The Thrombin Receptor Restricts Subventricular Zone Neural Stem Cell Expansion and Differentiation

Abstract

Thrombin is frequently increased in the CNS after injury yet little is known regarding its effects on neural stem cells. Here we show that the subventricular zone (SVZ) of adult mice lacking the high affinity receptor for thrombin, proteinase activated receptor 1 (PAR1), show increased numbers of Sox2+ and Ki-67+ self-renewing neural stem cells (NSCs) and Olig2+ oligodendrocyte progenitors. SVZ NSCs derived from PAR1-knockout mice, or treated with a PAR1 small molecule inhibitor (SCH79797), exhibited enhanced capacity for self-renewal in vitro, including increases in neurosphere formation and BrdU incorporation. PAR1-knockout SVZ monolayer cultures contained more Nestin, NG2+ and Olig2+ cells indicative of enhancements in expansion and differentiation towards the oligodendrocyte lineage. Cultures of NSCs lacking PAR1 also expressed higher levels of myelin basic protein, proteolipid protein and glial fibrillary acidic protein upon differentiation. Complementing these findings, the corpus callosum and anterior commissure of adult PAR1-knockout mice contained greater numbers of Olig2+ progenitors and CC1+ mature oligodendrocytes. Together these findings highlight PAR1 inhibition as a means to expand adult SVZ NSCs and to promote an increased number of mature myelinating oligodendrocytes in vivo that may be of particular benefit in the context of neural injury where PAR1 agonists such as thrombin are deregulated.

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

“…Myelin repair then occurs spontaneously when mice are returned to regular chow (Matsushima and Morell, 2001). As expected based on our recent report (Choi et al, 2018), the density of Olig2 ϩ cells in the corpus callosum of PAR1 Ϫ/Ϫ mice after 6 weeks on a cuprizone-free diet was 1.7-fold greater than that of age-matched controls (n ϭ 4, df ϭ 8, t ϭ 5.38, Enhanced remyelination after lysolecithin demyelination in PAR1 knock-out mice is accompanied by increases in astroglial prorepair signatures. a, Immunofluorescence for GFAP (cyan) as a marker of astrogliosis showed higher levels in remyelinating lesions of PAR1 Ϫ/Ϫ mice at 28 dpi ( p ϭ 0.04).…”

“…Mice genetically deficient in PAR1 (PAR1 Ϫ/Ϫ , B6.129S4-F2r tm1Ajc /J, #002862) have been back crossed to C57BL/6J mice (#000664) for Ͼ50 generations (Burda et al, 2013;Radulovic et al, 2016;Choi et al, 2018). All mice were obtained from The Jackson Laboratory.…”

“…Although the potential influence of a developmental benefit, including differences in myelin aging as a result of PAR1 knock-out cannot be fully ruled out (Yoon et al, 2015;Choi et al, 2018), beneficial effects on the process of remyelination are suggested by several observations. First, whereas lysolecithin and cuprizone caused equal loss of mature myelinating cells and MBP across genotypes, signs of myelin replacement, including increased numbers of mature oligodendrocytes were greater in PAR1 knock-outs.…”

“…We previously showed that in excess serine proteases such thrombin or kallikrein 6 (Klk6) actively suppress oligodendrocyte differentiation, including process outgrowth and stability, and production of the major myelin proteins myelin basic protein (MBP) and proteolipid protein (PLP) (Scarisbrick et al, 2002;Burda et al, 2013). We discovered that these myelin suppressive effects are governed by their ability for cleavage-induced activation of a G-protein-coupled receptor referred to as the thrombin receptor or Protease Activated Receptor 1 (PAR1) (Burda et al, 2013;Choi et al, 2018). Through this mechanism, serine proteases signal in a hormone-like fashion, effectively communicating information to a cell about the extracellular microenvironment.…”

“…PAR1 plays critical roles in myelin development with PAR1 knock-out mice showing an accelerated pattern of myelin development and higher numbers of myelinating cells in the adult brain and spinal cord (Yoon et al, 2015;Choi et al, 2018). Moreover, PAR1 knock-out OPCs differentiate more readily in cell culture (Yoon et al, 2015).…”