Demyelination arrest and remyelination induced by glatiramer acetate treatment of experimental autoimmune encephalomyelitis.

The interplay between demyelination and remyelination is critical in the progress of multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE). In the present study, we explored the capacity of glatiramer acetate (GA, Copaxone) to affect the demyelination process and/or lead to remyelination in mice inflicted by chronic EAE, using both scanning electron microscopy and immunohistological methods. Spinal cords of untreated EAE mice revealed substantial demyelination accompanied by tissue destruction and axonal loss. In contrast, in spinal cords of GA-treated mice, in which treatment started concomitantly with disease induction (prevention), no pathology was observed. Moreover, when treatment was initiated after the appearance of clinical symptoms (suppression) or even in the chronic disease phase (delayed suppression) when substantial demyelination was already manifested, it resulted in a significant decrease in the pathological damage. Detection of oligodendrocyte progenitor cells (OPCs) expressing the NG2 or O4 markers via colocalization with the proliferation marker BrdU indicated their elevated levels in spinal cords of GA-treated mice. The mode of action of GA in this system is attributed to increased proliferation, differentiation, and survival of OPCs along the oligodendroglial maturation cascade and their recruitment into injury sites, thus enhancing repair processes in situ.

Serine phosphorylation proximal to its phosphotyrosine binding domain inhibits insulin receptor substrate 1 function and promotes insulin resistance.

Ser/Thr phosphorylation of insulin receptor substrate (IRS) proteins negatively modulates insulin signaling. Therefore, the identification of serine sites whose phosphorylation inhibit IRS protein functions is of physiological importance. Here we mutated seven Ser sites located proximal to the phosphotyrosine binding domain of insulin receptor substrate 1 (IRS-1) (S265, S302, S325, S336, S358, S407, and S408) into Ala. When overexpressed in rat hepatoma Fao or CHO cells, the mutated IRS-1 protein in which the seven Ser sites were mutated to Ala (IRS-1(7A)), unlike wild-type IRS-1 (IRS-1(WT)), maintained its Tyr-phosphorylated active conformation after prolonged insulin treatment or when the cells were challenged with inducers of insulin resistance prior to acute insulin treatment. This was due to the ability of IRS-1(7A) to remain complexed with the insulin receptor (IR), unlike IRS-1(WT), which underwent Ser phosphorylation, resulting in its dissociation from IR. Studies of truncated forms of IRS-1 revealed that the region between amino acids 365 to 430 is a main insulin-stimulated Ser phosphorylation domain. Indeed, IRS-1 mutated only at S408, which undergoes phosphorylation in vivo, partially maintained the properties of IRS-1(7A) and conferred protection against selected inducers of insulin resistance. These findings suggest that S408 and additional Ser sites among the seven mutated Ser sites are targets for IRS-1 kinases that play a key negative regulatory role in IRS-1 function and insulin action. These sites presumably serve as points of convergence, where physiological feedback control mechanisms, which are triggered by insulin-stimulated IRS kinases, overlap with IRS kinases triggered by inducers of insulin resistance to terminate insulin signaling.

Common inhibitory serine sites phosphorylated by IRS-1 kinases, triggered by insulin and inducers of insulin resistance.

The Insulin Receptor Substrate (IRS) proteins are key players in insulin signal transduction and are the best studied targets of the insulin receptor. Ser/Thr phosphorylation of IRS proteins negatively modulates insulin signaling; therefore, the identification of IRS kinases and their target Ser phosphorylation sites is of physiological importance. Here we show that in Fao rat hepatoma cells, the IkappaB kinase beta (IKKbeta) is an IRS-1 kinase activated by selected inducers of insulin resistance, including sphingomyelinase, ceramide, and free fatty acids. Moreover, IKKbeta shares a repertoire of seven potential target sites on IRS-1 with protein kinase C zeta (PKCzeta), an IRS-1 kinase activated both by insulin and by inducers of insulin resistance. We further show that mutation of these seven sites (Ser-265, Ser-302, Ser-325, Ser-336, Ser-358, Ser-407, and Ser-408) confers protection from the action of IKKbeta and PKCzeta when they are overexpressed in Fao cells or primary hepatocytes. This enables the mutated IRS proteins to better propagate insulin signaling. These findings suggest that insulin-stimulated IRS kinases such as PKCzeta overlap with IRS kinases triggered by inducers of insulin resistance, such as IKKbeta, to phosphorylate IRS-1 on common Ser sites.