Targeting demyelination via α-secretases promoting sAPPα release to enhance remyelination in central nervous system.

Remyelination is an endogenous regenerative process of myelin repair in the central nervous system (CNS) with limited efficacy in demyelinating disorders. As strategies enhancing endogenous remyelination become a therapeutic challenge, we have focused our study on α-secretase-induced sAPPα release, a soluble endogenous protein with neuroprotective and neurotrophic properties. However, the role of sAPPα in remyelination is not known. Therefore, we investigated the remyelination potential of α-secretase-induced sAPPα release following CNS demyelination in mice. Acute demyelination was induced by feeding mice with cuprizone (CPZ) for 5weeks. To test the protective effect and the remyelination potential of etazolate, an α-secretase activator, we designed two treatment protocols. Etazolate was administrated either during the last two weeks or at the end of the CPZ intoxication. In both protocols, etazolate restored the number of myelinated axons in corpus callosum with a corresponding increase in the amount of MBP, one of the major myelin proteins in the brain. We also performed ex vivo studies to decipher etazolate's mechanism of action in a lysolecithin-induced demyelination model using organotypic culture of cerebellar slices. Etazolate treatment was able to i) enhance the release of sAPPα in the culture media of demyelinated slices, ii) protect myelinated axons from demyelination, iii) increase the number of mature oligodendrocytes, iv) promote the reappearance of the paired Caspr+ adjacent to the nodes of Ranvier and v) increase the percentage of myelinated axons with short internodes, an indicator of remyelination. Etazolate failed to promote all the aforementioned effects in the presence of GI254023X, an α-secretase inhibitor. Moreover, the protective effects of etazolate in demyelinated slices were mimicked by sAPPα treatment in a dose-dependent manner. In conclusion, etazolate-induced sAPPα release protects myelinated axons from demyelination while also promoting remyelination. This work, thus, highlights the therapeutic potential of strategies that enhance sAPPα release in demyelinating disorders.

CB1 and CB2 cannabinoid receptor antagonists prevent minocycline-induced neuroprotection following traumatic brain injury in mice.

Traumatic brain injury (TBI) and its consequences represent one of the leading causes of death in young adults. This lesion mediates glial activation and the release of harmful molecules and causes brain edema, axonal injury, and functional impairment. Since glial activation plays a key role in the development of this damage, it seems that controlling it could be beneficial and could lead to neuroprotective effects. Recent studies show that minocycline suppresses microglial activation, reduces the lesion volume, and decreases TBI-induced locomotor hyperactivity up to 3 months. The endocannabinoid system (ECS) plays an important role in reparative mechanisms and inflammation under pathological situations by controlling some mechanisms that are shared with minocycline pathways. We hypothesized that the ECS could be involved in the neuroprotective effects of minocycline. To address this hypothesis, we used a murine TBI model in combination with selective CB1 and CB2 receptor antagonists (AM251 and AM630, respectively). The results provided the first evidence for the involvement of ECS in the neuroprotective action of minocycline on brain edema, neurological impairment, diffuse axonal injury, and microglial activation, since all these effects were prevented by the CB1 and CB2 receptor antagonists.

Wnt and lithium: a common destiny in the therapy of nervous system pathologies?

Wnt signaling is required for neurogenesis, the fate of neural progenitors, the formation of neuronal circuits during development, neuron positioning and polarization, axon and dendrite development and finally for synaptogenesis. This signaling pathway is also implicated in the generation and differentiation of glial cells. In this review, we describe the mechanisms of action of Wnt signaling pathways and their implication in the development and correct functioning of the nervous system. We also illustrate how a dysregulated Wnt pathway could lead to psychiatric, neurodegenerative and demyelinating pathologies. Lithium, used for the treatment of bipolar disease, inhibits GSK3ß, a central enzyme of the Wnt/ß-catenin pathway. Thus, lithium could, to some extent, mimic Wnt pathway. We highlight the possible dialogue between lithium therapy and modulation of Wnt pathway in the treatment of the diseases of the nervous system.

A Disintegrin And Metalloprotease 10 expression within the murine central nervous system.

A Disintegrin And Metalloprotease 10 (ADAM10), is able to control several important physiopathological processes through the shedding of a large number of protein substrates. Although ADAM10 plays a crucial role in the central nervous system (CNS) development and function, its protein distribution in the CNS has not been fully addressed. Here, we described the regional and cellular ADAM10 protein expression in C57BL/6 mice examined by immunofluorescence 1) throughout the adult mouse brain, cerebellum and spinal cord in vivo and 2) in different cell types as neurons, astrocytes, oligodendrocytes and microglia in vitro. We observed ADAM10 expression through the whole CNS, with a strong expression in the hippocampus, in the hypothalamus and in the cerebral and piriform cortex in the brain, in the Purkinje and in granular cell layers in the cerebellum and in the spinal cord to a lower extent. In vivo, ADAM10 protein expression was mainly found in neurons and in some oligodendroglial cell populations. However, in primary cultures we observed ADAM10 expression in neurons, oligodendrocytes, astrocytes and microglia. Interestingly, ADAM10 was not only found in the membrane but also in cytoplasmic vesicles and in the nucleus of primary cultured cells. Overall, this work highlights a wide distribution of ADAM10 throughout the CNS. The nuclear localization of ADAM10, probably due to its intracellular domain, emphasizes its role in cell signalling in physiological and pathological conditions. Further investigations are required to better elucidate the role of ADAM10 in glial cells.

Effect of Etazolate upon Cuprizone-induced Demyelination In Vivo: Behavioral and Myelin Gene Analysis.

Demyelination is a well-known pathological process in CNS disorders such as multiple sclerosis (MS). It provokes progressive axonal degeneration and functional impairments and no efficient therapy is presently available to combat such insults. Recently, we have shown that etazolate, a pyrazolopyridine compound and an α-secretase activator, was able to promote myelin protection and remyelination after cuprizone (CPZ)-induced acute demyelination in C57Bl/6 mice. In continuation of this work, here we have further investigated the effects of etazolate treatment after acute cuprizone-induced demyelination at the molecular level (expression of myelin genes Plp, Mbp and Mag and inflammatory markers Il-1ß, Tnf-α) and at the functional level (locomotor and spatial memory skills) in vivo. To this end, we have employed two protocols which consists of administering etazolate (10 mg/kg/d) for a period of 2 weeks either during (Protocol #1) or after (Protocol #2) 5-weeks of CPZ-induced demyelination. At the molecular level, we observed that CPZ intoxication altered inflammatory and myelin gene expression and it was not restored with either of the etazolate treatment protocols. At the functional level, the locomotor activity was impaired after 3-weeks of CPZ intoxication (Protocol #1) and our data indicates a modest but beneficial effect of etazolate treatment. Spatial memory evaluated was not affected either by CPZ intake or etazolate treatment in both protocols. Altogether, this study shows that the beneficial effect of etazolate upon demyelination does not occur at the gene expression level at the time points studied. Furthermore, our results also highlight the difficulty in revealing functional sequelae following CPZ intoxication.

A novel model of trauma-induced cerebellar injury and myelin loss in mouse organotypic cerebellar slice cultures using live imaging.

BACKGROUND: Traumatic brain injury (TBI) induces significant cognitive deficits correlated with white matter injury, involving both axonal and myelin damage. Several models of TBI ex vivo are available to mimic focal impact on brain tissue. However, none of them addressed the study of trauma-induced myelin damage. NEW METHOD: The aim of this study was to set up a novel ex vivo weight-drop model on organotypic cultures obtained from mouse cerebellum, a highly myelinated structure, in order to study the temporal evolution of cerebellar lesion and demyelination. The extent of injury was measured by propidium iodide (PI) fluorescence and demyelination was evaluated by loss of GFP-fluorescence in cerebellar slices from PLP-eGFP mice. RESULTS: Live imaging of slices showed an increase of PI-fluorescence and a significant loss of GFP-fluorescence at 6 h, 24 h and 72 h post-injury. At the impact site, we observed a loss of Purkinje cells and myelin sheaths with a marked loss of myelin protein MBP at 72 h following injury. Etazolate, a known protective compound, was able to reduce both the PI-fluorescence increase and the loss of GFP-fluorescence, emphasizing its protective effect on myelin loss. COMPARISON WITH EXISTING METHODS AND CONCLUSIONS: In line with the existing models of focal injury, we characterized trauma-induced cerebellar lesion with an increase of PI fluorescence by live imaging. Our findings describe a novel tool to study trauma-induced myelin damage in cerebellar slices and to test biomolecules of therapeutic interest for myelin protection.

Mechanical Stretch of High Magnitude Provokes Axonal Injury, Elongation of Paranodal Junctions, and Signaling Alterations in Oligodendrocytes.

Increasing findings suggest that demyelination may play an important role in the pathophysiology of brain injury, but the exact mechanisms underlying such damage are not well known. Mechanical tensile strain of brain tissue occurs during traumatic brain injury. Several studies have investigated the cellular and molecular events following a static tensile strain of physiological magnitude on individual cells such as oligodendrocytes. However, the pathobiological impact of high-magnitude mechanical strain on oligodendrocytes and myelinated fibers remains under investigated. In this study, we reported that an applied mechanical tensile strain of 30% on mouse organotypic culture of cerebellar slices induced axonal injury and elongation of paranodal junctions, two hallmarks of brain trauma. It was also able to activate MAPK-ERK1/2 signaling, a stretch-induced responsive pathway. The same tensile strain applied to mouse oligodendrocytes in primary culture induced a profound damage to cell morphology, partial cell loss, and a decrease of myelin protein expression. The lower tensile strain of 20% also caused cell loss and the remaining oligodendrocytes appeared retracted with decreased myelin protein expression. Finally, high-magnitude tensile strain applied to 158N oligodendroglial cells altered myelin protein expression, dampened MAPK-ERK1/2 and MAPK-p38 signaling, and enhanced the production of reactive oxygen species. The latter was accompanied by increased protein oxidation and an alteration of anti-oxidant defense that was strain magnitude-dependent. In conclusion, mechanical stretch of high magnitude provokes axonal injury with significant alterations in oligodendrocyte biology that could initiate demyelination.

Characterization of a rat model to study acute neuroinflammation on histopathological, biochemical and functional outcomes.

Neuroinflammation is one of the events occurring after acute brain injuries. The aim of the present report was to characterize a rat model to study acute neuroinflammation on the histopathological, biochemical and functional outcomes. Lipopolysaccharide (LPS), known as a strong immunostimulant, was directly injected into the hippocampus. The spatiotemporal evolution of inducible NOS (iNOS) and cell death was studied from 6 h to 7 days. A perfect time course correlation was observed between iNOS immunoreactivity and iNOS activity showing an acute, expansive and transient iNOS induction in the hippocampus with a peak at 24 h. It was associated with a marked increase in NO metabolite (NO(x)) levels, and a high level of myeloperoxidase (MPO) activity. This inflammation precedes a massive cellular loss including at least neurons and astrocytes, and a drop of constitutive NOS activity, restrictive to the ipsilateral hippocampus from 48 h after LPS injection. Moreover, sensorimotor function impairment occurred from 24 h to 7 days with a maximum at 24 h post-LPS injection. Therefore, we characterized an in vivo model of acute neuroinflammation and neurodegeneration, in relation with a neurological deficit, which may be a powerful tool for mechanistic studies and for further evaluation of the potential neuroprotective agents.

Cortical calcium increase following traumatic brain injury represents a pitfall in the evaluation of Ca2+-independent NOS activity.

In this report, our findings highlighted the presence of a high level of calcium in the cortex following traumatic brain injury (TBI) in a rat model of fluid percussion-induced brain injury. This calcium increase represents a pitfall in the assessment of Ca2+-independent nitric oxide synthase (NOS) activity supposed to play a role in the secondary brain lesion following TBI. The so-called Ca2+-independent NOS activity measured in the injured cortex 72 h after TBI had the pharmacological profile of a Ca2+-dependent NOS and was therefore inhibited with a supplement of calcium chelator. The remaining activity was very low and iNOS protein was hardly immunodetected on the same sample used for NOS activity assay. The concentration of calcium chelator used in the assay should be revised and adjusted consequently to make sure that the calcium-free condition is achieved for the assay. Otherwise, the findings tend towards an overestimation of Ca2+-independent and underestimation of Ca2+-dependent NOS activities. The revised Ca2+-independent NOS activity assay was then tested, in relation with the amount of iNOS protein, in a model of LPS-induced neuroinflammation. Taken together, precautions should be taken when assessing the Ca2+-independent enzymatic activity in cerebral tissue after a brain insult.

Etazolate, an α-secretase activator, reduces neuroinflammation and offers persistent neuroprotection following traumatic brain injury in mice.

Traumatic brain injury (TBI) evokes an intense neuroinflammatory reaction that is essentially mediated by activated microglia and that has been reported to act as a secondary injury mechanism that further promotes neuronal death. It involves the excessive production of inflammatory cytokines and the diminution of neuroprotective and neurotrophic factors, such as the soluble form alpha of the amyloid precursor protein (sAPPα), generated by the activity of α-secretases. Hence, the aim of this study was to examine the effects of etazolate, an α-secretase activator, on acute and belated post-TBI consequences. The mouse model of TBI by mechanical percussion was used and injured mice received either the vehicle or etazolate at the dose of 1, 3 or 10 mg/kg at 2 h post-TBI. Neurological score, cerebral œdema, IL-1ß and sAPPα levels, microglial activation and lesion size were evaluated from 6 to 24 h post-TBI. Spontaneous locomotor activity was evaluated from 48 h to 12 weeks post-TBI, memory function at 5 weeks and olfactory bulb lesions at 13 weeks post-TBI. A single administration of etazolate exerted a dose-dependent anti-inflammatory and anti-œdematous effect accompanied by lasting memory improvement, reduction of locomotor hyperactivity and olfactory bulb tissue protection, with a therapeutic window of at least 2 h. These effects were associated with the restoration of the levels of the sAPPα protein post-TBI. Taken together, these results highlight for the first time the therapeutic interest of an α-secretase activator in TBI.

Minocycline restores olfactory bulb volume and olfactory behavior after traumatic brain injury in mice.

Permanent olfactory dysfunction can often arise after traumatic brain injury (TBI) and while one of the main causes is the immediate loss of neurons in the olfactory bulb (OB), the emergent neuroinflammatory environment following TBI may further promote OB deterioration. Therefore, we examined the effects of acute anti-inflammatory treatment with minocycline on post-TBI olfactory behavior and on OB surface. The mouse model of closed-head injury by mechanical percussion was applied to anesthetized Swiss mice. The treatment protocol included three injections of minocycline (i.p.) at 5 min (90 mg/kg), 3 h, and 9 h (45 mg/kg) post-TBI. An olfactory avoidance test was run up to 12 weeks post-TBI. The mice were then sacrificed and their OB surface was measured. Our results demonstrated a post-TBI olfactory behavior deficit that was significant up to at least 12 weeks post-TBI. Additionally, substantial post-TBI OB atrophy was observed that was strongly correlated with the behavioral impairment. Minocycline was able to attenuate both the olfactory lesions and corresponding functional deficit in the short and long term. These results emphasize the potential role of minocycline as a promising neuroprotective agent for the treatment of TBI-related olfactory bulb lesions and deficits.

Evaluation of late cognitive impairment and anxiety states following traumatic brain injury in mice: the effect of minocycline.

Comorbidity of cognitive and stress disorders is a common clinical sequel of traumatic brain injury (TBI) that is essentially determined by the site and severity of the insult, but also by the extent of the ensuing neuroinflammatory response. The present study sought to examine the late effects of closed-head TBI on memory function and anxiety in mice, in order to further examine the potential efficacy of an acute anti-inflammatory treatment with minocycline. The mouse model of closed-head injury by mechanical percussion was applied on anesthetized Swiss mice. The treatment protocol included three injections of minocycline (i.p.) at 5 min (90 mg/kg), 3 h and 9 h (45 mg/kg) post-TBI. The Novel Object Recognition Test as well as the Elevated Plus Maze (EPM) and Elevated Zero Maze (EZM) tasks were employed to assess post-TBI memory and anxiety respectively. Our results revealed a recognition memory deficit that was significant up to at least 13 weeks post-TBI. However, neither EPM nor EZM revealed any alteration in post-TBI anxiety levels albeit some mild disinhibition. Most importantly, minocycline was able to attenuate the memory impairment in an effective and lasting manner, highlighting its therapeutic potential in TBI.

Minocycline restores sAPPα levels and reduces the late histopathological consequences of traumatic brain injury in mice.

Traumatic brain injury (TBI) induces both focal and diffuse lesions that are concurrently responsible for the ensuing morbidity and mortality and for which no established treatment is available. It has been recently reported that an endogenous neuroprotector, the soluble form α of the amyloid precursor protein (sAPPα), exerts neuroprotective effects following TBI. However, the emergent post-traumatic neuroinflammatory environment compromises sAPPα production and may promote neuronal degeneration and consequent brain atrophy. Hence, the aim of this study was to examine the effects of the anti-inflammatory drug minocycline on sAPPα levels, as well as on long-term histological consequences post-TBI. The weight-drop model was used to induce TBI in mice. Minocycline or its vehicle were administered three times: at 5 min (90 mg/kg, i.p.) and at 3 and 9 h (45 mg/kg, i.p.) post-TBI. The levels of sAPPα, the extent of brain atrophy, and reactive gliosis were evaluated by ELISA, cresyl violet, and immunolabeling of GFAP and CD11b, respectively. Our results revealed a post-TBI sAPPα decrease that was significantly attenuated by minocycline. Additionally, corpus callosum and striatal atrophy, ventriculomegaly, astrogliosis, and microglial activation were observed at 3 months post-TBI. All the above consequences were significantly reduced by minocycline. In conclusion, inhibition of the acute phase of post-TBI neuroinflammation was associated with the sparing of sAPPα and the protection of brain tissue in the long-term, emphasizing the potential role of minocycline as an effective treatment for TBI.

Blockade of acute microglial activation by minocycline promotes neuroprotection and reduces locomotor hyperactivity after closed head injury in mice: a twelve-week follow-up study.

Traumatic brain injury (TBI) causes a wide spectrum of consequences, such as microglial activation, cerebral inflammation, and focal and diffuse brain injury, as well as functional impairment. In this study we aimed to investigate the effects of acute treatment with minocycline as an inhibitor of microglial activation on cerebral focal and diffuse lesions, and on the spontaneous locomotor activity following TBI. The weight-drop model was used to induce TBI in mice. Microglial activation and diffuse axonal injury (DAI) were detected by immunohistochemistry using CD11b and ss-amyloid precursor protein (ss-APP) immunolabeling, respectively. Focal injury was determined by the measurement of the brain lesion volume. Horizontal and vertical locomotor activities were measured for up to 12 weeks post-injury by an automated actimeter. Minocycline or vehicle were administered three times post-insult, at 5 min (90 mg/kg i.p.), 3 h, and 9 h post-TBI (45 mg/kg i.p.). Minocycline treatment attenuated microglial activation by 59% and reduced brain lesion volume by 58%, yet it did not affect DAI at 24 h post-TBI. More interestingly, minocycline significantly decreased TBI-induced locomotor hyperactivity at 48 h post-TBI, and its effect lasted for up to 8 weeks. Taken together, the results indicate that microglial activation appears to play an important role in the development of TBI-induced focal injury and the subsequent locomotor hyperactivity, and its short-term inhibition provides long-lasting functional recovery after TBI. These findings emphasize the fact that minocycline could be a promising new therapeutic strategy for head-injured patients.

Minocycline effects on cerebral edema: relations with inflammatory and oxidative stress markers following traumatic brain injury in mice.

One of the severe complications following traumatic brain injury (TBI) is cerebral edema and its effective treatment is of great interest to prevent further brain damage. This study investigated the effects of minocycline, known for its anti-inflammatory properties, on cerebral edema and its respective inflammatory markers by comparing different dose regimens, on oxidative stress and on neurological dysfunction following TBI. The weight drop model was used to induce TBI in mice. The brain water content was measured to evaluate cerebral edema. Inflammatory markers were detected by ELISA (IL-1beta), zymography and Western blot (MMP-9). The oxidative stress marker (glutathione levels) and neurological function were measured by Griffith technique and string test, respectively. Minocycline was administered i.p. once (5 min), twice (5 min and 3 h) or triple (5 min, 3 h and 9 h) following TBI. The first dose of minocycline only varied (45 or 90 mg/kg), whereas the following doses were all at 45 mg/kg. The single and double administrations of minocycline reduced the increase of inflammatory markers at 6 h post-TBI. Minocycline also reduced cerebral edema at this time point, only after double administration and at the high dose regimen, although with no effect on the TBI-induced oxidized glutathione increase. The anti-edematous effect of minocycline persisted up to 24 h, upon a triple administration, and accompanied by a neurological recovery. In conclusion, we reported an anti-edematous effect of minocycline after TBI in mice according to a specific treatment regimen. These findings emphasize that the beneficial effects of minocycline depend on the treatment regimen following a brain injury.

Lack of iNOS induction in a severe model of transient focal cerebral ischemia in rats.

Calcium-independent nitric oxide synthase (NOS) activity has been reported in ischemic brains and usually attributed to the inducible isoform, iNOS. Because calcium-independent mechanisms have recently been shown to regulate the constitutive calcium-dependent NOS, we proposed to confirm the presence of iNOS activity in our model of transient focal cerebral ischemia in rats. Our initial results showed that, in our model, ischemia induced an important increase in brain calcium concentration. Consequently, the determination of calcium-independent NOS activity required a higher concentration of calcium chelator than classically used in the NOS assay. In these conditions, calcium-independent NOS activity was not observed after ischemia. Moreover, our ischemia was associated with neither iNOS protein expression, measured by Western blotting, nor increased NO production, evaluated by its metabolites (nitrate/nitrite). Our results demonstrate that iNOS activity may be overestimated due to increased brain calcium concentration in ischemic conditions and also that iNOS is not systematically induced after cerebral ischemia.