The Role of NO/cGMP Signaling on Neuroinflammation: A New Therapeutic Opportunity

The nitric oxide/cyclic guanosine monophosphate (NO/cGMP) signaling appears to play a key role in inhibiting neuroinflammation and preventing the activation of a proapoptotic pathway, thereby promoting neural cell survival. In addition, evidence indicates that cGMP/protein kinase G (PKG) pathway is involved in the modulation of glial cell activity. Phosphodiesterase 5 (PDE5), which hydrolyzes cGMP in the inactive form, 5ʹGMP, is present throughout the body and brain and has emerged as a potential therapeutic target for diseases related to neuroinflammatory and neurodegenerative processes, since their inhibition leads to accumulation of cGMP. The objective of this chapter is to review current knowledge of NO/cGMP signaling pathways on neuroinflammation and the potential therapeutic use of PDE5 inhibitors (PDE5-Is) in neurological diseases. The extensive, while recent, literature on the effects of PDE-Is on Alzheimer’s disease (AD), multiple sclerosis (MS), Parkinson’s disease (PD), Huntington’s disease (HD), and stroke has been reviewed

Regulation of injury-induced neurogenesis by Nitric Oxide

The finding that neural stem cells (NSCs) are able to divide, migrate, and differentiate into several cellular types in the adult brain raised a new hope for restorative neurology. Nitric oxide (NO), a pleiotropic signaling molecule in the central nervous system (CNS), has been described to be able to modulate neurogenesis, acting as a pro-or antineurogenic agent. Some authors suggest that NO is a physiological inhibitor of neurogenesis, while others described NO to favor neurogenesis, particularly under inflammatory conditions. Thus, targeting the NO system may be a powerful strategy to control the formation of new neurons. However, the exact mechanisms by which NO regulates neural proliferation and differentiation are not yet completely clarified. In this paper we will discuss the potential interest of the modulation of the NO system for the treatment of neurodegenerative diseases or other pathological conditions that may affect the CNS.Foundation for Science and Technology, (FCT, Portugal); COMPETE

Studio del ruolo dell’infiammazione e delle alterazioni della barriera ematoencefalica (BBB) in tre modelli murini di danno cerebrale perinatale

Co-supervisori: Luigi Titomanlio, Anna Pusiol - Laboratorio di aggregazione: INSERM U1141 Paris, Université Paris-Diderot, diretta da Pierre GressensopenIl cervello in via di sviluppo presenta delle caratteristiche peculiari che lo rendono più vulnerabile o diversamente suscettibile ad eventuali danni perinatali. I più importanti danni cerebrali perinatali sono quelli di natura vascolare, come l’ipossi-ischemia, l’emorragia della matrice germinalee lo stroke neonatale, quelli di natura eccitotossica, che coinvolgono prevalentemente la sostanza bianca, che possiamo definire in termine generale danni diffusi della sostanza bianca,quelli infettivi, ed infine quelli traumatici. Indipendentemente dal tipo di insulto, è ormai riconosciuto che al di là del danno acuto, più o meno grave e più o meno reversibile, si associano tutta una serie di conseguenze a lungo termine, alla cui origine risiede l’attivazione delle cellule immunitarie, residenti o sistemiche. Tali cellule, attraverso la secrezione di citochine e/o fattori di crescita o chemotattici, non solo si sommano ai meccanismi fisiopatologici acuti, ma generano delle modificazioni strutturali che sono associate a problemi cognitivo-comportamentali a lungo termine. Mentre i meccanismi fisiopatologici acuti sono stati a lungo esplorati, in particolare nell’adulto, solo recentemente gli sforzi della comunità scientifica si sono rivolti allo studio dell’infiammazione e delle cellule immunomodulatrici del sistema nervoso centrale (SNC). Ancora minori sono le conoscenze riguardo a tali fenomeni nel cervello in via di sviluppo. Un altro argomento di studio che sta assumendo sempre maggiore importanza negli ultimi decenni è la cosiddetta Unità Neurovascolare (NeuroVascular Unit, NVU), la barriera che divide il microambiente cerebrale da quello sistemico. Le proprietà di impermeabilità selettiva della NVU sono garantite dalla Barriera Ematoencefalica (Blood-Brain Barrier, BBB), e dall’interazione di questa con la matrice extracellulare e le cellule del SNC, tra cui, per l’appunto, le cellule gliali. La compromissione delle proprietà di tale barriera agisce di pari passo con l’attivazione delle cellule infiammatorie nella creazione del danno perinatale, e spesso i due processi si influenzano reciprocamente. Da qui l’importanza del mio studio, che valuta, attraverso l’utilizzo di tre modelli murini di danno cerebrale perinatale (stroke perinatale, TBI e leucomalacia periventricolare), l’entità e la modificazione temporale del danno, ed esplora il ruolo delle cellule immunitarie residenti, ed in particolare dei mastociti e/o della microglia, e le modificazioni della BBB. Accanto ad uno studio puramente descrittivo, peraltro indispensabile per un’ottimale conoscenza dei meccanismi fisiopatologici alla base del danno e quindi per lo sviluppo di nuove e mirate tecniche di neuroprotezione, durante questi tre anni ho anche testato, nei vari rispettivi modelli, alcune possibili molecole neuroprotettrici (sildenafil, minociclina, cromoglicato e melatonina). Gli obiettivi di questa tesi sono stati i seguenti: 1)Caratterizzare l’emodinamica cerebrale e l’infiammazione cerebrale mediata dalle cellule gliali e valutare gli effetti di un inibitore selettivo della fosfodiesterasi V (vasodilatatore e modulatore dell’infiammazione), in un modello pediatrico d’ischemia cerebrale permanente (PMCAO) 2)Studiare il ruolo dei mastociti, e valutare gli effetti di una loro inibizione, in un modello pediatrico di traumatismo cranico per caduta di peso a cranio chiuso. 3)Caratterizzare l’infiammazione cerebrale mediata dalle cellule microgliali e valutare gli effetti di una loro inibizione, in un modello pediatrico di traumatismo cranico per caduta di peso a cranio chiuso. 4)Studiare le caratteristiche e il timing di apertura della Barriera Ematoencefalica in un modello pediatrico di traumatismo cranico per caduta di peso a cranio chiuso.Dottorato di ricerca in Medicina cellulare e molecolareopenMoretti, Raffaell

PDE4 gene inhibition:a novel approach to treat demyelinating disorders

Neurological disorders such as MS, spinal cord injury, and nerve damage can result in damage to the myelin sheath, the protective layer around nerve fibers in the body. This leads to disrupted signal transmission between the brain and other parts of the body, resulting in a range of symptoms, including paralysis and cognitive problems. Stimulating the regrowth of this protective layer, also known as remyelination, can therefore be an important therapeutic strategy for treating such disorders. In addition, the immune system also plays a significant role in neurological disorders, as it can attack the myelin sheath, leading to further nerve damage. Reducing the immune reaction against the nervous system can alleviate symptoms and slow disease progression. This thesis demonstrates that a specific class of proteins called phosphodiesterase 4 subtypes are potential new targets for developing therapies that can promote recovery and suppress the inflammatory response

Neural Stem Cells

Twenty years after the discovery of neural stem cells, the question whether the central nervous system can be considered among regenerative tissues is still open. On one side, deep characterization of neural stem and progenitor cells, their niches, and their progeny in brain neurogenic sites overtly showed that new neurons can be generated in the brain of adult mammals, including humans. On the other side, many problems arise when stem cells encounter the mature brain parenchyma, still hampering the development of efficacious therapeutic approaches with endogenous or exogenously-delivered neural stem cells. This book tries to make the point on these extremely promising, yet unresolved, issues

Phosphodiesterase 1 (PDE1) at the crossroads of calcium and cyclic nucleotide signaling in diabetic nephropathy

Indiana University-Purdue University Indianapolis (IUPUI)Diabetic Kidney Disease (DKD) is a major complication of diabetes. Incomplete understanding of its molecular mechanisms is highlighted by the limited treatments options. We hypothesized that inhibition of protective endogenous mechanisms plays major role in the pathogenesis of DKD. While renoprotection is mediated by cyclic nucleotides (cAMP and cGMP), phosphodiesterases (PDEs) lead to cyclic nucleotide degradation. Our investigation focused on the role of calcium/calmodulin activated PDE1 in DKD. Three isoforms of PDE1 are differentially expressed in vascular smooth muscle cells, renal tubular epithelial cells, podocytes, and mesangial cells. We used highly potent and selective PDE1 inhibitor LY1 to explore systemic hemodynamic and local renal role of PDE1. LY1 reduced systolic and diastolic blood pressure in normotensive and spontaneously hypertensive rats. Renal protection with PDE1 inhibition was tested in mouse model of DKD, featuring a combination of diabetes, nephron loss and arterial hypertension. In this model, a PDE1 inhibitor caused a significant improvement in renal function as evident by significant reduction of albuminuria, serum creatinine and several urine biomarkers of inflammation and injury. Histopathological analysis revealed substantial improvement in the pathology of DKD in the treated group that was associated with the reduction of gene expression related to inflammation and fibrosis. Thus, we revealed the role of calcium activated PDE1 in DKD. However, the source of calcium in this context remained obscure. Our bioinformatics analysis pointed out that calcium channel TRPC6 is likely to be involved. Further in vitro studies demonstrated that TRPC6 activation induced apoptosis in human mesangial cells and isolated rat glomeruli, which was attenuated by both TRPC6 and PDE1 inhibition, thereby suggesting a functional coupling between TRPC6 (as a source of calcium) and PDE1 activation. Moving upstream, we showed that several systemic risk factors of DKD (angiotensin II, endothelin 1 and glucose) activated TRPC6 in a different manner, through generation of either reactive oxygen species or diacylglycerol. The computational modeling to relate human transcriptomic and phenotype data demonstrated the pre-clinical findings of renal benefit upon PDE1 inhibition is translatable in human. Taken together, our results suggest mechanistic link among systemic risk factors, TRPC6, calcium flux and PDE1 activation in pathogenesis of DKD. As a corollary, PDE1 inhibition leads to direct and indirect renoprotective effects

Développement d’une nouvelle stratégie neuroprotectrice efficace et d’une méthode de quantification précoce non invasive des lésions de la matière blanche cérébrale immature sur un modèle animal

Les grands prématurés sont particulièrement vulnérables aux lésions inflammatoires de la substance blanche (WMI) qui augmentent le risque de troubles cognitifs et neurodéveloppementaux à long terme dans cette population. L’utilisation de l’imagerie par résonance magnétique (IRM) dans cette population a permis une évaluation non invasive de la progression des WMI et une meilleure compréhension de la pathologie. Les WMI sont associées une activation de la microglie et des astrocytes et la production de facteurs pro-inflammatoires, dont l’interleukine 1 (IL-1). En utilisant un modèle de WMI induite par injection intracérébrale de lipopolysaccharides (LPS), nous avons évalué dans un premier temps les changements de méthylation de l’ADN durant la phase aigüe (24 h) et la phase chronique (21 jours) de l’inflammation. Par la suite, nous avons déterminé la capacité de l’IRM multimodale de détecter la lésion et la réponse thérapeutique à un antagoniste du récepteur de l’IL-1. Finalement, par le biais d’un antagoniste et d’un modulateur allostérique du récepteur à l’IL-1, nous avons évalué in vitro le rôle de la signalisation IL-1 durant la phase aigüe de la modulation de l’activation de la microglie et des astrocytes par le LPS. Nous avons démontré la présence d’une altération du méthylome cérébral dans divers mécanismes liés au neurodéveloppement et à la réponse immunitaire. De plus, l’application de l’IRM multimodale dans notre modèle a permis d’évaluer in vivo la lésion et le début de la réponse thérapeutique durant la phase aigüe (24 h) de l’inflammation. L’évaluation à l’IRM corrèle aux changements observés par immunomarquage post mortem. In vitro, le LPS induit une réponse mixte de la microglie et des astrocytes qui évoluent dans le temps vers une réponse pro-inflammatoire et neurotoxique. Bien que l’IL-1 est hautement exprimée par la microglie et les astrocytes, son inhibition a un effet limité sur la modulation de l’activation gliale dû à la multitude de voies activées par le LPS durant la phase aigüe de l’inflammation.Very premature infants are particularly vulnerable to inflammatory white matter injury (WMI) which increases the risk of long-term cognitive and neurodevelopmental disorders in this population. The use of magnetic resonance imaging (MRI) in this population has allowed non-invasive assessment of the progression of WMI and a better understanding of the pathology. WMI is associated with activation of microglia and astrocytes and the production of pro-inflammatory mediators, including interleukin 1 (IL-1). Using a model of inflammatory WMI induced by intracerebral injection of lipopolysaccharides (LPS), we first evaluated the changes in DNA methylation during the acute phase (24 h) and the chronic phase (21 days) of inflammation. We then determined the ability of multimodal MRI to detect the lesion and the therapeutic response to an IL-1 receptor antagonist. Finally, using an antagonist and an allosteric modulator of the IL-1 receptor, we evaluated in vitro the contribution of IL-1 signaling during the acute phase of the modulation of microglia and astrocytes activation by LPS. We have shown the presence of persistent alteration DNA methylation profile in the brain that was associated with pathways involved in neurodevelopment and immune response. In addition, the application of multimodal MRI in our model made it possible to evaluate in vivo the lesion and the therapeutic response during the acute phase (24 h) of the inflammation. The changes at the MRI correlated to post-mortem evaluation by immunostaining. In vitro, LPS induce a mixed response of microglia and astrocytes which evolved over time toward a pro-inflammatory and neurotoxic phenotype. Although IL-1 is highly expressed by microglia and astrocytes, its inhibition has a limited effect on the modulation of glial activation due to the multitude of pathways activated by LPS during the acute phase of inflammation

Mecanismos neuroquímicos associados à ação antidepressiva e neuroprotetora da lectina isolada das sementes de Canavalia brasiliensis (ConBr)

Dissertação (mestrado) - Universidade Federal de Santa Catarina, Centro de Ciências Biológicas, Programa de Pós-Graduação em Bioquímica, Florianópolis, 2014.As lectinas são proteínas que reconhecem e se ligam reversivelmente a carboidratos ligados a proteínas e lipídeos. ConBr é uma lectina purificado a partir das sementes de Canavalia brasiliensis com afinidade por manose/glucose. Previamente mostramos que ConBr injetada i.c.v. pode produzir uma ação do tipo antidepressiva similar a fluoxetina. ConBr também apresenta efeito neuroprotetor contra a excitotoxicidade glutamatérgica em fatias de hipocampo e em modelo de convulsões induzidas por ácido quinolínico. No presente estudo investigamos o envolvimento do receptor N-metil-D-aspartato (NMDA) e da via L-arginina-óxido nítrico (NO·) na ação tipo antidepressiva de ConBr no teste do nado forçado (TNF). Para esse objetivo, camundongos machos adultos foram pré-tratados com NMDA (0,1 pmol/sítio, i.c.v), D-serina (30 mg/sítio, i.c.v. co-agonista de receptores NMDA), L-arginina (750 mg/kg, i.p. precursor de NO·) e sildenafil (5 mg/kg, i.p. inibidor de fosfodiesterase isoforma 5), os tratamentos bloquearam o efeito antidepressivo de ConBr. Do mesmo modo, a administração de ConBr em uma dose sub-ativa (0,1 µg/sítio, i.c.v.) combinada com doses sub-ativas de antagonistas de receptores NMDA, MK-801 (0,001 mg/kg, i.p.) e cetamina (0,1 mg/kg, i.p.) ou do inibidor da guanilato ciclase solúvel, 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 pmol/sítio, i.c.v.) foram capazes de produzir um efeito sinérgico do tipo antidepressivo. Esses resultados sugerem que o efeito antidepressivo de ConBr no TNF envolve a inibição do receptor NMDA, a redução de NO· e diminuição da síntese de GMPc. Adicionalmente, realizamos um estudo sistemático de vias de sinalização intracelular reguladas por ConBr e que podem estar associadas com os efeitos antidepressivos e neuroprotetores. ConBr (10 µg/sítio) foi injetada no ventrículo lateral (i.c.v.) de camundongos e os hipocampos foram retirados 0,5, 1, 3, 6, 8, 12, 18 e 24 h após o tratamento para análise por Western Blot e qRT-PCR. Os resultados mostraram que ConBr induziu a ativação das proteínas cinases Akt, ERK1 e PKA, bem como induziu aumento da fosforilação de CREB e da expressão do fator neurotrófico derivado do encéfalo (BDNF). Esses dados sugerem que a ativação destas vias poderia estar envolvida nos efeitos neuroprotetores e antidepressivos de ConBr. Por fim, avaliamos o potencial neuroprotetor de ConBr em um modelo de isquemia utilizando culturas organotípicas hipocampais (COH) submetidas a privação de oxigênio e glicose (POG). O tratamento com ConBr (0,1 µg/mL), preveniu a morte celular, reverteu o estresse oxidativo e impediu a redução na fosforilação de Akt e ERK1 induzidos por POG. Em culturas primárias de neurônios hipocampais,utilizando a sonda fluorescente Fluo-4/AM, ConBr bloqueou a entrada de Ca2+ dependente da despolarização por K+. Corroborando com este dado COHs submetidas a POG foram coincubadas com doses sub-ativas de ConBr e nifedipina, um inibidor dos canais de Ca2+ voltagem dependentes (CCDV) do tipo L. Os resultados demonstraram um efeito sinérgico de nifedipina e ConBr na redução da morte celular provocada por POG, sugerindo os CCDV como alvo do mecanismo neuroprotetor do ConBr. Dessa forma, em conjunto os resultados indicam a aplicação de lectinas de plantas como estratégia para neuroproteção e modulação da neuroplasticidade através da regulação de glicoproteínas da superfície celular e regulação da sinalização intracelular.Abstract : Lectins are proteins that recognize and reversibly bind to carbohydrates attached to proteins and lipids. ConBr is a lectin purified from Canavalia brasiliensis seeds that displays high affinity for mannose/glucose. We have previously shown that ConBr produced antidepressant-like effect and blocked hippocampal neurotoxicity induced by quinolinic acid and glutamate. The present study was undertaken to investigate the involvement of the N-methyl-D-aspartate (NMDA) receptor and the L-arginine-nitric oxide (NO·) pathway in the antidepressant-like action displayed by ConBr in the forced swimming test (FST). Moreover, it was evauated the intracellular signaling pathways regulated by ConBr that could be potentially associated with the antidepressant and neuroprotective effects. Finally, neuroprotective activity of ConBr was analised in a model of ischemia in vitro using organotypic hippocampal cultures (OHCs) submitted to oxygen/glucose deprivation (OGD). With the aim of verifying the involvement of NMDA receptors in the antidepressant-like effect of ConBr (10 µg/site, i.c.v.), a pretreatment with NMDA (0.1 pmol/site, i.c.v.), D-serine (30 µg/site, i.c.v.), L-arginine (750 mg/kg, i.p.) or sildenafil (5 mg/kg, i.p.) was carried out. The results show that all treatments blocked the effect of ConBr. Furthermore, the coadministration of subeffective doses of MK-801 (0.001 mg/kg, i.p.), ketamine (0.1 mg/kg, i.p.) or 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 30 pmol/site, i.c.v.) and ConBr (0.1 µg/site, i.c.v.), produced a synergistic antidepressant-like effect in the FST. Taken together, the results suggest that the antidepressant-like effect of ConBr involves NMDA receptor inhibition, reduction in NO· and cGMP synthesis. In order to elucidate intracellular signaling pathways regulated by ConBr that may be potentially associated with the antidepressant and neuroprotective effects previously reported, ConBr (10 µg/site) was injected into the ventricle (i.c.v.) of mice, and the hippocampi were removed 0.5, 1, 3, 6, 8, 12, 18 and 24 h after treatment. Our results indicate that centrally administered ConBr is able to activate the hippocampal neurotrophic pathway PKA/Akt/ERK1-CREB(Ser133)-BDNF, suggesting that these signaling pathways might be involved in the neuroprotective and antidepressant-like effects of ConBr. Finally, the potential neuroprotective activity of ConBr was assessed in OHCs submitted to OGD for 15 min, followed by 24 h of recovery when cell viability and neurochemical parameters were analyzed. In addition, the ability of ConBr to modulate intracellular Ca2+ was evaluated by a Ca2+-sensitive fluorescent dye (Fluo-4/AM) in primary cultures ofhippocampal neurons. Treatment with ConBr (0.1 µg/mL) prevented cell death, mitigated the oxidative stress and prevented the reduction of Akt and ERK1 phosphorylation induced by OGD. Furthermore, ConBr blocked K+-induced Ca2+ entrance in hippocampal neurons. To examine the role of voltage dependent Ca2+ channels (VDCC) in the neuroprotective effect of ConBr, OHCs subjected to OGD were submitted to a coincubation of sub-effectives doses of ConBr and nifedipine, an inhibitor of L-type VDCC. The results showed a synergistic effect of nifedipine plus ConBr counteracting the impairment of cell viability induced by OGD, suggesting VDCC as a target of neuroprotective mechanism of ConBr. These results corroborate with previous data that indicate the application of plant lectins for neuroprotective strategies through the regulation of cell surface glycoproteins and intracellular signaling