Myelin pallor is frequently reported in HIV patients, and can occur in the CNS prior to other evidence of disease process. Our exploratory studies showed that oligodendrocytes (OLs) are direct targets of HIV-1 Tat (transactivator of transcription). Tat induces a dose-dependent increase of intracellular Ca2+ level ([Ca2+]i) in cultured murine OLs, which can be attenuated by ionotropic glutamate receptor (iGluR) antagonists MK801 and CNQX. The Tat-induced [Ca2+]i increase leads to increased death in immature (O4+, MBP-), but not mature (O4+, MBP+) OLs, over 96 h. In addition, Tat-induced [Ca2+]i increase also reduced myelin-like membrane production by mature OLs. Calcium/Calmodulin dependent kinase IIβ (CaMKIIβ) and glycogen synthase kinase 3β (GSK3β) have been known to regulate differentiation, myelination, and apoptosis in OLs. Since both CaMKIIβ and GSK3β are important downstream modulators of [Ca2+]i change, we hypothesized that the detrimental effects of Tat on immature/mature OL viability and function are mediated via CaMKIIβ and GSK3β activation. Our results showed that Tat activates both CaMKIIβ and GSK3β in immature OLs, but only activates CaMKIIβ in mature OLs. MK801 completely blocks Tat-induced CaMKIIβ and GSK3β activation in both immature and mature OLs, while CNQX blocks GSK3β activation, but has only a partial effect on CaMKIIβ activity. Blocking iGluRs or inhibiting GSK3β both rescue Tat-induced immature OL death, but only MK801 reverses the membrane injury in mature OLs. Together, these data strongly suggest that 1) activity of CaMKIIβ and GSK3β in OLs can be regulated by Tat-induced iGluRs activation and 2) OLs at different developmental stages show different responses to Tat, possibly due to activation of different signaling pathways.

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Multiple sclerosis (MS) is a chronic central nervous system disease featuring exacerbations of inflammation and demyelination that cause progressively debilitating clinical effects over time. Current treatments for multiple sclerosis are limited in their ability to impact overall disease progression. Research aimed at generation of novel potential therapeutics for MS is needed. Recently, kallikrein 6 (KLK6), a member of the kallikrein (KLK) family of secreted serine proteases, was found to be elevated in the cerebrospinal fluid and brain of MS patients. The fifteen known tissue-based KLKs cleave proteins through a similar mechanism, but have different binding pocket specificity, diverse localization in human tissues, and multiple biological functions. KLKs have been linked to normal human physiology (e.g. KLK4, enamel formation) and disease (e.g. KLK3, prostate cancer). KLK6 is one of the highest expressed serine proteases in the healthy human brain and is expressed predominately in mature oligodendrocytes in both human and mouse brain. The role of KLK6 in oligodendrocyte maturation, myelination, and disease is not fully understood. To evaluate the role of KLK6 in oligodendrocyte maturation, I used a pluripotent in vitro primary cell system to assess the impact of exogenous KLK6 and modulators of the KLK6 pathway on oligodendrocyte maturation. I demonstrate that signaling through KLK6 decreases the number of mature oligodendrocytes in culture, whereas blockade of KLK6 signaling increases the number of mature oligodendrocytes in culture in the presence of triiodothyronine higher than either agent alone. This work suggests that KLK6 modulation impacts oligodendrocyte maturation. To understand the potential impact of KLK6 pathway inhibition on remyelination, I used the toxin cuprizone to induce demyelination in mice. I found that animals treated with a KLK6 inhibitor had increased myelin staining in the corpus callosum compared to vehicle-treated. This work suggests that KLK6 modulates oligodendrocyte maturation and myelination and may be relevant for improving myelin-related therapeutic outcomes, particularly in multiple sclerosis. / 2019-06-12T00:00:00Z

Multiple sclerosis (MS), a demyelinating disorder of the central nervous system (CNS), affects approximately 400,000 individuals in the United States, and 2.5 million people worldwide. It is a leading cause of disability in young adults. Current treatments for MS target the inflammatory aspects of the disease, but do not aid in remyelination. To address remyelination as a therapeutic strategy, it is imperative to identify mechanisms that regulate myelin formation, including epigenetic targets. In this study, we investigate the role of the LMNA, a gene encoding Lamins A and C, intermediate filaments of the nuclear lamina, in regulating oligodendrocyte development and myelination in the CNS. Using electron microscopic analyses, I examined levels of heterochromatin and its distribution in the oligodendrocyte nucleus as an indicator of gene expression, oligodendrocyte maturity, and myelin formation in the absence of A type lamins.. While overall levels of heterochromatin in oligodendrocytes were not altered in the absence of A type lamins, peripherally located heterochromatin was reduced and thinner myelin was observed in the spinal cord. My observations present novel findings for the role of LMNA in oligodendrocytes and myelination.

The extent of damage in animal models of spinal cord injury (SCI) can be reduced by various neuroprotective regimens that include maintaining levels of the second messenger, cyclic adenosine monophosphate (cAMP), via administration of the phosphodiesterase 4 inhibitor, Rolipram. The current study sought to determine the optimal neuroprotective dose, route and therapeutic window for Rolipram following thoracic contusive SCI injury in rat. Rolipram or vehicle control (10% ethanol) was given daily for 2 weeks post-injury (PI) after which the preservation of oligodendrocytes, neurons and central myelinated axons (CMAs) was stereologically assessed. Doses of 0.1 mg/kg to 1.0 mg/kg (2 h PI) increased neuronal survival; 0.5 mg- 1.0 mg/kg protected oligodendrocytes, 1.0 mg/kg produced optimal preservation of CMAs. Administration of 1.0 mg/kg Rolipram via different routes (intravenous [i.v.], subcutaneous [s.c.] or oral, 2 h PI) demonstrated that all routes allowed for significant protection following SCI; the i.v. route provided the best clinical translation. Examination of delayed treatment, initiated 1-48 h after SCI, revealed protective efficacy of Rolipram even when administered up to 48 h PI. With the optimal therapeutic protocol (1.0 mg/kg, i.v.), Rolipram reduced the levels of the chemokine, monocyte chemoattractant protein acutely post-injury and elevated the levels of the anti-inflammatory cytokine, interleukin-10, based on Enzyme-Linked ImmunoSorbent Assay (ELISA) results. Rolipram, when delivered within 48 h PI, was also able to significantly reduce the number of ED1-positive mononuclear phagocytes compared to vehicle-treated controls. This work supports the use of Rolipram as an acute neuroprotectant following SCI, defines an administration protocol, and investigates a potential mechanism for Rolipram-mediated protection.

Spinal cord injury (SCI) often results in irreversible paralysis and widespread oligodendrocyte death and white matter damage. While the mechanisms underlying this phenomenon are poorly understood, previous studies from our laboratory indicate that inhibition of astroglial-NF-κB activation reduces white matter damage and improves functional recovery in a mouse model of SCI. Here we provide novel evidence demonstrating that astrocytes directly regulate oligodendrocyte fate after trauma by a glutamate-mediated AMPA receptor dependent mechanism. Following trauma, elevated expression of the SLC39a10 zinc transporter correlated with an increase in zinc uptake by astrocytes, thereby reducing extracellular zinc concentrations required for AMPA receptor inhibition. Stimulation of AMPA receptors on oligodendrocytes by glutamate induced oligodendrocyte toxicity through the activation of the NADPH oxidase enzyme within oligodendrocytes. Genetic and pharmacological inhibition of active NADPH oxidase was sufficient to attenuate oligodendrocyte death in vitro. Following SCI, NADPH oxidase inhibition reduced oligodendrocyte death by ~75%, suggesting that glutamate-mediated oligodendrocyte death is dependent on the activation of the NADPH oxidase enzyme within oligodendrocytes. Combined treatment of the NADPH oxidase inhibitor apocynin and the AMPA receptor inhibitor NBQX significantly improved hind limb locomotor behavior, reduced white matter damage and lesion volume, and significantly spared descending serotonergic fibers. These studies provide a novel mechanism of oligodendrocyte death and may lead to clinically relevant therapeutics after SCI.

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In the central nervous system (CNS), oligodendrocytes (OLs) generate myelin to facilitate the rapid propagation of neuronal impulses. In multiple sclerosis (MS), chronic demyelination leads to irreversible neurodegeneration that eventually impairs physical and cognitive function. Much effort is directed at elucidating the mechanisms underlying OL development in hope to unveil therapeutic targets for promoting remyelination in MS. Many aspects of OL biology are regulated by the integrins, a large family of transmembrane extracellular matrix (ECM) receptors. ECM components such as laminin and fibronectin bind to OL integrin receptors and initiate downstream signaling cascades involved in survival, proliferation, differentiation/myelination and migration. Integrin-linked kinase (ILK), an adaptor protein that binds to integrin cytosolic tails, works to stabilize the ECM-integrin connection by indirectly targeting the actin cytoskeleton to ECM adhesion sites. We hypothesized that ILK played an important role in OL migration, differentiation and capacity to myelinate neuronal projections. To address this hypothesis, we developed three cell culture techniques to assess these cellular phenomena in vitro. Conditional knockout of Ilk compromised both the morphological and molecular differentiation of primary mouse OLs in vitro, and reduced their capacity to produce myelin-like membrane. ILK was required for proper OL ensheathment of neuronal extensions when co-cultured with primary neurons. Conditional ablation of Ilk in vivo produced a transient amyelination defect that was endogenously compensated for at later time points. Loss of ILK in primary OLs was associated with upregulated RhoA signaling, and pharmacological inhibition of the RhoA axis restored the morphology of a distinct subset of NG2+ OPCs. ILK depletion in OL precursor cells (OPCs) resulted in a substrate-dependent defect in migration velocity and migration initiation. Inhibition of the RhoA signaling pathway enhanced the migratory velocity of wild-type OPCs, an effect that was dependent on ILK expression. In sum, we established three primary mouse OL cell culture techniques, with which we defined roles for ILK in OL biology. Our work highlights the importance of integrin signaling in OLs and provides new experimental methods useful in MS research.