Targeted overexpression of glutamate transporter-1 reduces seizures and attenuates pathological changes in a mouse model of epilepsy.

Astrocytic glutamate transporters are crucial for glutamate homeostasis in the brain, and dysregulation of these transporters can contribute to the development of epilepsy. Glutamate transporter-1 (GLT-1) is responsible for the majority of glutamate uptake in the dorsal forebrain and has been shown to be reduced at epileptic foci in patients and preclinical models of temporal lobe epilepsy (TLE). Current antiepileptic drugs (AEDs) work primarily by targeting neurons directly through suppression of excitatory neurotransmission or enhancement of inhibitory neurotransmission, which can lead to both behavioral and psychiatric side effects. This study investigates the therapeutic capacity of astrocyte-specific AAV-mediated GLT-1 expression in the intrahippocampal kainic acid (IHKA) model of TLE. In this study, we used Western blot analysis, immunohistochemistry, and long-term-video EEG monitoring to demonstrate that cell-type-specific upregulation of GLT-1 in astrocytes is neuroprotective at early time points during epileptogenesis, reduces seizure frequency and total time spent in seizures, and eliminates large behavioral seizures in the IHKA model of epilepsy. Our findings suggest that targeting glutamate uptake is a promising therapeutic strategy for the treatment of epilepsy.

Increase in chemokine CXCL1 by ERß ligand treatment is a key mediator in promoting axon myelination.

Estrogen receptor ß (ERß) ligands promote remyelination in mouse models of multiple sclerosis. Recent work using experimental autoimmune encephalomyelitis (EAE) has shown that ERß ligands induce axon remyelination, but impact peripheral inflammation to varying degrees. To identify if ERß ligands initiate a common immune mechanism in remyelination, central and peripheral immunity and pathology in mice given ERß ligands at peak EAE were assessed. All ERß ligands induced differential expression of cytokines and chemokines, but increased levels of CXCL1 in the periphery and in astrocytes. Oligodendrocyte CXCR2 binds CXCL1 and has been implicated in normal myelination. In addition, despite extensive immune cell accumulation in the CNS, all ERß ligands promoted extensive remyelination in mice at peak EAE. This finding highlights a component of the mechanism by which ERß ligands mediate remyelination. Hence, interplay between the immune system and central nervous system may be responsible for the remyelinating effects of ERß ligands. Our findings of potential neuroprotective benefits arising from the presence of CXCL1 could have implications for improved therapies for multiple sclerosis.

Diffusion tensor imaging identifies aspects of therapeutic estrogen receptor ß ligand-induced remyelination in a mouse model of multiple sclerosis.

Diffusion tensor imaging (DTI) has been shown to detect white matter degeneration in multiple sclerosis (MS), a neurodegenerative autoimmune disease that presents with diffuse demyelination of the central nervous system. However, the utility of DTI in evaluating therapeutic remyelination has not yet been well-established. Here, we assessed the ability of DTI to distinguish between remyelination and neuroprotection following estrogen receptor ß ligand (Indazole chloride, IndCl) treatment, which has been previously shown to stimulate functional remyelination, in the cuprizone (CPZ) diet mouse model of MS. Adult C57BL/6 J male and female mice received a normal diet (control), demyelination-inducing CPZ diet (9wkDM), or CPZ diet followed by two weeks of a normal diet (i.e., remyelination period) with either IndCl (RM + IndCl) or vehicle (RM + Veh) injections. We evaluated tissue microstructure of the corpus callosum utilizing in vivo and ex vivo DTI and immunohistochemistry (IHC) for validation. Compared to control mice, the 9wkDM group showed decreased fractional anisotropy (FA), increased radial diffusivity (RD), and no changes in axial diffusivity (AD) both in vivo and ex vivo. Meanwhile, RM + IndCl groups showed increased FA and decreased RD ex vivo compared to the RM + Veh group, in accordance with the evidence of remyelination by IHC. In conclusion, the DTI technology used in the present study can identify some changes in myelination and is a valuable translational tool for evaluating MS pathophysiology and therapeutic efficacy.

Sex-specific Tau methylation patterns and synaptic transcriptional alterations are associated with neural vulnerability during chronic neuroinflammation.

The molecular events underlying the transition from initial inflammatory flares to the progressive phase of multiple sclerosis (MS) remain poorly understood. Here, we report that the microtubule-associated protein (MAP) Tau exerts a gender-specific protective function on disease progression in the MS model experimental autoimmune encephalomyelitis (EAE). A detailed investigation of the autoimmune response in Tau-deficient mice excluded a strong immunoregulatory role for Tau, suggesting that its beneficial effects are presumably exerted within the central nervous system (CNS). Spinal cord transcriptomic data show increased synaptic dysfunctions and alterations in the NF-kB activation pathway upon EAE in Tau-deficient mice as compared to wildtype animals. We also performed the first comprehensive characterization of Tau post-translational modifications (PTMs) in the nervous system upon EAE. We report that the methylation levels of the conserved lysine residue K306 are significantly decreased in the chronic phase of the disease. By combining biochemical assays and molecular dynamics (MD) simulations, we demonstrate that methylation at K306 decreases the affinity of Tau for the microtubule network. Thus, the down-regulation of this PTM might represent a homeostatic response to enhance axonal stability against an autoimmune CNS insult. The results, altogether, position Tau as key mediator between the inflammatory processes and neurodegeneration that seems to unify many CNS diseases.

Connexins and pannexins: At the junction of neuro-glial homeostasis & disease.

In the central nervous system (CNS), connexin (Cx)s and pannexin (Panx)s are an integral component of homeostatic neuronal excitability and synaptic plasticity. Neuronal Cx gap junctions form electrical synapses across biochemically similar GABAergic networks, allowing rapid and extensive inhibition in response to principle neuron excitation. Glial Cx gap junctions link astrocytes and oligodendrocytes in the pan-glial network that is responsible for removing excitotoxic ions and metabolites. In addition, glial gap junctions help constrain excessive excitatory activity in neurons and facilitate astrocyte Ca2+ slow wave propagation. Panxs do not form gap junctions in vivo, but Panx hemichannels participate in autocrine and paracrine gliotransmission, alongside Cx hemichannels. ATP and other gliotransmitters released by Cx and Panx hemichannels maintain physiologic glutamatergic tone by strengthening synapses and mitigating aberrant high frequency bursting. Under pathological depolarizing and inflammatory conditions, gap junctions and hemichannels become dysregulated, resulting in excessive neuronal firing and seizure. In this review, we present known contributions of Cxs and Panxs to physiologic neuronal excitation and explore how the disruption of gap junctions and hemichannels lead to abnormal glutamatergic transmission, purinergic signaling, and seizures.

p39, the primary activator for cyclin-dependent kinase 5 (Cdk5) in oligodendroglia, is essential for oligodendroglia differentiation and myelin repair.

Cyclin-dependent kinase 5 (Cdk5) plays key roles in normal brain development and function. Dysregulation of Cdk5 may cause neurodegeneration and cognitive impairment. Besides the well demonstrated role of Cdk5 in neurons, emerging evidence suggests the functional requirement of Cdk5 in oligodendroglia (OL) and CNS myelin development. However, whether neurons and OLs employ similar or distinct mechanisms to regulate Cdk5 activity remains elusive. We report here that in contrast to neurons that harbor high levels of two Cdk5 activators, p35 and p39, OLs express abundant p39 but negligible p35. In addition, p39 is selectively up-regulated in OLs during differentiation along with elevated Cdk5 activity, whereas p35 expression remains unaltered. Specific knockdown of p39 by siRNA significantly attenuates Cdk5 activity and OL differentiation without affecting p35. Finally, expression of p39, but not p35, is increased during myelin repair, and remyelination is impaired in p39(-/-) mice. Together, these results reveal that neurons and OLs harbor distinct preference of Cdk5 activators and demonstrate important functions of p39-dependent Cdk5 activation in OL differentiation during de novo myelin development and myelin repair.

Restoration of axon conduction and motor deficits by therapeutic treatment with glatiramer acetate.

Glatiramer acetate (GA; Copaxone) is an approved drug for the treatment of multiple sclerosis (MS). The underlying multifactorial anti-inflammatory, neuroprotective effect of GA is in the induction of reactive T cells that release immunomodulatory cytokines and neurotrophic factors at the injury site. These GA-induced cytokines and growth factors may have a direct effect on axon function. Building on previous findings that suggest a neuroprotective effect of GA, we assessed the therapeutic effects of GA on brain and spinal cord pathology and functional correlates using the chronic experimental autoimmune encephalomyelitis (EAE) mouse model of MS. Therapeutic regimens were utilized based on promising prophylactic efficacy. More specifically, C57BL/6 mice were treated with 2 mg/mouse/day GA for 8 days beginning at various time points after EAE post-induction day 15, yielding a thorough, clinically relevant assessment of GA efficacy within the context of severe progressive disease. Therapeutic treatment with GA significantly decreased clinical scores and improved rotorod motor performance in EAE mice. These functional improvements were supported by an increase in myelinated axons and fewer amyloid precursor protein-positive axons in the spinal cords of GA-treated EAE mice. Furthermore, therapeutic GA decreased microglia/macrophage and T cell infiltrates and increased oligodendrocyte numbers in both the spinal cord and corpus callosum of EAE mice. Finally, GA improved callosal axon conduction and nodal protein organization in EAE. Our results demonstrate that therapeutic GA treatment has significant beneficial effects in a chronic mouse model of MS, in which its positive effects on both myelinated and non-myelinated axons results in improved axon function.

Demyelination and neurodegeneration early in experimental autoimmune encephalomyelitis contribute to functional deficits in the anterior visual pathway.

Impaired visual function is a prevalent feature of optic neuritis (ON) in multiple sclerosis (MS). Abnormal visual evoked potential (VEP) findings of increased latencies, reduced amplitudes and abnormal waveforms as well as decreased retinal nerve fiber layer (RNFL) assessed by optical coherence tomography (OCT) are hallmarks of ON-induced visual dysfunction. Here we utilized the experimental autoimmune encephalomyelitis (EAE) mouse model of MS to investigate the functional and pathological progression during early (before any clinical symptoms), peak (initial maximal clinical symptoms), and late (chronic disease for > 3 weeks) disease stages. Demyelination and initial stages of axon damage were observed in early EAE. Significant demyelination, inflammation, increased axon damage and impaired P1/N2 amplitudes and latencies by VEP were seen in middle and late EAE groups. A decrease in RNFL thickness by OCT was observed only during late EAE. NanoString analysis of optic nerves from late EAE indicated elevated inflammation-related genes, reduced myelin-related genes, and changes in axon degeneration-related genes. Early inflammatory demyelination and functional deficits of the visual pathway, if untreated, may lead to severe irrecoverable axon damage in EAE. These studies potentially help explain the progression of visual dysfunction during MS.

Astrocytic Ephrin-B1 Regulates Oligodendrocyte Development and Myelination.

Astrocytes have been implicated in oligodendrocyte development and myelination, however, the mechanisms by which astrocytes regulate oligodendrocytes remain unclear. Our findings suggest a new mechanism that regulates astrocyte-mediated oligodendrocyte development through ephrin-B1 signaling in astrocytes. Using a mouse model, we examined the role of astrocytic ephrin-B1 signaling in oligodendrocyte development by deleting ephrin-B1 specifically in astrocytes during the postnatal days (P)14-P28 period and used mRNA analysis, immunohistochemistry, and mouse behaviors to study its effects on oligodendrocytes and myelination. We found that deletion of astrocytic ephrin-B1 downregulated many genes associated with oligodendrocyte development, myelination, and lipid metabolism in the hippocampus and the corpus callosum. Additionally, we observed a reduced number of oligodendrocytes and impaired myelination in the corpus callosum of astrocyte-specific ephrin-B1 KO mice. Finally, our data show reduced motor strength in these mice exhibiting clasping phenotype and impaired performance in the rotarod test most likely due to impaired myelination. Our studies provide new evidence that astrocytic ephrin-B1 positively regulates oligodendrocyte development and myelination, potentially through astrocyte-oligodendrocyte interactions.

Estrogen receptor ß ligand therapy activates PI3K/Akt/mTOR signaling in oligodendrocytes and promotes remyelination in a mouse model of multiple sclerosis.

The identification of a drug that stimulates endogenous myelination and spares axon degeneration during multiple sclerosis (MS) could potentially reduce the rate of disease progression. Using experimental autoimmune encephalomyelitis (EAE), a mouse model of MS, we have previously shown that prophylactic administration of the estrogen receptor (ER) ß ligand 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) decreases clinical disease, is neuroprotective, stimulates endogenous myelination, and improves axon conduction without altering peripheral cytokine production or reducing central nervous system (CNS) inflammation. Here, we assessed the effects of therapeutic DPN treatment during peak EAE disease, which represents a more clinically relevant treatment paradigm. In addition, we investigated the mechanism of action of DPN treatment-induced recovery during EAE. Given that prophylactic and therapeutic treatments with DPN during EAE improved remyelination-induced axon conduction, and that ER (α and ß) and membrane (m)ERs are present on oligodendrocyte lineage cells, a direct effect of treatment on oligodendrocytes is likely. DPN treatment of EAE animals resulted in phosphorylated ERß and activated the phosphatidylinositol 3-kinase (PI3K)/serine-threonine-specific protein kinase (Akt)/mammalian target of rapamycin (mTOR) signaling pathway, a pathway required for oligodendrocyte survival and axon myelination. These results, along with our previous studies of prophylactic DPN treatment, make DPN and similar ERß ligands immediate and favorable therapeutic candidates for demyelinating disease.

The complexities of investigating mitochondria dynamics in multiple sclerosis and mouse models of MS.

Multiple sclerosis (MS) is a demyelinating, degenerating disorder of the central nervous system (CNS) that is accompanied by mitochondria energy production failure. A loss of myelin paired with a deficit in energy production can contribute to further neurodegeneration and disability in patients in MS. Mitochondria are essential organelles that produce adenosine triphosphate (ATP) via oxidative phosphorylation in all cells in the CNS, including neurons, oligodendrocytes, astrocytes, and immune cells. In the context of demyelinating diseases, mitochondria have been shown to alter their morphology and undergo an initial increase in metabolic demand. This is followed by mitochondrial respiratory chain deficiency and abnormalities in mitochondrial transport that contribute to progressive neurodegeneration and irreversible disability. The current methodologies to study mitochondria are limiting and are capable of providing only a partial snapshot of the true mitochondria activity at a particular timepoint during disease. Mitochondrial functional studies are mostly performed in cell culture or whole brain tissue, which prevents understanding of mitochondrial pathology in distinct cell types in vivo. A true understanding of cell-specific mitochondrial pathophysiology of MS in mouse models is required. Cell-specific mitochondria morphology, mitochondria motility, and ATP production studies in animal models of MS will help us understand the role of mitochondria in the normal and diseased CNS. In this review, we present currently used methods to investigate mitochondria function in MS mouse models and discuss the current advantages and caveats with using each technique. In addition, we present recently developed mitochondria transgenic mouse lines expressing Cre under the control of CNS specific promoters to relate mitochondria to disease in vivo.

Lanthionine Ketimine Ethyl Ester Accelerates Remyelination in a Mouse Model of Multiple Sclerosis.

Although over 20 disease modifying therapies are approved to treat Multiple Sclerosis (MS), these do not increase remyelination of demyelinated axons or mitigate axon damage. Previous studies showed that lanthionine ketenamine ethyl ester (LKE) reduces clinical signs in the experimental autoimmune encephalomyelitis (EAE) mouse model of MS and increased maturation of oligodendrocyte (OL) progenitor cells (OPCs) in vitro. In the current study, we used the cuprizone (CPZ) demyelination model of MS to test if LKE could increase remyelination. The corpus callosum (CC) and somatosensory cortex was examined by immunohistochemistry (IHC), electron microscopy and for mRNA expression changes in mice provided 5 weeks of CPZ diet followed by 2 weeks of normal diet in the presence of LKE or vehicle. A significant increase in the number of myelinated axons, and increased myelin thickness was observed in the CC of LKE-treated groups compared to vehicle-treated groups. LKE also increased myelin basic protein and proteolipid protein expression in the CC and cortex, and increased the number of mature OLs in the cortex. In contrast, LKE did not increase the percentage of proliferating OPCs suggesting effects on OPC survival and differentiation but not proliferation. The effects of LKE on OL maturation and remyelination were supported by similar changes in their relative mRNA levels. Interestingly, LKE did not have significant effects on GFAP or Iba1 immunostaining or mRNA levels. These findings suggest that remyelinating actions of LKE can potentially be formulated to induce remyelination in neurological diseases associated with demyelination including MS.

Oestrogen receptor beta ligand: a novel treatment to enhance endogenous functional remyelination.

Demyelinating diseases, such as multiple sclerosis, are characterized by inflammatory demyelination and neurodegeneration of the central nervous system. Therapeutic strategies that induce effective neuroprotection and enhance intrinsic repair mechanisms are central goals for future therapy of multiple sclerosis. Oestrogens and oestrogen receptor ligands are promising treatments to prevent multiple sclerosis-induced neurodegeneration. In the present study we investigated the capacity of oestrogen receptor ß ligand treatment to affect callosal axon demyelination and stimulate endogenous myelination in chronic experimental autoimmune encephalomyelitis using electrophysiology, electron microscopy, immunohistochemistry and tract-tracing methods. Oestrogen receptor ß ligand treatment of experimental autoimmune encephalomyelitis mice prevented both histopathological and functional abnormalities of callosal axons despite the presence of inflammation. Specifically, there were fewer demyelinated, damaged axons and more myelinated axons with intact nodes of Ranvier in oestrogen receptor ß ligand-treated mice. In addition, oestrogen receptor ß ligand treatment caused an increase in mature oligodendrocyte numbers, a significant increase in myelin sheath thickness and axon transport. Functional analysis of callosal axon conduction showed a significant improvement in compound action potential amplitudes, latency and in axon refractoriness. These findings show a direct neuroprotective effect of oestrogen receptor ß ligand treatment on oligodendrocyte differentiation, myelination and axon conduction during experimental autoimmune encephalomyelitis.

Alleviation of extensive visual pathway dysfunction by a remyelinating drug in a chronic mouse model of multiple sclerosis.

Visual deficits are among the most prevalent symptoms in patients with multiple sclerosis (MS). To understand deficits in the visual pathway during MS and potential treatment effects, we used experimental autoimmune encephalomyelitis (EAE), the most commonly used animal model of MS. The afferent visual pathway was assessed in vivo using optical coherence tomography (OCT), electroretinography (ERG), and visually evoked cortical potentials (VEPs). Inflammation, demyelination, and neurodegeneration were examined by immunohistochemistry ex vivo. In addition, an immunomodulatory, remyelinating agent, the estrogen receptor ß ligand chloroindazole (IndCl), was tested for its therapeutic potential in the visual pathway. EAE produced functional deficits in visual system electrophysiology, including suppression of ERG and VEP waveform amplitudes and increased signal latencies. Therapeutic IndCl rescued overall visual system latency by VEP but had little impact on amplitude or ERG findings relative to vehicle. Faster VEP conduction in IndCl-treated mice was associated with enhanced myelin basic protein signal in all visual system structures examined. IndCl preserved retinal ganglion cells (RGCs) and oligodendrocyte density in the prechiasmatic white matter, but similar retinal nerve fiber layer thinning by OCT was noted in vehicle and IndCl-treated mice. Although IndCl differentially attenuated leukocyte and astrocyte staining signal throughout the structures analyzed, axolemmal varicosities were observed in all visual fiber tracts of mice with EAE irrespective of treatment, suggesting impaired axonal energy homeostasis. These data support incomplete functional recovery of VEP amplitude with IndCl, as fiber tracts displayed persistent axon pathology despite remyelination-induced decreases in latencies, evidenced by reduced optic nerve g-ratio in IndCl-treated mice. Although additional studies are required, these findings demonstrate the dynamics of visual pathway dysfunction and disability during EAE, along with the importance of early treatment to mitigate EAE-induced axon damage.

Astrocyte Glutamate Uptake and Water Homeostasis Are Dysregulated in the Hippocampus of Multiple Sclerosis Patients With Seizures.

While seizure disorders are more prevalent among multiple sclerosis (MS) patients than the population overall and prognosticate earlier death & disability, their etiology remains unclear. Translational data indicate perturbed expression of astrocytic molecules contributing to homeostatic neuronal excitability, including water channels (AQP4) and synaptic glutamate transporters (EAAT2), in a mouse model of MS with seizures (MS+S). However, astrocytes in MS+S have not been examined. To assess the translational relevance of astrocyte dysfunction observed in a mouse model of MS+S, demyelinated lesion burden, astrogliosis, and astrocytic biomarkers (AQP4/EAAT2/ connexin-CX43) were evaluated by immunohistochemistry in postmortem hippocampi from MS & MS+S donors. Lesion burden was comparable in MS & MS+S cohorts, but astrogliosis was elevated in MS+S CA1 with a concomitant decrease in EAAT2 signal intensity. AQP4 signal declined in MS+S CA1 & CA3 with a loss of perivascular AQP4 in CA1. CX43 expression was increased in CA3. Together, these data suggest that hippocampal astrocytes from MS+S patients display regional differences in expression of molecules associated with glutamate buffering and water homeostasis that could exacerbate neuronal hyperexcitability. Importantly, mislocalization of CA1 perivascular AQP4 seen in MS+S is analogous to epileptic hippocampi without a history of MS, suggesting convergent pathophysiology. Furthermore, as neuropathology was concentrated in MS+S CA1, future study is warranted to determine the pathophysiology driving regional differences in glial function in the context of seizures during demyelinating disease.

Purkinje cell loss in experimental autoimmune encephalomyelitis.

Gray matter atrophy observed by brain MRI is an important correlate to clinical disability and disease duration in multiple sclerosis. The objective of this study was to link brain atrophy visualized by neuroimaging to its underlying neuropathology using the MS model, experimental autoimmune encephalomyelitis (EAE). Volumetric changes in brains of EAE mice, as well as matched healthy normal controls, were quantified by collecting post-mortem high-resolution T2-weighted magnetic resonance microscopy and actively stained magnetic resonance histology images. Anatomical delineations demonstrated a significant decrease in the volume of the whole cerebellum, cerebellar cortex, and molecular layer of the cerebellar cortex in EAE as compared to normal controls. The pro-apoptotic marker caspase-3 was detected in Purkinje cells and a significant decrease in Purkinje cell number was found in EAE. Cross modality and temporal correlations revealed a significant association between Purkinje cell loss on neuropathology and atrophy of the molecular layer of the cerebellar cortex by neuroimaging. These results demonstrate the power of using combined population atlasing and neuropathology approaches to discern novel insights underlying gray matter atrophy in animal models of neurodegenerative disease.

Estrogen treatment decreases matrix metalloproteinase (MMP)-9 in autoimmune demyelinating disease through estrogen receptor alpha (ERalpha).

Matrix metalloproteinases (MMPs) have a crucial function in migration of inflammatory cells into the central nervous system (CNS). Levels of MMP-9 are elevated in multiple sclerosis (MS) and predict the occurrence of new active lesions on magnetic resonance imaging (MRI). This translational study aims to determine whether in vivo treatment with the pregnancy hormone estriol affects MMP-9 levels from immune cells in patients with MS and mice with experimental autoimmune encephalomyelitis (EAE). Peripheral blood mononuclear cells (PBMCs) collected from three female MS patients treated with estriol and splenocytes from EAE mice treated with estriol, estrogen receptor (ER) alpha ligand, ERbeta ligand or vehicle were stimulated ex vivo and analyzed for levels of MMP-9. Markers of CNS infiltration were assessed using MRI in patients and immunohistochemistry in mice. Supernatants from PBMCs obtained during estriol treatment in female MS patients showed significantly decreased MMP-9 compared with pretreatment. Decreases in MMP-9 coincided with a decrease in enhancing lesion volume on MRI. Estriol treatment of mice with EAE reduced MMP-9 in supernatants from autoantigen-stimulated splenocytes, coinciding with decreased CNS infiltration by T cells and monocytes. Experiments with selective ER ligands showed that this effect was mediated through ERalpha. In conclusion, estriol acting through ERalpha to reduce MMP-9 from immune cells is one mechanism potentially underlying the estriol-mediated reduction in enhancing lesions in MS and inflammatory lesions in EAE.

Functional Effects of Cuprizone-Induced Demyelination in the Presence of the mTOR-Inhibitor Rapamycin.

Persistent demyelination has been implicated in axon damage and functional deficits underlying neurodegenerative diseases such as multiple sclerosis. The cuprizone diet model of demyelination allows for the investigation of mechanisms underlying timed and reproducible demyelination and remyelination. However, spontaneous oligodendrocyte (OL) progenitor (OPC) proliferation, OPC differentiation, and axon remyelination during cuprizone diet may convolute the understanding of remyelinating events. The Akt (a serine/threonine kinase)/mTOR (the mammalian target of rapamycin) signaling pathway in OLs regulates intermediate steps during myelination. Thus, in an effort to inhibit spontaneous remyelination, the mTOR inhibitor rapamycin has been administered during cuprizone diet. Intrigued by the potential for rapamycin to optimize the cuprizone model by producing more complete demyelination, we sought to characterize the effects of rapamycin on axonal function and myelination. Functional remyelination was assessed by callosal compound action potential (CAP) recordings along with immunohistochemistry in mice treated with rapamycin during cuprizone diet. Rapamycin groups exhibited similar myelination, but significantly increased axonal damage and inflammation compared to non-rapamycin groups. There was minimal change in CAP amplitude between groups, however, a significant decrease in conduction velocity of the slower, non-myelinated CAP component was observed in the rapamycin group relative to the non-rapamycin group. During remyelination, rapamycin groups showed a significant decrease in OPC proliferation and mature OLs, suggesting a delay in OPC differentiation kinetics. In conclusion, we question the use of rapamycin to produce consistent demyelination as rapamycin increased inflammation and axonal damage, without affecting myelination.

Analogues of ERß ligand chloroindazole exert immunomodulatory and remyelinating effects in a mouse model of multiple sclerosis.

Pharmaceutical agents currently approved for the treatment of multiple sclerosis reduce relapse rates, but do not reverse or prevent neurodegeneration nor initiate myelin repair. The highly selective estrogen receptor (ER) ß ligand chloroindazole (IndCl) shows particular promise promoting both remyelination while reducing inflammatory cytokines in the central nervous system of mice with experimental autoimmune encephalomyelitis. To optimize these benefits, we developed and screened seven novel IndCl analogues for their efficacy in promoting primary oligodendrocyte (OL) progenitor cell survival, proliferation, and differentiation in vitro by immunohistochemistry. Two analogues, IndCl-o-chloro and IndCl-o-methyl, induced proliferation and differentiation equivalent to IndCl and were selected for subsequent in vivo evaluation for their impact on clinical disease course, white matter pathology, and inflammation. Both compounds ameliorated disease severity, increased mature OLs, and improved overall myelination in the corpus callosum and white matter tracts of the spinal cord. These effects were accompanied by reduced production of the OL toxic molecules interferon-γ and chemokine (C-X-C motif) ligand, CXCL10 by splenocytes with no discernable effect on central nervous system-infiltrating leukocyte numbers, while IndCl-o-methyl also reduced peripheral interleukin (IL)-17. In addition, expression of the chemokine CXCL1, which is associated with developmental oligodendrogenesis, was upregulated by IndCl and both analogues. Furthermore, callosal compound action potential recordings from analogue-treated mice demonstrated a larger N1 component amplitude compared to vehicle, suggesting more functionally myelinated fibers. Thus, the o-Methyl and o-Chloro IndCl analogues represent a class of ERß ligands that offer significant remyelination and neuroprotection as well as modulation of the immune system; hence, they appear appropriate to consider further for therapeutic development in multiple sclerosis and other demyelinating diseases.