BackgroundDemyelination and axonal damage are critical processes in the pathogenesis of multiple sclerosis (MS). Oxidative stress and pro-inflammatory cytokines elicited by inflammation mediates tissue damage.Methods/Principal FindingsTo monitor the demyelination and axonal injury associated with microglia activation we employed a model using cerebellar organotypic cultures stimulated with lipopolysaccharide (LPS). Microglia activated by LPS released pro-inflammatory cytokines (IL-1β, IL-6 and TNFα), and increased the expression of inducible nitric oxide synthase (iNOS) and production of reactive oxygen species (ROS). This activation was associated with demyelination and axonal damage in cerebellar cultures. Axonal damage, as revealed by the presence of non-phosphorylated neurofilaments, mitochondrial accumulation in axonal spheroids, and axonal transection, was associated with stronger iNOS expression and concomitant increases in ROS. Moreover, we analyzed the contribution of pro-inflammatory cytokines and oxidative stress in demyelination and axonal degeneration using the iNOS inhibitor ethyl pyruvate, a free-scavenger and xanthine oxidase inhibitor allopurinol, as well as via blockage of pro-inflammatory cytokines using a Fc-TNFR1 construct. We found that blocking microglia activation with ethyl pyruvate or allopurinol significantly decreased axonal damage, and to a lesser extent, demyelination. Blocking TNFα significantly decreased demyelination but did not prevented axonal damage. Moreover, the most common therapy for MS, interferon-beta, was used as an example of an immunomodulator compound that can be tested in this model. In vitro, interferon-beta treatment decreased oxidative stress (iNOS and ROS levels) and the release of pro-inflammatory cytokines after LPS stimulation, reducing axonal damage.ConclusionThe model of neuroinflammation using cerebellar culture stimulated with endotoxin mimicked myelin and axonal damage mediated by the combination of oxidative stress and pro-inflammatory cytokines. This model may both facilitate understanding of the events involved in neuroinflammation and aid in the development of neuroprotective therapies for the treatment of MS and other neurodegenerative diseases.

Primary progressive multiple sclerosis is a poorly understood disease entity with no specific prognostic biomarkers and scarce therapeutic options. We aimed to identify disease activity biomarkers in multiple sclerosis by performing an RNA sequencing approach in peripheral blood mononuclear cells from a discovery cohort of 44 untreated patients with multiple sclerosis belonging to different clinical forms and activity phases of the disease, and 12 healthy control subjects. A validation cohort of 58 patients with multiple sclerosis and 26 healthy control subjects was included in the study to replicate the RNA sequencing findings. The RNA sequencing revealed an interleukin 1 beta (IL1B) signature in patients with primary progressive multiple sclerosis. Subsequent immunophenotyping pointed to blood monocytes as responsible for the IL1B signature observed in this group of patients. Functional experiments at baseline measuring apoptosis-associated speck-like protein containing a CARD (ASC) speck formation showed that the NOD-leucine rich repeat and pyrin containing protein 3 (NLRP3) inflammasome was overactive in monocytes from patients with primary progressive multiple sclerosis, and canonical NLRP3 inflammasome activation with a combination of ATP plus lipopolysaccharide was associated with increased IL1B production in this group of patients. Primary progressive multiple sclerosis patients with high IL1B gene expression levels in peripheral blood mononuclear cells progressed significantly faster compared to patients with low IL1B levels based on the time to reach an EDSS of 6.0 and the Multiple Sclerosis Severity Score. In agreement with peripheral blood findings, both NLRP3 and IL1B expression in brain tissue from patients with primary progressive multiple sclerosis was mainly restricted to cells of myeloid lineage. Treatment of mice with a specific NLRP3 inflammasome inhibitor attenuated established experimental autoimmune encephalomyelitis disease severity and improved CNS histopathology. NLRP3 inflammasome-specific inhibition was also effective in reducing axonal damage in a model of lipopolysaccharide-neuroinflammation using organotypic cerebellar cultures. Altogether, these results point to a role of IL1B and the NLRP3 inflammasome as prognostic biomarker and potential therapeutic target, respectively, in patients with primary progressive multiple sclerosis.

Cytosolic heat shock proteins have received significant attention as emerging therapeutic targets. Much of this excitement has been triggered by the discovery that HSP90 plays a central role in the maintenance and stability of multifarious oncogenic membrane receptors and their resultant tyrosine kinase activity. Numerous studies have dealt with the effects of small molecules on chaperone-and stress-related pathways of the endoplasmic reticulum (ER). However, unlike cytosolic chaperones, relatively little emphasis has been placed upon translational avenues towards targeting of the ER for inhibition of folding/secretion of disease-promoting proteins. Here, we summarise existing small molecule inhibitors and potential future targets of ER chaperone-mediated inhibition. Client proteins of translational relevance in disease treatment are outlined, alongside putative future disease treatment modalities based on ER-centric targeted therapies. Particular attention is paid to cancer and autoimmune disorders via the effects of the GRP94 inhibitor geldanamycin and its population of client proteins, overloading of the unfolded protein response, and inhibition of members of the IL-12 family of cytokines by celecoxib and non-coxib analogues. AbbreviationsBAP, BiP-associated protein; CNX, calnexin; CRT, calreticulin; CST, castanospermine; dNJ, deoxynojirimycin; ERAD, endoplasmic reticulum associated degradation; ERdj, endoplasmic reticulum DNAJ-like; ERQC, endoplasmic reticulum quality control; GRP, glucose regulated protein; HSP, heat shock protein; IGF, insulin growth factor; IL, interleukin; PDI, protein disulphide isomerase; TFM-C, trifluoromethyl-celecoxib; TLR, toll-like receptor; UDP, uridine diphosphate; UPR, unfolded protein response Introduction to the ERThe ER is home to an array of interlinked chaperone proteins upon which correct folding, partner chain assimilation and final multimer assembly of secreted proteins depend. This can be broken down into a number of semi-distinct functional systems. The lectin-binding chaperone system, consisting of calreticulin (CRT) and the membrane-bound homologue calnexin (CNX) operate in tandem with the N-glycan processing enzymes glucosidase I, glucosidase II and quality control checkpoint uridine diphosphate (UDP)-glucose glycoprotein glucosyltransferase (UGGT), to facilitate glycoprotein folding (Moremen and Molinari, 2006). The ER is also home to a multichaperone 'glucose regulated protein (GRP)' complex homologous to the cytoplasmic heat shock protein (HSP) complex of HSP90/HSP70. This ER complex centres on the HSP70 homologue GRP78 (Hendershot, 2004) and the HSP90 homologue GRP94 (Argon and Simen, 1999), but has been found to associate with a collection of ER DNAJ like (ERdj) HSP40 like co-chaperones (Shen and Hendershot, 2005;Dong et al., 2008) and peptidylpropylisomerases (Meunier et al., 2002) of similar ilk to those present in the HSP90/HSP70 complex, as well as with the two GRP78 nucleotide exchange factors BiP-associated protein (BAP) and GRP170 (Weitzmann et al., 20...