A fluorescence microscopy-based protocol for volumetric measurement of lysolecithin lesion-associated de- and re-myelination in mouse brain

Lysolecithin injections into the white matter tracts of the central nervous system are a valuable tool to study remyelination, but evaluating the resulting demyelinating lesion size is challenging. Here, we present a protocol to consistently measure the volume of demyelination and remyelination in mice following brain lysolecithin injections. We describe serial sectioning of the lesion, followed by the evaluation of the demyelinated area in two-dimensional images. We then detail the computation of the volume using our own automated iPython script. For complete details on the use and execution of this profile, please refer to Bosch-Queralt et al. (2021)

Myelin insulation as a risk factor for axonal degeneration in autoimmune demyelinating disease

Axonal degeneration determines the clinical outcome of multiple sclerosis and is thought to result from exposure of denuded axons to immune-mediated damage. Therefore, myelin is widely considered to be a protective structure for axons in multiple sclerosis. Myelinated axons also depend on oligodendrocytes, which provide metabolic and structural support to the axonal compartment. Given that axonal pathology in multiple sclerosis is already visible at early disease stages, before overt demyelination, we reasoned that autoimmune inflammation may disrupt oligodendroglial support mechanisms and hence primarily affect axons insulated by myelin. Here, we studied axonal pathology as a function of myelination in human multiple sclerosis and mouse models of autoimmune encephalomyelitis with genetically altered myelination. We demonstrate that myelin ensheathment itself becomes detrimental for axonal survival and increases the risk of axons degenerating in an autoimmune environment. This challenges the view of myelin as a solely protective structure and suggests that axonal dependence on oligodendroglial support can become fatal when myelin is under inflammatory attack

Diet-dependent regulation of TGFβ impairs reparative innate immune responses after demyelination

Multiple sclerosis (MS) is one of the most common causes of chronic disability in young adults. In 85% of the cases, the disease starts with a relapsing-remitting course but, as age advances, the majority of patients enter a progressive phase of the disease characterized by neurological decline and brain atrophy. Treatments that delay, prevent or reverse this progression phase are an unmet need in MS research. The cause of progressive MS is not known, but remyelination failure may contribute. Hence, large efforts have been directed into identifying strategies to enhance endogenous remyelination, which can prevent neuronal death. Microglia are the immune cells of the central nervous system (CNS) and play a crucial role in orchestrating remyelination. With ageing, microglia do not respond adequately to myelin damage, leading to failed remyelination. Apart from ageing, clinical observations suggest that also obesity increases the risk of progression in MS. However, whether and how obesity might influence remyelination is not known. In this study, we use western diet (WD) to induce obesity in mice and investigate the impact of WD on microglia’s response to demyelination. With this, we aim to understand how obesity might affect the pro-regenerative functions of microglia. Since the metabolism of myelin-derived lipids by microglia is an essential step for successful remyelination, we further examine how WD changes the lipid composition of the plasma and brain and whether these changes have consequences on microglia’s response to demyelination. We find that WD consumption leads to impaired remyelination after toxin-induced demyelination due to deficient cholesterol efflux by microglia. Furthermore, we show that WD intake alters the lipid profile of the brain white and grey matter, is associated with modest microgliosis in the corpus callosum, and causes an increase in transforming growth factor-β (TGFβ) in the brain. Such excess TGFβ signalling leads to insufficient microglia response to damage and impaired cholesterol efflux, which ultimately prevents inflammation resolution and remyelination. By blocking TGFβ signalling or enhancing microglia activation through triggering-receptor expressed on myeloid cells 2 (TREM2), we could promote adequate microglia activation and successful resolution of damage in the CNS. Hence, we unravel a microglia immune checkpoint mechanism as a potential therapeutic target to promote a reparative inflammatory response after demyelinating injury. In conclusion, our study demonstrates that obesity leads to failed remyelination by disturbing the pro-regenerative functions of microglia. In addition, our findings expand the spectrum of potential therapeutic strategies to enhance endogenous remyelination

TREM2-dependent lipid droplet biogenesis in phagocytes is required for remyelination

Upon demyelinating injury, microglia orchestrate a regenerative response that promotes myelin repair, thereby restoring rapid signal propagation and protecting axons from further damage. Whereas the essential phagocytic function of microglia for remyelination is well known, the underlying metabolic pathways required for myelin debris clearance are poorly understood. Here, we show that cholesterol esterification in male mouse microglia/macrophages is a necessary adaptive response to myelin debris uptake and required for the generation of lipid droplets upon demyelinating injury. When lipid droplet biogenesis is defective, innate immune cells do not resolve, and the regenerative response fails. We found that triggering receptor expressed on myeloid cells 2 (TREM2)-deficient mice are unable to adapt to excess cholesterol exposure, form fewer lipid droplets, and build up endoplasmic reticulum (ER) stress. Alleviating ER stress in TREM2-deficient mice restores lipid droplet biogenesis and resolves the innate immune response. Thus, we conclude that TREM2-dependent formation of lipid droplets constitute a protective response required for remyelination to occur

Proteomic and lipidomic profiling of demyelinating lesions identifies fatty acids as modulators in lesion recovery

After demyelinating injury of the central nervous system, resolution of the mounting acute inflammation is crucial for the initiation of a regenerative response. Here, we aim to identify fatty acids and lipid mediators that govern the balance of inflammatory reactions within demyelinating lesions. Using lipidomics, we identify bioactive lipids in the resolution phase of inflammation with markedly elevated levels of n-3 polyunsaturated fatty acids. Using fat-1 transgenic mice, which convert n-6 fatty acids to n-3 fatty acids, we find that reduction of the n-6/n-3 ratio decreases the phagocytic infiltrate. In addition, we observe accelerated decline of microglia/macrophages and enhanced generation of oligodendrocytes in aged mice when n-3 fatty acids are shuttled to the brain. Thus, n-3 fatty acids enhance lesion recovery and may, therefore, provide the basis for pro regenerative medicines of demyelinating diseases in the central nervous system