Insights into the biological function 2',3'-cyclic nucleotide 3'-phosphodiesterase

Oligodendrocytes (OLs) produce myelin sheathes that wrap around axons, a critical component for saltatory conduction in nerve fibers. Myelin-afflicting diseases impair nerve conduction resulting in severe neurological symptoms. The myelin sheath is composed of a specific set of proteins and lipids that are expressed at high levels compared to other cell types. One such protein is 2',3'-cyclic nucleotide 3'-phosphodiesterase (CNP). Although the expression pattern is well-characterized, its functional role in myelinating cells is unknown. The work presented in my thesis aims to identify the physiological function of CNP. In vitro, CNP hydrolyzes 2',3'-cyclic nucleotides to produce 2'-nucleotides, but the relevance in vivo is unknown. Mutational studies identified histidine and threonine residues in the C-terminal catalytic domain that are essential for enzymatic activity. Structural determination of the catalytic domain by NMR revealed striking similarities with RNA/nucleotide-processing enzymes in bacteria and plants, thereby placing CNP in the 2H phosphodiesterase superfamily sharing two identical tetrapeptide HX(T/S)X motifs. Structural similarities and the evolutionary conservation of key active-site residues suggest a role for CNP in RNA/nucleotide metabolism. My thesis also examines the role of CNP in modulating the OL cytoskeleton for process outgrowth. Immunoprecipitation experiments and mass spectrometry identified tubulin as a major CNP-interacting candidate. In vitro, CNP binds preferentially to tubulin compared with MTs and induces MT assembly by copolymerizing with tubulin. CNP overexpression induces process outgrowth and dramatic morphology changes in different cell types due to MT and F-actin reorganization. These effects are attributed to CNP MT assembly activity. Formation of branched processes is abolished with the expression of loss-of-function CNP mutants, and cultured OLs from CNP-deficient mice extend smaller outgrowths with less arborized processes. CNP is likely an important component of the cytoskeletal machinery that directs process outgrowth in OLs. Lastly, my thesis examines the function of the larger CNP2 isoform. Although both isoforms are abundantly expressed in myelinating cells, CNP2 is expressed at low levels outside the nervous system. The functional role of CNP2, apart from CNP1, in myelinating cells and the significance for CNP2 expression in non-myelinating tissues are unknown. A mitochondrial targeting signal at the CNP2 N-terminus was identified to direct mitochondrial translocation. CNP2 distribution is regulated by phosphorylation of the targeting signal by PKC. Mitochondrial localization is inhibited upon phosphorylation, thus retaining CNP2 in the cytoplasm, whereas in the non-phosphorylated state, CNP2 is translocated and imported into mitochondria with subsequent cleavage of the targeting signal. The sole presence of CNP2 in its truncated form in embryonic brain and adult liver indicates that CNP2 is localized specifically to mitochondria in pre- or non-myelinating cells. This points to an additional, broader biological role for CNP2 in mitochondria