Hyperphosphorylation and aggregation of tau in experimental autoimmune encephalomyelitis

Axonal damage is a major morphological correlate and cause of permanent neurological deficits in patients with multiple sclerosis (MS), a multifocal, inflammatory and demyelinating disease of the central nervous system. Hyperphosphorylation and pathological aggregation of microtubule-associated protein tau is a common feature of many neurodegenerative diseases with axonal degeneration including Alzheimer's disease. We have therefore analyzed tau phosphorylation, solubility and distribution in the brainstem of rats with experimental autoimmune encephalomyelitis (EAE), an animal model of MS. Tau was hyperphosphorylated at several sites also phosphorylated in Alzheimer's disease and became partially detergent-insoluble in EAE brains. Morphological examination demonstrated accumulation of amorphous deposits of abnormally phosphorylated tau in the cell body and axons of neurons within demyelinating plaques. Hyperphosphorylation of tau was accompanied by up-regulation of p25, an activator of cyclin-dependent kinase 5. Phosphorylation of tau, activation of cdk5, and axonal pathology were significantly reduced when diseased rats were treated with prednisolone, a standard therapy of acute relapses in MS. Hyperphosphorylation of tau was not observed in a genetic or nutritional model of axonal degeneration or demyelination, suggesting that inflammation as detected in the brains of rats with EAE is the specific trigger of tau pathology. In summary, our data provide evidence that axonal damage in EAE and possibly MS is linked to tau pathology.     

Mechanisms of immunopathology in murine models of central nervous system demyelinating disease

Many disorders of the CNS, such as multiple sclerosis (MS), are characterized by the loss of the myelin sheath surrounding nerve axons. MS is associated with infiltration of inflammatory cells into the brain and spinal cord, which may be the primary cause of demyelination or which may be induced secondary to axonal damage. Both the innate and adaptive arms of the immune system have been reported to play important roles in myelin destruction. Numerous murine demyelinating models, both virus-induced and/or autoimmune, are available, which reflect the clinical and pathological variability seen in human disease. This review will discuss the immunopathologic mechanisms involved in these demyelinating disease models.     

Antigen-specific therapy promotes repair of myelin and axonal damage in established EAE

Inflammation results in CNS damage in multiple sclerosis (MS) and experimental autoimmune encephalomyelitis (EAE), an animal model of MS. It is uncertain how much repair of injured myelin and axons can occur following highly selective anti-inflammatory therapy in EAE and MS. In this study, SJL/J mice with established EAE were treated successfully with an antigen-specific recombinant T cell receptor ligand (RTL), RTL401, a mouse I-A(s)/PLP-139-151 construct, after the peak of EAE. To define the mechanisms by which late application of RTL401 inhibits EAE, we evaluated mice at different time points to assess the levels of neuroinflammation and myelin and axon damage in their spinal cords. Our results showed that RTL401 administered after the peak of acute EAE induced a marked reduction in inflammation in the CNS, associated with a significant reduction of demyelination, axonal loss and ongoing damage. Electron microscopy showed that RTL-treated mice had reduced pathology compared with mice treated with vehicle and mice at the peak of disease, as demonstrated by a decrease in continued degeneration, increase in remyelinating axons and the presence of an increased number of small, presumably regenerative axonal sprouts. These findings indicate that RTL therapy targeting encephalitogenic T cells may promote CNS neuroregenerative processes.     

Mediators of oligodendrocyte differentiation during remyelination

Myelin, a dielectric sheath that wraps large axons in the central and peripheral nervous systems, is essential for proper conductance of axon potentials. In multiple sclerosis (MS), autoimmune-mediated damage to myelin within the central nervous system (CNS) leads to progressive disability primarily due to limited endogenous repair of demyelination with associated axonal pathology. While treatments are available to limit demyelination, no treatments are available to promote myelin repair. Studies examining the molecular mechanisms that promote remyelination are therefore essential for identifying therapeutic targets to promote myelin repair and thereby limit disability in MS. Here, we present our current understanding of the critical extracellular and intracellular pathways that regulate the remyelinating capabilities of oligodendrocyte precursor cells (OPCs) within the adult CNS.     

Promoting myelin repair and return of function in multiple sclerosis

Multiple sclerosis (MS) is an inflammatory demyelinating disease of the CNS. Conduction block in demyelinated axons underlies early neurological symptoms, but axonal transection and neuronal loss are believed to be responsible for more permanent chronic deficits. Several therapies are approved for treatment of relapsing-remitting MS, all of which are immunoregulatory and clinically proven to reduce the rate of lesion formation and exacerbation. However, existing approaches are only partially effective in preventing the onset of disability in MS patients, and novel treatments to protect myelin-producing oligodendrocytes and enhance myelin repair may improve long-term outcomes. Studies in vivo in genetically modified mice have assisted in the characterization of mechanisms underlying the generation of neuropathology in MS patients, and have identified potential avenues for oligodendrocyte protection and myelin repair. However, no treatments are yet approved that target these areas directly, and in addition, the relationship between demyelination and axonal transection in the lesions of the disease remains unclear. Here, we review translational research targeting oligodendrocyte protection and myelin repair in models of autoimmune demyelination, and their potential relevance as therapies in MS.     

Mechanism of Myelin Breakdown in Experimental Demyelination: A Putative Role for Calpain

Although calpain has been extensively studied, its physiological function is poorly understood. In contrast, its role in the pathophysiology of various diseases has been implicated, including that of experimental allergic encephalomyelitis (EAE), an animal model of the demyelinating disease multiple sclerosis (MS). In EAE, calpain degrades myelin proteins, including myelin basic protein (MBP), suggesting a role for calpain in the breakdown of myelin in this disease. Subsequent studies revealed increased calpain activity and expression in the glial and inflammatory cells concomitant with loss of axon and myelin proteins. This suggested a crucial role for calpain in demyelinating diseases.     

Promotion of Remyelination by Sulfasalazine in a Transgenic Zebrafish Model of Demyelination

Most of the axons in the vertebrate nervous system are surrounded by a lipid-rich membrane called myelin, which promotes rapid conduction of nerve impulses and protects the axon from being damaged. Multiple sclerosis (MS) is a chronic demyelinating disease of the CNS characterized by infiltration of immune cells and progressive damage to myelin and axons. One potential way to treat MS is to enhance the endogenous remyelination process, but at present there are no available treatments to promote remyelination in patients with demyelinating diseases.Sulfasalazine is an anti-inflammatory and immune-modulating drug that is used in rheumatology and inflammatory bowel disease. Its anti-inflammatory and immunomodulatory properties prompted us to test the ability of sulfasalazine to promote remyelination. In this study, we found that sulfasalazine promotes remyelination in the CNS of a transgenic zebrafish model of NTR/MTZ-induced demyelination. We also found that sulfasalazine treatment reduced the number of macrophages/microglia in the CNS of demyelinated zebrafish larvae, suggesting that the acceleration of remyelination is mediated by the immunomodulatory function of sulfasalazine. Our data suggest that temporal modulation of the immune response by sulfasalazine can be used to overcome MS by enhancing myelin repair and remyelination in the CNS.     

CNTF is a major protective factor in demyelinating CNS disease: a neurotrophic cytokine as modulator in neuroinflammation

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system (CNS). So far, immunological mechanisms responsible for demyelination have been the focus of interest. However, mechanisms regulating axon maintenance as well as glial precursor-cell proliferation and oligodendrocyte survival might also influence disease outcome. The cytokine ciliary neurotrophic factor (CNTF), which was originally identified as a survival factor for isolated neurons, promotes differentiation, maturation and survival of oligodendrocytes. To investigate the role of endogenous CNTF in inflammatory demyelinating disease, we studied myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in CNTF-deficient and wild-type C57BL/6 mice. Disease was more severe in CNTF-deficient mice and recovery was poor, with a 60% decrease in the number of proliferating oligodendrocyte precursor cells (OPCs) and a more than 50% increase in the rate of oligodendrocyte apoptosis. In addition, vacuolar dystrophy of myelin and axonal damage were more severe in CNTF-deficient mice. These specific pathological features could be prevented by treatment with an antiserum against tumor necrosis factor-alpha, suggesting that endogenous CNTF may counterbalance this effect of TNF-alpha (ref. 7). Here we identify a factor that modulates, in an inflammatory environment, glial cell survival and is an outcome determinant of EAE.     

Delayed Nerve Stimulation Promotes Axon-Protective Neurofilament Phosphorylation,Accelerates Immune Cell Clearance and Enhances Remyelination In Vivo in Focally Demyelinated Nerves

Rapid and efficient axon remyelination aids in restoring strong electrochemical communication with end organs and in preventing axonal degeneration often observed in demyelinating neuropathies. The signals from axons that can trigger more effective remyelination in vivo are still being elucidated. Here we report the remarkable effect of delayed brief electrical nerve stimulation (ES; 1 hour @ 20 Hz 5 days post-demyelination) on ensuing reparative events in a focally demyelinated adult rat peripheral nerve. ES impacted many parameters underlying successful remyelination. It effected increased neurofilament expression and phosphorylation, both implicated in axon protection. ES increased expression of myelin basic protein (MBP) and promoted node of Ranvier re-organization, both of which coincided with the early reappearance of remyelinated axons, effects not observed at the same time points in non-stimulated demyelinated nerves. The improved ES-associated remyelination was accompanied by enhanced clearance of ED-1 positive macrophages and attenuation of glial fibrillary acidic protein expression in accompanying Schwann cells, suggesting a more rapid clearance of myelin debris and return of Schwann cells to a nonreactive myelinating state. These benefits of ES correlated with increased levels of brain derived neurotrophic factor (BDNF) in the acute demyelination zone, a key molecule in the initiation of the myelination program. In conclusion, the tremendous impact of delayed brief nerve stimulation on enhancement of the innate capacity of a focally demyelinated nerve to successfully remyelinate identifies manipulation of this axis as a novel therapeutic target for demyelinating pathologies.     

A reversible form of axon damage in experimental autoimmune encephalomyelitis and multiple sclerosis

In multiple sclerosis, a common inflammatory disease of the central nervous system, immune-mediated axon damage is responsible for permanent neurological deficits. How axon damage is initiated is not known. Here we use in vivo imaging to identify a previously undescribed variant of axon damage in a mouse model of multiple sclerosis. This process, termed 'focal axonal degeneration' (FAD), is characterized by sequential stages, beginning with focal swellings and progressing to axon fragmentation. Notably, most swollen axons persist unchanged for several days, and some recover spontaneously. Early stages of FAD can be observed in axons with intact myelin sheaths. Thus, contrary to the classical view, demyelination-a hallmark of multiple sclerosis-is not a prerequisite for axon damage. Instead, focal intra-axonal mitochondrial pathology is the earliest ultrastructural sign of damage, and it precedes changes in axon morphology. Molecular imaging and pharmacological experiments show that macrophage-derived reactive oxygen and nitrogen species (ROS and RNS) can trigger mitochondrial pathology and initiate FAD. Indeed, neutralization of ROS and RNS rescues axons that have already entered the degenerative process. Finally, axonal changes consistent with FAD can be detected in acute human multiple sclerosis lesions. In summary, our data suggest that inflammatory axon damage might be spontaneously reversible and thus a potential target for therapy.     

Multiple sclerosis - remyelination failure as a cause of disease progression

Multiple sclerosis (MS) is the most frequent demyelinating disease of the central nervous system (CNS) that affects worldwide about 2.5 million people. The morphological correlates of the disease are multiple lesions in brain and spinal cord which are characterized by demyelination, inflammation, gliosis and axonal damage. The underlying cause for the permanent neurological deficits in MS patients is axonal loss. Demyelinated axons are prone to damage due to the lack of trophic support by myelin sheaths and oligodendrocytes, as well as the increased vulnerability to immune mediated attacks. Remyelination occurs, but especially in chronic lesions is frequently limited to a small rim at the lesion border. Current treatment strategies are based on anti-inflammatory or immunomodulatory drugs and have the potential to reduce the numbers of newly evolving lesions, although as yet no treatment strategy exists to influence or prevent the progressive disease phase. Therefore, the development of neuroprotective treatment options, such as the promotion of endogenous remyelination is an attractive strategy. A prerequisite for the development of such new treatments is the understanding of the mechanisms leading to remyelination and the reasons for insufficient endogenous repair in chronic MS. This review will therefore provide an overview of the current concepts regarding remyelination in the rodent and human CNS. We will also summarize a selected number of inhibitory pathways and non-disease related factors which may contribute to remyelination failure in chronic MS.     

Early glial responses in murine models of multiple sclerosis

Investigations of functional interactions among axons and glia over the last decade have revealed the extent and complexity of glial-neuronal and glial-glial communication during development, adult function and recovery from injury. These data have profound implications for the understanding of central nervous system (CNS) disorders, which until recently, have been classified as either neuronal or glial diseases. Re-evaluation of the pathological processes in a number of conditions has clearly shown involvement of both neurons and glia in early pathology. In multiple sclerosis (MS), the myelin sheath has traditionally been regarded as the primary target. However, recent evidence has clearly demonstrated axonal damage in new lesions. We have addressed the question of the role of axonal pathology in early MS by using well-characterized murine models for the relapsing-remitting (RR) or the primary progressive (PP) forms of the disease. We performed a histopathological survey of the CNS, following induction of the disease, to determine the timing of appearance, as well as the development of lesions. Then we analysed the relationship between inflammation, demyelination and axonal damage together with responses from astrocytes and microglia in each model from the earliest evidence of inflammation. We found that axonal damage begins well ahead of the appearance of motor symptoms. Pathology appears to be more closely related to the degree of inflammation than to demyelination. We also show that early astrocyte responses and the degree of axonal loss are markedly different in the two models and relate to the severity of pathology. These data support the now widely accepted hypothesis that axonal damage begins early in the disease process, but also suggest modulation of axonal loss and disease progression by the astrocytic response.     

Increased activation of the epidermal growth factor receptor in transgenic mice overexpressing epigen causes peripheral neuropathy

In the mammalian nervous system, axons are commonly surrounded by myelin, a lipid-rich sheath that is essential for precise and rapid conduction of nerve impulses. In the peripheral nervous system (PNS), myelin sheaths are formed by Schwann cells which wrap around individual axons. While the tyrosine kinase receptors ERBB2 and ERBB3 are established mediators of peripheral myelination, less is known about the functions of the related epidermal growth factor receptor (EGFR) in the regulation of PNS myelination. Here, we report a peripheral neurodegenerative disease caused by increased EGFR activation. Specifically, we characterize a symmetric and distally pronounced, late-onset muscular atrophy in transgenic mice overexpressing the EGFR ligand epigen. Histological examination revealed a demyelinating neuropathy and axon degeneration, and molecular analysis of signaling pathways showed reduced protein kinase B (PKB, AKT) activation in the nerves of Epigen-tg mice, indicating that the muscular phenotype is secondary to PNS demyelination and axon degeneration. Crossing of Epigen-tg mice into an EGFR-deficient background revealed the pathology to be completely EGFR-dependent. This mouse line provides a new model for studying molecular events associated with early stages of peripheral neuropathies, an essential prerequisite for the development of successful therapeutic interventions.     

Time-Dependent Progression of Demyelination and Axonal Pathology in MP4-Induced Experimental Autoimmune Encephalomyelitis

BackgroundMultiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS) characterized by inflammation, demyelination and axonal pathology. Myelin basic protein/proteolipid protein (MBP-PLP) fusion protein MP4 is capable of inducing chronic experimental autoimmune encephalomyelitis (EAE) in susceptible mouse strains mirroring diverse histopathological and immunological hallmarks of MS. Limited availability of human tissue underscores the importance of animal models to study the pathology of MS.MethodsTwenty-two female C57BL/6 (B6) mice were immunized with MP4 and the clinical development of experimental autoimmune encephalomyelitis (EAE) was observed. Methylene blue-stained semi-thin and ultra-thin sections of the lumbar spinal cord were assessed at the peak of acute EAE, three months (chronic EAE) and six months after onset of EAE (long-term EAE). The extent of lesional area and inflammation were analyzed in semi-thin sections on a light microscopic level. The magnitude of demyelination and axonal damage were determined using electron microscopy. Emphasis was put on the ventrolateral tract (VLT) of the spinal cord.ResultsB6 mice demonstrated increasing demyelination and severe axonal pathology in the course of MP4-induced EAE. In addition, mitochondrial swelling and a decrease in the nearest neighbor neurofilament distance (NNND) as early signs of axonal damage were evident with the onset of EAE. In semi-thin sections we observed the maximum of lesional area in the chronic state of EAE while inflammation was found to a similar extent in acute and chronic EAE. In contrast to the well-established myelin oligodendrocyte glycoprotein (MOG) model, disease stages of MP4-induced EAE could not be distinguished by assessing the extent of parenchymal edema or the grade of inflammation.ConclusionsOur results complement our previous ultrastructural studies of B6 EAE models and suggest that B6 mice immunized with different antigens constitute useful instruments to study the diverse histopathological aspects of MS.     

Mitochondrial changes associated with demyelination: consequences for axonal integrity

The loss of myelin sheath (demyelination) renders axons vulnerable to a variety of insults. Axonal degeneration is well recognised in inflammatory demyelinating disorders of the central nervous system (CNS) such as multiple sclerosis (MS) and also certain neurodegenerative diseases. Energy required for nerve impulse conduction and maintenance of structural integrity of axons is met by mitochondria. Based on the distribution of ion channels and the Na(+)/K(+) ATPase, the energy requirements of demyelinated and dysmyelinated axons are likely to differ from myelinated axons. In this review we discuss the changes in mitochondrial presence within axons in relation to presence or absence of healthy myelin sheaths and propose the increase in mitochondrial presence following demyelination as an adaptive process. An energy deficit within demyelinated axons is likely to be more detrimental compared to myelinated axons, judging by the neuropathological findings in primary mitochondrial disorders due to mitochondrial and nuclear DNA mutations and the mitochondrial changes that follow demyelination. Agents that enhance and protect mitochondria, as potential therapy, need to be considered and investigated in earnest for demyelinating disorders of the CNS such as MS.     

Inhibition of 5-lipoxygenase activity in mice during cuprizone-induced demyelination attenuates neuroinflammation, motor dysfunction and axonal damage

Multiple sclerosis (MS) is a chronic inflammatory demyelinating disease of the central nervous system (CNS). Increased expression of 5-lipoxygenase (5-LO), a key enzyme in the biosynthesis of leukotrienes (LTs), has been reported in MS lesions and LT levels are elevated in the cerebrospinal fluid of MS patients. To determine whether pharmacological inhibition of 5-LO attenuates demyelination, MK886, a 5-LO inhibitor, was given to mice fed with cuprizone. Gene and protein expression of 5-LO were increased at the peak of cuprizone-induced demyelination. Although MK886 did not attenuate cuprizone-induced demyelination in the corpus callosum or in the cortex, it attenuated cuprizone-induced axonal damage and motor deficits and reduced microglial activation and IL-6 production. These data suggest that during cuprizone-induced demyelination, the 5-LO pathway contributes to microglial activation and neuroinflammation and to axonal damage resulting in motor dysfunction. Thus, 5-LO inhibition may be a useful therapeutic treatment in demyelinating diseases of the CNS.     

Direct visualization of membrane architecture of myelinating cells in transgenic mice expressing membrane‐anchored EGFP

Myelinogenesis is a complex process that involves substantial and dynamic changes in plasma membrane architecture and myelin interaction with axons. Highly ramified processes of oligodendrocytes in the central nervous system (CNS) make axonal contact and then extrapolate to wrap around axons and form multilayer compact myelin sheathes. Currently, the mechanisms governing myelin sheath assembly and axon selection by myelinating cells are not fully understood. Here, we generated a transgenic mouse line expressing the membrane‐anchored green fluorescent protein (mEGFP) in myelinating cells, which allow live imaging of details of myelinogenesis and cellular behaviors in the nervous systems. mEGFP expression is driven by the promoter of 2'‐3'‐cyclic nucleotide 3'‐phosphodiesterase (CNP) that is expressed in the myelinating cell lineage. Robust mEGFP signals appear in the membrane processes of oligodendrocytes in the CNS and Schwann cells in the peripheral nervous system (PNS), wherein mEGFP expression defines the inner layers of myelin sheaths and Schmidt‐Lanterman incisures in adult sciatic nerves. In addition, mEGFP expression can be used to track the extent of remyelination after demyelinating injury in a toxin‐induced demyelination animal model. Taken together, the membrane‐anchored mEGFP expression in the new transgenic line would facilitate direct visualization of dynamic myelin membrane formation and assembly during development and process remodeling during remyelination after various demyelinating injuries. genesis 52:341–349, 2014. © 2014 Wiley Periodicals, Inc.     

Heterogeneity of multiple sclerosis pathogenesis: implications for diagnosis and therapy

Multiple sclerosis is a chronic inflammatory disease of the nervous system in which a T-cell-mediated inflammatory process is associated with destruction of myelin sheaths. Although demyelination is the primary event, axons are also destroyed in the lesions, and the loss of axons correlates with permanent functional deficit. Here, we discuss evidence that demyelination and axonal destruction follow different pathogenetic pathways in subgroups of patients. This might, at least in part, explain the heterogeneity in genetic susceptibility, clinical presentation and response to treatment observed between individuals.     

Induction of Oligodendrocyte Differentiation and In Vitro Myelination by Inhibition of Rho-Associated Kinase

In inflammatory demyelinating diseases such as multiple sclerosis (MS), myelin degradation results in loss of axonal function and eventual axonal degeneration. Differentiation of resident oligodendrocyte precursor cells (OPCs) leading to remyelination of denuded axons occurs regularly in early stages of MS but halts as the pathology transitions into progressive MS. Pharmacological potentiation of endogenous OPC maturation and remyelination is now recognized as a promising therapeutic approach for MS. In this study, we analyzed the effects of modulating the Rho-A/Rho-associated kinase (ROCK) signaling pathway, by the use of selective inhibitors of ROCK, on the transformation of OPCs into mature, myelinating oligodendrocytes. Here we demonstrate, with the use of cellular cultures from rodent and human origin, that ROCK inhibition in OPCs results in a significant generation of branches and cell processes in early differentiation stages, followed by accelerated production of myelin protein as an indication of advanced maturation. Furthermore, inhibition of ROCK enhanced myelin formation in cocultures of human OPCs and neurons and remyelination in rat cerebellar tissue explants previously demyelinated with lysolecithin. Our findings indicate that by direct inhibition of this signaling molecule, the OPC differentiation program is activated resulting in morphological and functional cell maturation, myelin formation, and regeneration. Altogether, we show evidence of modulation of the Rho-A/ROCK signaling pathway as a viable target for the induction of remyelination in demyelinating pathologies.     

Demyelination induces transport of ribosome-containing vesicles from glia to axons: evidence from animal models and MS patient brains

Glial cells were previously proven capable of trafficking polyribosomes to injured axons. However, the occurrence of such transfer in the general pathological context, such as demyelination-related diseases, needs further evidence. Since this may be a yet unidentified universal contributor to axonal survival, we study putative glia–axonal ribosome transport in response to demyelination in animal models and patients in both peripheral and central nervous system. In the PNS we investigate whether demyelination in a rodent model has the potential to induce ribosome transfer. We also probe the glia–axonal ribosome supply by implantation of transgenic Schwann cells engineered to produce fluorescent ribosomes in the same demyelination model. We furthermore examine the presence of axonal ribosomes in mouse experimental autoimmune encephalomyelitis (EAE), a well-established model for multiple sclerosis (MS), and in human MS autopsy brain material. We provide evidence for increased axonal ribosome content in a pharmacologically demyelinated sciatic nerve, and demonstrate that at least part of these ribosomes originate in the transgenic Schwann cells. In the CNS one of the hallmarks of MS is demyelination, which is associated with severe disruption of oligodendrocyte–axon interaction. Here, we provide evidence that axons from spinal cords of EAE mice, and in the MS human brain contain an elevated amount of axonal ribosomes compared to controls. Our data provide evidence that increased axonal ribosome content in pathological axons is at least partly due to glia-to-axon transfer of ribosomes, and that demyelination in the PNS and in the CNS is one of the triggers capable to initiate this process.