Strategies for Controlling Apoptosis in Oligodendrocytes

Myelination is necessary for proper CNS function. Neurological deficits occur in spinal cord injury (SCI) and other conditions where oligodendrocyte (OL) death results in demyelination. It is not clear how the loss of OLs is initiated in SCI but several factors known to induce OL apoptosis are elevated in the extracellular environment. This suggests that OLs may be the target of multiple toxic agents. OLs, which die in SCI, show activation of post-mitochondrial events typical of apoptosis including caspase-3 activation. The goal of this proposal is to define events, which occur earlier in OL apoptosis, and to apply intervention strategies so that OLs survive and myelinating function is maintained. Our overall goal is to devise a therapeutic strategy to interrupt OL apoptosis regardless of the initiating agent so that it can be used effectively in many demyelinating conditions. The proposed studies use in vivo and culture methods to test the following hypotheses: 1) that soluble factors which induce OL apoptosis after SCI cause activation of the proapoptotic molecule BAD; 2) that OL apoptosis can be ameliorated by inactivating BAD, either by upregulating the enzymes which phosphorylate it (Akt, PI 3-kinase) or by using drugs which inhibiting its dephosphorylation (FK506); 3) that caspase-independent effectors such as endonuclease-G play an important role in cell loss after SCI. Specific Aim 1: Immunohistochemistry and immunoblots are used to show that soluble apoptotic agents result in BAD dephosphorylation and related biochemical events in OLs. Specific Aim 2: Adenoviral transfection and pharmacological approaches are used to test whether increasing levels of P-BAD through overexpression of Akt1 and PI 3-kinase or inhibition of calcineurin activity can promote survival and myelinating function in cultured OLs. Specific, Aim 3: Unbiased stereology is used to test whether mice with deletions in the genes for PTEN (an Akt inhibitor) and caspase-3 show an increase in tissue sparing and OL survival after SCI. Specific Aim 4: Biochemical and histological techniques are used to determine whether pathways which are independent of caspase-3 play a significant role in cell loss after SCI.