André Müller, Ph.D. is a research associate at the MSRCNY whose undergraduate work in technical biology at the University of Stuttgart, Germany, prepared him to earn a doctorate from the University of Regensburg, Germany in 2005 Before he joined the MSRCNY in 2008 he was studying the influence of several TNF family members on the pathogenesis of an MS animal model as well as the transcriptional changes in the CNS within the context of the MOG-induced experimental autoimmune encephalomyelitis of dark agouti rats between 2004 and 2008.
Disease Mechanisms of progressive MS
Severely disabled progressive MS patients are not responsive to anti-inflammatory therapies, but they seem to profit from an intrathecal administration of the anti-metabolite methotrexate (MTX) whose impact on progressive MS was not shown before (Gray et al., 2006, Sadiq et al., 2010). MTX is only used at our MS center as an off-label, inexpensive (medication costing approximately $2 plus the cost of the lumbar puncture) treatment for disabled patients with MS, who had no alternative therapeutic options, in an attempt to modify the otherwise dismal course of progressive disease.
The benefit derived from methotrexate is most likely independent of its well established anti-inflammatory properties, because those MS patients profiting from it were unresponsive to established anti-inflammatory therapies. Hence, methotrexate must be able to influence neurodegenerative processes in the CNS. Neurodegenerative mechanisms represent major challenges for therapeutic interventions. Characterization and targeting of the processes that initiate specific disease pathologies are clearly important areas for continued investigation (Dutta and Trapp, 2011). We noticed that methotrexate was able to reduce the activation of astrocytes in the context of two different non-inflammatory CNS demyelination models. As astrocytes are CNS-resident cells whose activation is supposed to contribute to the limited repair of CNS lesions seen in chronic MS (Colangelo et al., 2011), we inferred that the inhibition of astroglial activation is most likely the mechanism by which methotrexate helps severely disabled progressive MS patients.
Our current work aims to corroborate the pathogenic role of astrocytes within the context of neurodegenerative MS models. Additionally, we are establishing surrogate markers for astroglial activation in MS patients in order to assess which individual patients are likely to profit from methotrexate or from other substances targeting astroglial activation.
Biomarkers of MS
Several pathophysiological processes such as inflammation, demyelination, axonal damage and repair mechanisms contribute to the complex disease manifestation of MS. These processes are not uniformly represented across patient populations and can selectively predominate in individual patients. The varying degrees of involvement contribute to the heterogeneity in phenotypic expression of the disease, its prognosis and the response to therapies. Successful therapeutic intervention is assumed to require a combination of different therapeutic strategies that would target individual dominant pathophysiological processes. Such process-specific therapies will require the use of biomarkers in order to support the identification of the dominant mechanisms, the selection of appropriate patient populations, and the initial screening for efficacious agents. Although numerous potential biomarkers to guide MS therapy have been proposed, very few could be validated. Hence, there is a high need for tests which can aid in the MS diagnosis, prognosis of disease progression and treatment efficacy.
Direct investigation of cells form inflammatory lesions in the brain or spinal cord in inflammatory CNS diseases is hardly possible in humans, mostly because MS lesions are rarely biopsied. However, cells in the cerebrospinal fluid (CSF) likely have interacted with or have derived from inflammatory CNS lesions. Therefore, CSF cells may reflect on disease activity in the CNS and are for that reason of high interest. Considering CSF cells to be a surrogate for the target organ itself in CNS disorders, we are addressing the gene expression profile of CSF cells associated with MS. In our research center we have the unique opportunity to study cells derived from the CSF. We were able to establish the collection of the CSF under ideal conditions that maintain the integrity of messenger RNA (mRNA) which is crucial in such studies as RNA is highly unstable. We implemented the use of gene array technology in order to define changes that characterize the various stages of multiple sclerosis, inflammation, demyelination, remyelination, and neural genesis.
So far, we analyzed the CSF cell expression profile of 50 MS and control patients. In all cases, high-quality RNA could be isolated from CSF cells. The hybridization of HG U133 plus 2.0 microarrays (Affymetrix) with the processed RNA revealed that CSF cells from MS patients strongly express B cell-related genes and immunoproteasome components indicating the presence of highly active immune cells in the CSF of MS patients. Flow cytometric analyses of the same samples used for array hybridization confirmed high frequencies of B cells in the CSF of MS patients compared to control probands.
We established that the CSF cell expression of the hepatocyte growth factor (HGF) is significantly lower in MS patients than in patients with other neurological diseases as well as in MS patients with an active disease than in inactive MS patients. Importantly, we found corresponding differences in HGF protein levels in the CSF. HGF is a growth hormone with potent neuroprotective properties (Benkhoucha et al., Ebens et al., 1996). It was also reported to enhance the survival and maturation of myelin producing oligodendrocytes which are required for the repair of MS lesions (Lalive et al., 2005, Kitamura et al., 2007). The suppression of HGF in MS probably contributes to the imperfect repair of demyelinated CNS lesions frequently seen in MS patients. We currently evaluate if HGF can be established as a biomarker for the assessment of MS disease activity.
The diagnosis of MS is made well after initiation of the disease process, because it is based on the detection of plaques and of clinically evident neuropathy. The earlier MS patients are diagnosed the earlier they get the chance to profit from therapeutic interventions. Although only a very limited number of CSF cell expression profiles was analyzed, we could identify 18 genes whose expression by CSF cells allow an early diagnosis of MS with a very high sensitivity (91%) and a 100% specificity.
These results indicate that high quality mRNA can be isolated from CSF cells, and that the CSF cell transcriptome is representative for disease processes in the CNS of multiple sclerosis patients. Therefore, we conclude that new meaningful insights will be gained by a comprehensive study of the transcriptome of CSF cells from multiple sclerosis patients. Currently, we are screening for gene subsets facilitating the stratification of disease subtypes, the prognosis of the disease course, and the prediction of the efficacy of currently available MS therapies. We are aiming to develop a diagnostic and prognostic CSF cell transcriptome-based tool.
Promoting CNS repair processes by targeting CNS proteases
The role of proteases in MS pathogenesis is of high interest because they play crucial regulatory roles in inflammatory processes through a number of different mechanisms (Cuzner and Opdenakker, 1999), including modification of immune activators. Moreover, proteases can also directly contribute to inflammatory damage through causing direct CNS tissue injury (Rosenberg, 2002) and through disruption of the blood brain barrier (BBB) which separates the CNS from the periphery (Agrawal et al., 2006). Patients with acute MS have elevated protease levels in cerebrospinal fluid, blood, and brain tissue (Yong et al., 2007).
The secretory leukocyte protease inhibitor (SLPI), a potent inhibitor of proteases (Zitnik et al., 1997) was originally identified in bodily secretions such as saliva, seminal fluid, and in the mucus of cervical, nasal and bronchial passages (Fritz, 1988). Later it was shown to be also strongly produced in the CNS as a consequence of diseases including an animal model of multiple sclerosis (Mueller et al., 2008). SLPI exerts anti-inflammatory functions in several immune cell types which play a role in MS (Ward and Lentsch, 2002, Taggart et al., 2005) and is neuroprotective in an ischemic stroke model (Wang et al., 2003). SLPI may be able to promote repair processes because it stimulates the generation of myelin-producing oligodendrocytes (Mueller et al., 2008). Recently, we identified a prominent pro-inflammatory role of SLPI. It is interfering with the proteolytic activation of the anti-inflammatory molecule TGF-β which leads to a decreased generation of regulatory T cells. This effect was observed in both, in in vitro as well as in vivo experiments (Mueller et al. (in review)).
SLPI´s inhibitory activity towards pathogenic proteases, its neuroprotective and immunomodulatory properties and especially its influence on neural stem cells suggest that it might be an interesting candidate for future MS therapy. Therefore, we are highly interested in SLPI´s protective and repair enhancing properties.
We are currently determining if SLPI has any role in neurodegenerative disorders. In particular we are trying to elucidate if its immunomodulatory and inhibitory effect on pathogenic proteases by SLPI are more important than its interference with the proteolytic activation of latent TGF-β.
SLPI´s therapeutic influence is addressed in an inflammatory animal model of MS as well as in the toxic, non-inflammatory cuprizone-induced demyelination model. In the experiments, mostly transgenic mice which cannot produce SLPI are examined.
Our long-term aims are to establish SLPI´s role on repair processes and to target its activity for the development of MS therapy that is able to promote lesion repair in MS.