AimsAmyotrophic lateral sclerosis (ALS) is characterized by degeneration of motoneurons and progressive muscle wasting. Inflammatory processes, mediated by non‐neuronal cells, such as glial cells, are known to contribute to disease progression. Inflammasomes consist of pattern recognition receptors (PRRs), apoptosis‐associated speck‐like protein (ASC) and caspase 1 and are essential for interleukin (IL) processing and a rapid immune response after tissue damage. Recently, we described inflammasome activation in the spinal cord of ALS patients and in SOD1(G93A) ALS mice. Since pathological changes in the skeletal muscle are early events in ALS, we hypothesized that PRRs might be abnormally expressed in muscle fibre degeneration.MethodsWestern blot analysis, real‐time PCR and immunohistochemistry were performed with muscle tissue from presymptomatic and early‐symptomatic male SOD1(G93A) mice and with muscle biopsies of control and sporadic ALS (sALS) patients. Analysed PRRs include nucleotide‐binding oligomerization domain‐like (NOD‐like) receptor protein 1 (NLRP1), NLR protein 3 (NLRP3), NLR family CARD domain‐containing 4 (NLRC4) and absent in melanoma 2. Additionally, expression levels of ASC, caspase 1, interleukin 1 beta (IL1β) and interleukin 18 (IL18) were evaluated.ResultsExpression of PRRs and ASC was detected in murine and human tissue. The PRR NLRC4, caspase 1 and IL1β were significantly elevated in denervated muscle of SOD1(G93A) mice and sALS patients. Furthermore, levels of caspase 1 and IL1β were already increased in presymptomatic animals.ConclusionOur findings suggest that increased inflammasome activation may be involved in skeletal muscle pathology in ALS. Furthermore, elevated levels of NLRC4, caspase 1 and IL1β reflect early changes in the skeletal muscle and may contribute to the denervation process.

Alpha-motoneurons and muscle fibres are structurally and functionally interdependent. Both cell types particularly rely on endoplasmic reticulum (ER/SR) functions. Mutations of the ER proteins VAPB, SigR1 and HSP27 lead to hereditary motor neuron diseases (MNDs). Here, we determined the expression profile and localization of these ER proteins/chaperons by immunohistochemistry and immunoblotting in biopsy and autopsy muscle tissue of patients with amyotrophic lateral sclerosis (ALS) and other neurogenic muscular atrophies (NMAs) and compared these patterns to mouse models of neurogenic muscular atrophy. Postsynaptic neuromuscular junction staining for VAPB was intense in normal human and mouse muscle and decreased in denervated Nmd(2J) mouse muscle fibres. In contrast, VAPB levels together with other chaperones and autophagy markers were increased in extrasynaptic regions of denervated muscle fibres of patients with MNDs and other NMAs, especially at sites of focal myofibrillar disintegration (targets). These findings did not differ between NMAs due to ALS and other causes. G93A-SOD1 mouse muscle fibres showed a similar pattern of protein level increases in denervated muscle fibres. In addition, they showed globular VAPB-immunoreactive structures together with misfolded SOD1 protein accumulations, suggesting a primary myopathic change. Our findings indicate that altered expression and localization of these ER proteins and autophagy markers are part of the dynamic response of muscle fibres to denervation. The ER is particularly prominent and vulnerable in both muscle fibres and alpha-motoneurons. Thus, ER pathology could contribute to the selective build-up of degenerative changes in the neuromuscular axis in MNDs.

Oxidative stress critically contributes to the pathogenesis of a variety of neurodegenerative diseases such as multiple sclerosis. Astrocytes are the main regulators of oxidative homeostasis in the brain and dysregulation of these cells likely contributes to the accumulation of oxidative damage. The nuclear factor erythroid 2-related factor 2 (Nrf2) is the main transcriptional regulator of the anti-oxidant stress defense. In this study, we elucidate the effects of astrocytic Nrf2-activation on brain-intrinsic inflammation and lesion development. Cells deficient for the Nrf2 repressor kelch-like ECH-associated protein 1 (Keap1) are characterized by hyperactivation of Nrf2-signaling. Therefore, wild type mice and mice with a GFAP-specific Keap1-deletion were fed with 0.25% cuprizone for 1 or 3 weeks. Cuprizone intoxication induced pronounced oligodendrocyte loss, demyelination and reactive gliosis in wild type animals. In contrast, astrocyte-specific Nrf2-activation was sufficient to prevent oligodendrocyte loss and demyelination, to ameliorate brain intrinsic inflammation and to counteract axonal damage. Our results highlight the potential of the Nrf2/ARE system for the treatment of neuroinflammation in general and of multiple sclerosis in particular. © GLIA 2016;64:2219–2230

BACKGROUND AND PURPOSE:Modulation of the sphingosine 1-phosphate receptor is an approved treatment for relapsing multiple sclerosis because of its anti-inflammatory effect of retaining lymphocytes within the lymph nodes. Here, we evaluated the potential of an agonist at this receptor, FTY720 (fingolimod), to activate the promyelinating pathways within the brain to encourage remyelination and neuroprotection.EXPERIMENTAL APPROACH:In this study, we used the cuprizone model in male C57BL/6 mice and tested the promyelinating and neuroprotective effects of FTY720 after acute and chronic toxin-induced experimental demyelination. We used histological, immunohistochemical and gene expression methods.KEY RESULTS:The midline of the corpus callosum was severely demyelinated after acute and chronic cuprizone-induced demyelination. Robust endogenous remyelination was evident after acute, but impaired after chronic, demyelination. FTY720 treatment modestly accelerated myelin recovery after acute but not chronic cuprizone exposure. Markers of gliosis (astrocyte and microglia activation) were not affected by FTY720 treatment. Remarkably, the accumulation of amyloid precursor protein-positive spheroids in axons was less distinct in FTY720-treated animals, indicating that this compound alleviated ongoing axonal damage.CONCLUSIONS AND IMPLICATIONS:We show that even during endogenous remyelination, axonal degeneration continued at a low level, accumulating over time. This continuous neurodegenerative process was ameliorated by FTY720 treatment. FTY720 preserved CNS integrity by direct interaction with brain resident cells, the actions of which are still to be defined.

In multiple sclerosis (MS), activated microglia and macrophages have been linked to axonal damage that underlies neurodegeneration and progressive disease. This is exemplified by the finding that cortical grey matter (GM) lesions are characterised by less pronounced microglia activation and lymphocyte infiltration compared to white matter (WM) lesions. Such differences are clearly highlighted by leukocortical lesions that extend across the WM and GM offering an opportunity to examine differences in WM and GM microglia activation in one lesion. So far, dissection of microglia responses in MS has been hampered by an insufficient understanding of factors that trigger activation. Here we have examined leukocortical lesions in people with MS with respect to microglia activation, degree of axonal damage and phagocytosis of neuronal debris. To examine factors that may underlie the differential activation of microglia in the WM and GM in vitro studies were performed with mouse microglia. Our studies reveal a clear difference in the degree of microglia activation in the WM and GM of leukocortical lesions that correlated with the extent of axonal damage. Phagocytosis of neuronal debris was more frequent in the WM compared to the GM, but in both regions phagocytosis was associated with axonal damage. Morphometric analysis and gene array studies of mouse microglia showed significant differences between WM and GM-derived microglia in mice. In particular, expression of genes within the Toll-like receptor pathway as well as those associated with antigen processing and presentation pathways were significantly enriched in microglia derived from the WM. On the contrary, direct application of neuronal debris into the corpus callosum or cortex of mice induced profound and comparable inflammation in both regions. In summary, our data show that microglia activation is reduced in the GM region compared to the WM region of leukocortical MS lesions despite the degree of axonal damage. That we also show a strong differential expression of immune-related genes in WM and GM-derived microglia in mice may explain the differences in microglia activation in the WM and GM in leukocortical lesion in MS. Introduction Multiple sclerosis (MS) is a common disease of the central nervous system (CNS) of which demyelination and neurodegeneration are pathological hallmarks. Most research on MS has been performed on the demyelinating part of the disease notwithstanding that the strong neurodegenerative component contributes to the neurological disabilities seen in patients (1). Despite the correlation between inflammation and axonal damage in white matter (WM) MS lesions, the mechanisms contributing to neurodegeneration in MS are largely unknown (2-5). Proposed neurodegenerative mechanisms include chronic demyelination (2, 6), macrophage derived reactive oxygen and nitrogen species (7) and mitochondrial dysfunction (8). In addition, it is suggested that demyelinated axons are more vulnerable to the inflammatory microenvironment containing proteolytic enzymes, oxidative products, cytokines and free radicals, and thereby more susceptible to damage (2, 9). Neurodegeneration could also develop through autoreactivity to neuroaxonal antigens and subsequently targeting of axons, which has been shown in several other neurological disorders (9). Evidence for similar mechanisms in MS comes from antibodies and T cells direct to neuroaxonal antigens as well as axon-reactive B cells in the cerebrospinal fluid (CSF) in people with MS (10-14). It is shown that direct damage to axons, indicated by axonal end bulbs and myelin sheaths devoid of axons, occurs after immunisation with the neuronal cytoskeletal protein 3

Intracellular accumulations of mutant, misfolded proteins are major pathological hallmarks of amyotrophic lateral sclerosis (ALS) and related disorders. Recently, mutations in Sigma receptor 1 (SigR1) have been found to cause a form of ALS and frontotemporal lobar degeneration (FTLD). Our goal was to pinpoint alterations and modifications of SigR1 in ALS and to determine how these changes contribute to the pathogenesis of ALS. In the present study, we found that levels of the SigR1 protein were reduced in lumbar ALS patient spinal cord. SigR1 was abnormally accumulated in enlarged C-terminals and endoplasmic reticulum (ER) structures of alpha motor neurons. These accumulations co-localized with the 20s proteasome subunit. SigR1 accumulations were also observed in SOD1 transgenic mice, cultured ALS-8 patient's fibroblasts with the P56S-VAPB mutation and in neuronal cell culture models. Along with the accumulation of SigR1 and several other proteins involved in protein quality control, severe disturbances in the unfolded protein response and impairment of protein degradation pathways were detected in the above-mentioned cell culture systems. Furthermore, shRNA knockdown of SigR1 lead to deranged calcium signaling and caused abnormalities in ER and Golgi structures in cultured NSC-34 cells. Finally, pharmacological activation of SigR1 induced the clearance of mutant protein aggregates in these cells. Our results support the notion that SigR1 is abnormally modified and contributes to the pathogenesis of ALS.

Objectives: Mitochondria play a crucial role in cell function and survival and as such in preconditioning prior to a hypoxic insult.

The cuprizone model is a suitable animal model of de- and remyelination secondary to toxin-induced oligodendrogliopathy. From a pharmaceutical point of view, the cuprizone model is a valuable tool to study the potency of compounds which interfere with toxin-induced oligodendrocyte cell death or boost/inhibit remyelinating pathways and processes. The aim of this study was to analyze the vulnerability of neighboring white mater tracts (i.e., the fornix and cingulum) next to the midline of the corpus callosum which is the region of interest of most studies using this model. Male mice were fed cuprizone for various time periods. Different white matter areas were analyzed for myelin (anti-PLP), microglia (anti-IBA1), and astrocyte (anti-GFAP) responses by means of immunohistochemistry. Furthermore, Luxol fast blue–periodic acid Schiff stains were performed to validate loss of myelin-reactive fibers in the different regions. Cuprizone induced profound demyelination of the midline of the corpus callosum and medial parts of the cingulum that was paralleled by a significant astrocyte and microglia response. In contrast, lateral parts of the corpus callosum and the cingulum, as well as the fornix region which is just beneath the midline of the corpus callosum appeared to be resistant to cuprizone exposure. Furthermore, resistant areas displayed reduced astrogliosis and microgliosis. This study clearly demonstrates that neighboring white matter tracts display distinct vulnerability to toxin-induced demyelination. This important finding has direct relevance for evaluation strategies in this frequently used animal model for multiple sclerosis.

Brain inflammation plays a central role in multiple sclerosis (MS). Besides lymphocytes, the astroglia and microglia mainly contribute to the cellular composition of the inflammatory infiltrate in MS lesions. Several studies were able to demonstrate that cortical lesions are characterized by lower levels of inflammatory cells among activated microglia/macrophages. The underlying mechanisms for this difference, however, remain to be clarified. In the current study, we compared the kinetics and extent of microglia and astrocyte activation during early and late cuprizone-induced demyelination in the white matter tract corpus callosum and the telencephalic gray matter. Cellular parameters were related to the expression profiles of the chemokines Ccl2 and Ccl3. We are clearly able to demonstrate that both regions are characterized by early oligodendrocyte stress/apoptosis with concomitant microglia activation and delayed astrocytosis. The extent of microgliosis/astrocytosis appeared to be greater in the subcortical white matter tract corpus callosum compared to the gray matter cortex region. The same holds true for the expression of the key chemokines Ccl2 and Ccl3. The current study defines a model to study early microglia activation and to investigate differences in the neuroinflammatory response of white vs. gray matter.