The overall aim of our research is to study the signalling mechanisms that participate in the regulation of myelin biogenesis. The myelin sheath is a highly specialized membranous structure that surrounds axons of the central and peripheral nervous systems and is essential for normal saltatory axonal conduction. The disruption of this membrane, for example in multiple sclerosis, leads to irreparable consequences. Myelin in the central nervous system (CNS), arises from the cellular processes that extend from the oligodendrocyte perikaryon to wrap a segment of axon in a spiral manner. Myelin biogenesis is a highly regulated process that requires the coordination of several oligodendrocytic events including lipid and protein synthesis, intracellular membrane trafficking and changes in cell shape. Intracellular vesicle transport plays a major role in the formation and maintenance of myelin. Individual myelin components are synthesized in different cellular compartments, sorted out and transported to the site of myelin formation by several different mechanisms. Some of the myelin protein including proteolipid protein (PLP) and myelin associated glycoprotein (MAG), are synthesized in the endoplasmic reticulum and transported via intracellular vesicles first to the Golgi and then to myelin. The fundamental importance of intracellular vesicular transport is further indicated by the occurrence of endocytosis in oligodendrocyte processes and myelin. Strict control of this traffic is necessary for preserving the structural and functional organization of oligodendrocytes and myelin. Our research is oriented toward: 1) Defining the intracellular membrane transport pathways in the oligodendrocytes. 2) Dissecting the molecular mechanisms that regulate the different trafficking pathways.
3) Understanding how the different routes of intracellular trafficking are integrated. 4) Determining how intracellular transport of vesicles is related to the regulation of other cellular events, such as protein and lipid synthesis, and organization of the cytoskeleton.
We demonstrated the presence in the oligodendrocytes of several GTP-binding proteins including members of the Rab, Arf and Rho families. Evidence showed that Rab proteins are key components of the mechanisms that regulated intracellular traffic of membranes. Each Rab family member is located in a specific region (exocytic, endocytic, or transcytotic) and regulates a particular step of vesicular traffic. In our current studies, the different intracellular membrane trafficking pathways in living cells are visualized by fluorescent microcopy analysis of oligodendrocytes expressing fusion proteins of Rab proteins with EYFP (a fluorescent protein). The involvement of the different pathway in the myelin formation is assess by co-expression of Rab-EYFP and myelin proteins such as myelin associated glycoprotein (MAG) tagged with ECFP, and by comparing the distribution of ECFP-tagged myelin proteins co-expressed with dominant negative mutants of Rab proteins. In addition, to define the molecular mechanisms in which the oligodendrocyte Rab proteins participate, we are using molecular cloning in a two-hybrid system for identification of the proteins that interact with the oligodendrocyte Rab proteins.
1. Larocca, J.N. 20, 25 Diazocholesterol. In: Experimental and Clinical Neurotoxicology. (Editors: Spencer and H.S. Schaumburg). Oxford University Press. 472-474 (2000).
2. Ragheb F., Molina-Holgado E., Cui Q.J., Khorchid A, Liu H.N., Larocca J. N. and Almazan, G.. Pharmacological and functional characterization of muscarinic receptor subtypes in developing oligodendrocytes. J. Neurochem. 77: 1396 (2001).
3. Rodriguez-Gabin, A. G ; Cammer, M.; Almazan , G.; Charron, M and Larocca, J.N. Role of rRab22b, an oligodendrocyte protein, in regulation of transport of vesilces from trans Golgi to endocytic compartments. J. Neurosci. Res. 66: 1149 (2001).
4. Larocca J.N. and Rodriguez-Gabin A.G. Myelin Biogenesis: Vesicle transport in oligodendrocytes. Neurochem. Res. 27:1313-29 (2002)
5. Rodriguez-Gabin, A. G., Almazan, G. and Larocca, J. N. Vesicle Transport in Oligodendrocytes: Role of Rab40c. J. Neurosci. Res. 76:758-770 (2004).
6. Larocca, J. N., E. Ortiz, Demoliner, K., Si, Q and Rodriguez-Gabin. Vesicle transport in oligodendrocytes: Role of rRab22b and OCRL-1. Trans. Am. Soc. Neurochem. Madison Wisconsin, USA, (2005).
7. Larocca, J. N., E. Ortiz, Demoliner, K., Si, Q and Rodriguez-Gabin. Vesicle transport in oligodendrocytes: Interaction of rRab22b with OCRL-1. 20th Biennial Meeting International Society for Neurochemistry. Innsbruck, Austria, (2005).
8. Larocca, J. N. and Norton, W.T. Isolation of Myelin. Current Protocols in Cell Biology. (In Press)