Regina C. Armstrong

Noninvasive detection of cuprizone induced axonal damage and demyelination in the mouse corpus callosum.

Previously, we tested the prediction that axonal damage results in decreased axial diffusivity (λ∥) while demyelination leads to increased radial diffusivity (λ⟂). Cuprizone treatment of C57BL/6 mice was a highly reproducible model of CNS white matter demyelination and remyelination affecting the corpus callosum (CC). In the present study, six C57BL/6 male mice were fed 0.2% cuprizone for 12 weeks followed by 12 weeks of recovery on normal chow. The control mice were fed normal chow and imaged in parallel. Biweekly in vivo DTI examinations showed transient decrease of λ∥ in CC at 2–6 weeks of cuprizone treatment. Immunostaining for nonphosphorylated neurofilaments demonstrated corresponding axonal damage at 4 weeks of treatment. Significant demyelination was evident from loss of Luxol fast blue staining at 6–12 weeks of cuprizone ingestion and was paralleled by increased λ⟂ values, followed by partial normalization during the remyelination phase. The sensitivity of λ⟂ to detect demyelination may be modulated in the presence of axonal damage during the early stage of demyelination at 4 weeks of cuprizone treatment. Our results suggest that λ∥ and λ⟂ may be useful in vivo surrogate markers of axonal and myelin damage in mouse CNS white matter. Magn Reson Med, 2006. Published 2006 Wiley‐Liss, Inc.

In vivo proliferation of oligodendrocyte progenitors expressing PDGF?R during early remyelination

Endogenous oligodendrocyte lineage cells spontaneously remyelinate focal areas of demyelination induced by murine hepatitis virus A59 infection of C57Bl/6 mice. We used this model to examine the potential for platelet-derived growth factor (PDGF) to have a role in repopulating demyelinated lesions, and in doing so we also further characterized the in vivo responses of oligodendrocyte lineage cells following demyelination. Very early in the progress of remyelination, we administered a 4-h in vivo pulse of bromodeoxyuridine (BrdU) and subsequently performed in situ hybridization for PDGF-alpha receptor (PDGFalphaR), an established marker for oligodendrocyte progenitors in vivo, or for proteolipid protein (PLP), to identify oligodendrocytes. Sections of lesioned spinal cords had a 14.5-fold increase in the number of BrdU-labeled oligodendrocyte progenitor cells (PDGFalphaR+), while BrdU-labeled oligodendrocytes (PLP+) were extremely rare. Immunocytochemistry of similar sections demonstrated that immunoreactivity for both PDGFalphaR and NG2, another marker of oligodendrocyte progenitors, was locally increased in areas of white-matter lesions. High-resolution immunofluorescence imaging was used to detect oligodendrocyte progenitor cells expressing receptors for both PDGF and fibroblast growth factor. In addition, expression of PDGF-A mRNA transcripts was increased in sections of lesioned spinal cords and reactive astrocytes in lesions exhibited immunoreactivity for PDGF ligand. Our findings indicate that during the initial stages of remyelination, oligodendrocyte progenitors proliferate locally, and that this response may potentially involve PDGF.

Differential sensitivity of in vivo and ex vivo diffusion tensor imaging to evolving optic nerve injury in mice with retinal ischemia

Decreased axial (lambda(||)) and increased radial (lambda( perpendicular)) diffusivity have been shown to reflect axonal and myelin injury respectively. In the present study, evolving white matter injury within the optic nerves of mice with retinal ischemia was examined by in vivo and ex vivo measurements of lambda(||) and lambda( perpendicular). The results show that at 3 days after retinal ischemia, a 33% decrease in vivo and a 38% decrease ex vivo in lambda(||) without change in lambda( perpendicular) was observed in the injured optic nerve compared to the control, suggestive of axonal damage without myelin injury. At 14 days, both in vivo and ex vivo measured lambda( perpendicular) increased significantly to 220-240% of the control level in the injured optic nerve suggestive of myelin damage. In contrast, the axonal injury that was clearly detected in vivo as a significantly decreased lambda(||) (33% decrease) was not as clearly detected by ex vivo lambda(||) (17% decrease). The current findings suggest that ex vivo lambda( perpendicular) is comparable to in vivo lambda( perpendicular) in detecting myelin injury. However, the structural changes resulting from axonal damage causing the decreased in vivo lambda(||) may not be preserved ex vivo in the fixed tissues. Despite the accurate depiction of the pathology using lambda(||) and lambda( perpendicular) in vivo, the use of ex vivo lambda(||) to extrapolate the status of axonal injury in vivo would require further investigation.

Fibroblast growth factor 2 (FGF2) and FGF receptor expression in an experimental demyelinating disease with extensive remyelination.

Fibroblast growth factor 2 (FGF2) is an excellent candidate to regulate remyelination based on its proposed actions in oligodendrocyte lineage cell development in conjunction with its involvement in CNS regeneration. To assess the potential for FGF2 to play a role in remyelination, we examined the expression pattern of FGF2 and FGF receptors (FGFRs) in an experimental demyelinating disease with extensive remyelination. Adult mice were intracranially injected with murine hepatitis virus strain A‐59 (MHV‐A59) to induce focally demyelinated spinal cord lesions that spontaneously remyelinate, with corresponding recovery of motor function. Using kinetic RT‐PCR analysis of spinal cord RNA, we found significantly increased levels of FGF2 mRNA transcripts, which peaked during the initial stage of remyelination. Analysis of tissue sections demonstrated that increased levels of FGF2 mRNA and protein were localized within demyelinated regions of white matter, including high FGF2 expression associated with astrocytes. The expression of corresponding FGF receptors was significantly increased in lesion areas during the initial stage of remyelination. In normal and lesioned white matter, oligodendrocyte lineage cells, including progenitors and mature cells, were found to express multiple FGFR types (FGFR1, FGFR2, and/or FGFR3). In addition, in lesion areas, astrocytes expressed FGFR1, FGFR2, and FGFR3. These findings indicate that, during remyelination, FGF2 may play a role in directly regulating oligodendrocyte lineage cell responses and may also act through paracrine or autocrine effects on astrocytes, which are known to synthesize other growth factors and immunoregulatory molecules that influence oligodendrocyte lineage cells. J. Neurosci. Res. 62:241–256, 2000. Published 2000 Wiley‐Liss, Inc.

Rostrocaudal analysis of corpus callosum demyelination and axon damage across disease stages refines diffusion tensor imaging correlations with pathological features.

Noninvasive assessment of the progression of axon damage is important for evaluating disease progression and developing neuroprotective interventions in multiple sclerosis patients. We examined thecellular responses correlated with diffusion tensor imaging-derivedaxial (&lgr;∥) and radial (&lgr;⊥) diffusivity values throughout acute (4 weeks) and chronic (12 weeks) stages of demyelination and after 6 weeks of recovery using the cuprizone demyelination of the corpus callosum model in C57BL/6 and Thy1-YFP-16 mice. The rostrocaudal progression of pathological alterations in the corpus callosum enabled spatially and temporally defined correlations of pathological features with diffusion tensor imaging measurements. During acute demyelination, microglial/macrophage activation was most extensive and axons exhibited swellings, neurofilament dephosphorylation, and reduced diameters. Axial diffusivity values decreased in the acute phase but did not correlate with axonal atrophy during chronic demyelination. In contrast, radial diffusivity increased with the progression of demyelination but did not correlate with myelin loss orastrogliosis. Unlike other animal models with progressive neurodegeneration and axon loss, the acute axon damage did not progress to discontinuity or loss of axons even after a period of chronicdemyelination. Correlations of reversible axon pathology, demyelination, microglia/macrophage activation, and astrogliosis with regional axial and radial diffusivity measurements will facilitate the clinical application of diffusion tensor imaging in multiple sclerosis patients.

PDGF and FGF2 regulate oligodendrocyte progenitor responses to demyelination.

Abstract Acute demyelination of adult CNS, resulting from trauma or disease, is initially followed by remyelination. However, chronic lesions with subsequent functional impairment result from eventual failure of the remyelination process, as seen in multiple sclerosis. Studies using animal models of successful remyelination delineate a progression of events facilitating remyelination. A universal feature of this repair process is extensive proliferation of oligodendrocyte progenitor cells (OPs) in response to demyelination. To investigate signals that regulate OP proliferation in response to demyelination we used murine hepatitis virus‐A59 (MHV‐A59) infection of adult mice to induce focal demyelination throughout the spinal cord followed by spontaneous remyelination. We cultured glial cells directly from demyelinating and remyelinating spinal cords using conditions that maintain the dramatically enhanced OP proliferative response prior to CNS remyelination. We identify PDGF and FGF2 as significant mitogens regulating this proliferative response. Furthermore, we demonstrate endogenous PDGF and FGF2 activity in these glial cultures isolated from demyelinated CNS tissue. These findings correlate well with our previous demonstration of increased in vivo expression of PDGF and FGF2 ligand and corresponding receptors in MHV‐A59 lesions. Together these studies support the potential of these pathways to function in vivo as critical factors in regulating remyelination.

Intracellular signals and cytoskeletal elements involved in oligodendrocyte progenitor migration

We have examined the potential roles of intracellular Ca2+ regulation and of multiple cytoskeletal elements in control of the directed migration of cultured oligodendrocyte progenitor cells (OPs). OPs were found to migrate in response to platelet‐derived growth factor (PDGF) or to a lesser extent to basic fibroblast growth factor (FGF) in a non‐additive manner. This response was inhibited by chelation of intracellular Ca2+ by using BAPTA‐AM. OP migration was not evoked by the neurotransmitter agonists phenylephrine or methacholine, which elevate OP Ca2+ levels. Inhibition of the MAP kinase pathway with PD 098059 did not affect OP migration to PDGF. Within growth cone‐like leading edges of migratory OP processes, monomeric and filamentous actin were found to be colocalized with myosin and filamentous actin was prominent in filopodia extending beyond the leading edge. Tubulin was distributed throughout OP processes and cell bodies. Inhibition of actin or tubulin polymerization, by using cytochalasin B or nocodazole, respectively, altered OP morphology and markedly impaired migration. Inhibition of the myosin ATPase by BDM, which prevents force‐generating actin/myosin interactions, greatly inhibited the chemotaxic response at concentrations that did not disrupt cell morphology. These results indicate that growth factors stimulate OP migration by activating pathways which include intracellular Ca2+ regulation, and characterize the distribution of multiple cytoskeletal elements involved in the generation of directed OP movement. GLIA 26:22–35, 1999.

Endogenous cell repair of chronic demyelination.

In multiple sclerosis lesions, remyelination typically fails with repeated or chronic demyelinating episodes and results in neurologic disability. Acute demyelination models in rodents typically exhibit robust spontaneous remyelination that prevents appropriate evaluation of strategies for improving conditions of insufficient remyelination. In the current study, we used a mouse model of chronic demyelination induced by continuous ingestion of 0.2% cuprizone for 12 weeks. This chronic process depleted the oligodendrocyte progenitor population and impaired oligodendrocyte regeneration. Remyelination remained limited after removal of cuprizone from the diet. Fibroblast growth factor 2 (FGF2) expression was persistently increased in the corpus callosum of chronically demyelinated mice as compared with nonlesioned mice. We used FGF2−/− mice to determine whether removal of endogenous FGF2 promoted remyelination of chronically demyelinated areas. Wild-type and FGF2−/− mice exhibited similar demyelination during chronic cuprizone treatment. Importantly, in contrast to wild-type mice, the FGF2−/− mice spontaneously remyelinated completely during the recovery period after chronic demyelination. Increased remyelination in FGF2−/− mice correlated with enhanced oligodendroglial regeneration. FGF2 genotype did not alter the density of oligodendrocyte progenitor cells or proliferating cells after chronic demyelination. These findings indicate that attenuating FGF2 created a sufficiently permissive lesion environment for endogenous cells to effectively remyelinate viable axons even after chronic demyelination.

Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system

The establishment and operation of the nervous system requires genetic regulation by a network of DNA‐binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys‐Cys‐His‐Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1‐like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303–321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system. J. Neurosci. Res. 50:272–290, 1997.

Myelin transcription factor 1 (Myt1) modulates the proliferation and differentiation of oligodendrocyte lineage cells.

Myelin transcription factor 1 (Myt1) is a zinc finger DNA-binding protein that is expressed in neural progenitors and oligodendrocyte lineage cells. This study examines the role of Myt1 in oligodendrocyte lineage cells by overexpressing putative functional domains, a four-zinc finger DNA-binding region (4FMyt1) or a central protein-protein interaction domain (CDMyt1), without the predicted transcriptional activation domain. In the presence of mitogens, overexpression of 4FMyt1 inhibited proliferation of oligodendrocyte progenitors, but not cell types (astrocytes and NIH3T3 cells) lacking endogenous Myt1. Expression of 4FMyt1 inhibited the differentiation of oligodendrocyte progenitors into oligodendrocytes as assessed by morphology, immunostaining, and myelin gene expression. Progenitor differentiation was similarly inhibited by expression of CDMyt1 but only partially suppressed by overexpression of the intact Myt1. These data indicate that Myt1 may regulate a critical transition point in oligodendrocyte lineage development by modulating oligodendrocyte progenitor proliferation relative to terminal differentiation and up-regulation of myelin gene transcription.