It has become clear that expression of voltage-operated Ca++ channels (VOCCs) is highly regulated in the oligodendroglial lineage and is essential for proper OPC development. Understanding the mechanisms of the voltage-dependent Ca++ influx is important as changes in intracellular Ca++ are central to many cellular activities. For example, in oligodendrocyte progenitor cells (OPCs) voltage-dependent Ca++ influx plays a key role in multiple important mechanisms such as process extension and cell migration (Paez et al., 2009a;b). Despite these relevant findings, next to nothing is known about the role of VOCCs in OPC differentiation and myelination. We will test the hypothesis that voltage-gated Ca++ entry promotes OPC maturation and myelination in the postnatal brain and we will determine if oligodendroglial VOCCs play a key role in a model of myelin repair.
Three specific aims are proposed: 1) To examine the role of VOCCs on oligodendrocyte development in vitro. Using pharmacological tools and VOCC specific siRNAs we will test if VOCCs are centrally involved in triggering oligodendrocyte maturation through voltage-gated Ca++ uptake. We propose to knock down the in vitro expression of VOCCs in oligodendrocytes and measure cell death, proliferation and OPC differentiation. We will also test whether these Ca++ channels facilitate axo-glial signaling during the first steps of myelin formation in an in vitro co-culture system of OPCs and cortical neurons. 2) Test if voltage-gated Ca++ entry promotes OPC maturation and myelination in vivo by specifically deleting the L-type VOCC isoform in OPCs. Viral vectors expressing siRNAs for L-type VOCCs will be injected into the corpus callosum and the subventricular zone of newborn pups to analyze the in vivo migration and myelination capabilities of VOCC deficient OPCs. Additionally, a conditional knockout mouse for L-type VOCC in OPCs will be generated by crossing the floxed mutant CaV1.2 mice with the NG2CreERTM transgenic mice which express a tamoxifen-inducible Cre recombinase under the control of the mouse NG2 promoter. Injecting tamoxifen in newborn pups of the crossbred mice will result in the L-type VOCC isoform CaV1.2 being postnatally deleted specifically in NG2 positive OPCs. 3) To examine the effects of VOCC ablation in myelin loss and recovery. Our preliminary findings indicate a role for VOCCs as a potential modulator of OPC development in adult mouse brain in acute demyelination. Using the Cre-lox system to silence Ca++ channel expression specifically in OPCs, we will test if voltage-dependent Ca++ entry promotes OPC survival and maturation in the remyelinating adult brain. For that purpose, we will use the cuprizone model of demyelination which has proven to be a useful tool for the analysis of myelin loss and remyelination in the adult brain. Successful completion of these studies will define by which mechanisms VOCCs control OPC development and myelination, and the role of these Ca++ channels in myelin pathology. These findings could lead to novel approaches to intervene in neurodegenerative diseases in which myelin is lost or damaged.

Public Health Relevance

The objective of this proposal is to determine the mechanism by which voltage-operated Ca++ channels regulate oligodendrocytes development and myelination in the Central Nervous System. This work is relevant to developing means to induce remyelination in myelin degenerative diseases and for myelin repair in damaged nervous tissue.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
3R01NS078041-02S1
Application #
8786686
Study Section
Cellular and Molecular Biology of Glia Study Section (CMBG)
Program Officer
Morris, Jill A
Project Start
2013-06-01
Project End
2018-05-31
Budget Start
2014-06-01
Budget End
2015-05-31
Support Year
2
Fiscal Year
2014
Total Cost
$62,380
Indirect Cost
$19,122
Name
State University of New York at Buffalo
Department
Pharmacology
Type
Schools of Medicine
DUNS #
038633251
City
Buffalo
State
NY
Country
United States
Zip Code
14260