Neuroimaging to monitor worsening of multiple sclerosis: advances supported by the grant for multiple sclerosis innovation.

Key unmet needs in multiple sclerosis (MS) include detection of early pathology, disability worsening independent of relapses, and accurate monitoring of treatment response. Collaborative approaches to address these unmet needs have been driven in part by industry-academic networks and initiatives such as the Grant for Multiple Sclerosis Innovation (GMSI) and Multiple Sclerosis Leadership and Innovation Network (MS-LINK™) programs. We review the application of recent advances, supported by the GMSI and MS-LINK™ programs, in neuroimaging technology to quantify pathology related to central pathology and disease worsening, and potential for their translation into clinical practice/trials. GMSI-supported advances in neuroimaging methods and biomarkers include developments in magnetic resonance imaging, positron emission tomography, ocular imaging, and machine learning. However, longitudinal studies are required to facilitate translation of these measures to the clinic and to justify their inclusion as endpoints in clinical trials of new therapeutics for MS. Novel neuroimaging measures and other biomarkers, combined with artificial intelligence, may enable accurate prediction and monitoring of MS worsening in the clinic, and may also be used as endpoints in clinical trials of new therapies for MS targeting relapse-independent disease pathology.

Integrin/Chemokine receptor interactions in the pathogenesis of experimental autoimmune encephalomyelitis.

Excessive infiltration of leukocytes and the elaboration of inflammatory cytokines are believed to be responsible for the observed damage to neurons and oligodendrocytes during multiple sclerosis (MS). Blocking adhesion molecules or preventing the effects of chemotactic mediators such as chemokines can be exploited to prevent immune cell recruitment to inflamed tissues. An anti-α4 integrin antibody (anti-VLA-4mAb/natalizumab (Tysabri®)) has been used as a treatment for MS and reduces leukocyte influx into the brain. In patients, anti-VLA-4 reduces relapses and disability progression. However, its mechanism of action in the brain is not completely understood. The anti-VLA-4mAb was demonstrated to mobilize hematopoietic progenitor cells. Interestingly, the chemokine SDF-1/CXCL12 and its receptor CXCR4 are also key factors regulating the migration of hematopoietic stem cells. Moreover, studies have revealed a crosstalk between SDF-1/CXCR4 and VLA-4 signaling in regulating cell migration. In this study, we address the effects of anti-VLA-4 on chemokine signaling in the brain during MS. We assessed the ability of anti-VLA-4 to regulate Experimental Autoimmune Encephalomyelitis (EAE) and chemokine/receptor signaling. Preclinical administration of anti-VLA-4 delayed clinical signs of EAE. We found that anti-VLA-4 treatment reduced chemokine expression. In order to further explore the interaction of anti-VLA-4 with chemokine/receptor signaling we used dual color transgenic mice. After EAE induction, the expression of both SDF-1/CXCL12 and CXCR4 receptor was upregulated, treatment with anti-VLA-4 inhibited this effect. The effects of anti-VLA-4 on chemokine signaling in the CNS may be of importance when considering its mechanism of action and understanding the pathogenesis of EAE.

Epithelial V-like antigen mediates efficacy of anti-alpha4 integrin treatment in a mouse model of multiple sclerosis.

Natalizumab inhibits the transmigration of activated T lymphocytes into the brain and is highly efficacious in multiple sclerosis (MS). However, from a pharmacogenomic perspective, its efficacy and safety in specific patients remain unclear. Here our goal was to analyze the effects of epithelial V-like antigen (EVA) on anti-alpha4 integrin (VLA4) efficacy in a mouse model of MS, experimental autoimmune encephalomyelitis (EAE). EVA has been previously characterized in human CD4 T lymphocytes, mouse thymic development, and choroid plexus epithelial cells. Further analysis here demonstrated expression in B lymphocytes and an increase in EVA⁺ lymphocytes following immunization. Following active induction of EAE using the MOG³5⁻55 active immunization model, EVA deficient mice developed more severe EAE and white matter tissue injury as compared to wild type controls. This severe EAE phenotype did not respond to anti-VLA4 treatment. In both the control antibody and anti-VLA4 conditions, these mice demonstrated persistent CNS invasion of mature B lymphocyte (CD19⁺, CD21⁺, sIgG⁺), increased serum autoantibody levels, and extensive complement and IgG deposition within lesions containing CD5⁺IgG⁺ cells. Wild type mice treated with control antibody also demonstrated the presence of CD19⁺, CD21⁺, sIgG⁺ cells within the CNS during peak EAE disease severity and detectable serum autoantibody. In contrast, wild type mice treated with anti-VLA4 demonstrated reduced serum autoantibody levels as compared to wild type controls and EVA-knockout mice. As expected, anti-VLA4 treatment in wild type mice reduced the total numbers of all CNS mononuclear cells and markedly decreased CD4 T lymphocyte invasion. Treatment also reduced the frequency of CD19⁺, CD21⁺, sIgG⁺ cells in the CNS. These results suggest that anti-VLA4 treatment may reduce B lymphocyte associated autoimmunity in some individuals and that EVA expression is necessary for an optimal therapeutic response. We postulate that these findings could optimize the selection of treatment responders.

Interferon-beta-1a treatment increases CD56bright natural killer cells and CD4+CD25+ Foxp3 expression in subjects with multiple sclerosis.

Disease modifying effects of interferon (IFN)-beta therapy in patients with multiple sclerosis (MS) may be mediated in part through enhanced immunoregulation by the CD56(bright) subpopulation of natural killer (NK) cells and by Foxp3+ (not italicized) CD4+CD25+ regulatory T cells (Treg). We found that IFN-beta-1a(IM) treatment of relapsing-remitting (RR)MS subjects over 12 months significantly increased both percentage of CD56(bright) NK cells and Foxp3 mRNA expression compared to baseline values, untreated RRMS subjects and healthy controls (HC). This striking enhancement of two prominent immunoregulatory pathways lends support to the idea that beneficial effects of IFN-beta-1a in MS include control of pernicious autoimmunity.

Delayed accumulation of the yeast G1 cyclins Cln1 and Cln2 and the F-box protein Grr1 in response to glucose.

The ability to integrate nutrient availability into cell cycle regulation is critical for the viability of organisms. The Saccharomyces cerevisiae ubiquitin ligase SCF(Grr1) regulates the stability of several proteins that participate in cell division or nutrient sensing. Two of its targets, the cyclins Cln1 and Cln2, accumulate in the presence of glucose. When glucose is added to cells growing asynchronously, we show that the accumulation of the cyclins is a very slow response. We report that the F-box protein Grr1 also accumulates at higher levels in the presence of glucose, and that the response to glucose follows a delayed pattern strikingly similar to that described for Cln1 and Cln2. A model for the regulation of F-box proteins predicts that substrate accumulation could stabilize Grr1. While we found that Grr1 is more stable in cells growing with glucose, we show that the delayed responses to glucose occur independently: Grr1 accumulates in the absence of the cyclins, and vice versa. Thus, our results indicate that this model might not apply to the cyclins and Grr1. Glucose is known to strengthen the interaction of Grr1 with Skp1 in the SCF complex. We hypothesize that glucose could promote the accumulation of Grr1 and its assembly into a SCF complex as a feedback regulation that helps compensate for higher cyclins levels.

SCFGrr1-mediated ubiquitination of Gis4 modulates glucose response in yeast.

The F box protein Grr1 is the substrate specificity-determinant of the SCF(Grr1) E3 ubiquitin ligase complex. Genetic analyses of Grr1 mutants have implicated Grr1 in glucose repression, specifically with regard to expression of the SUC2 transcript. To better understand Grr1, we screened for substrates using a mutant version of Grr1 that should not associate with the SCF complex. We identified Gis4 as a novel Grr1 substrate. Gis4 was originally isolated as a multi-copy suppressor of a Gal--phenotype in the triple mutant snf1 mig1 srb8. Here, we show that Gis4 binds Grr1 in vivo and that Grr1 protein levels positively affect the protein levels of Gis4. The Gis4 protein is stable in wild-type cells and in grr1Delta cells; however, Gis4 is ubiquitinated in a Grr1-dependent manner. Furthermore, we show that Gis4 interacts with Snf1 in a Grr1-dependent fashion, and that Gis4 is involved in de-repression of SUC2 and in transcription of other Snf1-dependent transcripts. Gis4 appears to connect the glucose repression and de-repression pathways. We suggest that Gis4 may explain the glucose repression defects in carbon source metabolism for the grr1 mutants.

The ISG15 isopeptidase UBP43 is regulated by proteolysis via the SCFSkp2 ubiquitin ligase.

The Skp2 oncoprotein belongs to the family of F-box proteins that function as substrate recognition factors for SCF (Skp1, cullin, F-box protein) E3 ubiquitin-ligase complexes. Binding of the substrate to the SCFSkp2 complex catalyzes the conjugation of ubiquitin molecules to the bound substrate, resulting in multi-ubiquitination and rapid degradation by the 26 S proteasome. Using Skp2 as bait in a yeast two-hybrid screen, we have identified UBP43 as a novel substrate for Skp2. UBP43 belongs to the family of ubiquitin isopeptidases and specifically cleaves ISG15, a ubiquitin-like molecule that is induced by cellular stresses, such as type 1 interferons (IFN), nephrotoxic damage, and bacterial infection. UBP43 was originally identified as an up-regulated gene in knock-in mice expressing an acute myelogenous leukemia fusion protein, AML1-ETO, as well as in melanoma cell lines treated with IFN-beta. The phenotype of UBP43 knockout mice includes shortened life span, hypersensitivity to IFN, and neuronal damage, suggesting that tight regulation of ISG15 conjugation is critical for normal cellular function. In this study, we demonstrate that UBP43 is ubiquitinated in vivo and accumulates in cells treated with proteasome inhibitors. We also show that Skp2 promotes UBP43 ubiquitination and degradation, resulting in higher levels of ISG15 conjugates. In Skp2-/- mouse cells, levels of UBP43 are consistently up-regulated, whereas levels of ISG15 conjugates are reduced. Our results demonstrate that the SCFSkp2 is involved in controlling UBP43 protein levels and may therefore play an important role in modulating type 1 IFN signaling.

Transferable domain in the G(1) cyclin Cln2 sufficient to switch degradation of Sic1 from the E3 ubiquitin ligase SCF(Cdc4) to SCF(Grr1).

Degradation of Saccharomyces cerevisiae G(1) cyclins Cln1 and Cln2 is mediated by the ubiquitin-proteasome pathway and involves the SCF E3 ubiquitin-ligase complex containing the F-box protein Grr1 (SCF(Grr1)). Here we identify the domain of Cln2 that confers instability and describe the signals in Cln2 that result in binding to Grr1 and rapid degradation. We demonstrate that mutants of Cln2 that lack a cluster of four Cdc28 consensus phosphorylation sites are highly stabilized and fail to interact with Grr1 in vivo. Since one of the phosphorylation sites lies within the Cln2 PEST motif, a sequence rich in proline, aspartate or glutamate, serine, and threonine residues found in many unstable proteins, we fused various Cln2 C-terminal domains containing combinations of the PEST and the phosphoacceptor motifs to stable reporter proteins. We show that fusion of the Cln2 domain to a stabilized form of the cyclin-dependent kinase inhibitor Sic1 (Delta N-Sic1), a substrate of SCF(Cdc4), results in degradation in a phosphorylation-dependent manner. Fusion of Cln2 degradation domains to Delta N-Sic1 switches degradation of Sic1 from SCF(Cdc4) to SCF(Grr1). Delta N-Sic1 fused with a Cln2 domain containing the PEST motif and four phosphorylation sites binds to Grr1 and is unstable and ubiquitinated in vivo. Interestingly, the phosphoacceptor domain of Cln2 binds to Grr1 but is not ubiquitinated and is stable. In summary, we have identified a small transferable domain in Cln2 that can redirect a stabilized SCF(Cdc4) target for SCF(Grr1)-mediated degradation by the ubiquitin-proteasome pathway.