Transient demyelination causes long-term cognitive impairment, myelin alteration and network synchrony defects.

In the adult brain, activity-dependent myelin plasticity is required for proper learning and memory consolidation. Myelin loss, alteration, or even subtle structural modifications can therefore compromise the network activity, leading to functional impairment. In multiple sclerosis, spontaneous myelin repair process is possible, but it is heterogeneous among patients, sometimes leading to functional recovery, often more visible at the motor level than at the cognitive level. In cuprizone-treated mouse model, massive brain demyelination is followed by spontaneous and robust remyelination. However, reformed myelin, although functional, may not exhibit the same morphological characteristics as developmental myelin, which can have an impact on the activity of neural networks. In this context, we used the cuprizone-treated mouse model to analyze the structural, functional, and cognitive long-term effects of transient demyelination. Our results show that an episode of demyelination induces despite remyelination long-term cognitive impairment, such as deficits in spatial working memory, social memory, cognitive flexibility, and hyperactivity. These deficits were associated with a reduction in myelin content in the medial prefrontal cortex (mPFC) and hippocampus (HPC), as well as structural myelin modifications, suggesting that the remyelination process may be imperfect in these structures. In vivo electrophysiological recordings showed that the demyelination episode altered the synchronization of HPC-mPFC activity, which is crucial for memory processes. Altogether, our data indicate that the myelin repair process following transient demyelination does not allow the complete recovery of the initial myelin properties in cortical structures. These subtle modifications alter network features, leading to prolonged cognitive deficits in mice.

Inhomogeneous Magnetization Transfer (ihMT) imaging in the acute cuprizone mouse model of demyelination/remyelination.

BACKGROUND: To investigate the association of ihMT (inhom signals with the demyelination and remyelination phases of the acute cuprizone mouse model in comparison with histology, and to assess the extent of tissue damage and repair from MRI data. METHODS: Acute demyelination by feeding 0.2% cuprizone for five weeks, followed by a four-week remyelination period was applied on genetically modified plp-GFP mice. Animals were scanned at different time points of the demyelination and remyelination phases of the cuprizone model using a multimodal MRI protocol, including ihMT T1D-filters, MPF (Macromolecular Proton Fraction) and R1 (longitudinal relaxation rate). For histology, plp-GFP (proteolipid protein - Green Fluorescent Protein) microscopy and LFB (Luxol Fast Blue) staining were employed as references for the myelin content. Comparison of MRI with histology was performed in the medial corpus callosum (mCC) and cerebral cortex (CTX) at two brain levels whereas ROI-wise and voxel-based analyses of the MRI metrics allowed investigating in vivo the spatial extent of myelin alterations. RESULTS: IhMT high-pass T1D-filters, targeted toward long T1D components, showed significant temporal variations in the mCC consistent with the effects induced by the cuprizone toxin. In addition, the corresponding signals correlated strongly and significantly with the myelin content assessed by GFP fluorescence and LFB staining over the demyelination and the remyelination phases. The signal of the band-pass T1D-filter, which isolates short T1D components, showed changes over time that were poorly correlated with histology, hence suggesting a sensitivity to pathological processes possibly not related to myelin. Although MPF was also highly correlated to histology, ihMT high-pass T1D-filters showed better capability to characterize the spatial-temporal patterns during the demyelination and remyelination phases of the acute cuprizone model (e.g., rostro-caudal gradient of demyelination in the mCC previously described in the literature). CONCLUSIONS: IhMT sequences selective for long T1D components are specific and sensitive in vivo markers of demyelination and remyelination and have successfully captured the spatially heterogeneous pattern of the demyelination and remyelination mechanisms in the cuprizone model. Interestingly, differences in signal variations between the ihMT high-pass and band-pass T1D-filter, suggest a sensitivity of the ihMT sequences targeted to short T1Ds to alterations other than those of myelin. Future studies will need to further address these differences by examining more closely the origin of the short T1D components and the variation of each T1D component in pathology.

T1D -weighted ihMT imaging - Part I. Isolation of long- and short-T1D components by T1D -filtering.

PURPOSE: To identify T1D -filtering methods, which can specifically isolate various ranges of T1D components as they may be sensitive to different microstructural properties. METHODS: Modified Bloch-Provotorov equations describing a bi-T1D component biophysical model were used to simulate the inhomogeneous magnetization transfer (ihMT) signal from ihMTRAGE sequences at high RF power and low duty-cycle with different switching time values for the dual saturation experiment: Δt = 0.0, 0.8, 1.6, and 3.2 ms. Simulations were compared with experimental signals on the brain gray and white matter tissues of healthy mice at 7T. RESULTS: The lengthening of Δt created ihMT high-pass T1D -filters, which efficiently eliminated the signal from T1D components shorter than 1 ms, while partially attenuating that of longer components (≥ 1 ms). Subtraction of ihMTR images obtained with Δt = 0.0 ms and Δt = 0.8 ms generated a new ihMT band-pass T1D -filter isolating short-T1D components in the 100-µs to 1-ms range. Simulated ihMTR values in central nervous system tissues were confirmed experimentally. CONCLUSION: Long- and short-T1D components were successfully isolated with high RF power and low duty-cycle ihMT filters in the healthy mouse brain. Future studies should investigate the various T1D -range microstructural correlations in in vivo tissues.

T1D -weighted ihMT imaging - Part II. Investigating the long- and short-T1D components correlation with myelin content. Comparison with R1 and the macromolecular proton fraction.

PURPOSE: To investigate the long- and short-T1D components correlation with myelin content using inhomogeneous magnetization transfer (ihMT) high-pass and band-pass T1D -filters and to compare ihMT, R1 , and the macromolecular proton fraction (MPF) for myelin specific imaging. METHODS: The 3D ihMT rapid gradient echo (ihMTRAGE) sequences with increasing switching times (Δt) were used to derive ihMT high-pass T1D -filters with increasing T1D cutoff values and an ihMT band-pass T1D -filter for components in the 100 µs to 1 ms range. 3D spoiled gradient echo quantitative MT (SPGR-qMT) protocols were used to derive R1 and MPF maps. The specificity of R1 , MPF, and ihMT T1D -filters was evaluated by comparison with two histological reference techniques for myelin imaging. RESULTS: The higher contribution of long-T1D s as compared to the short components as Δt got longer led to an increase in the specificity to myelination. In contrast, focusing on the signal originating from a narrow range of short-T1D s (< 1 ms) as isolated by the band-pass T1D -filter led to lower specificity. In addition, the significantly lower r2 correlation coefficient of the band-pass T1D -filter suggests that the origin of short-T1D components is mostly associated with non-myelin protons. Also, the important contribution of short-T1D s to the estimated MPF, explains its low specificity to myelination as compared to the ihMT high-pass T1D -filters. CONCLUSION: Long-T1D components imaging by means of ihMT high-pass T1D -filters is proposed as an MRI biomarker for myelin content. Future studies should enable the investigation of the sensitivity of ihMT T1D -filters for demyelinating processes.

Netrin 1 contributes to vascular remodeling in the subventricular zone and promotes progenitor emigration after demyelination.

Neural stem cells are maintained in the adult brain, sustaining structural and functional plasticity and to some extent participating in brain repair. A thorough understanding of the mechanisms and factors involved in endogenous stem/progenitor cell mobilization is a major challenge in the promotion of spontaneous brain repair. The main neural stem cell niche in the adult brain is the subventricular zone (SVZ). Following demyelination insults, SVZ-derived progenitors act in concert with oligodendrocyte precursors to repopulate the lesion and replace lost oligodendrocytes. Here, we showed robust vascular reactivity within the SVZ after focal demyelination of the corpus callosum in adult mice, together with a remarkable physical association between these vessels and neural progenitors exiting from their niche. Endogenous progenitor cell recruitment towards the lesion was significantly reduced by inhibiting post-lesional angiogenesis in the SVZ using anti-VEGF blocking antibody injections, suggesting a facilitating role of blood vessels for progenitor cell migration towards the lesion. We identified netrin 1 (NTN1) as a key factor upregulated within the SVZ after demyelination and involved in local angiogenesis and progenitor cell migration. Blocking NTN1 expression using a neutralizing antibody inhibited both lesion-induced vascular reactivity and progenitor cell recruitment at the lesion site. We propose a model in which SVZ progenitors respond to a demyelination lesion by NTN1 secretion that both directly promotes cell emigration and contributes to local angiogenesis, which in turn indirectly facilitates progenitor cell emigration from the niche.

Ciliary neurotrophic factor controls progenitor migration during remyelination in the adult rodent brain.

Ciliary neurotrophic factor (CNTF) has been shown to be expressed after brain lesions and in particular after demyelination. Here, we addressed the role of this cytokine in the regulation of neural progenitor migration in the adult rodent brain. Using an acute model of demyelination, we show that CNTF is strongly re-expressed after lesion and is involved in the postlesional mobilization of endogenous progenitors that participate in the myelin regenerative process. We show that CNTF controls the migration of subventricular zone (SVZ)-derived neural progenitors toward the demyelinated corpus callosum. Furthermore, an ectopic source of CNTF in adult healthy brains changes SVZ-derived neural progenitors' migratory behavior that migrate toward the source by activation of the Janus kinase/signal transducer and activator of transcription 3 (JAK/STAT3) pathway. Using various in vitro assays (Boyden chambers, explants, and video time-lapse imaging), we demonstrate that CNTF controls the directed migration of SVZ-derived progenitors and oligodendrocyte precursors. Altogether, these results demonstrate that in addition to its neuroprotective activity and its role in progenitor survival and maturation, CNTF acts as a chemoattractant and participates in the recruitment of endogenous progenitors during myelin repair.

Sonic Hedgehog signaling is a positive oligodendrocyte regulator during demyelination.

The morphogen Sonic Hedgehog (Shh) controls the generation of oligodendrocyte (OLs) during embryonic development and regulates OL production in adulthood in the cortex and corpus callosum. The roles of Shh in CNS repair following lesions associated with demyelinating diseases are still unresolved. Here, we address this issue by using a model of focal demyelination induced by lysolecithin in the corpus callosum of adult mice. Shh transcripts and protein were not detected in control animals but were upregulated in a time-dependent manner in the oligodendroglial lineage within the lesion. We report an increased transcription of Shh target genes suggesting a broad reactivation of the Shh pathway. We show that the adenovirus-mediated transfer of Shh into the lesioned brain results in the attenuation of the lesion extent with the increase of OL progenitor cells (OPCs) and mature myelinating OL numbers due to survival, proliferation, and differentiation activities as well as the decrease of astrogliosis and macrophage infiltration. Furthermore, the blocking of Shh signaling during the lesion, using its physiological antagonist, Hedgehog interacting protein, results in a decrease of OPC proliferation and differentiation, preventing repair. Together, our findings identify Shh as a necessary factor playing a positive role during demyelination and indicate that its signaling activation stands as a potential therapeutic approach for myelin diseases.

Tubacin prevents neuronal migration defects and epileptic activity caused by rat Srpx2 silencing in utero.

Altered development of the human cerebral cortex can cause severe malformations with often intractable focal epileptic seizures and may participate in common pathologies, notably epilepsy. This raises important conceptual and therapeutic issues. Two missense mutations in the sushi repeat-containing protein SRPX2 had been previously identified in epileptic disorders with or without structural developmental alteration of the speech cortex. In the present study, we aimed to decipher the precise developmental role of SRPX2, to have a better knowledge on the consequences of its mutations, and to start addressing therapeutic issues through the design of an appropriate animal model. Using an in utero Srpx2 silencing approach, we show that SRPX2 influences neuronal migration in the developing rat cerebral cortex. Wild-type, but not the mutant human SRPX2 proteins, rescued the neuronal migration phenotype caused by Srpx2 silencing in utero, and increased alpha-tubulin acetylation. Following in utero Srpx2 silencing, spontaneous epileptiform activity was recorded post-natally. The neuronal migration defects and the post-natal epileptic consequences were prevented early in embryos by maternal administration of tubulin deacetylase inhibitor tubacin. Hence epileptiform manifestations of developmental origin could be prevented in utero, using a transient and drug-based therapeutic protocol.

Olesoxime accelerates myelination and promotes repair in models of demyelination.

OBJECTIVE: Multiple sclerosis is a neurodegenerative disease characterized by episodes of immune attack of oligodendrocytes leading to demyelination and progressive functional deficit. One therapeutic strategy to address disease progression could consist in stimulating the spontaneous regenerative process observed in some patients. Myelin regeneration requires endogenous oligodendrocyte progenitor migration and activation of the myelination program at the lesion site. In this study, we have tested the ability of olesoxime, a neuroprotective and neuroregenerative agent, to promote remyelination in the rodent central nervous system in vivo. METHODS: The effect of olesoxime on oligodendrocyte progenitor cell (OPC) differentiation and myelin synthesis was tested directly in organotypic slice cultures and OPC-neuron cocultures. Using naive animals and different mouse models of demyelination, we morphologically and functionally assessed the effect of the compound on myelination in vivo. RESULTS: Olesoxime accelerated oligodendrocyte maturation and enhanced myelination in vitro and in vivo in naive animals during development and also in the adult brain without affecting oligodendrocyte survival or proliferation. In mouse models of demyelination and remyelination, olesoxime favored the repair process, promoting myelin formation with consequent functional improvement. INTERPRETATION: Our observations support the strategy of promoting oligodendrocyte maturation and myelin synthesis to enhance myelin repair and functional recovery. We also provide proof of concept that olesoxime could be useful for the treatment of demyelinating diseases.

Neuroblasts contribute to oligodendrocytes generation upon demyelination in the adult mouse brain.

After demyelinating insult, the neuronal progenitors of the adult mouse sub-ventricular zone (SVZ) called neuroblasts convert into oligodendrocytes that participate to the remyelination process. We use this rare example of spontaneous fate conversion to identify the molecular mechanisms governing these processes. Using in vivo cell lineage and single cell RNA-sequencing, we demonstrate that SVZ neuroblasts fate conversion proceeds through formation of a non-proliferating transient cellular state co-expressing markers of both neuronal and oligodendrocyte identities. Transition between the two identities starts immediately after demyelination and occurs gradually, by a stepwise upregulation/downregulation of key TFs and chromatin modifiers. Each step of this fate conversion involves fine adjustments of the transcription and translation machineries as well as tight regulation of metabolism and migratory behaviors. Together, these data constitute the first in-depth analysis of a spontaneous cell fate conversion in the adult mammalian CNS.

RAE-1 is expressed in the adult subventricular zone and controls cell proliferation of neurospheres.

Improving and controlling the capacity of endogenous or grafted adult neural stem cells to repair the nervous system relies on a better knowledge of interactions between immune cells and neural stem cells. Class I major histocompatibility complex (MHC) family members comprise numerous proteins playing either immune or nonimmune function. Among the latter, MHC functions in the central nervous system has started to receive recent interest. Here, our first goal was to investigate the potential relationship between MHC class I molecules and neurogenesis. For the first time, we report the expression of two MHC class I-related members by neural stem/progenitor cells: retinoic acid early induced transcript (RAE)-1 and CD1d. The expression of RAE-1 but not CD1d disappears when differentiation of neurosphere cells is induced. Interestingly, RAE-1 transcripts are expressed in the brain during development, and we demonstrate they persist in one of the main area of adult neurogenesis, the subventricular zone (SVZ). So far, RAE-1 is only known for its immune functions as a ligand of the activating receptor NKG2D expressed by natural killer (NK) cells, natural killer T, Tγδ, and some T CD8 lymphocytes. Here, we do not detect any NKG2D expression in the SVZ either in physiological or in pathological conditions. Interestingly, inhibition of RAE-1 expression in neurosphere cells reduces cell proliferation without alteration of cell viability, which argues for a nonimmune role for RAE-1. These results reveal an unexpected role of RAE-1 in regulating adult SVZ neurogenesis by supporting stem/progenitor cells proliferation.

Control of planar divisions by the G-protein regulator LGN maintains progenitors in the chick neuroepithelium.

The spatio-temporal regulation of symmetrical as opposed to asymmetric cell divisions directs the fate and location of cells in the developing CNS. In invertebrates, G-protein regulators control spindle orientation in asymmetric divisions, which generate progeny with different identities. We investigated the role of the G-protein regulator LGN (also called Gpsm2) in spindle orientation and cell-fate determination in the spinal cord neuroepithelium of the developing chick embryo. We show that LGN is located at the cell cortex and spindle poles of neural progenitors, and that it regulates spindle movements and orientation. LGN promotes planar divisions in the early spinal cord. Interfering with LGN function randomizes the plane of division. Notably, this does not affect cell fate, but frequently leads one daughter of proliferative symmetric divisions to exit the neuroepithelium prematurely and to proliferate aberrantly in the mantle zone. Hence, tight control of planar spindle orientation maintains neural progenitors in the neuroepithelium, and regulates the proper development of the nervous system.

Myelin Repair: From Animal Models to Humans.

It is widely thought that brain repair does not occur, but myelin regeneration provides clear evidence to the contrary. Spontaneous remyelination may occur after injury or in multiple sclerosis (MS). However, the efficiency of remyelination varies considerably between MS patients and between the lesions of each patient. Myelin repair is essential for optimal functional recovery, so a profound understanding of the cells and mechanisms involved in this process is required for the development of new therapeutic strategies. In this review, we describe how animal models and modern cell tracing and imaging methods have helped to identify the cell types involved in myelin regeneration. In addition to the oligodendrocyte progenitor cells identified in the 1990s as the principal source of remyelinating cells in the central nervous system (CNS), other cell populations, including subventricular zone-derived neural progenitors, Schwann cells, and even spared mature oligodendrocytes, have more recently emerged as potential contributors to CNS remyelination. We will also highlight the conditions known to limit endogenous repair, such as aging, chronic inflammation, and the production of extracellular matrix proteins, and the role of astrocytes and microglia in these processes. Finally, we will present the discrepancies between observations in humans and in rodents, discussing the relationship of findings in experimental models to myelin repair in humans. These considerations are particularly important from a therapeutic standpoint.

Endogenous neural stem cells modulate microglia and protect against demyelination.

In response to corpus callosum (CC) demyelination, subventricular zone-derived neural progenitors (SVZdNPs) are mobilized and generate new myelinating oligodendrocytes (OLG). Here, we examine the putative immunomodulatory properties of endogenous SVZdNPs during demyelination in the cuprizone model. SVZdNP density was higher in the lateral and rostral CC regions, and demyelination was inversely correlated with activated microglial density and pro-inflammatory cytokine levels. Single-cell RNA sequencing showed that CC areas with high levels of SVZdNP mobilization were enriched in a microglial cell subpopulation with an immunomodulatory signature. We propose MFGE8 (milk fat globule-epidermal growth factor-8) and ß3 integrin as a ligand/receptor pair involved in dialogue between SVZdNPs and microglia. Immature SVZdNPs mobilized to the demyelinated CC were found highly enriched in MFGE8, which promoted the phagocytosis of myelin debris in vitro. Overall, these results demonstrate that, in addition to their cell replacement capacity, endogenous progenitors have immunomodulatory properties, highlighting a new role for endogenous SVZdNPs in myelin regeneration.

Mature oligodendrocytes bordering lesions limit demyelination and favor myelin repair via heparan sulfate production.

Myelin destruction is followed by resident glia activation and mobilization of endogenous progenitors (OPC) which participate in myelin repair. Here we show that in response to demyelination, mature oligodendrocytes (OLG) bordering the lesion express Ndst1, a key enzyme for heparan sulfates (HS) synthesis. Ndst1+ OLG form a belt that demarcates lesioned from intact white matter. Mice with selective inactivation of Ndst1 in the OLG lineage display increased lesion size, sustained microglia and OPC reactivity. HS production around the lesion allows Sonic hedgehog (Shh) binding and favors the local enrichment of this morphogen involved in myelin regeneration. In MS patients, Ndst1 is also found overexpressed in oligodendroglia and the number of Ndst1-expressing oligodendroglia is inversely correlated with lesion size and positively correlated with remyelination potential. Our study suggests that mature OLG surrounding demyelinated lesions are not passive witnesses but contribute to protection and regeneration by producing HS.

LIFR beta plays a major role in neuronal identity determination and glial differentiation in the mouse facial nucleus.

In the hindbrain, generation of the facial nucleus involves complex developmental processes that will lead to the formation of a structure composed of motor neurons, astrocytes and oligodendrocytes. The implication of LIF-related cytokines in the development of this nucleus came to light with the analysis of mice mutant for the receptor of these cytokines, LIFR beta, which exhibit a massive loss of facial branchiomotor (fbm) neurons at birth and a severe decrease in GFAP expression, a marker of astrocytes. To uncover the cellular mechanisms regulated by LIFR beta during facial nucleus development, we first analyzed its expression pattern in the hindbrain. lifr beta is first expressed at E11.5 in the hindbrain neuroepithelium. The receptor is absent during the migration of fbm post-mitotic neurons but is strongly expressed when fbm neurons have reached rhombomere 6 at E12.5, and its expression is maintained until E18.5. From the analysis of lifr beta mutant embryos, we established that LIFR beta is necessary for fbm neurons' identity determination. We also show that LIFR beta is implicated in astrocyte and oligodendrocyte differentiation, specifically within the facial nucleus.

In vitro migration assays of neural stem cells.

We describe three rapid procedures for the in vitro investigation of molecular factors influencing the migration of neural precursors derived from embryonic or postnatal neural stem cells. In the first assay, factors influencing chain migration from the anterior subventricular zone of perinatal mice can be analyzed after explantation and embedding in Matrigel, a three-dimensional substrate mimicking the in vivo extracellular matrix. The second assay enables to assess soluble factors influencing radial migration away from adherent neurospheres in which embryonic stem cells have been expanded. In this example, neurospheres have been derived from the striatum primordium of embryonic mice. Finally, the directed migration of these precursor cells can be analyzed using a chemotaxis chamber assay, in which the directional movement (chemotaxis) of cells across a membrane occurs in controlled conditions. These three assays are useful tools to evaluate the importance of surface molecules and environmental factors, such as the polysialylated form of neural cell adhesion molecule (NCAM) or chemokines such as CXCL12, in the directional migration of neural precursors.

Promoting Myelin Repair through In Vivo Neuroblast Reprogramming.

Demyelination is frequently observed in a variety of CNS insults and neurodegenerative diseases. In rodents, adult neural stem cells can generate oligodendrocytes and participate to myelin repair. However, these cells mainly produce migratory neuroblasts that differentiate in the olfactory bulb. Here, we show that, in the demyelination context, a small subset of these neuroblasts can spontaneously convert into myelinating oligodendrocytes. Furthermore, we demonstrate that the contribution of neuroblasts to myelin repair can be improved by in vivo forced expression of two transcription factors: OLIG2 and SOX10. These factors promote directed fate conversion of endogenous subventricular zone neuroblasts into mature functional oligodendrocytes, leading to enhanced remyelination in a cuprizone-induced mouse model of demyelination. These findings highlight the unexpected plasticity of committed neuroblasts and provide proof of concept that they could be targeted for the treatment of demyelinated lesions in the adult brain.

Necdin shapes serotonergic development and SERT activity modulating breathing in a mouse model for Prader-Willi syndrome.

Prader-Willi syndrome (PWS) is a genetic neurodevelopmental disorder that presents with hypotonia and respiratory distress in neonates. The Necdin-deficient mouse is the only model that reproduces the respiratory phenotype of PWS (central apnea and blunted response to respiratory challenges). Here, we report that Necdin deletion disturbs the migration of serotonin (5-HT) neuronal precursors, leading to altered global serotonergic neuroarchitecture and increased spontaneous firing of 5-HT neurons. We show an increased expression and activity of 5-HT Transporter (SERT/Slc6a4) in 5-HT neurons leading to an increase of 5-HT uptake. In Necdin-KO pups, the genetic deletion of Slc6a4 or treatment with Fluoxetine, a 5-HT reuptake inhibitor, restored normal breathing. Unexpectedly, Fluoxetine administration was associated with respiratory side effects in wild-type animals. Overall, our results demonstrate that an increase of SERT activity is sufficient to cause the apneas in Necdin-KO pups, and that fluoxetine may offer therapeutic benefits to PWS patients with respiratory complications.