Molecular mechanisms of enhanced susceptibility to apoptosis in differentiating oligodendrocytes.

Several studies have shown that the progression of oligodendrocyte progenitors along the lineage correlates with increased susceptibility to death stimuli. The molecular basis of this phenomenon remains unclear. This study demonstrates that the protein levels of several proapoptotic molecules, including Bax, Bad (nonphosphorylated form), and certain caspase proforms, increase during oligodendrocyte development. In contrast, the steady-state levels of antiapoptotic molecules, such as Bcl2 and Bcl(XL), remain constant. This altered equilibrium between proapoptotic and antiapoptotic molecules correlates with increased cytochrome C in the cytosol. We conclude that, as oligodendrocytes mature, their susceptibility to apoptosis increases because of a change in the balance between protective mechanisms and proapoptotic pathways. This suggests the possible existence of a death susceptibility program, which is intrinsic to differentiating oligodendrocyte progenitors.

p21cip1 is required for the differentiation of oligodendrocytes independently of cell cycle withdrawal.

Differentiation of most cell types requires both establishment of G1 arrest and the induction of a program related to achieving quiescence. We have chosen to study the differentiation of oligodendrocyte cells to determine the role of p27 and p21 in this process. Here we report that both p27 and p21 are required for the appropriate differentiation of these cells. p27 is required for proper withdrawal from the cell cycle, p21 is not. Instead, p21 is required for the establishment of the differentiation program following growth arrest. Similar observations were made in vivo. We show that p21-/- cells withdraw from the cell cycle similar to wild-type cells; however, early in animal life, the brain is hypomyelinated, inferring that the loss of p21 delayed myelination in the cerebellum. We found that we could complement or bypass the differentiation failure in p21-/- cells with either PD98059, an inhibitor of Mek1, or by transducing them with a tat-p16ink4a protein. We concluded that the two cdk inhibitors serve non-redundant roles in this program of differentiation, with p27 being responsible for arrest and p21 having a function in differentiation independent of its ability to control exit from the cell cycle.

Morphological differentiation of oligodendrocytes requires activation of Fyn tyrosine kinase.

In the central nervous system, myelination of axons occurs when oligodendrocyte progenitors undergo terminal differentiation and initiate process formation and axonal ensheathment. Although it is hypothesized that neuron-oligodendrocyte contact initiates this process, the molecular signals are not known. Here we find that Fyn tyrosine kinase activity is upregulated very early during oligodendrocyte progenitor cell differentiation. Concomitant with this increase is the appearance of several tyrosine phosphorylated proteins present only in differentiated cells. The increased tyrosine kinase activity is specific to Fyn, as other Src family members are not active in oligodendrocytes. To investigate the function of Fyn activation on differentiation, we used Src family tyrosine kinase inhibitors, PP1 and PP2, in cultures of differentiating oligodendrocyte progenitors. Treatment of progenitors with these compounds prevented activation of Fyn and reduced process extension and myelin membrane formation. This inhibition was reversible and not observed with related inactive analogues. A similar effect was observed when a dominant negative Fyn was introduced in progenitor cells. These findings strongly suggest that activation of Fyn is an essential signaling component for the morphological differentiation of oligodendrocytes.

Ectopic expression of p27Kip1 in oligodendrocyte progenitor cells results in cell-cycle growth arrest.

Oligodendrocyte differentiation is a complex process believed to be controlled by an intrinsic mechanism associated with cell-cycle arrest. Recently, the cell-cycle inhibitor protein p27 Kip1 has been proposed as a key element in causing growth arrest of oligodendrocyte precursor cells. To investigate the effects of p27 upon oligodendrocyte cell development, we have introduced the p27 cDNA in oligodendrocyte progenitor cells using an adenovirus vector. Progenitor cells normally express low levels of p27. After adenoviral infection and p27 overexpression, progenitor cells were able to undergo cell-cycle arrest, even in the presence of strong mitogens. The effects of p27 were shown to be directly upon cyclin-dependent kinase-2 (CDK2), the protein kinase complex responsible for G1/S transition, as immunodepletion of oligodendrocyte extracts of p27 protein resulted in the activation of CDK2 activity. However, cells that became growth arrested owing to infection with p27 adenovirus did not display conventional oligodendrocyte differentiation markers, such as O4 or O1. Taken together, these data provide mechanistic evidence indicating that p27 is primarily involved in oligodendroglial progenitor proliferation by inhibiting CDK2 activity and inducing oligodendrocyte cell-cycle arrest.

Transplanted oligodendrocyte progenitor cells expressing a dominant-negative FGF receptor transgene fail to migrate in vivo.

The proliferation, migration, survival, and differentiation of oligodendrocyte progenitor cells, precursors to myelin-forming oligodendrocytes in the CNS, are controlled by a number of polypeptide growth factors in vitro. The requirement and roles for individual factors in vivo, however, are primarily unknown. We have used a cell transplantation approach to examine the role of fibroblast growth factor (FGF) in oligodendrocyte development in vivo. A dominant-negative version of the FGF receptor-1 transgene was introduced into oligodendrocyte progenitors in vitro, generating cells that were nonresponsive to FGF but responsive to other mitogens. When transplanted into the brains of neonatal rats, mutant cells were unable to migrate and remained within the ventricles. These results suggest a role for FGF signaling in establishing a motile phenotype for oligodendrocyte progenitor cell migration in vivo and illustrate the utility of a somatic cell mutagenesis approach for the study of gene function during CNS development in vivo.

Thrombospondin promotes process outgrowth in neurons from the peripheral and central nervous systems.

Thrombospondin (TSP) is a prominent constituent of the extracellular matrix of the developing nervous system. We have examined the effects of TSP on the morphological differentiation of neurons. In short-term cultures (less than or equal to 24 hr) of embryonic rat sympathetic neurons, TSP stimulated neurite outgrowth, causing significant increase in the number of processes and their length. Similar effects were observed in cultures of rat dorsal root ganglion, hippocampal, and cerebral cortical neurons. Moreover, in cultures of central neurons, TSP was more effective than laminin in enhancing process extension. Analysis of long-term (5-7 days) cultures of sympathetic neurons indicated that processes formed in the presence of TSP had the cytochemical characteristics of axons. Thus, TSP can influence neuronal development by selectively enhancing axonal growth. The neurite-promoting region of the molecule was identified using a panel of monoclonal antibodies targeted to different regions of the protein. Process outgrowth could be totally inhibited with antibody A4.1, which recognizes the stalk region of TSP. These data suggest that the neurite-promoting activity is localized to a single region of the TSP molecule.

Phosphorylation of smooth muscle myosin by type II Ca2+/calmodulin-dependent protein kinase.

Brain type II Ca2+/calmodulin-dependent protein kinase was found to phosphorylate smooth muscle myosin, incorporating maximally approximately 2 mol of phosphoryl per mol of myosin, exclusively on the 20,000 dalton light chain subunit. After maximal phosphorylation of myosin or the isolated 20,000 dalton light chain subunit by myosin light chain kinase, the addition of type II Ca2+/calmodulin-dependent protein kinase led to no further incorporation indicating the two kinases phosphorylated a common site. This conclusion was supported by two dimensional mapping of tryptic digests of myosin phosphorylated by the two kinases. By phosphoamino acid analysis the phosphorylated residue was identified as a serine. The phosphorylation by type II Ca2+/calmodulin-dependent protein kinase of myosin resulted in enhancement of its actin-activated Mg2(+)-ATPase activity. Taken together, these data strongly support the conclusion that type II Ca2+/calmodulin-dependent protein kinase phosphorylates the same amino acid residue on the 20,000 dalton light chain subunit of smooth muscle myosin as is phosphorylated by myosin light chain kinase and suggest an alternative mechanism for the regulation of actin-myosin interaction.