Neuroprotective effect of lipopolysaccharides in a dual-hit rat pup model of preterm hypoxia-ischemia.

The combination of lipopolysaccharide (LPS) and hypoxia-ischemia (HI) has been used to model the brain injury sustained by sick pre-term infants in order to study the pathological conditions of diffuse white matter injury, which is a major cause of preterm morbidity. Prior studies have shown that the timing and dose of LPS administration will determine whether the injury is reduced or exacerbated. Here we show that administering a single injection of LPS (0.1 mg/kg) to postnatal-day-2 rat pups 14 h before inducing HI effectively protects the brain from HI-associated damage. We show that the LPS-treated HI rat pups have significantly less histopathology compared to the saline-treated HI rat pups. Apoptotic deaths were dramatically curtailed in both the neocortex and white matter when evaluated at 2 days of recovery. Microglial activation was reduced when the percentage of CD68+/Iba1+ cells was quantified in the neocortex of the LPS-treated vs the saline-treated HI rat pups. One mechanism through which LPS pre-treatment appears to be preventing injury is through the AKT-endothelial nitric oxide synthase (eNOS) pathway as LPS induced an increase in both the expression and phosphorylation of eNOS. Altogether these data show that the neocortex, as well as the white matter sustain damage after HI at this timepoint in forebrain development and that acutely activating the immune system can protect the brain from brain injury.

Cholesterol biosynthesis defines oligodendrocyte precursor heterogeneity between brain and spinal cord.

Brain and spinal cord oligodendroglia have distinct functional characteristics, and cell-autonomous loss of individual genes can result in different regional phenotypes. However, a molecular basis for these distinctions is unknown. Using single-cell analysis of oligodendroglia during developmental myelination, we demonstrate that brain and spinal cord precursors are transcriptionally distinct, defined predominantly by cholesterol biosynthesis. We further identify the mechanistic target of rapamycin (mTOR) as a major regulator promoting cholesterol biosynthesis in oligodendroglia. Oligodendroglia-specific loss of mTOR decreases cholesterol biosynthesis in both the brain and the spinal cord, but mTOR loss in spinal cord oligodendroglia has a greater impact on cholesterol biosynthesis, consistent with more pronounced deficits in developmental myelination. In the brain, mTOR loss results in a later adult myelin deficit, including oligodendrocyte death, spontaneous demyelination, and impaired axonal function, demonstrating that mTOR is required for myelin maintenance in the adult brain.

mTOR Signaling Regulates Metabolic Function in Oligodendrocyte Precursor Cells and Promotes Efficient Brain Remyelination in the Cuprizone Model.

In demyelinating diseases, such as multiple sclerosis, primary loss of myelin and subsequent neuronal degeneration throughout the CNS impair patient functionality. While the importance of mechanistic target of rapamycin (mTOR) signaling during developmental myelination is known, no studies have yet directly examined the function of mTOR signaling specifically in the oligodendrocyte (OL) lineage during remyelination. Here, we conditionally deleted Mtor from adult oligodendrocyte precursor cells (OPCs) using Ng2-CreERT in male adult mice to test its function in new OLs responsible for remyelination. During early remyelination after cuprizone-induced demyelination, mice lacking mTOR in adult OPCs had unchanged OL numbers but thinner myelin. Myelin thickness recovered by late-stage repair, suggesting a delay in myelin production when Mtor is deleted from adult OPCs. Surprisingly, loss of mTOR in OPCs had no effect on efficiency of remyelination after lysophosphatidylcholine lesions in either the spinal cord or corpus callosum, suggesting that mTOR signaling functions specifically in a pathway dysregulated by cuprizone to promote remyelination efficiency. We further determined that cuprizone and inhibition of mTOR cooperatively compromise metabolic function in primary rat OLs undergoing differentiation. Together, our results support the conclusion that mTOR signaling in OPCs is required to overcome the metabolic dysfunction in the cuprizone-demyelinated adult brain.SIGNIFICANCE STATEMENT Impaired remyelination by oligodendrocytes contributes to the progressive pathology in multiple sclerosis, so it is critical to identify mechanisms of improving remyelination. The goal of this study was to examine mechanistic target of rapamycin (mTOR) signaling in remyelination. Here, we provide evidence that mTOR signaling promotes efficient remyelination of the brain after cuprizone-mediated demyelination but has no effect on remyelination after lysophosphatidylcholine demyelination in the spinal cord or brain. We also present novel data revealing that mTOR inhibition and cuprizone treatment additively affect the metabolic profile of differentiating oligodendrocytes, supporting a mechanism for the observed remyelination delay. These data suggest that altered metabolic function may underlie failure of remyelination in multiple sclerosis lesions and that mTOR signaling may be of therapeutic potential for promoting remyelination.

Mechanistic Target of Rapamycin Regulates the Oligodendrocyte Cytoskeleton during Myelination.

During differentiation, oligodendrocyte precursor cells (OPCs) extend a network of processes that make contact with axons and initiate myelination. Recent studies revealed that actin polymerization is required for initiation of myelination whereas actin depolymerization promotes myelin wrapping. Here, we used primary OPCs in culture isolated from neonatal rat cortices of both sexes and young male and female mice with oligodendrocyte-specific deletion of mechanistic target of rapamycin (mTOR) to demonstrate that mTOR regulates expression of specific cytoskeletal targets and actin reorganization in oligodendrocytes during developmental myelination. Loss or inhibition of mTOR reduced expression of profilin2 and ARPC3, actin polymerizing factors, and elevated levels of active cofilin, which mediates actin depolymerization. The deficits in actin polymerization were revealed in reduced phalloidin and deficits in oligodendrocyte cellular branching complexity at the peak of morphologic differentiation and a delay in initiation of myelination. We further show a critical role for mTOR in expression and localization of myelin basic protein (Mbp) mRNA and MBP protein to the cellular processes where it is necessary at the myelin membrane for axon wrapping. Mbp mRNA transport deficits were confirmed by single molecule RNA FISH. Moreover, expression of the kinesin family member 1B, an Mbp mRNA transport protein, was reduced in CC1+ cells in the mTOR cKO and in mTOR inhibited oligodendrocytes undergoing differentiation in vitro These data support the conclusion that mTOR regulates both initiation of myelination and axon wrapping by targeting cytoskeletal reorganization and MBP localization to oligodendrocyte processes.SIGNIFICANCE STATEMENT Myelination is essential for normal CNS development and adult axon preservation and function. The mechanistic target of rapamycin (mTOR) signaling pathway has been implicated in promoting CNS myelination; however, there is a gap in our understanding of the mechanisms by which mTOR promotes developmental myelination through regulating specific downstream targets. Here, we present evidence that mTOR promotes the initiation of myelination through regulating specific cytoskeletal targets and cellular process expansion by oligodendrocyte precursor cells as well as expression and cellular localization of myelin basic protein.

Loss of Tuberous Sclerosis Complex1 in Adult Oligodendrocyte Progenitor Cells Enhances Axon Remyelination and Increases Myelin Thickness after a Focal Demyelination.

Although the mammalian target of rapamycin (mTOR) is an essential regulator of developmental oligodendrocyte differentiation and myelination, oligodendrocyte-specific deletion of tuberous sclerosis complex (TSC), a major upstream inhibitor of mTOR, surprisingly also leads to hypomyelination during CNS development. However, the function of TSC has not been studied in the context of remyelination. Here, we used the inducible Cre-lox system to study the function of TSC in the remyelination of a focal, lysolecithin-demyelinated lesion in adult male mice. Using two different mouse models in which Tsc1 is deleted by Cre expression in oligodendrocyte progenitor cells (OPCs) or in premyelinating oligodendrocytes, we reveal that deletion of Tsc1 affects oligodendroglia differently depending on the stage of the oligodendrocyte lineage. Tsc1 deletion from NG2+ OPCs accelerated remyelination. Conversely, Tsc1 deletion from proteolipid protein (PLP)-positive oligodendrocytes slowed remyelination. Contrary to developmental myelination, there were no changes in OPC or oligodendrocyte numbers in either model. Our findings reveal a complex role for TSC in oligodendrocytes during remyelination in which the timing of Tsc1 deletion is a critical determinant of its effect on remyelination. Moreover, our findings suggest that TSC has different functions in developmental myelination and remyelination.SIGNIFICANCE STATEMENT Myelin loss in demyelinating disorders such as multiple sclerosis results in disability due to loss of axon conductance and axon damage. Encouragingly, the nervous system is capable of spontaneous remyelination, but this regenerative process often fails. Many chronically demyelinated lesions have oligodendrocyte progenitor cells (OPCs) within their borders. It is thus of great interest to elucidate mechanisms by which we might enhance endogenous remyelination. Here, we provide evidence that deletion of Tsc1 from OPCs, but not differentiating oligodendrocytes, is beneficial to remyelination. This finding contrasts with the loss of oligodendroglia and hypomyelination seen with Tsc1 or Tsc2 deletion in the oligodendrocyte lineage during CNS development and points to important differences in the regulation of developmental myelination and remyelination.

Heterogeneity in oligodendroglia: Is it relevant to mouse models and human disease?

There are many lines of evidence indicating that oligodendrocyte progenitor cells and oligodendrocyte populations in the central nervous system (CNS) are heterogeneous based on their developmental origins as well as from morphological and molecular criteria. Whether these distinctions reflect functional heterogeneity is less clear and has been the subject of considerable debate. Recent findings, particularly from knockout mouse models, have provided new evidence for regional variations in myelination phenotypes, particularly between brain and spinal cord. These data raise the possibility that oligodendrocytes in these regions have different functional capacities and/or ability to compensate for loss of a specific gene. The goal of this review is to briefly revisit the evidence for oligodendrocyte heterogeneity and then to present data from transgenic and demyelinating mouse models suggesting functional heterogeneity in myelination, demyelination, and remyelination in the CNS and, finally, to discuss the implications of these findings for human diseases. © 2016 Wiley Periodicals, Inc.