Blocking the Thrombin Receptor Promotes Repair of Demyelinated Lesions in the Adult Brain

Abstract

Myelin loss limits neurological recovery and myelin regeneration and is critical for restoration of function. We recently discovered that global knockout of the thrombin receptor, also known as Protease Activated Receptor 1 (PAR1), accelerates myelin development. Here we demonstrate that knocking out PAR1 also promotes myelin regeneration. Outcomes in two unique models of myelin injury and repair, that is lysolecithin or cuprizone-mediated demyelination, showed that PAR1 knockout in male mice improves replenishment of myelinating cells and remyelinated nerve fibers and slows early axon damage. Improvements in myelin regeneration in PAR1 knockout mice occurred in tandem with a skewing of reactive astrocyte signatures toward a prorepair phenotype. In cell culture, the promyelinating effects of PAR1 loss of function are consistent with possible direct effects on the myelinating potential of oligodendrocyte progenitor cells (OPCs), in addition to OPC-indirect effects involving enhanced astrocyte expression of promyelinating factors, such as BDNF. These findings highlight previously unrecognized roles of PAR1 in myelin regeneration, including integrated actions across the oligodendrocyte and astroglial compartments that are at least partially mechanistically linked to the powerful BDNF-TrkB neurotrophic signaling system. Altogether, findings suggest PAR1 may be a therapeutically tractable target for demyelinating disorders of the CNS.

Critical role for astrocyte NAD+ glycohydrolase in myelin injury and regeneration

“…Although saturated fat inhibited markers of differentiation in vitro, it was not oligotoxic, suggesting a more complex mechanism at play in vivo (4). Considering the emerging role of astrocytes in the pathology of various neurological disorders (9,11,23) and in the CNS following chronic HFD (7,8,24), along with their known essential metabolic and functional links with oligodendrocytes (25,26), we decided to address the possibility of indirect effects of HFD mediated by astrocytes ( Figure 1A). NAD + is a key mediator of cellular energy status and is involved in mitochondrial function and quality control processes primarily through the activity of sirtuins that require NAD + as a cofactor (19,20,27).…”

“…Importantly, a significant reduction in 4HNE was found in the HFD-CD38ci mice compared to HFD-wildtype mice in the dorsal column and ventral spinal cord (grey and white matter). Superoxide dismutase 2 (SOD2) is a mitochondrial antioxidant that can protect from high fat-induced changes in oxidative stress and metabolism (33), while brain-derived neurotrophic factor (BDNF) is permissive to myelination (23) and S100a10 is a recently defined pro-repair astrocyte maker (9). Next, our histological findings were further substantiated by observed gene expression increases in SOD2 (F (1, 8) = 7.721, p=0.024 ; Figure 3D) and pro-repair astrocyte markers genes BDNF(F (1, 8) = 5.685, p= 0.044; Figure 3E) and S100a10 (F (1, 8) = 24.73, p= 0.0011; Figure F) in the CD38ci mice, as determined by qRT-PCR.…”

“…Immunohistochemical analysis of focal demyelinated lesions utilized overall neuropathological changes (hematoxylin and eosin) along with myelin loss (based on MBP staining) to determine the lesion core, while the lesion rim was defined as the region encompassing the entire border of neuroinflammatory markers (GFAP, IBA1) (70). Lesion areas between 50,000 -180,000 µm2 were included in the analysis based on our previous studies using this model (23,70). Analyses were performed with experimenter blinded to treatment group by assigning a random animal number.…”

“…Spinal cords were fixed in 4% paraformaldehyde, processed, paraffin-embedded, and cut to 6 µm for all immunoperoxidase and immunofluorescence procedures, as described previously (4,23). When necessary (all antibodies except GFAP and MBP),…”

“…Primary mixed glial cultures were prepared from P0-3 mice, as previously described (4,23). In brief, dissected cerebral cortices were dissociated and grown in DMEM (Fisher 11960-044) with 1 mM sodium pyruvate (Corning 11360070), 20 mM HEPES (Gibco 15630-080), 100 U/ml PenStrep (Life Technologies 15140122), 10% fetal bovine serum (FBS, Atlanta 337 Biologicals S11150), and 5 µg/ml insulin.…”