Andrew P. Robinson

Drug-based modulation of endogenous stem cells promotes functional remyelination in vivo

Multiple sclerosis involves an aberrant autoimmune response and progressive failure of remyelination in the central nervous system. Prevention of neural degeneration and subsequent disability requires remyelination through the generation of new oligodendrocytes, but current treatments exclusively target the immune system. Oligodendrocyte progenitor cells are stem cells in the central nervous system and the principal source of myelinating oligodendrocytes. These cells are abundant in demyelinated regions of patients with multiple sclerosis, yet fail to differentiate, thereby representing a cellular target for pharmacological intervention. To discover therapeutic compounds for enhancing myelination from endogenous oligodendrocyte progenitor cells, we screened a library of bioactive small molecules on mouse pluripotent epiblast stem-cell-derived oligodendrocyte progenitor cells. Here we show seven drugs function at nanomolar doses selectively to enhance the generation of mature oligodendrocytes from progenitor cells in vitro. Two drugs, miconazole and clobetasol, are effective in promoting precocious myelination in organotypic cerebellar slice cultures, and in vivo in early postnatal mouse pups. Systemic delivery of each of the two drugs significantly increases the number of new oligodendrocytes and enhances remyelination in a lysolecithin-induced mouse model of focal demyelination. Administering each of the two drugs at the peak of disease in an experimental autoimmune encephalomyelitis mouse model of chronic progressive multiple sclerosis results in striking reversal of disease severity. Immune response assays show that miconazole functions directly as a remyelinating drug with no effect on the immune system, whereas clobetasol is a potent immunosuppressant as well as a remyelinating agent. Mechanistic studies show that miconazole and clobetasol function in oligodendrocyte progenitor cells through mitogen-activated protein kinase and glucocorticoid receptor signalling, respectively. Furthermore, both drugs enhance the generation of human oligodendrocytes from human oligodendrocyte progenitor cells in vitro. Collectively, our results provide a rationale for testing miconazole and clobetasol, or structurally modified derivatives, to enhance remyelination in patients.

The experimental autoimmune encephalomyelitis (EAE) model of MS: utility for understanding disease pathophysiology and treatment

While no single model can exactly recapitulate all aspects of multiple sclerosis (MS), animal models are essential in understanding the induction and pathogenesis of the disease and to develop therapeutic strategies that limit disease progression and eventually lead to effective treatments for the human disease. Several different models of MS exist, but by far the best understood and most commonly used is the rodent model of experimental autoimmune encephalomyelitis (EAE). This model is typically induced by either active immunization with myelin-derived proteins or peptides in adjuvant or by passive transfer of activated myelin-specific CD4+ T lymphocytes. Mouse models are most frequently used because of the inbred genotype of laboratory mice, their rapid breeding capacity, the ease of genetic manipulation, and availability of transgenic and knockout mice to facilitate mechanistic studies. Although not all therapeutic strategies for MS have been developed in EAE, all of the current US Food and Drug Administration (FDA)-approved immunomodulatory drugs are effective to some degree in treating EAE, a strong indicator that EAE is an extremely useful model to study potential treatments for MS. Several therapies, such as glatiramer acetate (GA: Copaxone), and natalizumab (Tysabri), were tested first in the mouse model of EAE and then went on to clinical trials. Here we discuss the usefulness of the EAE model in understanding basic disease pathophysiology and developing treatments for MS as well as the potential drawbacks of this model.

Pharmaceutical integrated stress response enhancement protects oligodendrocytes and provides a potential multiple sclerosis therapeutic

Oligodendrocyte death contributes to the pathogenesis of the inflammatory demyelinating disease multiple sclerosis (MS). Nevertheless, current MS therapies are mainly immunomodulatory and have demonstrated limited ability to inhibit MS progression. Protection of oligodendrocytes is therefore a desirable strategy for alleviating disease. Here we demonstrate that enhancement of the integrated stress response using the FDA-approved drug guanabenz increases oligodendrocyte survival in culture and prevents hypomyelination in cerebellar explants in the presence of interferon-γ, a pro-inflammatory cytokine implicated in MS pathogenesis. In vivo, guanabenz treatment protects against oligodendrocyte loss caused by CNS-specific expression of interferon-γ. In a mouse model of MS, experimental autoimmune encephalomyelitis, guanabenz alleviates clinical symptoms, which correlates with increased oligodendrocyte survival and diminished CNS CD4+ T cell accumulation. Moreover, guanabenz ameliorates relapse in relapsing-remitting experimental autoimmune encephalomyelitis. Our results provide support for a MS therapy that enhances the integrated stress response to protect oligodendrocytes against the inflammatory CNS environment.

Nf1 Loss and Ras Hyperactivation in Oligodendrocytes Induce NOS-Driven Defects in Myelin and Vasculature

SUMMARY Patients with neurofibromatosis type 1 (NF1) and Costello syndrome Rasopathy have behavioral deficits. In NF1 patients, these may correlate with white matter enlargement and aberrant myelin. To model these features, we induced Nf1 loss or HRas hyperactivation in mouse oligodendrocytes. Enlarged brain white matter tracts correlated with myelin decompaction, downregulation of claudin-11, and mislocalization of connexin-32. Surprisingly, non-cell-autonomous defects in perivascular astrocytes and the blood-brain barrier (BBB) developed, implicating a soluble mediator. Nitric oxide (NO) can disrupt tight junctions and gap junctions, and NO and NO synthases (NOS1–NOS3) were upregulated in mutant white matter. Treating mice with the NOS inhibitor NG-nitro-L-arginine methyl ester or the antioxidant N-acetyl cysteine corrected cellular phenotypes. CNP-HRasG12V mice also displayed locomotor hyperactivity, which could be rescued by antioxidant treatment. We conclude that Nf1/Ras regulates oligodendrocyte NOS and that dysregulated NO signaling in oligodendrocytes can alter the surrounding vasculature. The data suggest that anti-oxidants may improve some behavioral deficits in Rasopathy patients.

ER Chaperone BiP/GRP78 Is Required for Myelinating Cell Survival and Provides Protection during Experimental Autoimmune Encephalomyelitis.

Myelinating cells synthesize large amounts of membrane protein through the secretory pathway, which makes these cells particularly sensitive to perturbations of the endoplasmic reticulum (ER). Ig binding protein (BiP), also known as glucose-regulated protein 78 (GRP78), is a critical ER chaperone that also plays a pivotal role in controlling the cellular response to ER stress. To examine the potential importance of BiP to myelinating cells, we used a conditional knock-out approach to BiP gene inactivation in oligodendrocytes during development, in adulthood, and in response to experimental autoimmune encephalomyelitis (EAE), an animal model of the inflammatory demyelinating disorder multiple sclerosis (MS). During development, mice lacking functional BiP gene expression in oligodendrocytes developed tremors and ataxia and died before reaching maturity. When BiP gene inactivation in oligodendrocytes was initiated in adulthood, the mice displayed severe neurological symptoms including tremors and hind-limb paralysis. The inactivation of BiP in oligodendrocytes during development or in adulthood resulted in oligodendrocyte loss and corresponding severe myelin abnormalities. Mice heterozygous for the oligodendrocyte-specific inactivation of BiP, which were phenotypically normal without evidence of neuropathology, displayed an exacerbated response to EAE that correlated with an increased loss of oligodendrocytes. Furthermore, mice in which the BiP gene was specifically inactivated in developing Schwann cells displayed tremor that progressed to hindlimb paralysis, which correlated with diminished numbers of myelinating Schwann cells and severe PNS hypomyelination. These studies demonstrate that BiP is critical for myelinating cell survival and contributes to the protective response of oligodendrocyte against inflammatory demyelination. SIGNIFICANCE STATEMENT The myelinating cells, oligodendrocytes in the CNS and Schwann cells in the PNS, are responsible for synthesizing an enormous amount of cellular membrane during the active phase of myelination. Therefore, these cells are particularly sensitive to insults that disrupt the function of the secretory pathway. Here, we show that the endoplasmic reticulum (ER) resident chaperone protein Ig binding protein (BiP) plays an essential role in the survival and function of myelinating cells both during the myelination process and in adult animals. Moreover, we demonstrate that BiP participates in the protective response of oligodendrocytes to inflammatory demyelinating insults. The work described here suggests that a compromised response to perturbations to the ER could contribute to myelin disorders of the CNS and PNS.

Characterization of oligodendroglial populations in mouse demyelinating disease using flow cytometry: Clues for MS pathogenesis

Characterizing and enumerating cells of the oligodendrocyte lineage (OLCs) is crucial for understanding demyelination and therapeutic benefit in models of demyelinating disease in the central nervous system. Here we describe a novel method for the rapid, unbiased analysis of mouse OLCs using flow cytometry. The assay was optimized to maximize viable yield of OLCs and maintain OLC antigen integrity. Panels of antibodies were assembled for simultaneous analysis of seven antigens on individual cells allowing for characterization of oligodendroglial cells throughout the lineage. We verified the utility of the assay with cultured OLCs and through a time course of developmental myelination. Next we employed the assay to characterize OLC populations in two well-characterized models of demyelination: cuprizone-induced demyelination and experimental autoimmune encephalomyelitis (EAE). In EAE we observed a dramatic loss of mature oligodendrocytes coincident with a dramatic expansion of oligodendrocyte progenitors cells (OPCs) at the onset of disease suggesting an attempt of the host to repair myelin. This expanded OPC pool was maintained through remission and relapse suggesting an arrest in differentiation in the face of the chronic autoimmune T cell-mediated inflammatory response. These robust, reproducible changes in OLCs through disease provide a rapid quantitative global analysis of myelin-producing cells in the adult mouse brain and important information regarding effects of disease on oligodendroglial proliferation/differentiation which is useful for defining the pathogenesis and therapy of MS.

IL‐17A activates ERK1/2 and enhances differentiation of oligodendrocyte progenitor cells

Inflammatory signals present in demyelinated multiple sclerosis lesions affect the reparative remyelination process conducted by oligodendrocyte progenitor cells (OPCs). Interferon‐γ (IFN‐γ), tumor necrosis factor‐α (TNF‐α), and interleukin (IL)−6 have differing effects on the viability and growth of OPCs, however the effects of IL‐17A are largely unknown. Primary murine OPCs were stimulated with IL‐17A and their viability, proliferation, and maturation were assessed in culture. IL‐17A‐stimulated OPCs exited the cell cycle and differentiated with no loss in viability. Expression of the myelin‐specific protein, proteolipid protein, increased in a cerebellar slice culture assay in the presence of IL‐17A. Downstream, IL‐17A activated ERK1/2 within 15 min and induced chemokine expression in 2 days. These results demonstrate that IL‐17A exposure stimulates OPCs to mature and participate in the inflammatory response. GLIA 2015;63:768–779

Peripherally derived T regulatory and γδ T cells have opposing roles in the pathogenesis of intractable pediatric epilepsy

The pathophysiology of drug-resistant pediatric epilepsy is unknown. Flow cytometric analysis of inflammatory leukocytes in resected brain tissues from 29 pediatric patients with genetic (focal cortical dysplasia) or acquired (encephalomalacia) epilepsy demonstrated significant brain infiltration of blood-borne inflammatory myeloid cells and memory CD4+ and CD8+ T cells. Significantly, proinflammatory (IL-17– and GM-CSF–producing) &ggr;&dgr; T cells were concentrated in epileptogenic lesions, and their numbers positively correlated with disease severity. Conversely, numbers of regulatory T (T reg) cells inversely correlated with disease severity. Correspondingly, using the kainic acid model of status epilepticus, we show ameliorated seizure activity in both &ggr;&dgr; T cell– and IL-17RA–deficient mice and in recipients of T reg cells, whereas T reg cell depletion heightened seizure severity. Moreover, both IL-17 and GM-CSF induced neuronal hyperexcitability in brain slice cultures. These studies support a major pathological role for peripherally derived innate and adaptive proinflammatory immune responses in the pathogenesis of intractable epilepsy and suggest testing of immunomodulatory therapies.

Characterizing oligodendrocyte lineage cell function by flow cytometry in animal models of demyelination

Background: The inadequate tissue repair of multiple sclerosis (MS) lesions is assumed to be, in part, due to dysregulated expression of signaling molecules that induce oligodendrogenesis and neurogenesis. Several bone morphogenic proteins (BMP) stimulate the differentiation of neuronal stem cells (NSCs) towards astogliogenesis on the expense of oligodendrogenesis and neurogenesis. BMP-2,4,5 are upregulated in immune cells of multiple sclerosis patients, while their antagonists, noggin and follistatin, are downregulated in these cells. Differential screening-selected gene aberrative in neuroblastoma (DAN) is a member of the DAN family of secreted glycoproteins that are putative BMP antagonists. Its role in multiple sclerosis has not yet been studied. Objective: The aim of this study was to study the expression of DAN levels in the sera, CSF and supernatants of immune cells of patients with relapsing-remitting MS (RRMS). Methods: Twenty four untreated RRMS patients, 25 interferon β (IFNβ) treated RRMS patients and 26 matched healthy controls (HC) participated in the study. The control group of CSF donors comprised patients with non-infectious and non-inflammatory disorders. DAN levels in the sera, CSF and the supernatants of peripheral blood mononuclear cells (PBMCs), that were either not stimulated or stimulated with anti-CD3/CD28 mAb, or LPS , were measured by ELISA with a sensitivity of 125 pg/ml. Results: In the CSF DAN levels only the RRMS patient group (mean ± S.D= 591.9 ± 147.3 pg/ml, p = 0.031) as compared to CSF from control group were detected. There were no significant differences in the sera levels of DAN between untreated patients with RRMS (757.9 ± 478.5) vs. HC (943.0 ± 591.6) and vs. IFNβ treated RRMS patients (936.5 ± 485.9). DAN levels were below the detection threshold in supernatants of all studied groups and stimulatory conditions. Discussion: Reduced production of BMP antagonists from PBMCs of MS patients was previously demonstrated and led to the suggestion that immune mediated neuroregenesis/oligidendrogenesis is defective in patients with MS. Our results show that DAN, a BMP antagonist, is not expressed by immune cells. However, the increased DAN levels in the CSF of RRMS patients with no differences in sera levels between RRMS patients and HC suggesting its production within the CNS to enhance neurogenesis and oligodendrogenesis.