The heparan sulfate proteoglycan agrin contributes to barrier properties of mouse brain endothelial cells by stabilizing adherens junctions.

Barrier characteristics of brain endothelial cells forming the blood-brain barrier (BBB) are tightly regulated by cellular and acellular components of the neurovascular unit. During embryogenesis, the accumulation of the heparan sulfate proteoglycan agrin in the basement membranes ensheathing brain vessels correlates with BBB maturation. In contrast, loss of agrin deposition in the vasculature of brain tumors is accompanied by the loss of endothelial junctional proteins. We therefore wondered whether agrin had a direct effect on the barrier characteristics of brain endothelial cells. Agrin increased junctional localization of vascular endothelial (VE)-cadherin, ß-catenin, and zonula occludens-1 (ZO-1) but not of claudin-5 and occludin in the brain endothelioma cell line bEnd5 without affecting the expression levels of these proteins. This was accompanied by an agrin-induced reduction of the paracellular permeability of bEnd5 monolayers. In vivo, the lack of agrin also led to reduced junctional localization of VE-cadherin in brain microvascular endothelial cells. Taken together, our data support the notion that agrin contributes to barrier characteristics of brain endothelium by stabilizing the adherens junction proteins VE-cadherin and ß-catenin and the junctional protein ZO-1 to brain endothelial junctions.

Schwann cell-like differentiation by adult oligodendrocyte precursor cells following engraftment into the demyelinated spinal cord is BMP-dependent.

The development of remyelinating strategies designed to enhance recruitment and differentiation of endogenous precursor cells available to a site of demyelination in the adult spinal cord will require a fundamental understanding of the potential for adult spinal cord precursor cells to remyelinate as well as an insight into epigenetic cues that regulate their mobilization and differentiation. The ability of embryonic and postnatal neural precursor cell transplants to remyelinate the adult central nervous system is well documented, while no transplantation studies to date have examined the remyelinating potential of adult spinal-cord-derived oligodendrocyte precursor cells (adult OPCs). In the present study, we demonstrate that, when transplanted subacutely into spinal ethidium bromide/X-irradiated (EB-X) lesions, adult OPCs display a limited capacity for oligodendrocyte remyelination. Interestingly, the glia-free environment of EB lesions promotes engrafted adult OPCs to differentiate primarily into cells with immunophenotypic and ultrastructural characteristics of myelinating Schwann cells (SCs). Astrocytes modulate this potential, as evidenced by the demonstration that SC-like differentiation is blocked when adult OPCs are co-transplanted with astrocytes. We further show that inhibition of bone morphogenetic protein (BMP) signaling through noggin overexpression by engrafted adult OPCs is sufficient to block SC-like differentiation within EB-X lesions. Present data suggest that the macroglial-free environment of acute EB lesions in the ventrolateral funiculus is inhibitory to adult spinal cord-derived OPC differentiation into remyelinating oligodendrocytes, while the presence of BMPs and absence of noggin promotes SC-like differentiation, thereby unmasking a surprising lineage fate for these cells.

Functional considerations of stem cell transplantation therapy for spinal cord repair.

Stem cells hold great promise for therapeutic repair after spinal cord injury (SCI). This review compares the current experimental approaches taken towards a stem cell-based therapy for SCI. It critically evaluates stem cell sources, injury paradigms, and functional measurements applied to detect behavioral changes after transplantation into the spinal cord. Many of the documented improvements do not exclusively depend on lineage-specific cellular differentiation. In most of the studies, the functional tests used cannot unequivocally demonstrate how differentiation of the transplanted cells contributes to the observed effects. Standardized cell isolation and transplantation protocols could facilitate the assessment of the true contribution of various experimental parameters on recovery. We conclude that at present embryonic stem (ES)-derived cells hold the most promise for therapeutic utility, but that non-neural cells may ultimately be optimal if the mechanism of possible transdifferentiation can be elucidated.

Consequences of noggin expression by neural stem, glial, and neuronal precursor cells engrafted into the injured spinal cord.

Bone morphogenetic proteins (BMPs) are a large class of secreted factors, which serve as modulators of development in multiple organ systems, including the CNS. Studies investigating the potential of stem cell transplantation for restoration of function and cellular replacement following traumatic spinal cord injury (SCI) have demonstrated that the injured adult spinal cord is not conducive to neurogenesis or oligodendrogenesis of engrafted CNS precursors. In light of recent findings that BMP expression is modulated by SCI, we hypothesized that they may play a role in lineage restriction of multipotent grafts. To test this hypothesis, neural stem or precursor cells were engineered to express noggin, an endogenous antagonist of BMP action, prior to transplantation or in vitro challenge with recombinant BMPs. Adult rats were subjected to both contusion and focal ischemic SCI. One week following injury, the animals were transplanted with either EGFP- or noggin-expressing neural stem or precursor cells. Results demonstrate that noggin expression does not antagonize terminal astroglial differentiation in the engrafted stem cells. Furthermore, neutralizing endogenous BMP in the injured spinal cord significantly increased both the lesion volume and the number of infiltrating macrophages in injured spinal cords receiving noggin-expressing stem cell grafts compared with EGFP controls. These data strongly suggest that endogenous factors in the injured spinal microenvironment other than the BMPs restrict the differentiation of engrafted pluripotent neural stem cells as well as suggest other roles for BMPs in tissue protection in the injured CNS.

Functional recovery in traumatic spinal cord injury after transplantation of multineurotrophin-expressing glial-restricted precursor cells.

Demyelination contributes to the physiological and behavioral deficits after contusive spinal cord injury (SCI). Therefore, remyelination may be an important strategy to facilitate repair after SCI. We show here that rat embryonic day 14 spinal cord-derived glial-restricted precursor cells (GRPs), which differentiate into both oligodendrocytes and astrocytes, formed normal-appearing central myelin around axons of cultured DRG neurons and had enhanced proliferation and survival in the presence of neurotrophin 3 (NT3) and brain-derived neurotrophin factor (BDNF). We infected GRPs with retroviruses expressing the multineurotrophin D15A (with both BDNF and NT3 activities) and then transplanted them into the contused adult thoracic spinal cord at 9 d after injury. Expression of D15A in the injured spinal cord is five times higher in animals receiving D15A-GRP grafts than ones receiving enhanced green fluorescent protein (EGFP)-GRP or DMEM grafts. Six weeks after transplantation, the grafted GRPs differentiated into mature oligodendrocytes expressing both myelin basic protein (MBP) and adenomatus polyposis coli (APC). Ultrastructural analysis showed that the grafted GRPs formed morphologically normal-appearing myelin sheaths around the axons in the ventrolateral funiculus (VLF) of spinal cord. Expression of D15A significantly increased the percentage of APC+ oligodendrocytes of grafted GRPs (15-30%). Most importantly, 8 of 12 rats receiving grafts of D15A-GRPs recovered transcranial magnetic motor-evoked potential responses, indicating that conduction through the demyelinated VLF axons was restored. Such electrophysiological recovery was not observed in rats receiving grafts of EGFP-GRPs, D15A-NIH3T3 cells, or an injection of an adenovirus expressing D15A. Recovery of hindlimb locomotor function was also significantly enhanced only in the D15A-GRP-grafted animals at 4 and 5 weeks after transplantation. Therefore, combined treatment with neurotrophins and GRP grafts can facilitate functional recovery after traumatic SCI and may prove to be a useful therapeutic strategy to repair the injured spinal cord.