Baudouin, L. | Search bioRxiv

bioRxiv2024

Pharmacogenomic screening identifies and repurposes leucovorin and dyclonine as pro-oligodendrogenic compounds in brain repair

Oligodendrocytes are the myelin-forming cells of the central nervous system (CNS), with oligodendroglial pathologies leading to strong disabilities, from early preterm-birth brain injury (PBI) to adult multiple sclerosis (MS). No medication presenting convincing repair capacity in humans has been approved for these pathologies so far. Here, we present a pharmacogenomic approach leading to the identification of small bioactive molecules with a large pro-oligodendrogenic activity, selected through an expert curation scoring strategy (OligoScore) of their large impact on transcriptional programs controlling oligodendrogenesis and (re)myelination. We demonstrate the pro-oligodendrogenic activity of these compounds in vitro, using neural and oligodendrocyte progenitor cell (OPC) cultures, as well as ex vivo, using organotypic cerebellar explant cultures. Focusing on the two most promising molecules, i.e. leucovorin and dyclonine, we tested their therapeutic efficacy using a mouse model of neonatal chronic hypoxia, which faithfully mimics aspects of PBI. In this model, both compounds promoted proliferation and oligodendroglial fate acquisition from neural stem/progenitor cells, with leucovorin also promoting their differentiation. We extended these findings to an adult focal de/remyelination mouse MS model, in which both compounds improved lesion repair by promoting OPC differentiation while maintaining the pool of OPCs, and in parallel, by accelerating the transition from pro-inflammatory to pro-regenerative microglial profiles and myelin debris clearance. This study paves the way for clinical trials aimed at repurposing these FDA-approved compounds to treat myelin pathologies such as PBI and MS. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/607135v1_ufig1.gif" ALT="Figure 1"> View larger version (46K): org.highwire.dtl.DTLVardef@83031eorg.highwire.dtl.DTLVardef@141d812org.highwire.dtl.DTLVardef@1d0a31corg.highwire.dtl.DTLVardef@848c67_HPS_FORMAT_FIGEXP M_FIG C_FIG Pharmacogenomic screening identifies and repurposes dyclonine and leucovorin as pro-oligodendrogenic and pro-myelinating compounds. Schematics of the pharmacogenomic approach leading to the identification of small bioactive molecules (compounds) with potential pro-oligodendrogenic activity, followed by the in vitro validation of the top compounds using neural and oligodendrocyte progenitor cell (OPC) cultures as well as organotypic cerebellar explants. The therapeutic efficacy of the top two compounds, leucovorin and dyclonine, both approved by the Food and Drug Administration (FDA), was assessed in vivo using two clinically relevant mouse models of myelin pathologies. In the neonatal hypoxia mouse model, mimicking some aspects of preterm brain injury, both leucovorin and dyclonine promoted neural stem cell (NSC) differentiation into OPCs and OPC proliferation, with leucovorin additionally restoring the density of myelinating OLs found in normoxic conditions. In an adult focal de/remyelination mouse model of multiple sclerosis, both compounds significantly improved lesion repair in adult mice by promoting OPC differentiation while preserving the pool of OPCs, and by accelerating myelin debris clearance and shifting microglia from pro-inflammatory to pro-regenerative profiles.

neuroscience↗

bioRxiv2023

PAK1 and NF2/Merlin jointly drive myelination by remodeling actin cytoskeleton in oligodendrocytes

In the central nervous system (CNS), myelin formation by oligodendrocytes (OLs) relies on actin dynamics. Actin polymerization supports the ensheathment step, when the OL process contacts the axon, while a drastic shift to actin depolymerization is required to enable the following step of wrapping and expansion of myelin membranes. The molecular mechanisms triggering this switch, essential for proper myelination, have yet to be elucidated. Here, we identify P21-activated kinase 1 (PAK1) as a major regulator of actin depolymerization in OLs. We show that PAK1 accumulates in OLs in a kinase inhibited form, triggering actin disassembly and, consequently, myelin expansion. Remarkably, we identify NF2/Merlin as an endogenous inhibitor of PAK1 by proteomics analysis of its binding partners. We found that Nf2 knockdown in OLs results in PAK1 activation and impairs myelin formation, and that pharmacological inhibition of PAK1 in Nf2-knockdown OLs rescues these defects. Moreover, we demonstrate that modulating PAK1 activity in OLs controls myelin expansion and provide compelling evidence indicating that specific Pak1 loss-of-function in oligodendroglia stimulates the thickening of myelin sheaths in vivo. Overall, our data indicate that PAK1-NF2/Merlin duo plays a key role in actin cytoskeleton remodeling in OLs, required for proper myelin formation. These findings have broad mechanistic and therapeutic implications for demyelinating diseases and neurodevelopmental disorders. SignificanceRemodeling actin cytoskeleton plays a crucial role in myelin formation by oligodendrocytes (OLs). Recent studies have shown that expansion and wrapping of myelin membranes around axons depends on actin depolymerization. However, the molecular mechanisms triggering this key step in myelination are not fully elucidated. Using genetic and pharmacological tools as well as proteomics analyses, we found that PAK1 (P21 Activated Kinase 1) kinase activity is maintained inhibited by NF2/Merlin in OLs to allow actin depolymerization and, consequently, myelin membrane expansion. Pak1 loss-of-function in OLs leads to an increase in myelin thickness in the white matter of adult mice, confirming the role of PAK1 inactivation in myelin membrane expansion.

neuroscience↗