A phenotypic screening platform for identifying chemical modulators of astrocyte reactivity.

Disease, injury and aging induce pathological reactive astrocyte states that contribute to neurodegeneration. Modulating reactive astrocytes therefore represent an attractive therapeutic strategy. Here we describe the development of an astrocyte phenotypic screening platform for identifying chemical modulators of astrocyte reactivity. Leveraging this platform for chemical screening, we identify histone deacetylase 3 (HDAC3) inhibitors as effective suppressors of pathological astrocyte reactivity. We demonstrate that HDAC3 inhibition reduces molecular and functional characteristics of reactive astrocytes in vitro. Transcriptional and chromatin mapping studies show that HDAC3 inhibition disarms pathological astrocyte gene expression and function while promoting the expression of genes associated with beneficial astrocytes. Administration of RGFP966, a small molecule HDAC3 inhibitor, blocks reactive astrocyte formation and promotes neuroprotection in vivo in mice. Collectively, these results establish a platform for discovering modulators of reactive astrocyte states, inform the mechanisms that control astrocyte reactivity and demonstrate the therapeutic benefits of modulating astrocyte reactivity for neurodegenerative diseases.

Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis.

Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by inflammation, demyelination, and neurodegeneration. The ideal MS therapy would both specifically inhibit the underlying autoimmune response and promote repair/regeneration of myelin as well as maintenance of axonal integrity. Currently approved MS therapies consist of non-specific immunosuppressive molecules/antibodies which block activation or CNS homing of autoreactive T cells, but there are no approved therapies for stimulation of remyelination nor maintenance of axonal integrity. In an effort to repurpose an FDA-approved medication for myelin repair, we chose to examine the effectiveness of digoxin, a cardiac glycoside (Na+ /K+ ATPase inhibitor), originally identified as pro-myelinating in an in vitro screen. We found that digoxin regulated multiple genes in oligodendrocyte progenitor cells (OPCs) essential for oligodendrocyte (OL) differentiation in vitro, promoted OL differentiation both in vitro and in vivo in female naïve C57BL/6J (B6) mice, and stimulated recovery of myelinated axons in B6 mice following demyelination in the corpus callosum induced by cuprizone and spinal cord demyelination induced by lysophosphatidylcholine (LPC), respectively. More relevant to treatment of MS, we show that digoxin treatment of mice with established MOG35-55 -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG35-55 (PLG-MOG35-55 ) completely ameliorated clinical disease symptoms and stimulated recovery of OL lineage cell numbers. These findings provide critical pre-clinical evidence supporting future clinical trials of myelin-specific tolerance with myelin repair/regeneration drugs, such as digoxin, in MS patients.

Astrocytes Are Required for Oligodendrocyte Survival and Maintenance of Myelin Compaction and Integrity.

Astrocytes have been implicated in regulating oligodendrocyte development and myelination in vitro, although their functions in vivo remain less well defined. Using a novel approach to locally ablate GFAP+ astrocytes, we demonstrate that astrocytes are required for normal CNS myelin compaction during development, and for maintaining myelin integrity in the adult. Transient ablation of GFAP+ astrocytes in the mouse spinal cord during the first postnatal week reduced the numbers of mature oligodendrocytes and inhibited myelin formation, while prolonged ablation resulted in myelin that lacked compaction and structural integrity. Ablation of GFAP+ astrocytes in the adult spinal cord resulted in the rapid, local loss of myelin integrity and regional demyelination. The loss of myelin integrity induced by astrocyte ablation was greatly reduced by NMDA receptor antagonists, both in vitro and in vivo, suggesting that myelin stability was affected by elevation of local glutamate levels following astrocyte ablation. Furthermore, targeted delivery of glutamate into adult spinal cord white matter resulted in reduction of myelin basic protein expression and localized disruption of myelin compaction which was also reduced by NMDA receptor blockade. The pathology induced by localized astrocyte loss and elevated exogenous glutamate, supports the concept that astrocytes are critical for maintenance of myelin integrity in the adult CNS and may be primary targets in the initiation of demyelinating diseases of the CNS, such as Neuromyelitis Optica (NMO).

Developmental ablation of mature oligodendrocytes exacerbates adult CNS demyelination.

Multiple sclerosis (MS) is a CNS neurodegenerative autoimmune disease characterized by loss of oligodendrocytes and myelin in the brain and the spinal cord that results in localized functional deficits. Several risk factors have been associated with MS, however none fully explain the enhanced susceptibility seen in older individuals. Epidemiological data, based on geographical prevalence studies suggest that susceptibility is established early in life and frequently long before the diagnosis of disease raising the possibility that developmental events influence adult disease onset and progression. Here we test the hypothesis that selective loss of mature oligodendrocytes during postnatal development results in enhanced susceptibility to a demyelinating insult to the mature CNS. A transgenic mouse model was utilized to specifically induce apoptotic cell death in a subset of mature oligodendrocytes (MBP-iCP9) during the first 2 postnatal weeks followed by either a local LPC spinal cord injection or the induction of EAE in the adult animal. Immunostaining, immunoblotting, behavioral testing, and electron microscopy were utilized to examine the differences in the response between animals with developmental loss of oligodendrocytes and controls. We show that during development, oligodendrocyte apoptosis results in transient reductions in myelination and functional deficits that recover after 10-14 days. Compared to animals in which oligodendrocyte development was unperturbed, animals subjected to postnatal oligodendrocyte loss showed delayed recovery from an LPC lesion to the mature spinal cord. Unexpectedly, the induction and severity of MOG induced EAE was not significantly altered in animals following oligodendrocyte developmental loss even though there was a substantial increase in spinal cord tissue damage and CNS inflammation. It is unclear why the elevated glial responses seen in developmentally compromised animals were not reflected in enhanced functional deficits. These observations suggest that developmental loss of oligodendrocytes results in long lasting tissue changes that alter its response to subsequent insults and the capacity for repair in the adult.

Induction of myelinating oligodendrocytes in human cortical spheroids.

Cerebral organoids provide an accessible system for investigations of cellular composition, interactions, and organization but have lacked oligodendrocytes, the myelinating glia of the central nervous system. Here we reproducibly generated oligodendrocytes and myelin in 'oligocortical spheroids' derived from human pluripotent stem cells. Molecular features consistent with those of maturing oligodendrocytes and early myelin appeared by week 20 in culture, with further maturation and myelin compaction evident by week 30. Promyelinating drugs enhanced the rate and extent of oligodendrocyte generation and myelination, and spheroids generated from human subjects with a genetic myelin disorder recapitulated human disease phenotypes. Oligocortical spheroids provide a versatile platform for studies of myelination of the developing central nervous system and offer new opportunities for disease modeling and therapeutic development.

Accumulation of 8,9-unsaturated sterols drives oligodendrocyte formation and remyelination.

Regeneration of myelin is mediated by oligodendrocyte progenitor cells-an abundant stem cell population in the central nervous system (CNS) and the principal source of new myelinating oligodendrocytes. Loss of myelin-producing oligodendrocytes in the CNS underlies a number of neurological diseases, including multiple sclerosis and diverse genetic diseases1-3. High-throughput chemical screening approaches have been used to identify small molecules that stimulate the formation of oligodendrocytes from oligodendrocyte progenitor cells and functionally enhance remyelination in vivo4-10. Here we show that a wide range of these pro-myelinating small molecules function not through their canonical targets but by directly inhibiting CYP51, TM7SF2, or EBP, a narrow range of enzymes within the cholesterol biosynthesis pathway. Subsequent accumulation of the 8,9-unsaturated sterol substrates of these enzymes is a key mechanistic node that promotes oligodendrocyte formation, as 8,9-unsaturated sterols are effective when supplied to oligodendrocyte progenitor cells in purified form whereas analogous sterols that lack this structural feature have no effect. Collectively, our results define a unifying sterol-based mechanism of action for most known small-molecule enhancers of oligodendrocyte formation and highlight specific targets to propel the development of optimal remyelinating therapeutics.

Transcriptional Profiling of Mesenchymal Stem Cells Identifies Distinct Neuroimmune Pathways Altered by CNS Disease.

BACKGROUND AND OBJECTIVES: Bone marrow mesenchymal stem cells (BM-MSCs) are an attractive cell based therapy in the treatment of CNS demyelinating diseases such as multiple sclerosis (MS). Preclinical studies demonstrate that BM-MSCs can effectively reduce clinical burden and enhance recovery in experimental autoimmune encephalomyelitis (EAE), a commonly used animal model of MS. However, a number of recent clinical trials have not shown significant functional benefit following BM-MSC infusion into MS patients. One possibility for the discrepancy between animal and human studies is the source of the cells, as recent studies suggest BM-MSCs from MS patients or animals with EAE lack reparative efficacy compared to naïve cells. We sought to define important transcriptional and functional differences between diseased and naïve MSCs. METHODS AND RESULTS: We utilized RNA Sequencing (RNA-Seq) to assess changes in gene expression between BM-MSCs derived from EAE animals and those derived from healthy controls. We show that EAE alters the expression of a large number of genes in BM-MSCs and changes in gene expression are more pronounced in chronic versus acute disease. Bioinformatic analysis revealed extensive perturbations in BM-MSCs in pathways related to inflammation and the regulation of neural cell development. These changes suggest that signals from EAE derived BM-MSCs inhibit rather than enhance remyelination, and in-vitro studies showed that conditioned medium from EAE MSCs fails to support the development of mature oligodendrocytes, the myelinating cells of the CNS. CONCLUSIONS: These data provide insight into the failure of autologous BM-MSCs to promote recovery in MS and support the concept of utilizing non-autologous MSCs in future clinical trials.

CNS disease diminishes the therapeutic functionality of bone marrow mesenchymal stem cells.

Mesenchymal stem cells (MSCs) have emerged as a potentially powerful cellular therapy for autoimmune diseases including multiple sclerosis (MS). Based on their success in treating animal models of MS like experimental autoimmune encephalomyelitis (EAE), MSCs have moved rapidly into clinical trials for MS. The majority of these trials use autologous MSCs derived from MS patients, although it remains unclear how CNS disease may affect these cells. Here, we report that bone marrow MSCs derived from EAE mice lack therapeutic efficacy compared to naïve MSCs in their ability to ameliorate EAE. Treatment with conditioned medium from EAE-MSCs also fails to modulate EAE, and EAE-MSCs secrete higher levels of many pro-inflammatory cytokines compared to naïve MSCs. Similarly, MSCs derived from MS patients have less therapeutic efficacy than naïve MSCs in treating EAE and secrete higher levels of some of the same pro-inflammatory cytokines. Thus diseases like EAE and MS diminish the therapeutic functionality of bone marrow MSCs, prompting reevaluation about the ongoing use of autologous MSCs as a treatment for MS.