytes andhave used these transgenic animals to track PLPgene expression in developing oligodendrocytesfrom normal and mutant animals. The EGFPtransgenic mice are being used because PLPfluoresces in live cells, so we can identify oligodendrocytesin live tissue. In the PLP-EGFP mice, allcells in the lineage from oligodendrocyte progenitorto mature myelinating oligodendrocytes aredetected. In addition, we detect strong expressionin the sciatic nerve and the developing embryo.The embryo expression is in nonmyelinating cellsof both the central and peripheral nervous systems.Thus, we are able to track these cells to investigatenormal development of glial cells in both the PNSand CNS, and to study their differentiation inmutant animals or other pathological environments.We study their migration and differentiationby live confocal imaging of optic nerve.We are investigating why the PLP gene isexpressed in nonmyelinating, migrating cells earlyin development. We have demonstrated that thePLP protein interacts with integrins, an interactioncontrolled by neurotransmitter receptors on thesurface of oligodendrocytes. This inside-outsignaling of integrins in response to neurotransmittersinduces a complex of PLP withα v-integrin and reduces binding of fibronectin tooligodendrocytes. This altered binding to anextracellular matrix protein also alters themigration of oligodendrocyte progenitor cells.In the PLP mutant mice, the oligodendrocytesdie by apoptosis. Other problems in additionto the PLP mutations can induce oligodendrocyteapoptosis, e.g., growth factor deprivation. Survivalfactors can protect cells from apoptosis by blockingcell death signals. Thus, the balance of death andsurvival signals determines cell fate. We havestudied the neuregulin/erbB receptor system,which is an ideal candidate to provide survivalsignals from neurons to oligodendrocytes in vivo. Wehave established that the survival function ofneuregulins acts through the PI3 kinase/Aktpathway. We are currently investigating Aktsubstrates in oligodendrocytes and haveoverexpressed Akt in transgenic mice, which arejust now being analyzed.In other studies on oligodendrocytedifferentiation, we are studying oligodendrocyte/neuron interactions during development, using anewly identified mouse mutant with defectivePurkinje cell development. Purkinje cells are amajor set of neurons in the cerebellum, and theonly neurons that are myelinated. These cells beginto differentiate, but even at 6 days of age, somedifferences are noted between these cells and thePurkinje cells in wild-type animals. Our particularinterest in these animals is in the interactionbetween the oligodendrocytes and their targets, thePurkinje cells. What happens to oligodendrocyteswhen their target is altered in development? Wehave demonstrated a delay in oligodendrocytedifferentiation and an overall downregulation inmyelin gene expression in oligodendrocytes thatwould normally have myelinated the Purkinje cellaxons.In overview, these studies all focus ondifferent aspects of the ability of cells in thedeveloping nervous system to differentiatecorrectly and to find the appropriate site and targetfor their functions.Wendy B. Macklin, Ph.D.Mallon, B.S., Shick, H.E., Kidd, G.J., and W.B. Macklin (2002) Proteolipid promoter activity distinguishestwo populations of NG2-positive cells throughout neonatal cortical development. J. Neurosci. 22:876-887.Gudz, T.I., Schneider, T.E., Haas, T.A., and W.B. Macklin (2002) Myelin proteolipid protein participates in integrinreceptor signaling in oligodendrocytes. J. Neurosci. 22:7398-7407.Kahle, P.J., Neumann, M., Ozmen, L., Mueller, V., Jacobsen, H., Spooren, W., Fuss, B., Mallon, B., Macklin,W.B. Fujiwara, H., Hasegawa, M., Iwatsubo, T., Kretzschmar, H.A. and C. Haass (2002) Hyperphosphorylationand insolubility of a-synuclein transgenic mouse oligodendrocytes, EMBO Rep. 3:583-588.Mallon, B.S., and W.B. Macklin (2002) Overexpression of the 3’-untranslated region of myelin proteolipid proteinmRNA leads to reduced levels of endogenous proteolipid transcripts. Neurochem. Res. 27:1349-1360.Baracskay, K.L., Duchala, C.S., Miller, R.H., Macklin, W.B., and B.D. Trapp (2002) Oligodendrogenesis isdifferentially regulated in gray and white matter of jimpy mice. J. Neurosci. Res. 70:645-654.140