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The cannabinoid system exerts functional regulation of neural stem cell (NSC) selfrenewal, proliferation, and differentiation during both homeostatic and pathologic conditions. Recent evidence suggests that cannabinoid signaling is neuroprotective against reduction in NSC proliferation and neurogenesis caused by a multitude of conditions including injury due to HIV-1 associated neurotoxic proteins, neuroinflammation, and stroke. Yet not all effects of cannabinoids or cannabinoid-like compounds on neurogenesis can be attributed to signaling through either of the classical cannabinoid receptors CB1 or CB2. The recently de-orphaned GPR55 is targeted by numerous cannabinoid compounds suggesting GPR55 may be causing these aberrant effects. Activation of GPR55 has shown immune-modulatory effects outside the central nervous system (CNS) and anti-inflammatory actions on microglia, the resident immune cells within the CNS. New evidence has confirmed that both human and murine NSCs express functional levels of GPR55 yet the effects that GPR55 activation has on adult neurogenesis or NSC responses to inflammation has not been elucidated. In the present study we sought to determine the role GPR55 signaling has on NSC proliferation and neurogenesis as well as possible neuroprotective mechanisms within the NSC pool in response to inflammatory insult. Activation of GPR55 increased human NSC proliferation in vitro as assessed by BrdU incorporation and flow cytometry. Neuronal differentiation was also upregulated by signaling through GPR55 under homeostatic conditions in both human and murine NSC samples. Expression of NSC differentiation markers (nestin, sox2, GFAP, S100b, DCX, bIII-tubulin) in vitro was determined by immunohistochemistry, qPCR, and flow cytometry. In vivo, C57BL/6 and GPR55-/- mice were administered the GPR55 agonist O-1602 (4 μg/kg/day) directly into the left hippocampus via stainless steel cannula connected to an osmotic mini-pump for a continuous 14 days. O-1602 treatment increased hippocampal NSC proliferation, survival, and immature neuron formation as compared to vehicle treated animals. These results were determined to be dependent on GPR55 activation as GPR55-/- animals did not show any response to agonist treatment. Interestingly, GPR55-/- mice displayed significantly reduced rates of hippocampal NSC proliferation and neuroblast formation as compared to C57BL/6 animals. Chronic production of inflammatory mediators, such as IL-1b seen in neuroinflammation, to NSCs is known to reduce proliferation rates and attenuate neurogenesis both in vitro and in vivo. Addition of GPR55 agonists to IL-1b (10 ng/mL) treated human and murine NSC samples in vitro protected against reductions in neuron formation as assessed by immunohistochemistry and flow cytometry. Moreover, inflammatory cytokine receptor mRNA expression was down regulated by GPR55 activation in a neuroprotective manner. To determine inflammatory responses in vivo, we treated C57BL/6 and GPR55-/- mice with LPS (0.2 mg/kg/day) continuously for 14 days via osmotic mini-pump. Reductions in NSC survival (as determined by BrdU incorporation), immature neurons, and neuroblast formation due to LPS were attenuated by concurrent direct intrahippocampal administration of the GPR55 agonist, O-1602 (4μg/kg/day) in C57BL/6 mice but not in GPR55-/-mice. Neuroprotection by O-1602 treatment was not found to be microglia dependent as microglia activation was not altered by agonist administration. Molecular analysis of the hippocampal region showed a suppressed ability to regulate immune responses by GPR55-/- animals manifesting in a prolonged inflammatory response (IL-1b, IL-6, TNFa) after chronic, systemic inflammation as compared to C57BL/6 animals. Taken together, these results suggest a neuroprotective role of GPR55 activation on NSCs in vitro and in vivo and that GPR55 provides a novel therapeutic target against negative regulation of hippocampal neurogenesis by inflammatory insult. / Biomedical Sciences

Background and Purpose –The mammalian target of rapamycin (mTOR) pathway has been implicated in cellular responses to hypoxia and inflammation. Cerebral palsy (CP) is a neurodevelopmental disorder often linked to hypoxic and inflammatory injury to the brain, however, a role for mTOR modulation in CP has not been investigated. We hypothesized that mTOR inhibition would prevent neuronal death and diminish inflammation in a mouse model of CP. Methods – Post-natal day 6 mouse pups were subjected to hypoxia-ischemia and lipopolysaccharide-induced inflammation (HIL), a model of CP causing injury to several brain areas. Mice received rapamycin (5mg/kg) following HIL, and then daily for 3 subsequent days. The phospho-activation of the mTOR effector mTOR effector proteins S6, S6K and 4EBP as well as upstream negative regulators, TSC1 and Redd1, were assessed as an in vivo measure of the mTOR signaling cascade. Expression of hypoxia inducible factor 1 (HIF-1 alpha) was assayed as an indicator of hypoxia-mediated cellular injury. Neuronal cell death was defined with Fluoro-Jade C (FJC) and cleaved-caspase 3 (CC3), a marker of apoptosis. Autophagy was measured using Beclin-1 and LC3II expression. Lastly, neuroninflammation following HIL was evaluated by examining Iba-1 labeled microglia number and morphology, as well as P-STAT3 expression. Results – Neuronal death, HIF-1alpha expression, and numerous Iba-1 labeled microglia were evident at 24 and 48 hours following HIL. Basal mTOR signaling was unchanged by HIL. Coincident with persistent mTOR signaling, a decreased in Redd1 expression but not TSC1 was observed in HIL. Increased P-STAT3 expression was observed at 24 and 48 hours post-HIL. Rapamycin treatment following HIL significantly reduced neuronal death, decreased HIF-1 alpha and P-STAT3 expression, and microglial activation, coincident with enhanced expression of Beclin-1 and LC3II, markers of autophagy induction. Increase in neuronal death was observed with concomitant administration of rapamycin and chloroquine, an autophagy inhibitor. Administration of a S6K inhibitor, PF-4708671, following HIL also decreased FJC staining further supporting an mTOR-dependent effect of HIL. Conclusions – mTOR inhibition prevented neuronal cell death and diminished neuroinflammation in this model of CP. Persistent mTOR signaling following HIL suggests a failure of autophagy induction, which may contribute to neuronal death in CP. These results suggest that mTOR signaling may be a novel therapeutic target to reduce neuronal cell death in CP. / Biomedical Neuroscience

Individuals living with depression and anxiety show systemic increases of the bacterial endotoxin lipopolysaccharide (LPS), which induces an inflammatory cascade, resulting in negative effects across the gut-brain axis (GBA). LPS administration in mice has previously been used as a rodent model of depression/anxiety. Flaxseed (FS) contains key bioactives, including an omega-3 fatty-acid, dietary fibre, and a poly-phenolic compound which all may attenuate the effects of LPS through modulation of the GBA. The objectives of this thesis were to examine the effects of LPS on the GBA in C57Bl/6 mice and to determine if dietary supplementation with FS and/or FS oil (FO) provided protection against the LPS challenge. The LPS-induced negative effects across the GBA were partially attenuated by dietary supplementation with FS, but not FO, through changes in microbiota composition/function and systemic-/neuro-inflammation. Therefore, the potential benefits of FS are independent of the oil or are synergistic of all bioactives.

Yes / Chemerin is an adipokine involved in inflammation, adipogenesis, angiogenesis and energy metabolism, and has been hypothesized as a link between obesity and type II diabetes. In humans affected by obesity, chemerin gene expression in peripheral tissues and circulating levels are elevated. In mice, plasma levels of chemerin are upregulated by high-fat feeding and gain and loss of function studies show an association of chemerin with body weight, food intake and glucose homeostasis. Therefore, chemerin is an important blood-borne mediator that, amongst its other functions, controls appetite and body weight. Almost all studies of chemerin to date have focused on its release from adipose tissue and its effects on peripheral tissues with the central effects largely overlooked. To demonstrate a central role of chemerin, we manipulated chemerin signaling in the hypothalamus, a brain region associated with appetite regulation, using pharmacological and genetic manipulation approaches. Firstly, the selective chemerin receptor CMKLR1 antagonist α-NETA was administered i.c.v. to rats to test for an acute physiological effect. Secondly, we designed a short-hairpin-RNA (shRNA) lentivirus construct targeting expression of CMKLR1. This shRNA construct, or a control construct was injected bilaterally into the arcuate nucleus of male Sprague Dawley rats on high-fat diet (45%). After surgery, rats were maintained on high-fat diet for 2 weeks and then switched to chow diet for a further 2 weeks. We found a significant weight loss acutely and inhibition of weight gain chronically. This difference became apparent after diet switch in arcuate nucleus-CMKLR1 knockdown rats. This was not accompanied by a difference in blood glucose levels. Interestingly, appetite-regulating neuropeptides remained unaltered, however, we found a significant reduction of the inflammatory marker TNF-α suggesting reduced expression of CMKLR1 protects from high-fat diet induced neuroinflammation. In white and brown adipose tissue, mRNA expression of chemerin, its receptors and markers of adipogenesis, lipogenesis and brown adipocyte activation remained unchanged confirming that the effects are driven by the brain. Our behavioral analyses suggest that knockdown of CMKLR1 had an impact on object recognition. Our data demonstrate that CMKLR1 is functionally important for the central effects of chemerin on body weight regulation and neuroinflammation. / This work was funded in part by the Academy of Medical Sciences, the Wellcome Trust, the Government of Business, Energy and Industrial Strategy and the British Heart Foundation and Diabetes United Kingdom [SBF004/1063] (GH), the Society for Endocrinology Equipment Grant (GH, RD), the University of Bradford (GH, KP, SK) and Nottingham Trent University (RD).

Over 15 million contact sports players and military veterans are at risk for the development of chronic traumatic encephalopathy (CTE), a neurodegenerative disease associated with exposure to repetitive head impacts (RHI) that sometimes presents with parkinsonian motor symptoms, although very little is understood about how these individuals develop parkinsonism. CTE is characterized by accumulation of hyperphosphorylated tau protein (p-tau), and diagnosis requires the presence of neuronal tau in the form of neurofibrillary tangles at the depth of cortical sulci. We performed quantitative immunoassays for markers of neurovascular inflammation within the postmortem dorsolateral frontal cortex of participants with and without a history of RHI and CTE (n = 156), and tested for associations with RHI, microgliosis, and tau pathology measures. Levels of vascular injury-associated markers were increased in CTE compared to RHI-exposed and -naïve controls. Markers increased with RHI exposure duration and were associated with increased microglial density and tau pathology. Histologically, there was significantly increased ICAM1 staining of the microvasculature, extracellular space, and astrocytes at the sulcal depths in high stage CTE compared to both low stage CTE and controls. Multifocal perivascular immunoreactivity for serum albumin was present in all RHI-exposed individuals. These findings demonstrate that vascular injury markers are associated with RHI exposure, duration, and microgliosis, are elevated in CTE, and increase with disease severity. We next performed a cross-sectional analysis of all brain donors with CTE and without comorbid neurodegenerative disease (n=495) in the UNITE Brain Bank. Participants with parkinsonism (CTE-p, n=119) had a higher mean age at death (71.5 years (y)) than participants without parkinsonism (CTE-np, n=362, 54.1 y) and exhibited a higher rate of dementia than CTE-np participants. CTE-p participants had a more severe CTE stage and nigral pathology (NFTs, neuronal loss, and more frequent Lewy bodies), though the majority of cases were negative for nigral Lewy bodies. In American football players, simultaneous regression analysis demonstrated that nigral NFTs and neuronal loss mediate a connection between years of play and parkinsonism in CTE. In this cross-sectional study of contact sports athletes with CTE, years of contact sports participation was associated with SN proteinopathy and neuronal loss, and these pathologies were associated with parkinsonism. Finally, in a postmortem cohort (n=392) of brain donors with CTE without comorbid neurodegenerative disease, we used linear regression modelling to analyze the associations between isodendritic core nuclei pathology (semiquantitative neurofibrillary tangles (NFTs), neurites, and neuronal loss scores) and CTE disease severity, RHI exposure duration (years of contact sport play), and informant-reported cognitive and daily function as assessed by the Cognitive Difficulties Scale (CDS) and Functional Activities Questionnaire (FAQ), respectively. Overall, isodendritic core (IC) NFT scores increase with disease stage, Initially in the locus coeruleus and finally in the median raphe nuclei. Neuronal loss occurred at later disease stages than NFT accumulation. RHI exposure was associated with p-tau pathology for all IC regions. NFTs and neuronal loss in the substantial nigra were associated with increased CDS scores (i.e., worse cognitive function), and neuronal loss in the substantia nigra and locus coeruleus were associated with increased FAQ scores (i.e., worse daily function). We are able to show CTE is similar in distribution of p-tau pathology to progressive supranuclear palsy (PSP), a disease that is thought to primarily affect subcortical regions, especially by end stage disease. These results demonstrate the vulnerability of the isodendritic core nuclei to p-tau pathology and neuronal loss in CTE, and suggest that their involvement contributes to cognitive and functional symptoms during life. This work highlights the possible linkage between neuroinflammation leading to nigral p-tau accumulation and neuron loss which likely underlies the development and progression of parkinsonian motor symptoms in CTE.

No / Cryptolepis sanguinolenta and its bioactive alkaloid, cryptolepine have shown anti-inflammatory activity. However, the underlying mechanism of anti-inflammatory action in neuronal cells has not been investigated. In the present study we evaluated an extract of C. sanguinolenta (CSE) and cryptolepine (CAS) on neuroinflammation induced with IL-1β in SK-N-SH neuroblastoma cells. We then attempted to elucidate the mechanisms underlying the anti-neuroinflammatory effects of CAS in SK-N-SH cells. Cells were stimulated with 10 U/ml of IL-1β in the presence or absence of different concentrations of CSE (25–200 μg/ml) and CAS (2.5–20 μM). After 24 h incubation, culture media were collected to measure the production of PGE2 and the pro-inflammatory cytokines (TNFα and IL-6). Protein and gene expressions of cyclooxygenase (COX-2) and microsomal prostaglandin synthase-1 (mPGES-1) were studied by immunoblotting and qPCR, respectively. CSE produced significant (p < 0.05) inhibition of TNFα, IL-6 and PGE2 production in SK-N-SH cells. Studies on CAS showed significant and dose-dependent inhibition of TNFα, IL-6 and PGE2 production in IL-1β-stimulated cells without affecting viability. Pre-treatment with CAS (10 and 20 μM) was also found to inhibit IL-1β-induced protein and gene expressions of COX-2 and mPGES-1. Further studies to determine the mechanism of action of CAS showed inhibition of NF-κBp65 nuclear translocation, but not IκB phosphorylation. At 10 and 20 μM, CAS inhibited IL-1β-induced phosphorylation of p38 MAPK. Studies on the downstream substrate of p38, MAPK-activated protein kinase 2 (MAPKAPK2) showed that CAS produced significant (p < 0.05) and dose dependent inhibition of MAPKAPK2 phosphorylation in IL-1β-stimulated SK-N-SH cells. This study clearly shows that cryptolepine (CAS) inhibits neuroinflammation through mechanisms involving inhibition of COX-2 and mPGES-1. It is suggested that these actions are probably mediated through NF-κB and p38 signalling.

Yes

L’état fébrile et le vieillissement normal sont deux processus physiologiques conduisant à un déséquilibre hydrominéral de l'organisme. Ce déséquilibre se traduit par une déshydratation sévère qui peut être aggravée par des conditions climatiques comme nous l'avons vu durant l'été 2003. Dans les deux cas, fièvre et vieillissement, l'organisme répond par une stimulation du système hypothalamo-neurohypophysaire conduisant à l’augmentation de la libération de vasopressine ou hormone antidiurétique, qui pourrait prévenir une déshydratation possiblement critique. Cependant, les modalités d’activation des neurones vasopressinergiques (AVP) dans ces conditions restent inconnues.Le but des recherches réalisées dans cette thèse, a été de déterminer les mécanismes cellulaires et moléculaires responsables de l’activation des neurones vasopressinergiques (AVP) lors d’une réponse inflammatoire et au cours du vieillissement.Nous avons pu démontrer dans la première partie de ce travail que lors d’un épisode inflammatoire (mimé par une injection de lypopolysaccharide LPS) l’activité des neurones AVP est rapidement augmentée et cette activation est soutenue pendant plus de six heures. De plus, cette activation n’est pas due à un effet potentiel secondaire du LPS sur l'osmolarité plasmatique ou la pression artérielle. L’activation précoce des neurones AVP par le LPS semble être soutenue par l’IL-6 (qui mime les effets du LPS), puisque l’activation par le LPS est bloquée par une injection préalable d’anticorps anti-IL-6.Dans la seconde partie de ce travail, nous avons pu montrer le traitement chronique d’IGF-I chez le rat âgé permet de restaurer une fonction urinaire comparable à celle observée chez l’adulte, en agissant vraisemblablement directement sur les neurones AVP puisque le taux plasmatique d’AVP chez les rats âgés traités par l’IGF-I revient à des valeurs normales, i.e., équivalente à celle de rats adultes. Cette hypothèse est confortée par le fait que (i) les neurones AVP expriment le récepteur de l’IGF-I et qu’il n’y a pas de différence dans l’expression de ces récepteurs entre rats âgés et adultes, et (ii) les neurones AVP sont inhibés par l’IGF-I.Enfin, dans la dernière partie de ce travail, nous avons pu montrer que lors du vieillissement, les neurones AVP sont activés, ce qui se traduit par un taux plasmatique d’AVP élevé et un taux d’apeline très faible. De même, les astrocytes sont activés et ne présentent plus de plasticité morphofonctionelle. La microglie, en état d’alerte, ne semble pas jouer un rôle prépondérant dans cette suractivation neuronale et astrocytaire. De plus, cette suractivation neuronale est palliée par un traitement central par un anticorps anti-IL-6 ou un inhibiteur non sélectif des canaux TRPV. Cependant, un traitement central par un anticorps anti-IL-6 n’affecte pas l’expression des TRPV2 dans le noyau supra-optique (NSO). En conclusion générale, il apparait que :1/ L’IL-1 n’est pas le chef d’orchestre de tous les processus inflammatoires. En effet, dans le NSO, l’activation des neurones AVP est soutenue par l’IL-62/ La balance pro- / anti-inflammatoire est un élément importante du dysfonctionnement neuronal. Cependant, le facteur critique du dysfonctionnement des neurones AVP n’est pas la production excessive de facteurs inflammatoires mais l’insuffisante production compensatoire de facteurs anti-inflammatoires.3/ lors du vieillissement, la neuroinflammation responsable du dysfonctionnement des neurones AVP peut être qualifiée de type « chronique à bas bruit », processus dans lequel (i) la microglie, en alerte, voit sa réactivité décuplée lors d'une sollicitation inflammatoire supplémentaire; (ii) le cross-talk astrocytes-neurones est figé dans une configuration d'hyperactivité, semblable à celle observée à l'âge adulte en condition de stimulation physiologique soutenue (comme lors d'une déshydratation), mais qui empêche toute réponse appropriée du réseau à toute demande physiologique supplémentaire, quelle soit transitoire (comme la réponse à une injection aigüe de LPS ou de NaCl 9%) ou soutenue (déshydratation de 48h).Cependant, les données de la littérature montrent le rôle majeur de la microglie dans d'autres types de neuroinflammation dites à « haut bruit », et dont les effets délétères - qui vont du dysfonctionnement neuronal à la neuro-dégénérescence – trouvent leur origine dans la surexpression de molécules microgliales telles l'IL-1 ou le TNF. Pour tenter de comprendre les mécanismes cellulaires et moléculaires impliqués dans un tel dysfonctionnement et pour caractériser la nature du dysfonctionnement neuronal, nous avons mis au point un modèle pharmacologique de neuroinflammation à haut bruit, en injectant directement dans les NSO de l'IL-1. Nos données préliminaires montrent que le dysfonctionnement neuronal ainsi que les mécanismes cellulaires et moléculaires à l’origine de ce dysfonctionnement diffèrent de ceux observés lors du vieillissement : la microglie est activée et surexprime de nombreuses molécules inflammatoires, probablement à l’origine du dysfonctionnement neuronal (absence de pattern phasique, même lors d’une stimulation osmotique), puisque les astrocytes ne semblent pas être affectés. L’absence de pattern phasique à l’origine du faible taux d’AVP plasmatique traduit une perturbation des propriétés électrophysiologiques intrinsèques sous-tendant ce pattern phasique (récepteurs ; canaux ioniques) et/ou des afférences excitatrices (Glu ; ACh ; Na) ou inhibitrices (GABA) modulant cette activité phasique. / The fever and normal aging are two physiological processes leading to water and mineral imbalance in the body. This imbalance results in severe dehydration which can be aggravated by climatic conditions as we saw during the summer of 2003. In both cases, fever and age, the body responds by stimulating the hypothalamic-neurohypophysial system leading to increased release of vasopressin or antidiuretic hormone, which could possibly prevent dehydration criticism. However, the modalities of activation of vasopressinergic neurons (AVP) in these conditions remain unknown. The aim of the research done in this thesis was to determine the cellular and molecular mechanisms responsible for the activation of vasopressinergic neurons (AVP) during an inflammatory response and during aging. We showed ,in the first part of this work, that during an inflammatory episode (mimicked by an injection of lypopolysaccharide LPS) the activity of AVP neurons is rapidly increased and this activation is sustained for more than six hours. Moreover, this activation is not due to a potential secondary effect of LPS on plasma osmolarity and blood pressure. The early activation of AVP neurons by LPS seems to be supported by IL-6 (which mimics the effects of LPS), since activation by LPS is blocked by prior injection of anti-IL-6. In the second part of this work, we showed chronic treatment of IGF-I in old rats can restore bladder function similar to that observed in adults, presumably by acting directly on neurons AVP as the rate plasma AVP in aged rats treated with IGF-I returned to normal values, ie, equivalent to that of adult rats. This hypothesis is supported by the fact that (i) AVP neurons express the receptor for IGF-I and there is no difference in the expression of these receptors between adult and aged rats, and (ii) AVP neurons are inhibited by IGF-I. Finally, in the latter part of this work, we showed that during aging, the AVP neurons are activated, which results in increased serum AVP level and a very low rate of apelin. Similarly, astrocytes are activated and show more morphofunctional plasticity. Microglia does not seem to play a role in neuronal and astrocytic overactivation. Moreover, this neuronal overactivation is overcome by a central processing by an anti-IL-6 or a nonselective TRPV channels. However, an icv treatment by an anti-IL-6 does not affect the expression of TRPV2 in the supraoptic nucleus (SON). In general conclusion, it appears that: 1 / IL-1  is not the conductor of all inflammatory processes. Indeed, in the NSO, the activation of AVP neurons is sustained by IL-6 2 / the balance of pro-/ anti-inflammatory is significant in neuronal dysfunction. However, the critical factor in the dysfunction of AVP neurons is not the excessive production of inflammatory factors, but the insufficient production of compensatory anti-inflammatory factors. 3 / during aging, neuroinflammation responsible for the dysfunction of AVP neurons can be classified as type "chronic and low-grade" process in which (i) microglia, in alert, saw its reactivity increased tenfold during inflammatory additional solicitation; (ii) cross-talk astrocyte-neuron is stuck in a pattern of hyperactivity, similar to that observed in adulthood under conditions of sustained physiological arousal (such as in dehydration), but that would prevent the proper response network to any additional physiological demand, which is transient (as the response to acute injection of LPS or NaCl 9%) or sustained (48 h dehydration). However, literature data show the important role of microglia in other types of neuroinflammation called "high grade", and whose deleterious effects - ranging from neuronal dysfunction to neurodegeneration - are rooted in Microglial overexpression of molecules such as IL-1 or TNF  . In an attempt to understand the cellular and molecular mechanisms involved in such dysfunction and to characterize the nature of neuronal dysfunction, we have developed a pharmacological model of neuroinflammation high grade by injecting IL-1  directly into the SON. Our preliminary data show that neuronal dysfunction and the cellular and molecular mechanisms behind this dysfunction differ from those observed during aging: activated microglia overexpressing many inflammatory molecules, probably at the origin of neuronal dysfunction ( absence of phasic pattern, even during osmotic stimulation), since astrocytes do not appear to be affected. The absence of phasic pattern causing the low plasma AVP reflects a disturbance of intrinsic electrophysiological properties underlying the phasic pattern (receptors, ion channels) and / or afferent excitatory (Glu, ACh, Na) or inhibitory (GABA) modulating the phasic activity.