Inflammatory Cascades in the Pathogenesis of Multiple Sclerosis Lesions

Multiple sclerosis (MS) is a disease of the human central nervous system (CNS) characterised by inflammation and demyelination. Initially the MS lesion has a distinct histopathological picture with myelin-positive microglia in the midst of apparently intact myelin but minimal perivascular inflammation. Inflammatory mediators produced by these activated microglia may precipitate the infiltration of mononuclear cells and the overt myelin loss seen in actively demyelinating MS lesions. Nuclear factor-κB (NF-κB) is a transcriptional regulator of proteolytic enzymes, adhesion molecules and inflammatory cytokines which rapidly translates extracellular signals into protein synthesis. The immunocytochemical detection of the transcriptionally active form of NF-κB, but not the inhibitory protein IκBα, in the nuclei of microglia in normal human CNS white matter indicates the capability of microglia to respond rapidly to pathological stimuli in the CNS. Activation of NF-κB in MS plaques, evident from the nuclear localisation of the NF-κB subunits RelA, c-Rel and p50 in macrophages, may propagate inflammatory demyelination through upregulation of NF-κB-controlled macrophage genes for inflammatory mediators. In demyelinating disease the plasmin-matrix metalloprotease (MMP) enzymatic cascade promotes blood-brain barrier (BBB) damage, generation of encephalitogenic myelin peptides and activation of pro-inflammatory cytokines. Constitutive expression of MMPs 1, 2, 3 and 9 in glial cells in normal control white matter was demonstrated by immunocytochemistry. However, the lack of tissue (t-PA) and urokinase (u-PA) plasminogen activators in glial cells and the absence of caseinolytic activity as shown by in situ zymography emphasises the latent nature of the plasmin-MMP cascade in normal CNS tissue. In contrast, the co-localisation of t-PA and u-PA, rate-limiting serine-proteases, and MMPs in macrophages and astrocytes in active MS lesions forms the basis of a functional enzymatic cascade. Furthermore, increased amounts and activity of u-PA and MMP-9 in homogenates of active MS plaques coupled with the presence of caseinolytic activity in foamy macrophages implicates these cells as the major source of MMPs, which cause proteolytic damage in MS. Insulin-like growth factors (IGFs) play an important role in development and myelination in the CNS but can also stimulate phagocytosis and production of inflammatory mediators by macrophages. In active MS lesions binding of IGF-II to the IGF receptor on foamy macrophages may induce mitogenic responses and invasiveness of macrophages which can be further enhanced by MMP-mediated proteolytic removal of inhibitory IGF-binding proteins. Similarly, the potent mitogens IGF-I and insulin may stimulate astrocytosis and gliosis. In contrast, oligodendrocytes in normal-appearing white matter do not express IGFs or IGF-I receptor which implies that the oligodendrocyte response to these remyelinating growth factors is impaired. Therefore, the prevailing role of IGFs in MS lesions may be in line with pro-inflammatory mediators promoting macrophage and astrocyte responses to tissue damage. In conclusion, NF-κB activation in microglia and macrophages upregulates the production of PAs and inflammatory cytokines which trigger the plasmin-MMP cascade, leading to BBB damage and enhanced inflammatory cell migration and demyelination in white matter. Influx of IGFs through the damaged BBB and their increased local production may promote myelin phagocytosis and reactive astrocytosis. In turn IGF-mediated upregulation of PAs in glial cells could provide a feedback amplification of the MMP cascade. Therefore, the findings from these studies bring together three systems of mediators, NF-κB, MMPS and IGFs, into a hypothetical model for the propagation of demyelination in MS lesions

Production of IL-16 correlates with CD4+ Th1 inflammation and phosphorylation of axonal cytoskeleton in multiple sclerosis lesions

BACKGROUND: Multiple sclerosis (MS) is a central nervous system-specific autoimmune, demyelinating and neurodegenerative disease. Infiltration of lesions by autoaggressive, myelin-specific CD4+Th1 cells correlates with clinical manifestations of disease. The cytokine IL-16 is a CD4+ T cell-specific chemoattractant that is biased towards CD4+ Th1 cells. IL-16 precursor is constitutively expressed in lymphocytes and during CD4+ T cell activation; active caspase-3 cleaves and releases C-terminal bioactive IL-16. Previously, we used an animal model of MS to demonstrate an important role for IL-16 in regulation of autoimmune inflammation and subsequent axonal damage. This role of IL-16 in MS is largely unexplored. Here we examine the regulation of IL-16 in relation to CD4+ Th1 infiltration and inflammation-related changes of axonal cytoskeleton in MS lesions. METHODS: We measured relative levels of IL-16, active caspase-3, T-bet, Stat-1 (Tyr (701)), and phosphorylated NF(M+H), in brain and spinal cord lesions from MS autopsies, using western blot analysis. We examined samples from 39 MS cases, which included acute, subacute and chronic lesions, as well as adjacent, normal-appearing white and grey matter. All samples were taken from patients with relapsing remitting clinical disease. We employed two-color immunostaining and confocal microscopy to identify phenotypes of IL-16-containing cells in frozen tissue sections from MS lesions. RESULTS: We found markedly increased levels of pro- and secreted IL-16 (80 kD and 22 kD, respectively) in MS lesions compared to controls. Levels of IL-16 peaked in acute, diminished in subacute, and were elevated again in chronic active lesions. Compared to lesions, lower but still appreciable IL-6 levels were measured in normal-appearing white matter adjacent to active lesions. Levels of IL-16 corresponded to increases in active-caspase-3, T-bet and phosphorylated Stat-1. In MS lesions, we readily observed IL-16 immunoreactivity confined to infiltrating CD3+, T-bet+ and active caspase-3+ mononuclear cells. CONCLUSION: We present evidence suggesting that IL-16 production occurs in MS lesions. We show correlations between increased levels of secreted IL-16, CD4+ Th1 cell inflammation, and phosphorylation of axonal cytoskeleton in MS lesions. Overall, the data suggest a possible role for IL-16 in regulation of inflammation and of subsequent changes in the axonal cytoskeleton in MS

Unveiling the olfactory proteostatic dissangement in Parkinson's disease by proteome-wide profiling

Olfactory dysfunction is one of the earliest features in Lewy-type alphasynucleinopathies (LTS) such as Parkinson´s disease (PD). However, the underlying molecular mechanisms associated to smell impairment are poorly understood. Applying mass spectrometry-based quantitative proteomics in postmortem olfactory bulbs (OB) across limbic, early-neocortical, and neocortical LTS stages of parkinsonian subjects, a proteostasis impairment was observed, identifying 268 differentially expressed proteins between controls and PD phenotypes. In addition, network-driven proteomics revealed a modulation in ERK1/2, MKK3/6, and PDK1/PKC signalling axis. Moreover, a crossdisease study of selected olfactory molecules in sporadic Alzheimer's disease (AD) cases, revealed different protein derangements in the modulation of Secretagogin (SCGN), Calcyclin binding protein (CACYBP), and Glucosamine 6 phosphate isomerase 2 (GNPDA2) between PD and AD. An inverse correlation between GNPDA2 and α-synuclein protein levels was also reflected in PD cerebrospinal fluid (CSF). Interestingly, PD patients exhibited significantly lower serum GNPDA2 levels than controls (n=82/group). Our study provides important avenues for understanding the OB proteostasis imbalance in PD, deciphering mechanistic clues to the equivalent smell deficits observed in AD and PD pathologies

Considerations in establishing a post-mortem brain and tissue bank for the study of myalgic encephalomyelitis/chronic fatigue syndrome: a proposed protocol.

BACKGROUND: Our aim, having previously investigated through a qualitative study involving extensive discussions with experts and patients the issues involved in establishing and maintaining a disease specific brain and tissue bank for myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), was to develop a protocol for a UK ME/CFS repository of high quality human tissue from well characterised subjects with ME/CFS and controls suitable for a broad range of research applications. This would involve a specific donor program coupled with rapid tissue collection and processing, supplemented by comprehensive prospectively collected clinical, laboratory and self-assessment data from cases and controls. FINDINGS: We reviewed the operations of existing tissue banks from published literature and from their internal protocols and standard operating procedures (SOPs). On this basis, we developed the protocol presented here, which was designed to meet high technical and ethical standards and legal requirements and was based on recommendations of the MRC UK Brain Banks Network. The facility would be most efficient and cost-effective if incorporated into an existing tissue bank. Tissue collection would be rapid and follow robust protocols to ensure preservation sufficient for a wide range of research uses. A central tissue bank would have resources both for wide-scale donor recruitment and rapid response to donor death for prompt harvesting and processing of tissue. CONCLUSION: An ME/CFS brain and tissue bank could be established using this protocol. Success would depend on careful consideration of logistic, technical, legal and ethical issues, continuous consultation with patients and the donor population, and a sustainable model of funding ideally involving research councils, health services, and patient charities. This initiative could revolutionise the understanding of this still poorly-understood disease and enhance development of diagnostic biomarkers and treatments

GENOME-WIDE ASSOCIATION FOR THE PARK10 LOCUS IN IDIOPATHIC, AUTOPSY-PROVEN, LEWY BODY PARKINSON DISEASE AND AUTOPSY-PROVEN CONTROLS. (P1.001)

Objective: To obtain genome-wide significance for the PARK10 locus for autopsy-confirmed Lewy body (LB) Parkinson disease (PD). Background: Previous autopsy studies have found that up to 20% of clinically-diagnosed PD have other/additional neuropathology other than LB-PD on autopsy. This suggests that genetic and pathologic heterogeneity exists that could reduce the power of association analysis in clinical PD. The autopsy confirmed PD genetic consortium (APDGC) was formed, and we recently reported a strong association with PD risk with the previously described PARK10 locus using both autopsy-confirmed (AC)-Lewy body (LB)-PD and AC-controls (Beecham et al, 2015). However, as much larger studies using only clinically-defined cases and controls, have not seen this association, some authors have questioned the validity of the findings of Beecham et al. Design/Methods: DNA on 137 AC-PD cases and 55 AC-controls was obtained from the Parkinson’s UK brain bank. These were genotyped for rs10788972, the SNP demonstrating the strongest previous association. This dataset was analyzed individually for association and then jointly with the previous dataset collected by the Autopsy-confirmed PD Genetics Consortium (APDGC) (Beecham et al, 2015). Results: Using the Parkinson’s UK brain bank samples we demonstrated positive association for rs10788972, with p=0.02. Joint analysis (621 cases, 1200 controls) demonstrated genome-wide association (p = 4.43E-09) with rs10788972. Conclusions: This finding has several important implications for studies in both PD and neurodegeneration. First this confirms PARK10 as a major locus for idiopathic, Lewy-body PD. Second, the reason for the absence of genetic association with PARK10 in clinically-defined cases and controls is unclear. While the most likely mechanism is neuropathic and genetic heterogeneity, it would seem that heterogeneity in BOTH PD cases as well as controls are contributing to this finding. This raises the question whether using clinically identified controls is adding more genetic heterogeneity to current genetic analyses in neurodegenerative studies than previously thought. Study Supported by: NIH/NINDS grant P50NS0171674 (J.Vance P.I.) Disclosure: Dr. Vance has received personal compensation in an editorial capacity for Neurology Genetics. Dr. Vance9s institution has received royalty payments from Athena Diagnostics. Dr. Nuytemans has nothing to disclose. Dr. Gomez has nothing to disclose. Dr. Scott has nothing to disclose. Dr. Beecham has nothing to disclose. Dr. Gveric has nothing to disclose. Dr. Martin has nothing to disclose. Dr. Consortium has nothing to disclose

Phosphorylation and compactness of neurofilaments in multiple sclerosis: Indicators of axonal pathology☆

Aims: Axonal pathology extends to the axonal cytoarchitecture leaving its Signature on axoskeletal proteins. This study investigated whether neurofilament (NfH) phosphorylation would relate to the dynamics of axonal pathology in multiple sclerosis (MS).Methods: NfH phosphoforms (SM132, SM134, SM135) were quantified by ELISA from microdissected samples of control and MS brain and spinal cord. Individual axons were analysed by electron microscopy, densitometrically and morphologically in adjacent tissue sections. Experiments were Carried Out pre- and post enzymatic dephosphorylation.Results: In control tissue a rostro-caudal gradient of NfH indicated an increase in axonal density from the brain gray matter towards the spinal cord. The highest levels of phosphorylated and hyperphosphorylated NfH were found in acute lesions of brain and spinal cord, in contrast to chronic lesions where levels were lower than in white matter, consistent with axonal loss. Dephosphorylated NfH was higher, but less densly packed in MS white matter axons compared to control tissue.Conclusions: The findings suggest that a less organised/compact axoskeleton or impaired axonal transport may represent an early sign of axonal pathology within the normal appearing white matter in MS. Subsequently a proportional increase of dephosphorylated NfH, aberrant phosphorylation and/or aggregation may occur whilst the protein is transported through the white matter towards the MS plaque, where hyperphosphorylated NfH dominates. (c) 2008 Elsevier Inc. All rights reserved

Longitudinally worsening MSIS-29-PHYS scores are associated with reduced survival time.

<p>Four subgroups are presented in this figure: Subgroup 1—initial MSIS-29-PHYS score 20–84, no worsening after 1 year (solid line); Subgroup 2—initial MSIS-29-PHYS score 20–84, worsening after 1 year (short-dashed line); Subgroup 3—initial MSIS-29-PHYS score 85–100, no worsening after 1 year (dot-dashed line); Subgroup 4—initial MSIS-29-PHYS score 85–100, worsening after 1 year (long-dashed line). (A) Table: Subgroups 1 and 2 had no statistically significant difference in survival time (HR = 1.289, 95% CI 0.843–1.972, p = 0.241). Subgroup 4 had reduced survival time compared with subgroup 3 (HR = 2.266, 95% CI 1.163–4.413, p = 0.016). p < 0.001 for differences between the 4 subgroups. (B) Kaplan–Meier failure curves (n = 872). Note that Kaplan–Meier curves do not account for the effect of age and sex on survival time. HR, hazard ratio; MSIS-29, Multiple Sclerosis Impact Scale–29; MSIS-29-PHYS, MSIS-29 physical.</p

Higher MSIS-29-PSYCH scores are associated with reduced survival time.

<p>(A) Table: Higher MSIS-29-PSYCH score was associated with reduced survival time (greater hazard ratio for death), as were older age at first MSIS-29 completion and male sex. (B) Kaplan–Meier failure curves (n = 2,119). Note that Kaplan–Meier curves do not account for the effect of age and sex on survival time. MSIS-29, Multiple Sclerosis Impact Scale–29; MSIS-29-PSYCH, MSIS-29 psychological.</p