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Isolation of Pure Astrocytes and Microglia from the Adult Mouse Spinal Cord For <em>In Vitro</em> Assays and Transcriptomic Studies

Astrocytes and microglia play pivotal roles in central nervous system development, injury responses, and neurodegenerative diseases. These highly dynamic ...

The George Washington University School of Medicine

The concept of the oligodendrocyte lineage as simply a source of myelinating cells in the vertebrate CNS is undergoing radical revision. Elucidation of the origins of oligodendrocytes in the CNS has led to identification of important signaling pathways, the timing and mechanism of lineage commitments and overlapping as well as redundant functionality among oligodendrocytes. The realization that a significant proportion of the oligodendrocyte lineage cells remain in a proliferative and immature state suggests they have roles other than as a reservoir of myelinating cells. While early studies were focused on understanding the development of oligodendrocytes, more recent work has begun to define the role of oligodendrocyte lineage cells in CNS functionality and the identification of new avenues for neural repair. A relatively unexplored aspect of the oligodendrocyte lineage is their contribution either directly or indirectly to the pathology of neurodegenerative diseases such as ALS and Alzheimer's disease. Here we briefly consider the potential role of oligodendrocyte lineage cells as mediators of neural repair and neurodegeneration in the vertebrate CNS. This article is part of the Special Issue entitled 'Oligodendrocytes in Health and Disease'.

Contribution of the oligodendrocyte lineage to CNS repair and neurodegenerative pathologies.

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.

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

Calcium control of myelin sheath growth.

Myelination is critical for the normal functioning of the central nervous system (CNS) in vertebrates. Conditions in which the development of myelin is perturbed result in severely compromised individuals often with shorter lifespans, while loss of myelin in the adult results in a variety of functional deficits. Although some form of spontaneous remyelination often takes place, the repair process as a whole often fails. Several lines of evidence suggest it is feasible to develop strategies that enhance the capacity of the CNS to undergo remyelination and potentially reverse functional deficits. Such strategies include cellular therapies using either neural or mesenchymal stem cells as well as molecular regulators of oligodendrocyte development and differentiation. Given the prevalence of demyelinating diseases and their effects on the quality of life for affected individuals it is imperative that effective therapies are developed. Here we discuss some of the new approaches to CNS myelin repair that hold promise for reducing the burden of diseases characterized by myelin loss.

Emerging Cellular and Molecular Strategies for Enhancing Central Nervous System (CNS) Remyelination.

The optic nerve represents one of the simplest regions of the CNS and has been useful in developing an understanding of glial development and myelination. While the visual system is frequently affected in demyelinating conditions, utilizing the optic nerve to model demyelination/remyelination studies has been difficult due to its accessibility, relatively small size, and dense nature that makes direct injections challenging. Taking advantage of the lack of oligodendrocytes and myelination in the mouse retina, we have developed a model in which the induction of apoptosis in mature oligodendrocytes allows for the selective, non-invasive generation of demyelinating lesions in optic nerve. Delivery of an inducer of oligodendrocyte apoptosis by intravitreous injection minimizes trauma to the optic nerve and allows for the assessment of oligodendrocyte death in the absence of injury related factors. Here we show that following induction of apoptosis, oligodendrocytes are lost within 3 days. The loss of oligodendrocytes is associated with limited microglial and astrocyte response, is patchy along the nerve, and results in localized myelin loss. Unlike in other regions of the murine CNS, where local demyelination stimulates activation of local oligodendrocyte precursors and remyelination, optic nerve demyelination induced by oligodendrocyte apoptosis fails to recover and results in persistent areas of myelin loss. Over time these chronic lesions change cellular composition and ultimately become devoid of GFAP+ astrocytes and OPCs. Why the optic nerve lesions fail to repair may reflect the lack of early immune responsiveness and provide a novel model of chronic demyelination.

Targeted Oligodendrocyte Apoptosis in Optic Nerve Leads to Persistent Demyelination.

Retinal ganglion cells generate a pattern of action potentials to communicate visual information from the retina to cortical areas. Myelin, an insulating sheath, wraps axonal segments to facilitate signal propagation and when deficient, can impair visual function. Optic nerve development and initial myelination has largely been considered completed by the fifth postnatal week. However, the relationship between the extent of myelination and axonal signaling in the maturing optic nerve is not well characterized. Here, we examine the relationship between axon conduction and elements of myelination using extracellular nerve recordings, immunohistochemistry, western blot analysis, scanning electron microscopy, and simulations of nerve responses. Comparing compound action potentials from mice aged 4-12 weeks revealed five functional distinct axonal populations, an increase in the number of functional axons, and shifts toward fast-conducting axon populations at 5 and 8 weeks postnatal. At these ages, our analysis revealed increased myelin thickness, lower g-ratios and changes in the 14 kDa MBP isoform, while the density of axons and nodes of Ranvier remained constant. At 5 postnatal weeks, axon diameter increased, while at 8 weeks, increased expression of a mature sodium ion channel subtype, Na 1.6, was observed at nodes of Ranvier. A simulation model of nerve conduction suggests that ion channel subtype, axon diameter, and myelin thickness are more likely to be key regulators of nerve function than g-ratio. Such refinement of axonal function and myelin rearrangement identified an extended period of maturation in the normal optic nerve that may facilitate the development of visual signaling patterns.

Refinement of axonal conduction and myelination in the mouse optic nerve indicate an extended period of postnatal developmental plasticity.

Astrocytes and microglia are essential cellular elements of the CNS that are critical for normal development, function, and injury responses. Both cell types are highly pleiotropic and respond rapidly to environmental changes, making them challenging to characterize. One approach is to develop efficient isolation paradigms of distinct cell populations, allowing for characterization of their roles in distinct CNS regions and in pathological states.

Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord.

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 MOG -induced Th1/Th17-mediated chronic EAE combined with tolerance induced by the i.v. infusion of biodegradable poly(lactide-co-glycolide) nanoparticles coupled with MOG (PLG-MOG ) 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.

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

The myelin sheath facilitates signal conduction along axons in white matter tracts, and when disrupted, can result in significant functional deficits. Demyelination, observed in diseases like multiple sclerosis and optic neuritis, are associated with neural degeneration, however the extent of this damage on upstream circuitry is not well understood. Here we use the MBP-iCP9 mouse model to induce selective oligodendrocyte ablation in the optic nerve at P14 via a chemical inducer of dimerization (CID), resulting in partial demyelination of retinal ganglion cell (RGC) axons with minimal inflammation after two weeks. Oligodendrocyte loss reduced axon diameter and altered compound action potential waveforms, blocking conduction in the slowest-conducting axon populations. Demyelination resulted in disruptions to the normal composition of the retina, including reduced density of RBPMS+, Brn3a+, and OFF-transient RGCs, thinning of the IPL, and reduced density of displaced amacrine cells. The INL and ONL were unaffected by oligodendrocyte loss, suggesting that demyelination-induced deficits in this model are specific to the IPL and GCL. These results show that a partial demyelination of a subpopulation of RGC axons disrupts optic nerve function and affects the organization of the retinal network. This study highlights the significance of myelination in maintaining upstream neural connectivity and provides support for targeting neuronal degeneration in treatments of demyelinating diseases.

Loss of optic nerve oligodendrocytes during maturation alters retinal organization.

Multiple sclerosis (MS) is characterized by a compromised blood-brain barrier (BBB) resulting in central nervous system (CNS) entry of peripheral lymphocytes, including T cells and B cells. While T cells have largely been considered the main contributors to neuroinflammation in MS, the success of B cell depletion therapies suggests an important role for B cells in MS pathology. Glial cells in the CNS are essential components in both disease progression and recovery, raising the possibility that they represent targets for B cell functions. Here, we examine astrocyte and microglia responses to B cell depleting treatments in an animal model of MS, experimental autoimmune encephalomyelitis (EAE). B cell depleted EAE animals had markedly reduced disease severity and myelin damage accompanied by reduced microglia and astrocyte reactivity 20 days after symptom onset. To identify potential initial mechanisms mediating functional changes following B cell depletion, astrocyte and microglia transcriptomes were analyzed 3 days following B cell depletion. In control EAE animals, transcriptomic analysis revealed astrocytic inflammatory pathways were activated and microglial influence on neuronal function were inhibited. Following B cell depletion, initial functional recovery was associated with an activation of astrocytic pathways linked with restoration of neurovascular integrity and of microglial pathways associated with neuronal function. These studies reveal an important role for B cell depletion in influencing glial function and CNS vasculature in an animal model of MS.

Article	Year
Contribution of the oligodendrocyte lineage to CNS repair and neurodegenerative pathologies. Neuropharmacology\| PubMed ID: 27108096	2016
CNS disease diminishes the therapeutic functionality of bone marrow mesenchymal stem cells. Experimental neurology\| PubMed ID: 28602834	2017
Calcium control of myelin sheath growth. Nature neuroscience\| PubMed ID: 29269759	2018
Emerging Cellular and Molecular Strategies for Enhancing Central Nervous System (CNS) Remyelination. Brain sciences\| PubMed ID: 29914096	2018
Targeted Oligodendrocyte Apoptosis in Optic Nerve Leads to Persistent Demyelination. Neurochemical research\| PubMed ID: 30848441	2020
Refinement of axonal conduction and myelination in the mouse optic nerve indicate an extended period of postnatal developmental plasticity. Developmental neurobiology\| PubMed ID: 35403346	2022
Cell type specific isolation of primary astrocytes and microglia from adult mouse spinal cord. Journal of neuroscience methods\| PubMed ID: 35460698	2022
Repurposing the cardiac glycoside digoxin to stimulate myelin regeneration in chemically-induced and immune-mediated mouse models of multiple sclerosis. Glia\| PubMed ID: 35809238	2022
Loss of optic nerve oligodendrocytes during maturation alters retinal organization. Experimental eye research\| PubMed ID: 37364629	2023
B cell depletion modulates glial responses and enhances blood vessel integrity in a model of multiple sclerosis. Neurobiology of disease\| PubMed ID: 37709209	2023

Robert H. Miller

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