Brain Metabolite Changes in Patients with Relapsing-Remitting and Secondary Progressive Multiple Sclerosis: A Two-Year Follow-Up Study.

Magnetic resonance spectroscopy (MRS) provides the unique ability to monitor several disease-related pathological processes via their characteristic metabolic markers in vivo. In the present study metabolic compositions were assessed every six months over the period of two years in 36 patients with Multiple Sclerosis (MS) including 21 relapsing-remitting (RR), 15 secondary progressive (SP) patients and 12 normal subjects. The concentrations of the main MRS-detectable metabolites N-acetylaspartate and N-acetylaspartylglutamate (tNAA), creatine and phosphocreatine (tCr), choline containing compounds (Cho), myo-Inositol (Ins), glutamine and glutamate (Glx) and their ratios were calculated in the normal appearing white matter (NAWM) and in selected non-enhancing white matter (WM) lesions. Association between metabolic concentrations in the NAWM and disability were investigated. Concentration of tNAA, a marker for neuroaxonal integrity, did not show any difference between the investigated groups. However, the patients with SPMS showed significant reduction of tNAA in the NAWM over the investigation period of two years indicating diffuse neuroaxonal loss during the disease course. Furthermore, we found a significant increase of Ins, Ins/tCr and Ins/tNAA in WM lesions independently from the course of the disease suggesting ongoing astrogliosis in silent-appearing WM lesions. Analyzing correlations between MRS metabolites in the NAWM and patients clinical status we found the positive correlation of Ins/tNAA with disability in patients with RRMS. In SPMS positive correlation of Cho with disability was found.

The Use of Oral Disease-Modifying Therapies in Multiple Sclerosis.

Three oral disease-modifying drugs-fingolimod, teriflunomide, and dimethyl fumarate (DMF)-are available for treatment of relapsing forms of multiple sclerosis (MS). All three agents were approved in the last decade, primarily on the basis of a moderate to substantial reduction in the occurrence of MS relapses and central nervous system lesion formation detected by MRI. In the trials leading to approval, the first oral disease-modifying drug, fingolimod, reduced the annualized relapse rate (ARR) from 0.40 in placebo-treated patients to 0.18 (FREEDOMS) and from 0.33 in patients treated with interferon ß1a intramuscularly to 0.16 (TRANSFORMS). Teriflunomide, approved on the basis of the two placebo-controlled trials TEMSO and TOWER, demonstrated a reduction in the ARR from 0.54 to 0.37 and from 0.50 to 0.32 respectively. The latest oral MS medication, approved in 2014, is DMF, which had been used in a different formulation for treatment of psoriasis for decades. In the 2-year DEFINE study, the proportion of patients with a relapse was reduced to 27 %, compared with 46 % in placebo arm, whereas in the CONFIRM trial, the ARR was reduced from 0.40 (placebo) to 0.22 in the DMF-treated group of patients. In this review, we will elucidate the mechanisms of action of these three medications and compare their efficacy, safety, and tolerability as a practical guideline for their use. We will further discuss effects other than relapse reduction these small molecules may exert, including potential activities within the central nervous system, and briefly summarize emerging data on new oral MS drugs in clinical development.

Neurodegeneration in Autoimmune Optic Neuritis Is Associated with Altered APP Cleavage in Neurons and Up-Regulation of p53.

Multiple Sclerosis (MS) is a chronic autoimmune inflammatory disease of the central nervous system (CNS). Histopathological and radiological analysis revealed that neurodegeneration occurs early in the disease course. However, the pathological mechanisms involved in neurodegeneration are poorly understood. Myelin oligodendrocyte glycoprotein (MOG)-induced experimental autoimmune encephalomyelitis (EAE) in Brown Norway rats (BN-rats) is a well-established animal model, especially of the neurodegenerative aspects of MS. Previous studies in this animal model indicated that loss of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve, occurs in the preclinical phase of the disease and is in part independent of overt histopathological changes of the optic nerve. Therefore, the aim of this study was to identify genes which are involved in neuronal cell loss at different disease stages of EAE. Furthermore, genes that are highly specific for autoimmune-driven neurodegeneration were compared to those regulated in RGCs after optic nerve axotomy at corresponding time points. Using laser capture micro dissection we isolated RNA from unfixed RGCs and performed global transcriptome analysis of retinal neurons. In total, we detected 582 genes sequentially expressed in the preclinical phase and 1150 genes in the clinical manifest EAE (P < 0.05, fold-induction >1.5). Furthermore, using ingenuity pathway analysis (IPA), we identified amyloid precursor protein (APP) as a potential upstream regulator of changes in gene expression in the preclinical EAE but neither in clinical EAE, nor at any time point after optic nerve transection. Therefore, the gene pathway analysis lead to the hypothesis that altered cleavage of APP in neurons in the preclinical phase of EAE leads to the enhanced production of APP intracellular domain (AICD), which in turn acts as a transcriptional regulator and thereby initiates an apoptotic signaling cascade via up-regulation of the target gene p53.

Beneficial effect of chronic Staphylococcus aureus infection in a model of multiple sclerosis is mediated through the secretion of extracellular adherence protein.

BACKGROUND: Bacterial infections have been assumed to worsen multiple sclerosis (MS) disease symptoms and to lead to increased neurodegeneration. However, the underlying biological mechanisms for these effects are complex and poorly understood. Here, we assessed the disease-modulating effects of chronic infection with Staphylococcus aureus, a common human pathogen, on the clinical course and the extent of neurodegeneration in experimental autoimmune encephalomyelitis (EAE), an animal model of MS. METHODS: To conduct this study, we established a persistent chronic infection in female brown Norway rats by inoculating Staphylococcus aureus (S. aureus) bacteria in a subcutaneously implanted tissue cages. RESULTS: In this study, we observed that the introduction of a localized S. aureus infection during the subclinical phase of EAE induced a chronic systemic inflammatory response, consisting of increased T- and B-cell counts and systemic production of proinflammatory cytokines. Unexpectedly, the S. aureus infection completely prevented the development of clinical EAE, and markedly reduced inflammatory infiltration and demyelination of the optic nerve, while it increased the number of surviving retinal neurons. Using a S. aureus strain that lacked the extracellular adherence protein (Eap), we determined that the extracellular adherence protein is at least partially responsible for the inhibitory effect of S. aureus infection on autoimmune inflammation of the central nervous system. CONCLUSIONS: Our results demonstrate for the first time that chronic infection with S. aureus has a beneficial effect on EAE, indicating a dual role of infection in the pathogenesis of MS. We also showed that secretion of Eap by S. aureus plays a major role in preventing autoimmune inflammation of the CNS. Moreover, we identified Eap as a factor responsible for this protective effect.

Chemokine-mediated redirection of T cells constitutes a critical mechanism of glucocorticoid therapy in autoimmune CNS responses.

Glucocorticoids (GCs) are the standard therapy for treating multiple sclerosis (MS) patients suffering from an acute relapse. One of the main mechanisms of GC action is held to be the induction of T cell apoptosis leading to reduced lymphocyte infiltration into the CNS, yet our analysis of experimental autoimmune encephalomyelitis (EAE) in three different strains of genetically manipulated mice has revealed that the induction of T cell apoptosis is not essential for the therapeutic efficacy of GCs. Instead, we identified the redirection of T cell migration in response to chemokines as a new therapeutic principle of GC action. GCs inhibited the migration of T cells towards CCL19 while they enhanced their responsiveness towards CXCL12. Importantly, blocking CXCR4 signaling in vivo by applying Plerixafor(®) strongly impaired the capacity of GCs to interfere with EAE, as revealed by an aggravated disease course, more pronounced CNS infiltration and a more dispersed distribution of the infiltrating T cells throughout the parenchyma. Our observation that T cells lacking the GC receptor were refractory to CXCL12 further underscores the importance of this pathway for the treatment of EAE by GCs. Importantly, methylprednisolone pulse therapy strongly increased the capacity of peripheral blood T cells from MS patients of different subtypes to migrate towards CXCL12. This indicates that modulation of T cell migration is an important mechanistic principle responsible for the efficacy of high-dose GC therapy not only of EAE but also of MS.

Treatment with atacicept enhances neuronal cell death in a rat model of optic neuritis.

We investigated the effect of atacicept, a recombinant fusion protein blocking BLyS and APRIL and acting on B cells, on degeneration of retinal ganglion cells (RGCs) during experimental autoimmune encephalomyelitis (EAE). We used myelin oligodendrocyte glycoprotein in Brown Norway rats to induce a variant of EAE which involves B cells and leads to severe optic neuritis. Intraperitoneal treatment with atacicept at some of the studied dose levels (100 or 200 µg) resulted in increased apoptosis of retinal ganglion cells whereas at a tenfold lower dose or in vehicle-treated animals no such effect became apparent. Also the extent of inflammation, demyelination, and axonal loss of the optic nerve was more pronounced in rats treated with the higher atacicept dose level. The present study describes observational evidence for adverse effects of atacicept on neuronal survival during EAE.

Acute onset of adult Alexander disease.

Adult-onset Alexander disease (AOAD) is a rare leukoencephalopathy affecting predominantly the brainstem and cervical cord with insidious onset of clinical features. Acute onset is very rare and has yet been described only twice, to our knowledge. We report a 32-year-old hitherto healthy male who, after excessive consumption of alcohol, presented with stroke-like onset of symptoms including rigidospasticity, loss of consciousness, and bulbar dysfunction. MRI features comprised bilateral T2-hyperintensities of frontal white matter and basal ganglia as well as atrophy of medulla oblongata with a peculiar "tadpole" appearance, a pattern characteristic of AOAD. Mutation analysis of the GFAP gene revealed a heterozygous de novo 9-bp microduplication in exon 1. Adult Alexander disease may present with stroke-like features. MRI patterns of chronic neurodegenerative conditions may be recognizable even in acute neurological emergencies.

An optical coherence tomography study on degeneration of retinal nerve fiber layer in rats with autoimmune optic neuritis.

PURPOSE: The aim of the present study was to evaluate the ability and accuracy of spectral domain optical coherence tomography (OCT) for in vivo monitoring of retinal ganglion cell degeneration in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. METHODS: First, OCT imaging was established for imaging of all retinal layers in Brown Norway rats. Second, thickness measurements of retinal nerve fiber layer (RNFL) were performed by periodically imaging during the development and progression of autoimmune optic neuritis. Third, the reproducibility of OCT measurements was determined by comparing RNFL measurements of two independent investigators. Fourth, OCT data were correlated with histopathology obtained ex vivo after the final imaging session. RESULTS: Results showed that RNFL thickness declined significantly before clinical manifestation of the disease and decline progresses continuously during the disease course. RNFL thickness measured by OCT had good repeatability and also corresponded with histomorphometric measurements. The reproducibility was limited because of the post-processing analyses performed by manual measurements. CONCLUSIONS: In summary, it is shown here for the first time that OCT can reliably monitor neurodegeneration in an experimental model of autoimmune optic neuritis in rodents. Moreover, in comparing RNFL thickness decline with histopathological analyses of the optic nerve, these results suggest an early, and in part, inflammation-independent process of RNFL degeneration in autoimmune optic neuritis.

Anti-inflammatory effects of FTY720 do not prevent neuronal cell loss in a rat model of optic neuritis.

In multiple sclerosis, long-term disability is caused by axonal and neuronal damage. Established therapies target primarily the inflammatory component of the disease, but fail to prevent neurodegeneration. Fingolimod (codenamed FTY720) is an oral sphingosine 1-phosphate (S1P) receptor modulator with promising results in phase II trials in multiple sclerosis patients and is under further development as a novel treatment for multiple sclerosis. To evaluate whether FTY720 has neuroprotective properties, we tested this drug in a rat model of myelin oligodendrocyte glycoprotein-induced optic neuritis. FTY720 exerted significant anti-inflammatory effects during optic neuritis and reduced inflammation, demyelination, and axonal damage; however, FTY720 treatment did not prevent apoptosis of retinal ganglion cells (RGCs), the neurons that form the axons of the optic nerve. Consistent with this lack of effect on RGC survival, FTY720 treatment did not improve visual function, nor did it prevent apoptosis of RGCs in vitro. We observed a persistent activation of apoptotic signaling pathways in RGCs under FTY720 treatment, a possible underlying mechanism for the lack of neuroprotection in the presence of strong anti-inflammatory effects, Furthermore, FTY720 shifted the remaining inflammation in the optic nerve toward neurotoxicity by modest up-regulation of potential neurotoxic cytokines. We conclude that FTY720-induced anti-inflammation and axon protection did not of itself protect neurons from apoptotic cell death.

AZT resistance of simian foamy virus reverse transcriptase is based on the excision of AZTMP in the presence of ATP.

Azidothymidine (AZT, zidovudine) is one of the few nucleoside inhibitors known to inhibit foamy virus replication. We have shown previously that up to four mutations in the reverse transcriptase gene of simian foamy virus from macaque (SFVmac) are necessary to confer high resistance against AZT. To characterize the mechanism of AZT resistance we expressed two recombinant reverse transcriptases of highly AZT-resistant SFVmac in Escherichia coli harboring three (K211I, S345T, E350K) or four mutations (K211I, I224T, S345T, E350K) in the reverse transcriptase gene. Our analyses show that the polymerization activity of these mutants is impaired. In contrast to the AZT-resistant reverse transcriptase of HIV-1, the AZT resistant enzymes of SFVmac reveal differences in their kinetic properties. The SFVmac enzymes exhibit lower specific activities on poly(rA)/oligo(dT) and higher K(M)-values for polymerization but no change in K(D)-values for DNA/DNA or RNA/DNA substrates. The AZT resistance of the mutant enzymes is based on the excision of the incorporated inhibitor in the presence of ATP. The additional amino acid change of the quadruple mutant appears to be important for regaining polymerization efficiency.

AZT-resistant foamy virus.

Azidothymidine (AZT) is a reverse transcriptase (RT) inhibitor that efficiently blocks the replication of spumaretroviruses or foamy viruses (FVs). To more precisely elucidate the mechanism of action of the FV RT enzyme, we generated an AZT-resistant FV in cell culture. Biologically resistant virus was obtained for simian foamy virus from macaque (SFVmac), which was insensitive to AZT concentrations of 1 mM, but not for FVs derived from chimpanzees. Nucleotide sequencing revealed four non-silent mutations in the pol gene. Introduction of these mutations into an infectious molecular clone identified all changes to be required for the fully AZT-resistant phenotype of SFVmac. The alteration of individual sites showed that AZT resistance in SFVmac was likely acquired by consecutive acquisition of pol mutations in a defined order, because some alterations on their own did not result in an efficiently replicating virus, neither in the presence nor in the absence of AZT. The introduction of the mutations into the RT of the closely related prototypic FV (PFV) did not yield an AZT-resistant virus, instead they significantly impaired the viral fitness.