NRF2 and RIP140 as new therapeutic targets for X-linked adrenoleukodystrophy (X-ALD): Control of redox/metabolic homeostasis and inflammation

[eng] X-linked adrenoleukodystrophy (X-ALD) is a rare neurometabolic disease characterized by the loss of function of the peroxisomal transporter ABCD1, which leads to an accumulation of very long-chain fatty acids, inducing mitochondrial reactive oxygen species. Clinical phenotypes in humans range from adrenal insufficiency to fatal inflammatory cerebral demyelination. Abcd1-null mice (Abcd1- mice) develop late onset axonal degeneration in the spinal cord and locomotor disability resembling the most common phenotype in humans, adrenomyeloneuropathy (AMN). Oxidative stress and mitochondrial dysfunction are key common features in X-ALD patients as well as in Abcd1- mouse. In this thesis, we sought to explore novel therapeutic targets that would contribute to better understand the pathophysiology of the disease, based on the existing knowledge on these hallmarks of X-ALD. First, we studied the nuclear factor erythroid-derived 2, like 2 (NFE2L2, also known as NRF2), in X-ALD mouse models (Abcd1- and Abcd1-/Abcd2-/- mice) and in fibroblasts derived from healthy subjects and X-ALD patients. Here, we identify that NRF2, the master regulator of endogenous antioxidant response, and its target genes are impaired in X-ALD due to an aberrant activity of the AKT/GSK-3β axis. Moreover, GSK-3β inhibitors reactivated the blunted NRF2-dependent response upon oxidative stress in X-ALD fibroblasts (Chapter I). In a second study, we sought to determine the role of RIP140 (receptor interacting protein 140), a transcriptional coregulator essential for metabolic homeostasis and inflammatory response, in X-ALD pathophysiology. To address this objective we studied RIP140 in Abcd1- mouse, organotypic spinal cord slice cultures (OSCSC) from mice and normal appearing white matter (NAWM) from healthy subjects and cerebral X-ALD patients. We found out a redox-dependent increase of RIP140 in the spinal cord and OSCSC from Abcd1- mice, as well as an induction of RIP140 in the NAWM of childhood cerebral ALD (ccALD) patients (Chapter II). Finally, we explored the therapeutic potential of targeting NRF2 and RIP140, independently, in X-ALD mice (Abcd1- and Abcd1-/Abcd2-/- mice). Regarding NRF2, we followed a pharmacologic approach, by treating Abcd1- and Abcd1-/Abcd2-/- mice with dimethyl fumarate, an FDA-approved NRF2 activator (Chapter I). In the case of RIP140, we followed a genetic approach, by crossing RIP140-deficient mice with X-ALD mouse models (Chapter II). In both cases, the therapeutic intervention led to an amelioration of i) mitochondrial dysfunction, ii) bioenergetic failure, iii) oxidative damage and iv) dysregulated inflammatory profile, and most importantly, halted axonal degeneration and behavioural abnormalities in X-ALD mice (Chapters I and II). Collectively, these findings reveal an impairment of the AKT/GSK-3β/NRF2 axis that controls endogenous response against oxidative stress, as well as point to RIP140 as a candidate for the impaired mitochondrial biogenesis and induction of proinflammatory response in X-ALD. Finally, the results of this doctoral thesis indicate that therapies based on NRF2 activation and RIP140 inhibition may be valuable strategies to treat X-ALD and other neurodegenerative disorders which share impaired redox homeostasis, mitochondrial dysfunction and neuroinflammation among their hallmarks.[spa] La adrenoleuocodistrofia ligada al cromosoma X (X-ALD) es una enfermedad neurometabólica rara, que se caracteriza por la pérdida de función del transportador peroxisomal ABCD1. Como consecuencia se acumulan ácidos grasos de cadena muy larga, que inducen la producción de especies reactivas de oxígeno en la mitocondria. El cuadro clínico de X-ALD en humanos es variable, desde la insuficiencia adrenal hasta una desmielinización inflamatoria cerebral que suele ser fatal. Los ratones nulos para el gen Abcd1 (ratones Abcd1-) desarrollan una degeneración axonal de aparición tardía en la médula espinal, además de presentar incapacidad locomotora, un fenotipo similar al más común en humanos, la adrenomieloneuropatía (AMN). El estrés oxidativo y la disfunción mitocondrial son unas características claves de X-ALD, tanto en el modelo de ratón como en humanos. En esta tesis, hemos decidido explorar nuevas dianas terapéuticas que contribuyan a una mejor comprensión de la fisiopatología de esta enfermedad, basándonos en el conocimiento existente sobre estas alteraciones en X-ALD. En primer lugar, estudiamos el factor nuclear NRF2 (nuclear factor, erythroid-derived 2, like 2; también NFE2L2) en los modelos animales de X-ALD (ratones Abcd1- y Abcd1-/Abcd2-/-) y en fibroblastos de pacientes con X-ALD y sujetos sanos. Así, identificamos que NRF2, el regulador maestro de la respuesta antioxidante endógena, así como sus genes diana, están inhibidos en X-ALD, debido a una actividad aberrante del eje AKT/GSK-3β. Además, inhibidores de GSK-3β reactivaron la respuesta frente al estrés oxidativo dependiente de NRF2, que estaba bloqueada en los fibroblastos de pacientes con X-ALD (Capítulo I). En el segundo estudio, pretendemos determinar el papel de RIP140 (receptor interacting protein 140) en la fisiopatología de X-ALD. RIP140 es un coregulador transcripcional esencial para la homeostasis metabólica y la respuesta inflamatoria. Para lograr este objetivo, primero estudiamos RIP140 en los ratones Abcd1-, en cultivos organotípicos de láminas de médula espinal (OSCSC) de ratón, y en sustancia blanca cerebral de apariencia normal (NAWM) de pacientes X-ALD. De este modo, encontramos una inducción mediada por estrés oxidativo de RIP140 en la médula espinal y en OSCSC de los ratones Abcd1-, además de una activación de RIP140 en NAWM de pacientes con ALD cerebral infantil (ccALD) (Capítulo II). Por último, investigamos el potencial terapéutico de estas vías para tratar X-ALD, mediante la administración en la dieta de dimetil fumarato, un activador de NRF2 aprobado por la FDA, a los ratones X-ALD (Capítulo I); y a través de la deleción del gen Rip140 en los ratones X-ALD (Capítulo II). En ambos casos, la intervención terapéutica conllevó una mejora de i) la disfunción mitocondrial, ii) el fallo bioenergético, iii) el daño oxidativo, iv) la alteración del perfil inflamatorio, y sobre todo, detuvo la degeneración axonal y previno las alteraciones en el comportamiento en los ratones X-ALD (Capítulos I y II). En conjunto, estos resultados muestran una disfunción del eje AKT/GSK-3β/NRF2, que controla la respuesta antioxidante endógena, así como apuntan a RIP140 como responsable de la disminuida biogenesis mitocondrial y la inducción de la respuesta pro-inflamatoria que observamos en X-ALD. Finalmente, los resultados derivados de esta tesis doctoral indican que terapias basadas en la activación de NRF2 o la inhibición de RIP140 tienen un valor potencial como estrategias terapéuticas para tratar pacientes con X-ALD u otras enfermedades neurodegenerativas, que compartan como sello distintivo, un fallo en la homeostasis redox, disfunción mitocondrial y neuroinflamación.[cat] L’adrenoleuocodistròfia lligada al cromosoma X (X-ALD) és una malaltia neurometabòlica rara, que es caracteritza per la pèrdua de funció del transportador peroxisomal ABCD1. Com a conseqüència s'acumulen àcids grassos de cadena molt llarga, que indueixen la producció d'espècies reactives d'oxigen en el mitocondri. El quadre clínic de X-ALD en humans és variable, des de la insuficiència adrenal fins a una desmielinització inflamatòria cerebral que sol ser fatal. Els ratolins nuls per al gen Abcd1 (ratolins Abcd1-) desenvolupen una degeneració axonal d'aparició tardana en la medul·la espinal, a més de presentar incapacitat locomotora, un fenotip similar al més comú en humans, l’adrenomieloneuropatia (AMN). L'estrès oxidatiu i la disfunció mitocondrial són unes característiques claus de X-ALD, tant en el model de ratolí com en humans. En aquesta tesi, hem decidit explorar noves dianes terapèutiques que contribueixin a una millor comprensió de la fisiopatologia d'aquesta malaltia, basant-nos en el coneixement existent sobre aquestes alteracions en X-ALD. En primer lloc, hem estudiat el factor nuclear NRF2 (nuclear factor, erythroid-derived 2, like 2; també NFE2L2) en els models animals de X-ALD (ratolins Abcd1- i Abcd1-/ Abcd2-/-) i en fibroblasts de pacients amb X-ALD i subjectes sans. Així, hem identificat que NRF2, el regulador mestre de la resposta antioxidant endògena, així com els seus gens diana, estan inhibits en X-ALD, a causa d'una activitat aberrant de l'eix AKT/GSK-3β. A més, inhibidors de GSK-3β van reactivar la resposta enfront de l'estrès oxidatiu dependent de NRF2, que estava bloquejada en els fibroblasts de pacients amb X-ALD (Capítol I). En el segon estudi, pretenem determinar el paper de RIP140 (receptor interacting protein 140) en la fisiopatologia de X-ALD. RIP140 és un coregulador transcripcional essencial per a l'homeòstasi metabòlica i la resposta inflamatòria. Per aconseguir aquest objectiu, primer hem estudiat RIP140 en els ratolins Abcd1-, en cultius organotípics de làmines de medul·la espinal (OSCSC) de ratolí, i en substància blanca cerebral d'aparença normal (NAWM) de pacients X-ALD. D'aquesta manera, hem trobat una inducció de RIP140 intervinguda per estrès oxidatiu en la medul·la espinal i en OSCSC dels ratolins Abcd1-, a més d'una activació de RIP140 en NAWM de pacients amb ALD cerebral infantil (ccALD) (Capítol II). Finalment, hem investigat el potencial terapèutic d'aquestes vies per tractar X-ALD, mitjançant l'administració en la dieta de dimetil fumarat, un activador de NRF2 aprovat per la FDA, als ratolins X-ALD (Capítol I); i a través de la deleció del gen Rip140 en els ratolins X-ALD (Capítol II). En tots dos casos, la intervenció terapèutica va comportar una millora de i) la disfunció mitocondrial, ii) la fallada bioenergètica, iii) el dany oxidatiu, iv) l'alteració del perfil inflamatori i, sobretot, va aturar la degeneració axonal i va prevenir les alteracions en el comportament en els ratolins X-ALD (Capítols I i II). En conjunt, aquests resultats mostren una disfunció de l'eix AKT/GSK-3β/NRF2, que controla la resposta antioxidant endògena, així com senyalen a RIP140 com a responsable de la disminuïda biogènesis mitocondrial i la inducció de la resposta pro- inflamatòria que observem en X-ALD. Finalment, els resultats derivats d'aquesta tesi doctoral indiquen que teràpies basades en l'activació de NRF2 o la inhibició de RIP140 tenen un valor potencial com a estratègies terapèutiques per tractar pacients amb X-ALD o altres malalties neurodegeneratives, que comparteixin com a segell distintiu, una fallada en l'homeòstasi redox, disfunció mitocondrial i neuroinflamació

The effects of Omega-3, Omega-6 Polyunsaturated Fatty Acids (PUFAs) and Vitamin E on patients with the remitting-relapsing (RR) form of Multiple Sclerosis (MS) in Cyprus

Multiple sclerosis is an inflammatory and demyelinating multifactorial disease that results from the interplay between environmental factors and a susceptible genetic background. Omega (ω)-3/ω-6 polyunsaturated fatty acids (PUFAs) and some vitamins have been shown to reduce the number and severity of relapses and the overall progression of disability in multiple sclerosis patients, however clinical trials remain inconclusive due to a plethora of reasons. In this randomized, double blinded, placebo controlled trial aiming to provide concrete conclusions for the role of PUFAs and vitamins A and E (both gamma (γ) and alpha (α)) in multiple sclerosis. By measuring the incorporation and changes of the lipid composition in red blood cell (RBC) membranes before and after the dietary intervention, and by correlating the efficacy of the different interventions with disease progression, it was shown that supplementation with these specific molecules (that can either act on their own or synergistically) could probably cause the decrease of arachidonic acid (AA) and linoleic acid (LA) from the RBC membranes and the subsequent substitution by eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). These events parallel the clinical observations where this can be correlated with the increased number of relapse activity occurring in the first six months of treatment and later with a prolonged period of remission. Supplementation with the mixture of PUFAs (both ω3 and ω6), vitamin E as gamma tocopherol significantly reduced the annualised relapse rate (ARR) and the risk of sustained disability progression without any reported serious adverse events

The effects of parenteral or dietary omega-3 polyunsaturated fatty acids in rat models of spinal cord injury

PhDThere is currently no effective treatment for spinal cord injury (SCI). Long chain omega-3 polyunsaturated fatty acids (PUFA) such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) have beneficial effects in various neurological disorders. DHA and EPA have neuroprotective effects when given intravenously (i.v.) after SCI, but dietary enrichment with these fatty acids is less well-characterized. It is important to characterize the effect of these compounds after parenteral and oral administration, as both regimes could be used clinically. The aims of this thesis were to: i) characterize the inflammatory response in the rat after T12 compression SCI, ii) characterize the effects of acute i.v. injection of DHA or EPA on inflammation after SCI, iii) explore the effects of i.v. DHA in a rat contusion model of SCI, iv) assess the effects of dietary enrichment with DHA or EPA before and/or after SCI. Compression SCI led to acute infiltration of neutrophils and delayed accumulation of macrophages/microglia in the spinal cord, and a systemic inflammatory response in plasma and liver. DHA i.v. injection reduced neutrophil infiltration to the epicentre and C-reactive protein in the plasma, whereas EPA had no significant effect. There was no effect of i.v. EPA or DHA on the increase in cytokines/chemokines following injury. Acute DHA restored stepping ability after contusion SCI, but there was no effect on histological markers. Dietary enrichment with EPA after compression SCI had a detrimental effect on recovery, but this was not correlated with changes in neurones, oligodendrocytes or macrophages/microglia. Dietary pre-treatment with DHA had no effect on locomotor outcome after compression SCI. Therefore, the inflammatory response after SCI is not changed significantly by acute administration of EPA and DHA. The study did not reveal a beneficial prophylactic effect of dietary DHA, but highlighted a possible risk associated with dietary EPA after SCI

Understanding progressive CNS autoimmunity using transgenic mouse models

La sclérose en plaques (SP) est une maladie auto-immune chronique du système nerveux central (SNC) caractérisée par une neurodégénérescence et une invalidité croissante avec le temps. Au Canada, plus de 100 000 personnes sont atteintes de SP. Plusieurs facteurs peuvent contribuer à l’aggravation de la maladie, mais la cause exacte n’est toujours pas connue. Il est largement admis que les lymphocytes T et B franchissent la barrière hémato-encéphalique et invoquent une attaque inflammatoire contre la myéline du SNC. Le rôle des cellules T et B dans l'auto-immunité du SNC peut être étudié à l'aide d'un modèle animal appelé encéphalomyélite auto-immune expérimentale (EAE). Dans la première partie de la thèse, j'ai utilisé une souris transgénique sur un fond de diabète non obèse (DNO) appelé 1C6, dont les cellules T réagissent spécifiquement à un peptide de la myéline, la glycoprotéine de la myéline provenant des oligodendrocytes 35-55 (MOG[35-55]). Les cellules T CD4 de souris 1C6 mâles et femelles sont différenciées en cellules Th17 et sont transférées de manière adoptive à des souris DNOscid déficientes en lymphocytes. Les cellules 1C6 Th17 mâles sont devenues très pathogéniques par rapport aux femelles et ont induit une maladie évolutive sévère chez les receveurs. Les cellules Th17 des deux sexes présentaient une plasticité phénotypique telle que mesurée par leur expression de la cytokine Th1 classique IFN-©. Cependant, les Th17 mâles affichent une production accrue d’ IFN-© par les cellules Th17 mâles, ce qui est corrélé à la gravité de la maladie chez les souris receveuses. L'utilisation d'un modèle de génotype à quatre noyaux nous a permis de séparer l'effet des hormones sexuelles et des chromosomes sexuels dans l'EAE. Nous avons découvert qu'un gène de régulation immunitaire dans le chromosome X, appelé Jarid1c, s'est avéré être régulé négativement dans les cellules Th17 mâles ayant causé une plus grande sévérité, ainsi que dans les cellules T CD4+ provenant du sang périphérique d'hommes atteints de SP. Dans la deuxième partie de la thèse, j'ai utilisé une souris transgénique appelée IgH[MOG] sur un fond DNO dont les cellules B sont spécifiques de la protéine MOG. Lors de l'immunisation avec MOG[35-55], les souris IgH[MOG] ont présenté une EAE rapide et létale, qui est corrélée à l'inflammation et à la démyélinisation du système nerveux central de ces souris. Ceci est accompagné par l'infiltration de cellules B et de cellules T dans le système nerveux central. Chez les souris IgH[MOG], les cellules T CD4+ infiltrantes dans le SNC sont devenues très pro-inflammatoires, comme le montre leur production d'IL-17 et de GM-CSF dans le SNC. Par conséquent, nos données fournissent un aperçu des contributions des réponses des cellules T Th17, du sexe masculin et des cellules B dans l'auto-immunité chronique du SNC. À l’avenir, ces travaux pourraient nous permettre d’identifier des molécules et des voies pouvant être ciblées pour le traitement de la SP.Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) marked by neurodegeneration and accumulating disability over time. Over 100,000 people in Canada are affected by MS. Multiple factors could contribute to the worsening of the disease and yet the exact cause is still unknown. It is widely accepted that T and B lymphocytes cross the blood-brain barrier and invoke an inflammatory attack against CNS myelin. The role of T and B cells in CNS autoimmunity can be studied using an animal model called experimental autoimmune encephalomyelitis (EAE). In the first part of this thesis, I have used a transgenic mouse on a non-obese diabetic (NOD) background called 1C6 whose T cells possess specificity for a myelin-derived peptide, myelin oligodendrocyte glycoprotein 35-55 (MOG[35-55]). 1C6 CD4+ T cells from both male and female mice are differentiated into Th17 cells and are adoptively transferred into lymphocyte deficient NOD.Scid mice. Male 1C6 Th17 cells became highly pathogenic compared to the females and induced a severe progressive disease in the recipients. Th17 cells from both sexes exhibited phenotypic plasticity as measured by their expression of the classic Th1 cytokine IFN-©. However, male Th17 display increased production of IFN-© by male Th17 cells and this is correlated with disease severity in recipient mice. The use of four core genotype model has allowed us to segregate the effect of sex hormones and sex chromosomes in EAE. We uncovered an immune regulatory gene in the X chromosome called Jarid1c is found to be downregulated in both male Th17 cells that caused greater severity as well as in CD4+ T cells from the peripheral blood of men with MS. In the second part of the thesis, I utilized a transgenic mouse called IgH[MOG] on a NOD background whose B cells are specific for MOG protein. Upon immunization with MOG[35-55], IgH[MOG] mice displayed a rapid and lethal EAE, which is correlated to the inflammation and demyelination in the CNS of these mice. This is accompanied by the infiltration of B cells and T cells into the CNS. In IgH[MOG] mice, CNSinfiltrating CD4+ T cells became highly proinflammatory as measured by their production of IL-17 and GM-CSF in the CNS. Hence, our data provide insight into the contributions of Th17 T cell responses, male sex and B cells in chronic CNS autoimmunity. In the future, this work may permit us to identify targetable molecules and pathways for the treatment of MS

CB2, PPAR-γ y GPR55 como dianas farmacológicas para un tratamiento anti-inflamatorio y neuroprotector en la enfermedad de Parkinson

Tesis inédita de la Universidad Complutense de Madrid, Facultad de Medicina, leída el 15-07-2022Parkinson’s disease (PD) is a chronic neurodegenerative disorder which courses with hypokinetic symptoms due to the selective death of the dopaminergic neurons of the substantia nigra pars compacta (SNpc) and the consequent dopaminergic denervation of the striatum. This disease is also a proteinopathy, as one of its features is the presence of aggregates of misfolded proteins, mainly α-synuclein, known as Lewy bodies (LB). The etiology of PD remains unknown, but several environmental and genetic risk factors have been described, together with aging as the main one. The main symptoms of PD are bradykinesia, rigidity, and resting tremor, usually determinant for the diagnosis, which appear when more than 50% of the dopaminergic neurons are already dead. However, there are non-motor symptoms such as sleep disturbances, mood symptoms, gastrointestinal problems, and olfactory dysfunction, which may be prodromal and could be useful for an early diagnosis. The pathological events that contribute to the neuronal death include not only protein aggregation, but also neuroinflammation, mitochondrial dysfunction, oxidative stress, and excitotoxicity. These mechanisms are known to be interconnected, worsening the dopaminergic neurodegeneration which disturbs the basal ganglia circuitry, preventing a correct motor functioning. The most frequent treatment is the replacement of dopamine levels, mainly with levodopa (L-DOPA), which recovers the mobility of the patients in the short term but causes irreversible dyskinesias after several years. This makes urgent the need of a disease-modifying treatment, and given the multifactorial pathophysiology of PD, we propose the use of pleiotropic molecules which can be effective at several levels, such as cannabinoids...La enfermedad de Parkinson (EP) es una enfermedad neurodegenerativa crónica que cursa con pérdida de movilidad por la muerte selectiva de las neuronas dopaminérgicas de la sustancia nigra pars compacta (SNpc) y la consiguiente denervación dopaminérgica del cuerpo estriado. Esta enfermedad es también una proteinopatía, ya que presenta agregados de proteínas mal plegadas, principalmente α-sinucleína, conocidos como cuerpos de Lewy (LB). La etiología de la EP sigue siendo desconocida, pero se han descrito varios factores de riesgo ambientales y genéticos, además del envejecimiento. Los principales síntomas de la EP son la bradiquinesia, la rigidez y el temblor en reposo, que son determinantes para el diagnóstico, y aparecen cuando más del 50% de las neuronas dopaminérgicas ya están muertas. Sin embargo, hay síntomas no motores, como alteraciones del sueño, trastornos del ánimo, problemas gastrointestinales y disfunción olfativa, que pueden ser prodrómicas y útiles para un diagnóstico precoz. Los eventos patológicos que contribuyen a la muerte neuronal incluyen la agregación proteica, pero también la neuroinflamación, la disfunción mitocondrial, el estrés oxidativo y la excitotoxicidad. Estos mecanismos están interconectados, agravando la neurodegeneración dopaminérgica que altera el circuito de los ganglios basales, impidiendo un correcto funcionamiento motor. El tratamiento más frecuente es la reposición de los niveles de dopamina con levodopa, que recupera la movilidad de los pacientes a corto plazo, pero provoca disquinesias irreversibles tras varios años. Esto hace necesario un tratamiento modificador de la enfermedad, y dada la fisiopatología multifactorial de la EP, se propone el uso de moléculas pleiotrópicas, como los cannabinoides...Fac. de MedicinaTRUEunpu

Development of a novel model of optic neuritis to assess neuroprotective and repair strategies in multiple sclerosis

PhDMultiple sclerosis (MS) is a putative autoimmune disease of the central nervous system (CNS), which often affects the optic nerve pathway. Optic neuritis (ON) is a clinical feature of MS that can cause loss of vision due to conduction block and demyelination. Visual function may not recover due to axonal loss in the optic nerve and subsequent loss of retinal ganglion cells (RGC) in the retina. The visual system is the most accessible and best studied part of the CNS and provides an ideal target to monitor the efficacy of strategies aimed at neuroprotection and repair. A C57BL/6 mouse expressing a T cell receptor (TCR) transgene specific for 35-55 residues of myelin oligodendrocyte glycoprotein (MOG), which develops ON spontaneously (approximately 5%) was characterised and an immunising protocol developed with a combination of immune adjuvants (Pertussis toxin, MOG-specific Z12 monoclonal antibody) to give a high incidence of disease. ON is associated with extensive axonal loss in the optic nerve and RGC loss in the retina. These animals were crossed with C57BL/6.Thy1 CFP mice, which express cyan fluorescent protein (CFP) under control of a Thy1 promoter that limits expression of CFP to the RGC in the eye. The resultant MOGTCRxThy1CFP mice develop ON leading to neuronal loss that can be monitored longitudinally in “real-time” in the living animal using techniques that correlate with studies undertaken in humans (visually evoked potentials, scanning laser ophthalmoscopy and optical coherence tomography). These techniques were used in the MOGTCRxThy1CFP to study neuroprotective and repair therapies for their potential in human trials. This novel model of optic neuritis will be invaluable for the study of neuroprotective and repair strategies in autoimmune diseases and offers a refinement of previous models of MS, such as “classical” EAE

Rôle du NKG2D et ses ligands dans un modèle murin de la sclérose en plaques

La sclérose en plaques (SEP) est considérée comme la maladie inflammatoire prototypique du système nerveux central (SNC). Les lésions des patients atteints de la SEP sont caractérisées par une destruction de la gaine de myéline, des dommages axonaux et neuronaux et une activation des cellules gliales. Il est bien établi que le système immunitaire (SI) participe à la pathogénèse de la SEP. Néanmoins, la contribution des médiateurs immunitaires spécifiques aux insultes reste à définir. Le NKG2D est un récepteur activateur exprimé par de multiples cellules immunitaires effectrices incluant des sous populations des lymphocytes T (LT) CD4 et CD8. Le NKG2D se lie à plusieurs ligands (NKG2DL) comprenant Rae-1 (alpha à epsilon), MULT1 et H60 (a à c) chez la souris. Les NKG2DL sont induits par des déclencheurs environnementaux (ex : inflammation) et alertent le SI de la présence de cellules anormales. Notre laboratoire a préalablement montré que des oligodendrocytes humains expriment au moins un NKG2DL dans les lésions de SEP mais pas dans les cerveaux témoins. De plus, les LT CD8 dans les lésions de SEP sont détectés à proximité des cellules exprimant les NKG2DL. Nous avons aussi établi qu’empêcher l’interaction entre le NKG2D et ses ligands inhibe la mort des oligodendrocytes humains par les cellules immunitaires effectrices activées, in vitro. Un autre groupe a montré que bloquer le NKG2D diminue la sévérité de la maladie dans un modèle murin de la SEP: l’encéphalomyélite auto-immune expérimentale (EAE). L’ensemble de ces résultats suggère que l’interaction NKG2D-NKG2DL peut contribuer à la pathogénèse de la SEP et de l’EAE. Cependant, il n’est pas encore établi, si des NKG2DL spécifiques sont augmentés in vivo pendant le développement de l’EAE et pourraient devenir une cible thérapeutique. Nous avons utilisé l’EAE pour évaluer l’expression des NKG2DL aux différents stades du développement de l’EAE et étudier la contribution de NKG2D. Nous avons utilisé la cytométrie en flux, la qRT-PCR, de l’immunobuvardage de type western et l’ELISA. Nous avons observé que le MULT1, un ligand spécifique du NKG2D, était augmenté dans le SNC des souris EAE et que cette expression élevée corrèlait avec la sévérité de la maladie. De plus, MULT1 était relâché dans le liquide cérébro-spinal (LCS) pendant l’EAE et la forme soluble du MULT1 avait augmenté les propriétés effectrices des cellules immunitaires. Au contraire, l’expression des autres ligands n’avait pas varié au cours de la maladie. Finalement, le transfert de cellules T auto-immunes activées dans des souris receveuses déficientes pour le NKG2D avait induit une maladie significativement moins sévère que lorsque ces cellules avaient été transférées à des souris sauvages. La proportion de cellules ayant infiltré le SNC corrélait avec la sévérité de la maladie. Nos résultats suggèrent que le NKG2D et un de ses ligands MULT1 participent à la pathogenèse de l’EAE et potentiellement de la SEP.Multiple sclerosis (MS) is considered the prototypic inflammatory disease of the central nervous system (CNS). Lesions in the CNS of MS patients are characterized by myelin sheath destruction, oligodendrocyte death, axonal and neuronal damage, and activation of glial cells. It is well established that the immune system (IS) participates in the pathogenesis of MS. Nevertheless, the contribution of specific immune mediators to injury remains to be defined. NKG2D is an activating receptor expressed by numerous immune effector cells including subsets of CD8 and CD4 T lymphocytes. NKG2D binds to various ligands (NKG2DL), which include Rae-1(alpha to epsilon), MULT1, and H60 (a to c) in mice. NKG2DL are induced by environmental triggers (e.g. inflammation) suggesting that these proteins alert the IS to abnormal cells. Our laboratory has previously shown that human oligodendrocytes express at least one NKG2DL in MS lesions but not in control brains. Moreover, CD8 T cells in MS lesions are detected in close proximity to NKG2DL expressing cells. We have also established that disruption of the NKG2D-NKG2DL interaction inhibits killing of human oligodendrocytes by activated immune effector cells in vitro. One group showed that blockade of NKG2D diminished disease severity in the MS mouse model: experimental autoimmune encephalomyelitis (EAE). Overall, these results imply that the NKG2D-NKG2DL interaction can contribute to the pathogenesis of MS and its animal model EAE. However, whether specific NKG2DL are upregulated in vivo during the development of EAE and could potentially be targeted is still unresolved.  We used EAE models, to assess the expression of NKG2DL during different disease stages and to study NKG2D contribution. We used flow cytometry, qRT-PCR, western blot and ELISA approaches.  We observed that MULT1, one specific ligand of NKG2D, is upregulated in the CNS of EAE mice and such elevated expression correlated with disease severity. Moreover, MULT1 was released in the cerebrospinal fluid during EAE and soluble MULT1 increased the effector properties of immune cells. In contrast, expression of other ligands did not vary throughout disease. Finally, adoptive transferred of activated autoimmune T cells into NKG2D deficient recipient mice induced a significantly less severe disease than the transfer into wild type counterparts. The proportion of infiltrated cells in the CNS correlated with disease severity. Our results suggest that NKG2D and specific ligands could play a role in the pathogenesis of EAE and potentially MS

Microglia regulate myelin growth and integrity in the central nervous system white matter

Disruption of myelin structure occurs with ageing and neurodegenerative disease, and involves myelin which is outfolding, unravelling, less compact, and thicker. This is associated with nerve dysfunction and cognitive decline; however, the mechanisms underpinning appropriate myelin structure, i.e. myelin integrity, are unclear. The central nervous system (CNS)-resident macrophages microglia are prime candidates, as they are considered to instruct maturation of the myelinproducing oligodendrocytes and thus, myelin formation in development and following demyelination, based on studies of microglial depletion following loss-of-function of the pro-survival colony stimulating factor 1 receptor (CSF1R). As this approach also targets other CNS macrophages which may contribute to these processes, I sought to investigate the specific roles of microglia in regulating myelin health. To achieve this, I utilised a recently developed transgenic mouse model, in which deletion of the FIRE super-enhancer of the Csf1r gene (FIREΔ/Δ) leads to an absence of microglia, while other CNS macrophages are present. FIREΔ/Δ mice had no impairment in oligodendrocyte maturation or myelin formation in the white matter, yet showed a loss of its integrity, with impaired compaction, increased thickness and outfoldings and unravelling of myelin, culminating in demyelination. Results were recapitulated by depleting microglia in adulthood, indicating a role for microglia in myelin maintenance rather than development. These myelin changes were associated with impaired cognitive flexibility. Loss of myelin integrity was also observed in a human condition (ALSP) where CSF1R mutations result in reduced white matter microglia and dementia. To identify the mechanism by which microglia regulate myelin integrity, singlecell RNA sequencing of FIREΔ/Δ mice was performed, which revealed a new oligodendrocyte subpopulation. The genes upregulated in this oligodendrocyte population were predicted to be regulated by transforming growth factor β 1 (TGFβ1), a factor primarily produced by microglia, which regulates expression ii of its receptors e.g., TGFβR1. Accordingly, TGFβ1 levels in FIREΔ/Δ white matter were reduced, and oligodendroglial TGFβR1 expression was downregulated. Additionally, the conditional knockout of Tgfbr1 in mature oligodendrocytes was sufficient to cause a loss of myelin integrity, mirroring the results in the FIREΔ/Δ mice. Reinstating TGFβ downstream signalling via administration of a small molecule agonist (SRI-011381) rescued the loss of myelin integrity in FIREΔ/Δ mice, significantly reducing inner tongue enlargement and myelin thickness versus vehicle-treated mice such that these were comparable to wildtype controls. My findings reveal that microglia regulate myelin health at later stages than previously thought, preserving the structural integrity of myelin rather than driving initial myelin formation. These findings have important implications for understanding the pathological mechanisms underpinning loss of myelin integrity in ageing and neurodegenerative diseases, where dysregulated microglia may represent key therapeutic targets to restore CNS health