Linking immune-mediated damage to neurodegeneration in multiple sclerosis: could network-based MRI help?

Inflammatory demyelination characterizes the initial stages of multiple sclerosis, while progressive axonal and neuronal loss are coexisting and significantly contribute to the long-term physical and cognitive impairment. There is an unmet need for a conceptual shift from a dualistic view of multiple sclerosis pathology, involving either inflammatory demyelination or neurodegeneration, to integrative dynamic models of brain reorganization, where, glia-neuron interactions, synaptic alterations and grey matter pathology are longitudinally envisaged at the whole-brain level. Functional and structural MRI can delineate network hallmarks for relapses, remissions or disease progression, which can be linked to the pathophysiology behind inflammatory attacks, repair and neurodegeneration. Here, we aim to unify recent findings of grey matter circuits dynamics in multiple sclerosis within the framework of molecular and pathophysiological hallmarks combined with disease-related network reorganization, while highlighting advances from animal models (in vivo and ex vivo) and human clinical data (imaging and histological). We propose that MRI-based brain networks characterization is essential for better delineating ongoing pathology and elaboration of particular mechanisms that may serve for accurate modelling and prediction of disease courses throughout disease stages

CSF markers of blood-brain barrier integrity forecast disease progression in early MS [Abstract]

Background and aim: Cortical atrophy, reflecting neuronal loss, is highly associated with long-term disability in patients with multiple sclerosis (MS). In this longitudinal study we link cerebrospinal fluid (CSF) markers of blood-brain barrier (BBB) integrity to longitudinal cortical atrophy and clinical disability. Methods: 71 relapsing-remitting multiple sclerosis (RRMS) patients (31.2 ± 9.4 (mean ± SD), 25 males) were included in this longitudinal study. CSF and serum samples were obtained from each patient at the time of first clinical event. We analyzed CSF levels of albumin (AlbCSF), immunoglobulin A (IgACSF), IgG (IgGCSF) and IgM (IgMCSF). All patients underwent MR imaging twice with the same standardized protocol (follow-up after 12 ± 1 months) at 3T (Siemens Magnetom Trio). Longitudinal changes of cortical thickness (CT) were extracted using the FreeSurfer processing stream. The Expanded Disability Status Scale (EDSS) score at follow-up was used as a clinical outcome measure to quantify clinical disability. The rate of cortical atrophy was assessed in relation to CSF variables. Results: Baseline AlbCSF and IgACSF were highly associated with the rate of cortical atrophy over one year. Significant correlations were found in the precuneus (PrC), rostral middle frontal, precentral and inferior parietal gyri of both hemispheres. The regions with the highest atrophy rates were the right PrC (R = 0.604, p < 0.001) and left fusiform gyrus (R = 0.539, p < 0.001). IgACSF and IgMCSF (IgA: 1.67 ± 0.69 mg/l vs 2.03 ± 0.71 mg/l, IgM: 9.87 ± 2.38 mg/l vs 11.5 ± 2.03 mg/l; p = 0.04 and p = 0.003, respectively) significantly differed between patients with no disability (EDSS 0 - 1.5) and those with mild to moderate disability (EDSS 2 - 6) at the second time point. Conclusion: Our data show that widespread cortical atrophy is highly associated with increased baseline CSF Albumin and IgA mirroring a permeable BBB. Patients with this BBB pattern showed higher functional disability at follow-up. These parameters could be addressed to dichotomize patients at risk of rapid disease progression

Longitudinal structural network reorganisation in early relapsing-remitting multiple sclerosis [Abstract]

Background: Multiple sclerosis (MS) is characterized by relapses and remissions indicating damage and compensatory processes occurring early in the disease. Over time, cortical pathology is highly relevant for disability, while brain networks evolve towards a disconnected organization as the disease progresses. However, it is poorly understood how and when pathology impacts cortical networks and in particular, how the network responds to damage in the very beginning of the disease. Aim: To address cortical pathology by quantifying structural connectivity patterns over 12 months in patients with early relapsing-remitting MS. Methods: Here we investigated cortical grey matter networks longitudinally as derived from structural 3 Tesla MRI in 92 patients in the initial phase of the disease (65 female / 27 male; mean age: 32.9 ± 9.9 years; mean disease duration: 12.1 ± 14.5 months) and in 101 healthy controls (59 female / 42 male; mean age: 19.7 ± 0.9 years). Longitudinal brain volume atrophy was analyzed using SIENA and cortical thickness changes were quantified using FreeSurfer. Brain networks were computed based on cortical thickness inter-regional correlations between anatomical regions and fed into graph theoretical analysis. Finally, subgroup analyses were performed between patients with “no evidence of disease activity” (NEDA) during this period and those with disease activity (EDA). Results: Over one year, increased local cortical connectivity and an emerging modular-constructed network were detected in patients - a pattern reported to be associated with adaptation, efficiency and compensation. These longitudinal dynamics were attested in both patients with NEDA and EDA, indicating continuous cortical reorganisation independent of disease activity. This local and modular cortical reorganisation was not detected in healthy controls over the same period of time and emerged beyond measureable signs of atrophy using established morphometric tools. Conclusion: Our findings demonstrate that despite initiation of neuroinflammatory damage, substantial structural adaptation processes emerge cortically in the early disease stage. This subtle reorganisation of the cortex architecture is quantifiable by structural MRI in patients with and without disease activity, suggesting a principal response of the network evolving from the onset of this chronic disease. Disclosure: The authors declare no conflict of interests

Excitotoxic neuronal cell death during an oligodendrocyte-directed CD8+ T cell attack in the CNS gray matter

Background: Neural-antigen reactive cytotoxic CD8+ T cells contribute to neuronal dysfunction and degeneration in a variety of inflammatory CNS disorders. Facing excess numbers of target cells, CNS-invading CD8+ T cells cause neuronal cell death either via confined release of cytotoxic effector molecules towards neurons, or via spillover of cytotoxic effector molecules from 'leaky’ immunological synapses and non-confined release by CD8+ T cells themselves during serial and simultaneous killing of oligodendrocytes or astrocytes. Methods: Wild-type and T cell receptor transgenic CD8+ T cells were stimulated in vitro, their activation status was assessed by flow cytometry, and supernatant glutamate levels were determined using an enzymatic assay. Expression regulation of molecules involved in vesicular glutamate release was examined by quantitative real-time PCR, and mechanisms of non-vesicular glutamate release were studied by pharmacological blocking experiments. The impact of CD8+ T cell-mediated glutamate liberation on neuronal viability was studied in acute brain slice preparations. Results: Following T cell receptor stimulation, CD8+ T cells acquire the molecular repertoire for vesicular glutamate release: (i) they upregulate expression of glutaminase required to generate glutamate via deamination of glutamine and (ii) they upregulate expression of vesicular proton-ATPase and vesicular glutamate transporters required for filling of vesicles with glutamate. Subsequently, CD8+ T cells release glutamate in a strictly stimulus-dependent manner. Upon repetitive T cell receptor stimulation, CD25high CD8+ T effector cells exhibit higher estimated single cell glutamate release rates than CD25low CD8+ T memory cells. Moreover, glutamate liberation by oligodendrocyte-reactive CD25high CD8+ T effector cells is capable of eliciting collateral excitotoxic cell death of neurons (despite glutamate re-uptake by glia cells and neurons) in intact CNS gray matter. Conclusion: Glutamate release may represent a crucial effector pathway of neural-antigen reactive CD8+ T cells, contributing to excitotoxicity in CNS inflammation.<br

Continuous reorganisation of cortical information flow in MS patients: a longitudinal effective connectivity study [Abstract]

Background: Brain reorganisation processes are essential for the long-term outcome in patients with multiple sclerosis (MS). Effective connectivity (EC) as derived from functional MRI, can be analysed to estimate reorganisation processes and directional information flows between cortical regions. These measures could provide the missing link for modelling the long-term disease course between tissue damage and repair or adaptation. Aim: To obtain longitudinal measurements of EC and information flows in MS patients at short-term intervals focusing on the main anatomical brain regions and to investigate the link between the connectivity strength and clinical impairment. Methods: Twelve MS patients (mean age: 41.7 ± 11.5 years) underwent 3 Tesla structural and resting state functional MRI at five different time points over one year (approximately every 12 weeks). Twelve healthy subjects (mean age: 33.5 ± 9.6 years) served as controls (HC). For the analytical framework, two novel approaches for EC quantification were used. Causal Bayesian Network (CBN) and Time Domain Partial Directed Coherence (TPDC) were applied for the description of the information flows between frontal, prefrontal, temporal, occipital, and parietal lobe; cerebellum and deep grey matter nuclei (DGMN) were also analysed. Results: Specific longitudinal EC patterns have been attested in the studied regions. Information flows from DGMN, frontal, prefrontal and temporal to the other studied regions showed a continuous increase over time, whereas the directed connections from parietal and occipital lobes and from the cerebellum did not change over time as confirmed by both applied methods. No longitudinal changes of EC were attested in HC. The longitudinal connectivity increase in the prefrontal-frontal and fronto-cerebellar pathway showed a significant inverse correlation to EDSS (Expanded Disability Status Scale). Moreover, the EC change from the frontal lobe to the cerebellum showed a significant inverse correlation to patients’ fatigue score. Conclusion: Our data depicts a continuous longitudinal increase in EC in patients with MS substantiated by two novel methodological approaches. Furthermore, the dynamics of the fronto-cerebellar connections are linked to clinical impairment and possibly essential for the long-term outcome

Post-stroke inhibition of induced NADPH oxidase type 4 prevents oxidative stress and neurodegeneration

Ischemic stroke is the second leading cause of death worldwide. Only one moderately effective therapy exists, albeit with contraindications that exclude 90% of the patients. This medical need contrasts with a high failure rate of more than 1,000 pre-clinical drug candidates for stroke therapies. Thus, there is a need for translatable mechanisms of neuroprotection and more rigid thresholds of relevance in pre-clinical stroke models. One such candidate mechanism is oxidative stress. However, antioxidant approaches have failed in clinical trials, and the significant sources of oxidative stress in stroke are unknown. We here identify NADPH oxidase type 4 (NOX4) as a major source of oxidative stress and an effective therapeutic target in acute stroke. Upon ischemia, NOX4 was induced in human and mouse brain. Mice deficient in NOX4 (Nox4(-/-)) of either sex, but not those deficient for NOX1 or NOX2, were largely protected from oxidative stress, blood-brain-barrier leakage, and neuronal apoptosis, after both transient and permanent cerebral ischemia. This effect was independent of age, as elderly mice were equally protected. Restoration of oxidative stress reversed the stroke-protective phenotype in Nox4(-/-) mice. Application of the only validated low-molecular-weight pharmacological NADPH oxidase inhibitor, VAS2870, several hours after ischemia was as protective as deleting NOX4. The extent of neuroprotection was exceptional, resulting in significantly improved long-term neurological functions and reduced mortality. NOX4 therefore represents a major source of oxidative stress and novel class of drug target for stroke therapy

CD4+ CD25+ FoxP3+ regulatory T cells suppress cytotoxicity of CD8+ effector T cells: implications for their capacity to limit inflammatory central nervous system damage at the parenchymal level

<p>Abstract</p> <p>Background</p> <p>CD4⁺CD25⁺forkhead box P3 (FoxP3)⁺regulatory T cells (T reg cells) are known to suppress adaptive immune responses, key control tolerance and autoimmunity.</p> <p>Methods</p> <p>We challenged the role of CD4⁺T reg cells in suppressing established CD8⁺T effector cell responses by using the OT-I/II system <it>in vitro </it>and an OT-I-mediated, oligodendrocyte directed <it>ex vivo </it>model (ODC-OVA model).</p> <p>Results</p> <p>CD4⁺T reg cells dampened cytotoxicity of an ongoing CD8⁺T effector cell attack <it>in vitro </it>and within intact central nervous system tissue <it>ex vivo</it>. However, their suppressive effect was limited by the strength of the antigen signal delivered to the CD8⁺T effector cells and the ratio of regulatory to effector T cells. CD8⁺T effector cell suppression required T cell receptor-mediated activation together with costimulation of CD4⁺T reg cells, but following activation, suppression did not require restimulation and was antigen non-specific.</p> <p>Conclusions</p> <p>Our results suggest that CD4⁺T reg cells are capable of suppressing CD8⁺T effector cell responses at the parenchymal site, that is, limiting parenchymal damage in autoimmune central nervous system inflammation.</p

Altered grey matter networks in young patients with MS at genetic risk for Alzheimer's disease [Abstract]

Background: The Apolipoprotein E (APOE) ε4 is the major susceptibility factor for cognitive impairment and Alzheimer’s disease. Cognitive decline is also a concern in patients with multiple sclerosis (MS). Whether APOE ε4 exerts an effect on brain structure and grey matter (GM) networks in MS patients that could potentiate the long-term cognitive disabilities is unclear. Moreover the description of the exact link between genetic markers and MR driven measures of brain integrity are of essential importance to study cognition in patients with MS and for interventions to prevent longitudinal deterioration. Methods: MS Patients with no immunomodulatory treatment were enrolled in the “Krankheitsbezogene Kompetenznetz Multiple Sclerosis (KKNMS)”. From this multicenter dataset 37 heterozygous APOE ε4 carriers (i.e. having the genotype ε3/ε4) and 37 non-carriers (ε3/ε3) were matched for demographics (mean age: 38.4±9.2 yrs, mean EDSS 1.23±0.99) from one site. A replication study was performed in a cohort (n=46) from a second site. Cortical thickness (CT) was derived from 3T MRI using FreeSurfer. GM connectivity networks were reconstructed from the CT correlation between the 68 regions of the Desikan-Killiany atlas. Cortical integrity and network connectivity -derived from graph theoretical approaches- were compared between the groups in both cohorts. Results corrected for multiple comparisons were considered (p< 0.05 FDR). Results: No regional or global cortical atrophy differences were attested between the two groups in both cohorts. In the network connectivity analysis a decreased local connectivity pattern (reduced transitivity, t=-3.24 p=0.008) was evident in APOE ε4 carriers. Regions with decreased connectivity were consistently seen in the medial part of the left temporal lobe. APOE ε4 status was further associated with raised whole brain connectivity, reflected by increased global efficiency (t=4.34 p=0.005) and reduced modularity (t=-2.84 p=0.02). This network pattern was shown in the frontal, parietal and lateral temporal associative cortices. The results were entirely replicated in the second cohort. Conclusion: We found that MS patients at genetic risk for cognitive decline have significant abnormalities of local GM networks and possibly compensatory increased long-range connectivity patterns. Chronic or focal neuroinflammation could lead to behaviourally relevant memory impairments in these patients through a specific break-down of the long-range paths

Novel multiple sclerosis susceptibility loci implicated in epigenetic regulation

We conducted a genome-wide association study (GWAS) on multiple sclerosis (MS) susceptibility in German cohorts with 4888 cases and 10,395 controls. In addition to associations within the major histocompatibility complex (MHC) region, 15 non-MHC loci reached genome-wide significance. Four of these loci are novel MS susceptibility loci. They map to the genes L3MBTL3, MAZ, ERG, and SHMT1. The lead variant at SHMT1 was replicated in an independent Sardinian cohort. Products of the genes L3MBTL3, MAZ, and ERG play important roles in immune cell regulation. SHMT1 encodes a serine hydroxymethyltransferase catalyzing the transfer of a carbon unit to the folate cycle. This reaction is required for regulation of methylation homeostasis, which is important for establishment and maintenance of epigenetic signatures. Our GWAS approach in a defined population with limited genetic substructure detected associations not found in larger, more heterogeneous cohorts, thus providing new clues regarding MS pathogenesis

Hierarchical Anatomical Brain Networks for MCI Prediction: Revisiting Volumetric Measures

Owning to its clinical accessibility, T1-weighted MRI (Magnetic Resonance Imaging) has been extensively studied in the past decades for prediction of Alzheimer's disease (AD) and mild cognitive impairment (MCI). The volumes of gray matter (GM), white matter (WM) and cerebrospinal fluid (CSF) are the most commonly used measurements, resulting in many successful applications. It has been widely observed that disease-induced structural changes may not occur at isolated spots, but in several inter-related regions. Therefore, for better characterization of brain pathology, we propose in this paper a means to extract inter-regional correlation based features from local volumetric measurements. Specifically, our approach involves constructing an anatomical brain network for each subject, with each node representing a Region of Interest (ROI) and each edge representing Pearson correlation of tissue volumetric measurements between ROI pairs. As second order volumetric measurements, network features are more descriptive but also more sensitive to noise. To overcome this limitation, a hierarchy of ROIs is used to suppress noise at different scales. Pairwise interactions are considered not only for ROIs with the same scale in the same layer of the hierarchy, but also for ROIs across different scales in different layers. To address the high dimensionality problem resulting from the large number of network features, a supervised dimensionality reduction method is further employed to embed a selected subset of features into a low dimensional feature space, while at the same time preserving discriminative information. We demonstrate with experimental results the efficacy of this embedding strategy in comparison with some other commonly used approaches. In addition, although the proposed method can be easily generalized to incorporate other metrics of regional similarities, the benefits of using Pearson correlation in our application are reinforced by the experimental results. Without requiring new sources of information, our proposed approach improves the accuracy of MCI prediction from (of conventional volumetric features) to (of hierarchical network features), evaluated using data sets randomly drawn from the ADNI (Alzheimer's Disease Neuroimaging Initiative) dataset