Functional recovery of callosal axons following demyelination: a critical window.

Axonal dysfunction as a result of persistent demyelination has been increasingly appreciated as a cause of functional deficit in demyelinating diseases such as multiple sclerosis. Therefore, it is crucial to understand the ultimate causes of ongoing axonal dysfunction and find effective measures to prevent axon loss. Our findings related to functional deficit and functional recovery of axons from a demyelinating insult are important preliminary steps towards understanding this issue. Cuprizone diet for 3-6 wks triggered extensive corpus callosum (CC) demyelination, reduced axon conduction, and resulted in loss of axon structural integrity including nodes of Ranvier. Replacing cuprizone diet with normal diet led to regeneration of myelin, but did not fully reverse the conduction and structural deficits. A shorter 1.5 wk cuprizone diet also caused demyelination of the CC, with minimal loss of axon structure and nodal organization. Switching to normal diet led to remyelination and restored callosal axon conduction to normal levels. Our findings suggest the existence of a critical window of time for remyelination, beyond which demyelinated axons become damaged beyond the point of repair and permanent functional loss follows. Moreover, initiating remyelination early within the critical period, before prolonged demyelination-induced axon damage ensues, will improve functional axon recovery and inhibit disease progression.

Yeast Mps1p phosphorylates the spindle pole component Spc110p in the N-terminal domain.

The yeast spindle pole body (SPB) component Spc110p (Nuf1p) undergoes specific serine/threonine phosphorylation as the mitotic spindle apparatus forms, and this phosphorylation persists until cells enter anaphase. We demonstrate that the dual-specificity kinase Mps1p is essential for the mitosis-specific phosphorylation of Spc110p in vivo and that Mps1p phosphorylates Spc110p in vitro. Phosphopeptides generated by proteolytic cleavage were identified and sequenced by mass spectrometry. Ser(60), Thr(64), and Thr(68) are the major sites in Spc110p phosphorylated by Mps1p in vitro, and alanine substitution at these sites abolishes the mitosis-specific isoform in vivo. This is the first time that phosphorylation sites of an SPB component have been determined, and these are the first sites of Mps1p phosphorylation identified. Alanine substitution for any one of these phosphorylated residues, in conjunction with an alanine substitution at residue Ser(36), is lethal in combination with alleles of SPC97, which encodes a component of the Tub4p complex. Consistent with a specific dysfunction for the alanine substitution mutations, simultaneous mutation of all four serine/threonine residues to aspartate does not confer any defect. Sites of Mps1p phosphorylation and Ser(36) are located within the N-terminal globular domain of Spc110p, which resides at the inner plaque of the SPB and binds the Tub4p complex.

A genetic analysis of interactions with Spc110p reveals distinct functions of Spc97p and Spc98p, components of the yeast gamma-tubulin complex.

The spindle pole body (SPB) in Saccharomyces cerevisiae functions as the microtubule-organizing center. Spc110p is an essential structural component of the SPB and spans between the central and inner plaques of this multilamellar organelle. The amino terminus of Spc110p faces the inner plaque, the substructure from which spindle microtubules radiate. We have undertaken a synthetic lethal screen to identify mutations that enhance the phenotype of the temperature-sensitive spc110-221 allele, which encodes mutations in the amino terminus. The screen identified mutations in SPC97 and SPC98, two genes encoding components of the Tub4p complex in yeast. The spc98-63 allele is synthetic lethal only with spc110 alleles that encode mutations in the N terminus of Spc110p. In contrast, the spc97 alleles are synthetic lethal with spc110 alleles that encode mutations in either the N terminus or the C terminus. Using the two-hybrid assay, we show that the interactions of Spc110p with Spc97p and Spc98p are not equivalent. The N terminus of Spc110p displays a robust interaction with Spc98p in two different two-hybrid assays, while the interaction between Spc97p and Spc110p is not detectable in one strain and gives a weak signal in the other. Extra copies of SPC98 enhance the interaction between Spc97p and Spc110p, while extra copies of SPC97 interfere with the interaction between Spc98p and Spc110p. By testing the interactions between mutant proteins, we show that the lethal phenotype in spc98-63 spc110-221 cells is caused by the failure of Spc98-63p to interact with Spc110-221p. In contrast, the lethal phenotype in spc97-62 spc110-221 cells can be attributed to a decreased interaction between Spc97-62p and Spc98p. Together, these studies provide evidence that Spc110p directly links the Tub4p complex to the SPB. Moreover, an interaction between Spc98p and the amino-terminal region of Spc110p is a critical component of the linkage, whereas the interaction between Spc97p and Spc110p is dependent on Spc98p.