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

Astrocytic Atrophy as a Pathological Feature of Parkinson's Disease with LRRK2 Mutation

The principal hallmark of Parkinson's disease (PD) is the selective neurodegeneration of dopaminergic neurones. Mounting evidence suggests that astrocytes may contribute to dopaminergic neurodegeneration through decreased homoeostatic support and deficient neuroprotection. In this study, we generated induced pluripotent stem cells (iPSC)-derived astrocytes from PD patients with LRRK2(G2019S) mutation and healthy donors of the similar age. In cell lines derived from PD patients, astrocytes were characterised by a significant decrease in S100B and GFAP-positive astrocytic profiles associated with marked decrease in astrocyte complexity. In addition, PD-derived astrocytes demonstrated aberrant mitochondrial morphology, decreased mitochondrial activity and ATP production along with an increase of glycolysis and increased production of reactive oxygen species. Taken together, our data indicate that astrocytic asthenia observed in patient-derived cultures with LRRK2(G2019S) mutation may contribute to neuronal death through decreased homoeostatic support, elevated oxidative stress and failed neuroprotectionThis work was supported by BIOEF (BIO17/ND/008 to FC), Euskampus, CIBERNED (CB06/0005/0076 to C.M.), the Ministry of Economy and Competitiveness, Government of Spain (SAF2016-75292-R to C.M. and PID2019-109724RB-I00 to C.M.), FEDER and ISCIII (AES 2018-PI18/00513 to S.M.) and the Basque Government (IT1203-19 to C.M.; PIBA19-0059 to S.M.). P.R.G. was supported by a fellowship from the Basque Governmen

Clemastine Induces an Impairment in Developmental Myelination

[EN] Abnormalities in myelination are associated to behavioral and cognitive dysfunction in neurodevelopmental psychiatric disorders. Thus, therapies to promote or accelerate myelination could potentially ameliorate symptoms in autism. Clemastine, a histamine H1 antagonist with anticholinergic properties against muscarinic M1 receptor, is the most promising drug with promyelinating properties. Clemastine penetrates the blood brain barrier efficiently and promotes remyelination in different animal models of neurodegeneration including multiple sclerosis, ischemia and Alzheimer's disease. However, its role in myelination during development is unknown. We showed that clemastine treatment during development increased oligodendrocyte differentiation in both white and gray matter. However, despite the increase in the number of oligodendrocytes, conduction velocity of myelinated fibers of corpus callosum decreased in clemastine treated mice. Confocal and electron microscopy showed a reduction in the number of myelinated axons and nodes of Ranvier and a reduction of myelin thickness in corpus callosum. To understand the mechanisms leading to myelin formation impairment in the presence of an excess of myelinating oligodendrocytes, we focused on microglial cells that also express muscarinic M1 receptors. Importantly, the population of CD11c(+) microglia cells, necessary for myelination, as well as the levels of insulin growth factor-1 decrease in clemastine-treated mice. Altogether, these data suggest that clemastine impact on myelin development is more complex than previously thought and could be dependent on microglia-oligodendrocyte crosstalk. Further studies are needed to clarify the role of microglia cells on developmental myelination.This work was supported by Spanish Ministry of Education and Science (SAF 2016-75292- R); Spanish Ministry of Science andInnovation (PID 2019-109724RB-I00); Basque Government (PI-2016-1-0016); the University of the Basque Country (UPV/EHU);and Centro de Investigacion Biomedica en Red, EnfermedadesNeurodegenerativas (CIBERNED; grant CB06/05/0076). AP hasa predoctoral fellowship from the University of the Basque Country (UPV/EHU), AM has a predoctoral fellowship fromthe Spanish Ministry of Education and Science and AO-de-A hasa postdoctoral fellowship from the Basque Governmen

Genetically modified macrophages accelerate myelin repair

[EN] Preventing neurodegeneration-associated disability progression in patients with multiple sclerosis (MS) remains an unmet therapeutic need. As remyelination prevents axonal degeneration, promoting this process in patients might enhance neuroprotection. In demyelinating mouse lesions, local overexpression of semaphorin 3F (Sema3F), an oligodendrocyte progenitor cell (OPC) attractant, increases remyelination. However, molecular targeting to MS lesions is a challenge. A clinically relevant paradigm for delivering Sema3F to demyelinating lesions could be to use blood-derived macrophages as vehicles. Thus, we chose transplantation of genetically modified hematopoietic stem cells (HSCs) as means of obtaining chimeric mice with circulating Sema3F-overexpressing monocytes. We demonstrated that Sema3F-transduced HSCs stimulate OPC migration in a neuropilin 2 (Nrp2, Sema3F receptor)-dependent fashion, which was conserved in middle-aged OPCs. While demyelinating lesions induced in mice with Sema3F-expressing blood cells showed no changes in inflammation and OPC survival, OPC recruitment was enhanced which accelerated the onset of remyelination. Our results provide a proof of concept that blood cells, particularly monocytes/macrophages, can be used to deliver pro-remyelinating agents "at the right time and place," suggesting novel means for remyelination-promoting strategies in MS.This work was supported by the French National Institute of Health and Medical Research (INSERM), French National Research Agency (ANR, project Stemimus ANR-12-BSV4-0002-02), the European Leukodystrophy Association (ELA, project 2016-004C5B), NeurATRIS, the program "Investissements d'avenir" (ANR-10-IAIHU-06), CIBERNED (CB06/0005/0076), and Gobierno Vasco (IT1203-19). VT was a recipient of the Spanish Ministry of Economy Young Investigator Grant (SAF2015-74332-JIN)

Cannabinoid CB1 receptor gene inactivation in oligodendrocyte precursors disrupts oligodendrogenesis and myelination in mice

[EN] Cannabinoids are known to modulate oligodendrogenesis and developmental CNS myelination. However, the cell-autonomous action of these compounds on oligodendroglial cells in vivo, and the molecular mechanisms underlying these effects have not yet been studied. Here, by using oligodendroglial precursor cell (OPC)-targeted genetic mouse models, we show that cannabinoid CB1 receptors exert an essential role in modulating OPC differentiation at the critical periods of postnatal myelination. We found that selective genetic inactivation of CB1 receptors in OPCs in vivo perturbs oligodendrogenesis and postnatal myelination by altering the RhoA/ROCK signaling pathway, leading to hypomyelination, and motor and cognitive alterations in young adult mice. Conversely, pharmacological CB1 receptor activation, by inducing E3 ubiquitin ligase-dependent RhoA proteasomal degradation, promotes oligodendrocyte development and CNS myelination in OPCs, an effect that was not evident in OPC-specific CB1 receptor-deficient mice. Moreover, pharmacological inactivation of ROCK in vivo overcomes the defects in oligodendrogenesis and CNS myelination, and behavioral alterations found in OPC-specific CB1 receptor-deficient mice. Overall, this study supports a cell-autonomous role for CB1 receptors in modulating oligodendrogenesis in vivo, which may have a profound impact on the scientific knowledge and therapeutic manipulation of CNS myelination by cannabinoids.This work was supported by the MINECO grants SAF2017-83516 and PID2020-112640RB-I00, and the Comunidad de Madrid grants 2016-T1/BMD-1060 and 2020-5 A/BMD-19728, Atraccion del Talento Investigador Program, to JP. AHG and TA. AHG was also supported by the Comunidad de Madrid contract PEJD-2017PRE/BMD-3703, and A.S.T by Fundacion Tatiana Perez de Guzman el Bueno. Support was also provided by MINECO (grants RTI2018-095311-B-I00 to MG. and SAF2016-75292-R to CM), CIBERNED (grants CB06/05/0005 to M.G. and CB06/0005/0076 to C.M.), FEDER and ISCIII (AES 2018 grants PI18-00941 to IG-R and PI18/00513 to SM), Basque Government (grants IT1203-19 to C.M. and PIBA19-0059 to SM), and ARSEP Foundation (grant to SM)

Δ 9 ‐Tetrahydrocannabinol promotes functional remyelination in the mouse brain

Background and purpose: Research on demyelinating disorders aims to find novel molecules that are able to induce oligodendrocyte precursor cell differentiation to promote central nervous system remyelination and functional recovery. Δ9 -Tetrahydrocannabinol (THC), the most prominent active constituent of the hemp plant Cannabis sativa, confers neuroprotection in animal models of demyelination. However, the possible effect of THC on myelin repair has never been studied. Experimental approach: By using oligodendroglia-specific reporter mouse lines in combination with two models of toxin-induced demyelination, we analysed the effect of THC on the processes of oligodendrocyte regeneration and functional remyelination. Key results: We show that THC administration enhanced oligodendrocyte regeneration, white matter remyelination and motor function recovery. THC also promoted axonal remyelination in organotypic cerebellar cultures. THC remyelinating action relied on the induction of oligodendrocyte precursor differentiation upon cell cycle exit and via CB1 cannabinoid receptor activation. Conclusions and implications: Overall, our study identifies THC administration as a promising pharmacological strategy aimed to promote functional CNS remyelination in demyelinating disorders

Osimertinib in advanced EGFR-T790M mutation-positive non-small cell lung cancer patients treated within the Special Use Medication Program in Spain : OSIREX-Spanish Lung Cancer Group

AURA study reported 61% objective response rate and progression-free survival of 9.6 months with osimertinib in patients with EGFR/T790M+ non-small cell lung cancer. Due to lack of real-world data, we proposed this study to describe the experience with osimertinib in Spain. Post-authorization, non-interventional Special Use Medication Program, multicenter, retrospective study in advanced EGFR/T790M+ non-small cell lung cancer. One hundred-fifty five patients were enrolled (August 2016-December 2018) from 30 sites. Primary objective: progression-free survival. Secondary objectives: toxicity profile, objective response rate, and use of health service resources. 70% women, median age 66. 63.9% were non-smokers and 99% had adenocarcinoma. Most patients had received at least one prior treatment (97%), 91.7% had received previous EGFR-tyrosine kinase inhibitors and 2.8% osimertinib as first-line treatment. At data cutoff, median follow-up was 11.8 months. One hundred-fifty five patients were evaluable for response, 1.3% complete response, 40.6% partial response, 31% stable disease and 11.6% disease progression. Objective response rate was 42%. Median progression-free survival was 9.4 months. Of the 155 patients who received treatment, 76 (49%) did not reported any adverse event, 51% presented some adverse event, most of which were grade 1 or 2. The resource cost study indicates early use is warranted. This study to assess the real-world clinical impact of osimertinib showed high drug activity in pretreated advanced EGFR/T790M+ non-small cell lung cancer, with manageable adverse events. Clinical trial registration number : NCT03790397

Treatment with tocilizumab or corticosteroids for COVID-19 patients with hyperinflammatory state: a multicentre cohort study (SAM-COVID-19)

Objectives: The objective of this study was to estimate the association between tocilizumab or corticosteroids and the risk of intubation or death in patients with coronavirus disease 19 (COVID-19) with a hyperinflammatory state according to clinical and laboratory parameters. Methods: A cohort study was performed in 60 Spanish hospitals including 778 patients with COVID-19 and clinical and laboratory data indicative of a hyperinflammatory state. Treatment was mainly with tocilizumab, an intermediate-high dose of corticosteroids (IHDC), a pulse dose of corticosteroids (PDC), combination therapy, or no treatment. Primary outcome was intubation or death; follow-up was 21 days. Propensity score-adjusted estimations using Cox regression (logistic regression if needed) were calculated. Propensity scores were used as confounders, matching variables and for the inverse probability of treatment weights (IPTWs). Results: In all, 88, 117, 78 and 151 patients treated with tocilizumab, IHDC, PDC, and combination therapy, respectively, were compared with 344 untreated patients. The primary endpoint occurred in 10 (11.4%), 27 (23.1%), 12 (15.4%), 40 (25.6%) and 69 (21.1%), respectively. The IPTW-based hazard ratios (odds ratio for combination therapy) for the primary endpoint were 0.32 (95%CI 0.22-0.47; p < 0.001) for tocilizumab, 0.82 (0.71-1.30; p 0.82) for IHDC, 0.61 (0.43-0.86; p 0.006) for PDC, and 1.17 (0.86-1.58; p 0.30) for combination therapy. Other applications of the propensity score provided similar results, but were not significant for PDC. Tocilizumab was also associated with lower hazard of death alone in IPTW analysis (0.07; 0.02-0.17; p < 0.001). Conclusions: Tocilizumab might be useful in COVID-19 patients with a hyperinflammatory state and should be prioritized for randomized trials in this situatio