FTY720 attenuates excitotoxicity and neuroinflammation

Background: FTY720 (fingolimod, Gilenya(TM)), a structural analog of sphingosine-1-phosphate (S1P), is the first oral drug approved for treatment the relapsing-remitting form of multiple sclerosis (MS), and its efficacy has been related to induced lymphopenia and consequent immunosuppression via modulation of S1P(1) receptors (S1P(1)R). However, due to its lipophilic nature, FTY720 crosses the blood brain barrier (BBB) and could act directly on neural cells. In this study, we investigated the effectiveness of FTY720 as a neuroprotective agent using in vitro and in vivo models of excitotoxic neuronal death and examined if FTY720 exerts a direct action on neurons, or/and an indirect modulation of inflammation-mediated neurodegeneration as a possible mechanism of neuroprotection. Methods: Primary neuronal and organotypic cortical cultures were treated with N-methyl-D-aspartic acid (NMDA) to induce excitotoxic cell death (measured by lactate dehydrogenase (LDH) assay or propidium iodide uptake, respectively). The effects of FTY720 treatment (10, 100 and 1,000 nM) on neuronal survival were examined. As an in vivo model of neuronal death and inflammation, we used intracerebroventricular (icv) administration of kainic acid (KA; 0.5 mu g/2 mu l) in Sprague-Dawley rats. FTY720 was applied icv (1 mu g/2 mu l), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 3 days after icv. Rats were evaluated for neurological score, neuronal loss in CA3 hippocampal region and activation of microglia at the lesion site. In addition, we tested FTY720 as a modulator of microglia responses using microglial cell cultures activated with lipopolysaccharide (LPS) and its effects in stress signalling pathways using western blotting for p38 and JNK1/2 mitogen-activated protein kinases (MAPKs). Results: FTY720 was able to reduce excitotoxic neuronal death in vitro. Moreover, in vivo repeated FTY720 administration attenuated KA-induced neurodegeneration and microgliosis at the CA3 lesion site. Furthermore, FTY720 negatively modulates p38 MAPK in LPS-activated microglia, whereas it had no effect on JNK1/2 activation. Conclusions: These data support a role for FTY720 as a neuroprotective agent against excitotoxin-induced neuronal death and as a negative modulator of neuroinflammation by targeting the p38 MAPK stress signalling pathway in microglia.This study was funded by Novartis Farmaceutica SA, Gobierno Vasco and CIBERNED

Effects of FTY720 on brain neurogenic niches in vitro and after kainic acid-induced injury

Background: FTY720 (fingolimod, Gilenya (TM)) is an oral, blood-brain barrier (BBB)-passing drug approved as immunomodulatory treatment for relapsing-remitting form of the multiple sclerosis (MS). In addition, FTY720 exerts several effects in the central nervous system (CNS), ranging from neuroprotection to reduction of neuroinflammation. However, the neurogenic and oligodendrogenic potential of FTY720 has been poorly investigated. In this study, we assessed the effect of FTY720 on the production of new neurons and oligodendrocytes from neural stem/precursor cells both in vitro and in vivo. Methods: Neural stem cells (NSCs) derived from the young rat subventricular zone (SVZ) were exposed to FTY720 (10, 100 nM), and their differentiation into neurons and oligodendrocytes was measured using immunofluorescence for anti-beta-III tubulin or CNPase (2',3'-cyclic nucleotide 3'-phosphodiesterase) as markers of mature neurons or oligodendrocytes, respectively. In addition, intracerebroventricular (icv) administration of kainic acid (KA; 0.5 mu g/2 mu l) in Sprague-Dawley rats was used as an in vivo model of neuronal death and inflammation. FTY720 was applied icv (1 mu g/2 mu l), together with KA, plus intraperitoneally (ip; 1 mg/kg) 24 h before, and daily, until sacrifice 8 days after KA injection. To visualize cell proliferation in the hippocampus and in white matter regions, rats were administered 5-bromo-2-deoxyuridine (BrdU) 100 mg/kg, ip injected every 2 days. Immunohistochemical analyses were performed on rat brain slices to measure the production of new neuronal precursors (doublecortin/DCX+ cells) and new oligodendrocytes precursors (proteoglycan/NG2(+) cells). Results: In this study, we observed that FTY720 increased postnatal NSCs differentiation into both neurons and oligodendrocytes in vitro. In turn, in adult animals, FTY720 enhanced the percentage of BrdU(+) cells coexpressing DCX marker, both in basal (FTY720 alone) and in neurodegenerative (FTY720 + KA) conditions. However, FTY720 had only a partial effect on proliferation and differentiation of oligodendrocyte progenitor cell (OPC) population in vivo. Conclusions: FTY720 promotes neurogenesis and oligodendrogenesis in vitro under basal conditions. In addition, it increases the generation of neuroblasts and oligodendrocytes after excitotoxic brain injury. This suggests that FTY720 has the potential to activate the neurogenic niche and thus favour tissue repair after lesion.This study was supported by the Novartis Farmaceutica SA, Ministerio de Economia y Competitividad (MINECO) and Centro de Investigacion Biomedica en Red sobre Enfermedades Neurodegenerativas (CIBERNED). RC was funded by Novartis Farmaceutica SA

Gene Expression Analysis of Astrocyte and Microglia Endocannabinoid Signaling during Autoimmune Demyelination

The endocannabinoid system is associated with protective effects in multiple sclerosis (MS) that involve attenuated innate immune cell responses. Astrocytes and microglia are modulated by endocannabinoids and participate in the biosynthesis and metabolism of these compounds. However, the role of neuroglial cells as targets and mediators of endocannabinoid signaling in MS is poorly understood. Here we used a microfluidic RT-qPCR screen to assess changes in the expression of the main endocannabinoid signaling genes in astrocytes and microglia purified from female mice during the time-course of experimental autoimmune encephalomyelitis (EAE). We show that astrocytes and microglia upregulate the expression of genes encoding neurotoxic A1 and pro-inflammatory molecules at the acute disease with many of these transcripts remaining elevated during the recovery phase. Both cell populations exhibited an early onset decrease in the gene expression levels of 2-arachidonoylglycerol (2-AG) hydrolytic enzymes that persisted during EAE progression as well as cell-type-specific changes in the transcript levels for genes encoding cannabinoid receptors and molecules involved in anandamide (AEA) signaling. Our results demonstrate that astrocytes and microglia responses to autoimmune demyelination involve alterations in the expression of multiple endocannabinoid signaling-associated genes and suggest that this system may regulate the induction of neurotoxic and pro-inflammatory transcriptional programs in both cell types during MS.This research was funded by FEDER and ISCIII (AES 2018—PI18/00513 to S.M. and A.R.-A.), the Basque Government (PIBA19-0059 to S.M. and IT1203-19 to C.M.), the Spanish Ministry of Economy and Competitiveness (SAF2016-75292-R to C.M.) and CIBERNED (CB06/0005/0076 to C.M.). A.M.-G. and A.B.-C. held fellowships from the UPV/EHU and the Basque Government, respectively

Assessing care-related regret among nurses specialized in multiple sclerosis: A psychometric analysis of a new assessment battery

Experiences of regret associated with caring for patients with multiple sclerosis (MS) can affect medical decisions. A non-interventional study was conducted to assess the dimensionality and item characteristics of a battery including the Regret Intensity Scale (RIS-10) and 15 items evaluating common situations experienced by nurses in MS care. A total of 97 nurses were included. The RIS-10 showed good internal reliability and a unidimensional structure according to Mokken analysis. All-item homogeneity coefficients exceeded 0.30, whereas scalability for the overall RIS-10 was 0.66, indicating a strong scale. This battery showed adequate psychometric properties to evaluate regret among MS nurseThe author(s) disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This study was funded by the Medical Department of Roche Farma Spain (SL42129)

Quality of Life in an Adult Population of More than 60 Years of Age without Cognitive Impairment

Objectives: To analyse the quality of life (QoL) of persons over 60 years of age in Bizkaia (Basque Country, Spain), not institutionalised and without cognitive impairment, and the relation and possible influence of the perception of the state of health with the cognitive level and the activities that they carry out. Subjects and Methods: Evaluation of a sample of 317 persons that belong to a longitudinal study. In 269 cases, there was a reliable informant. The general cognitive state was evaluated by means of the Mini-Mental State Examination (MMSE) and the Global Deterioration Scale (GDS). Two GDS, one of the family member (GDSf) and another of the evaluator (GDSe), were used and memory was evaluated by the Text Memory subtest of the Barcelona Test. The QoL was assessed with the SF-36 Health Survey and the mental, physical and social activities that they carry out were recorded. Results: In the SF-36, the participants showed values that exceed the reference values of the Spanish population in all dimensions of the SF-36, except in that of bodily pain. There were low correlations between the dimensions of the SF-36 and the mental, physical and social activities. A low correlation was observed between the memory tests and the SF-36. In 59.3% of the cases, there were no differences between the GDSf and the GDSe. Conclusions: The participants of this study present a better perception of their state of health than the Spanish population, but this perception is not observed to be influenced by the type or by the quantity of activities that they carry out or by their cognitive state. The informants present a vision very close to the cognitive state of their family members

An Overground Robotic Gait Training Program for People With Multiple Sclerosis: A Protocol for a Randomized Clinical Trial

Maintaining the ability to walk is one of the significant challenges in people with multiple sclerosis (MS) for keeping a good quality of life as the disease and the aging process progresses. Overground robotic (OR) wearable exoskeletons are promising tools for gait rehabilitation, but currently there is no evidence of their clinical effects on patients with MS. The present study aims to determine the effects of an OR intervention in people with MS and moderate to severe walking disabilities and ascertain if benefits are maintained over a follow-up period of 3 months. This randomized controlled trial will include 36 participants with MS. Inclusion criteria are: older than 18 years, definitive diagnosis of MS, 4.5-7 points on the EDSS (Expanded Disability Status Scale), and needing one or two canes or crutches for walking outdoors. Subjects in the control group will receive conventional physiotherapy sessions at ADEMBI (Asociacion de Esclerosis Multiple de Bizkaia) provided to control spasticity, maintain articular range and exercise balance. Subjects in the intervention group will receive the same physiotherapy but also participate in a progressive OR gait training program assisted by the EksoTM exoskeleton. The program consists of twice a week individually supervised sessions in two setting modalities: PreGait and ProStepPlus. The training parameters (duration, speed, cadence, length of steps) will be set during the first session and the progression and intensity of the intervention will be adapted to the tolerance of each participant. The primary outcome of this study is gait speed. Secondary outcomes will include physical and cognitive performance tests, clinical, fatigue and quality of life assessments, and changes in the plasma levels of inflammatory cytokines. The present trial is the first analyzing the effectiveness of an OR intervention for gait training in patients with MS. It will help clarify the applicability of robotic technologies to clinical practice, extending the functionality and quality of life of people with MS to face a successful aging process. (ACTRN12619000014156; https://anzctr.org.au/Trial/Registration/TrialReview.aspx?id=376548).This research was partially supported by Bilbao Bizkaia Kutxa Bank Foundation, Euskampus Fundazioa Foundation and by the Ministry of Science of Spain under Grant No. DPI2017-82694-R (AEI/FEDER, UE)

Dangerous liaisons: the ecology of private interest and common good

Multiple sclerosis (MS) is an inflammatory disease of the central nervous system characterized by myelin loss and neuronal dysfunction. Although the majority of patients do not present familial aggregation, Mendelian forms have been described. We performed whole-exome sequencing analysis in 132 patients from 34 multi-incident families, which nominated likely pathogenic variants for MS in 12 genes of the innate immune system that regulate the transcription and activation of inflammatory mediators. Rare missense or nonsense variants were identified in genes of the fibrinolysis and complement pathways (PLAU, MASP1, C2), inflammasome assembly (NLRP12), Wnt signaling (UBR2, CTNNA3, NFATC2, RNF213), nuclear receptor complexes (NCOA3), and cation channels and exchangers (KCNG4, SLC24A6, SLC8B1). These genes suggest a disruption of interconnected immunological and pro-inflammatory pathways as the initial event in the pathophysiology of familial MS, and provide the molecular and biological rationale for the chronic inflammation, demyelination and neurodegeneration observed in MS patients. Author summary Although the majority of patients diagnosed with multiple sclerosis do not have a family history of disease, 13% report having a close relative also diagnosed with multiple sclerosis. In these families, the cause of multiple sclerosis can be largely attributed to a single genetic variant that is transmitted through generations. In this study we analyzed DNA from 132 patients from 34 families, resulting in the identification of 12 rare genetic variants that are largely responsible for the onset of multiple sclerosis in these families. These variants are located in genes implicated in specific immunological pathways, and suggest the biological mechanisms that trigger the onset of multiple sclerosis. These genes and variants provide the means for the generation of cellular and animal models of human disease, and highlight biological targets for the development of novel treatments.This research was undertaken thanks to funding from the Canada Research Chair program (950-228408), Michael Smith Foundation for Health Research (16827), the Canadian Institutes of Health Research (MOP-137051), the Vancouver Coastal Health Research Institute, the Milan & Maureen Ilich Foundation (11-32095000), and the Vancouver Foundation (ADV14-1597) to CVG. Additional funds were provided by "Red Espanola de Esclerosis Multiple (REEM)" (grant to KV was RD12/0032/0013; RETICS, ISCIII), Project FIS PI13/0879 Grant RETICS-REEM RD07/0060/0019; Ministerio de Economia y Competitividad-FEDER SAF2016-80595-C2-1-P to AA and FM, Junta de Andalucia-FEDER to FM, and the Ricerca Finalizzata of the Italian Ministry of Health (RF-201102350347). EU, LL, LEP, and PUR are members of the Spanish Network of Multiple Sclerosis REEM RD16/0015/0010, supported by Institute of Health "Carlos III" of the Ministry of Economy and Competitiveness (grants cofunded by European Regional Development Fund). LL holds a Nicolas Monardes contract (C-0014-2015) from the Andalusian Health Ministry. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript