Randall D. McKinnon

Regulation of oligodendrocyte development and CNS myelination by growth factors: prospects for therapy of demyelinating disease.

Multiple sclerosis (MS), the most common neurological disorder diagnosed in young adults, is characterized by autoimmune demyelination in the central nervous system (CNS). Promotion of remyelination in the brain and spinal cord is a potential strategy for therapeutic intervention in MS and other demyelinating diseases. Recent studies have shown that the development of oligoden‐drocytes, the myelin‐forming cells of the CNS, is extensively controlled by growth factors. These factors regulate the proliferation, migration, differentiation, survival and regeneration of oligoden‐drogtial cells and the synthesis of myelin, and often interact in a complex manner. Moreover, insulin‐like growth factor 1 (IGF‐I) has proven effective for therapy of experimental autoimmune encephalomyelitis (EAE), an animal model of autoimmune demyelination. In this review we summarize recent findings on the regulation of oligodendrocyte development and CNS myelination by growth factors, and discuss these findings in the context of possible clinical application for the therapy of neurological disease in humans.

Distinct roles for PI3K in proliferation and survival of oligodendrocyte progenitor cells

Phosphoinositol 3‐kinase (PI3K) is a downstream effector for multiple ligand‐activated receptors and modulates cell responses through activation of its target protein kinase B (Akt). We examined the roles of PI3K‐Akt signaling in a primary glial (oligodendrocyte) progenitor cell culture system that is ligand‐dependent for cell proliferation, survival, and prevention of differentiation. We demonstrate that PI3K and Akt (Ser‐473 phosphorylation) are activated in response to platelet‐derived growth factor but not basic fibroblast growth factor‐2 (FGF2) and that distinct forms of PI3K are activated in early progenitors and later‐maturation pro‐oligodendroblasts as identified by their sensitivity to wortmannin. By establishing conditions to examine effects on cell proliferation and survival independently, we demonstrate that PI3K is necessary for a full mitogenic response and that PI3K is also necessary for early progenitor survival. Our results therefore demonstrate that PI3K‐Akt signaling independently regulates proliferation and survival, that the form of PI3K is distinct in early progenitors and pro‐oligodendroblasts, and that FGF2 does not activate this pathway in either primary glial cell population. J. Neurosci. Res. 62:336–345, 2000.

The Homeobox Transcription Factor Even-skipped Regulates Netrin-Receptor Expression to Control Dorsal Motor-Axon Projections in Drosophila

Homeobox transcription-factor codes control motor-neuron subtype identity and dorsal versus ventral axon guidance in both vertebrate and invertebrate nervous systems; however, the specific axon guidance-receptors that are regulated by these transcription factors to control pathfinding are poorly defined. In Drosophila, the Even-skipped (Eve) transcription factor specifies dorsal motor-axon projection through the regulation of unidentified guidance molecules. The Netrins and their attractive and repulsive receptors DCC and Unc-5, respectively, define important conserved cue and receptor families that control growth-cone guidance. In Drosophila, the Netrins and frazzled (the fly homolog of DCC) contribute to motor-axon guidance. Here, using genetics and single-cell mRNA-expression analysis, we show that expression and requirement of different Netrin receptor combinations correlate with distinct dorsal and ventral motor-axon projections in Drosophila. Mis-expression of eve dorsalizes ventral axons in part through the upregulation of Unc-5, whereas loss of eve function in two dorsally projecting motor neurons results in aberrant axon projections and a failure to express Unc-5. Our results support a functional link between the expression of distinct Netrin receptor combinations and the transcriptional control of dorsal motor-axon guidance.

PDGF α-Receptor Signal Strength Controls an RTK Rheostat That Integrates Phosphoinositol 3′-Kinase and Phospholipase Cγ Pathways during Oligodendrocyte Maturation

Receptors with tyrosine kinase activity (RTKs) control tissue growth and development in metazoans. How they generate cell-specific responses remains essentially unknown; one model proposes that distinct RTKs activate different second-messenger pathways, whereas a second proposes that all RTKs deliver a generic “go” signal to these pathways that is uniquely interpreted by downstream, cell-specific response competence factors. We examine pathway activation and pathway-specific responses downstream of PDGFα receptors, whose expression in the developing CNS identifies oligodendrocyte progenitor cells (OPCs) and whose activation controls OPC proliferation, migration, survival, and maturation. PDGFRα-null mice die in utero, and OPCs that emerge before their demise have migration and proliferation defects and rapidly differentiate into postmitotic oligodendrocytes in vitro. OPCs from hemizygous mice also undergo precocious differentiation, indicating a role for PDGFRα gene dosage in timing OPC maturation. The rescue of PDGFRα-null OPCs with PDGFRα transgenes revealed specific roles for the phosphatidylinositol 3-kinase (PI3K) and phospholipase Cγ (PLCγ) pathways and a distinct ligand concentration dependence. Activation of the PI3K pathway is required for PDGFRα-induced migration, whereas activation of both PI3K and PLCγ are required for PDGFRα-induced proliferation. For proliferation, PI3K activation is required at low ligand concentration, whereas PLCγ is required at high signal strength. Dose-response studies further demonstrate that PDGFRα activates PI3K at low ligand concentrations, whereas PLCγ is activated at high signal strength. Thus, PDGFRα signaling acts like a rheostat rather than generic ON switch, with signal strength dictating pathway activation during OPC maturation.

From Stem Cells to Oligodendrocytes: Prospects for Brain Therapy

Multiple sclerosis is an autoimmune disease that destroys myelin-forming oligodendrocytes of the CNS. While the damage can be partially controlled using anti-inflammatory cytokines and steroids, endogenous repair is insufficient to replace lost cells. Until now cell replenishment (transplant therapy) has been viewed as unlikely to succeed due to allograft rejection in this sensitized immune environment. However, advances in stem cell biology give new hope for deriving patient-specific, autologous oligodendrocytes which may tip the balance to favor repair. The challenge will be to engineer these cells to respond to cues that can target their migration into lesions for brain and spinal cord repair.

An inhibitor of cell proliferation associated with adhesion formation is suppressed by N,O-carboxymethyl chitosan.

Surgical adhesions are a major cause of morbidity and mortality. The ideal barrier agent will both minimize adhesions and provide a milieu for the regeneration of the mesothelium lining of the abdominal and thoracic cavities. N,O-Carboxymethylchitosan (NOCC), a derivation of chitin that markedly reduces adhesions, may function to modulate intracellular signals such as growth factors and cytokines in the inflammatory exudate. Since transforming growth factor-beta is implicated in the fibrotic process, we investigated the possibility that NOCCs effects on adhesion formation reflects a modulation of TGF-beta activity. Using a biological assay for inhibition of cell proliferation to detect TGF-beta activity, we demonstrate that NOCC suppresses the levels of an inhibitor of cell proliferation released into serum and peritoneal exudates after cecal abrasion in the rat. However, this activity was distinct from known forms of TGF-beta as determined using both TGF-beta-neutralizing antisera and a TGF-beta-resistant cell proliferation assay. Thus at least one potential effect of NOCC involves a mechanism distinct from TGF-beta inhibition.

Adjunctive MSCs enhance myelin formation by xenogenic oligodendrocyte precursors transplanted in the retina.

Adjunctive MSCs enhance myelin formation by xenogenic oligodendrocyte precursors transplanted in the retina

Teratogenic potential in cultures optimized for oligodendrocyte development from mouse embryonic stem cells.

We describe a rapid and efficient 5-step program of defined factors for the genesis of brain myelin-forming oligodendrocytes (OLs) from embryonic stem cells (ESCs). The OLs emerge on the same time frame in vitro as seen in vivo. Factors promoting neural induction (retinoids, noggin) are required, while exogenous Sonic hedgehog is not. In contrast we were unable to generate OLs by trans-differentiation of ethically neutral mesenchymal stem cells, indicating a requirement for cis-differentiation via neural ectoderm for OL genesis. In the ESC-derived cultures, our optimized protocol generated a mixed population with 49% O4(+), Olig2(+) OL lineage cells. These cultures also retained pluripotential markers including Oct4, and an analysis of embryoid body formation in vitro, and allogeneic grafts in vivo, revealed that the ESC-derived cultures also retained teratogenic cells. The frequency of embryoid body formation from terminal differentiated OL cultures was 0.001%, 100-fold lower than that from ESCs. Our results provide the first quantitative measurement of teratogenicity in ESC-derived, exhaustively differentiated allogeneic grafts, and demonstrate the unequivocal need to purify ESC-derived cells in order to generate a safe population for regenerative therapy.

Prevention of postoperative adhesions with the chitin derivative N-O-carboxymethylchitosan

The ideal barrier agent for the prevention of surgical adhesions has remained elusive. We have examined the ability of a new hydrogel N‐O‐carboxymethylchitosan, a derivative of chitin with properties similar to the extracellular matrix, to prevent adhesions when applied topically to traumatized mesothelial surfaces. In two rodent adhesion models (pericardial and peritoneal), the application of N‐O‐carboxymethylchitosan significantly prevented or minimized the formation of scar and fibrosis. According to a scoring system from 0 to 3 (0 = no adhesions and 3 = severe dense adhesions), control groups in each model consistently produced severe dense adhesions (2.9 ± 0.2, 2.7 ± 0.3). All treated groups consistently scored less than 1.0, indicating minimal or no fibrosis. The differences between the control and treated groups were statistically significant (p < 0.05). Thus, the application of N‐O‐carboxymethylchitosan to traumatized mesothelial surfaces may have significant potential in the prevention of postoperative adhesion formation.

Stem Cell‐Derived Therapeutic Myelin Repair Requires 7% Cell Replacement

Embryonic stem cells (ESCs) hold great potential for therapeutic regeneration and repair in many diseases. However, many challenges remain before this can be translated into effective therapy. A principal and significant limit for outcome evaluations of clinical trials is to define the minimal graft population necessary for functional repair. Here we used a preclinical model for quantitative analysis of stem cell grafts, with wild‐type ESC grafted into myelin mutant shiverer hosts, to determine minimum graft levels for therapeutic benefit. Using a timed motor function test we identified three groups, including recipients indistinguishable from nongrafted shiverer controls (time [t] = 20.1 ± 1.1 seconds), mice with marginal improvement (t = 15.7 ± 1 seconds), and mice with substantial phenotype rescue (t = 5.7 ± 0.9 seconds). The motor function rescued chimeras also had a considerably extended life span (T50 > 128 days) relative to both shiverer (T50 = 108 days) and the nonrescued chimeras. Retrospective genotype analysis identified a strong correlation (r2 = 0.85) between motor function and ESC‐derived chimerism, with > 7% chimerism required for rescue in this murine model of central nervous system myelin pathology. These results establish the minimal levels of engraftment to anticipate therapeutic repair of a cell‐autonomous defect by cell transplant therapy.