In vivo multimodal imaging of adenosine A1 receptors in neuroinflammation after experimental stroke

Adenosine A(l) receptors (A(l)ARs) are promising imaging biomarkers and targets for the treatment of stroke. Nevertheless, the role of A(l)ARs on ischemic damage and its subsequent neuroinflammatory response has been scarcely explored so far. Methods: In this study, the expression of A(1)ARs after transient middle cerebral artery occlusion (MCAO) was evaluated by positron emission tomography (PET) with [F-18]CPFPX and immunohistochemistry (IHC). In addition, the role of AIARs on stroke inflammation using pharmacological modulation was assessed with magnetic resonance imaging (MRI), PET imaging with [F-18]DPA-714 (TSPO) and [F-18]FLT (cellular proliferation), as well as IHC and neurofunctional studies. Results: In the ischemic territory, [F-18]CPFPX signal and IHC showed the overexpression of A(l)ARs in microglia and infiltrated leukocytes after cerebral ischemia. Ischemic rats treated with the AAR agonist ENBA showed a significant decrease in both [F-18]DPA-714 and [F-18]FLT signal intensities at day 7 after cerebral ischemia, a feature that was confirmed by IHC results. Besides, the activation of A(l)AR promoted the reduction of the brain lesion, as measured with T2W-MRI, and the improvement of neurological outcome including motor, sensory and reflex responses. These results show for the first time the in vivo PET imaging of A(l)AR expression after cerebral ischemia in rats and the application of [F-18]FLT to evaluate glial proliferation in response to treatment. Conclusion: Notably, these data provide evidence for A(l)AR playing a key role in the control of both the activation of resident glia and the de novo proliferation of microglia and macrophages after experimental stroke in rats.The authors would like to thank A. Leukona and V. Salinas for technical support in the radiosynthesis. This study was funded by grants from the Spanish Ministry of Education and Science/FEDER RYC-201722412, SAF2016-75292-R, SAF2017-87670-R and PID2019-107989RB-I00, the Basque Government (IT1203/19, BIO18/IC/006) and CIBERNED. Maria Ardaya holds a fellowship from the University of Pais Vasco. Ana Joya acknowledges funding from Fundacio La Marato de TV3 (17/C/2017). Juan Jose Gutierrez acknowledges funding from Euskampus Fundazioa. Jordi Llop also acknowledges The Spanish Ministry of Economy and Competitiveness (Grant CTQ2017-87637-R). Part of the work has been performed under the Maria de Maeztu Units of Excellence Program from the Spanish State Research Agency (Grant No. MDM-2017-0720)

Adenosine receptor signaling: a key to opening the blood–brain door

International audienceAbstractThe aim of this review is to outline evidence that adenosine receptor (AR) activation can modulate blood–brain barrier (BBB) permeability and the implications for disease states and drug delivery. Barriers of the central nervous system (CNS) constitute a protective and regulatory interface between the CNS and the rest of the organism. Such barriers allow for the maintenance of the homeostasis of the CNS milieu. Among them, the BBB is a highly efficient permeability barrier that separates the brain micro-environment from the circulating blood. It is made up of tight junction-connected endothelial cells with specialized transporters to selectively control the passage of nutrients required for neural homeostasis and function, while preventing the entry of neurotoxic factors. The identification of cellular and molecular mechanisms involved in the development and function of CNS barriers is required for a better understanding of CNS homeostasis in both physiological and pathological settings. It has long been recognized that the endogenous purine nucleoside adenosine is a potent modulator of a large number of neurological functions. More recently, experimental studies conducted with human/mouse brain primary endothelial cells as well as with mouse models, indicate that adenosine markedly regulates BBB permeability. Extracellular adenosine, which is efficiently generated through the catabolism of ATP via the CD39/CD73 ecto-nucleotidase axis, promotes BBB permeability by signaling through A1 and A2A ARs expressed on BBB cells. In line with this hypothesis, induction of AR signaling by selective agonists efficiently augments BBB permeability in a transient manner and promotes the entry of macromolecules into the CNS. Conversely, antagonism of AR signaling blocks the entry of inflammatory cells and soluble factors into the brain. Thus, AR modulation of the BBB appears as a system susceptible to tighten as well as to permeabilize the BBB. Collectively, these findings point to AR manipulation as a pertinent avenue of research for novel strategies aiming at efficiently delivering therapeutic drugs/cells into the CNS, or at restricting the entry of inflammatory immune cells into the brain in some diseases such as multiple sclerosis

Investigating Inflammatory Pathways as Therapeutic Targets and Biomarkers using Functional Imaging and Pharmacological Interventions in Epilepsy Models.

Epilepsy is a neurological disorder that is characterised by spontaneous seizures. After various epileptogenic injuries, astrocytes become dysfunctional and experimental evidence indicates that these cells contribute to seizure mechanisms. The generation of inflammatory molecules in astrocytes appears to play a key pathogenic role in seizures. However, astrocytes may also contribute to repair the hyperexcitable neuronal networks underlying seizures. We focused our studies on understanding the role of astrocytes in epilepsy by (1) developing a new in vivo imaging method to monitor astrocytic cell activation during epileptogenesis and coupled this with their phenotypic characterization; (2) studying the role of Toll-like receptor 3 (TLR3) signaling in seizure mechanisms. We report that in vivo bioluminescence imaging is a powerful tool for monitoring astrocytic activation in diseased conditions. Characterization of astrocytic activation during epileptogenesis showed a rapid cell activation corresponding to their inflammatory phenotype while homeostatic (neuroprotective) mechanisms were activated with a delay. Moreover, we demonstrate that in vivo imaging of astrocyte activation allows to study the potential involvement of these cells in the therapeutic effects of anti-inflammatory drugs. We also show that priming TLR3 activation in astrocytes with the use of a synthetic agonist results in remarkable anti-inflammatory and anti-ictogenic effects. Mechanistic studies revealed that interferon regulatory factor (IRF)-3/Interferon-β mediated cascade is likely responsible for the inhibitory effects of TLR3 priming on seizures and neuronal excitability. In summary, astroglia activation during the critical epileptogenesis phase provides a potential target for interfering in a timely manner with the inflammatory phenotype of these cells contributing to seizures. Importantly, there are astrocytic cell functions that mediate decreased neuronal excitability and they should be carefully considered when developing treatments targeting these cells for therapeutic purposes

Contribution of P2X4 Receptors to CNS Function and Pathophysiology

The release and extracellular action of ATP are a widespread mechanism for cell-to-cell communication in living organisms through activation of P2X and P2Y receptors expressed at the cell surface of most tissues, including the nervous system. Among ionototropic receptors, P2X4 receptors have emerged in the last decade as a potential target for CNS disorders such as epilepsy, ischemia, chronic pain, anxiety, multiple sclerosis and neurodegenerative diseases. However, the role of P2X4 receptor in each pathology ranges from beneficial to detrimental, although the mechanisms are still mostly unknown. P2X4 is expressed at low levels in CNS cells including neurons and glial cells. In normal conditions, P2X4 activation contributes to synaptic transmission and synaptic plasticity. Importantly, one of the genes present in the transcriptional program of myeloid cell activation is P2X4. Microglial P2X4 upregulation, the P2X4+ state of microglia, seems to be common in most acute and chronic neurodegenerative diseases associated with inflammation. In this review, we summarize knowledge about the role of P2X4 receptors in the CNS physiology and discuss potential pitfalls and open questions about the therapeutic potential of blocking or potentiation of P2X4 for different pathologies.The authors work are supported by grants from Merck Serono (a business of Merck KGaA, Darmstadt, Germany), Spanish Ministry of Education and Science (SAF2016-75292-R and PID2019-109724RB-I00), Basque Government (IT1203/19 and PI-2016-1-0016) and Centro de Investigación Biomédica en Red en Enfermedades Neurodegenerativas (CIBERNED)

Astrocytes: Initiators of and Responders to Inflammation

We are in the midst of a glial renaissance; astrocytes, essential for brain homeostasis and neuroprotection, have experienced resurgence in focused analyses. New roles in synaptic plasticity, innate immunity and control of recruited immune cells have placed astrocytes at the center of central nervous system functions. Astrocytes have been shown to receive and convey information to all neural cell types in a coordinated effort to respond to injury and infection, initiating reparative mechanisms. Astrocytes detect injury and infection signals from neurons, microglia, oligodendrocytes and endothelial cells, responding by secreting cytokines, chemokines and growth factors, which may activate immune defenses. While regional heterogeneity in astrocyte form and function has been appreciated since the early 1990s, technologic advances have allowed scientists to show only that astrocytes may be as individualized as neurons. Adult astrocytes may undergo a morphological and functional transformation referred to as astrogliosis. Newly generated astrocytes exhibit heterogenous phenotypes; thus, some remove toxic molecules, restore blood-brain barrier function, and promote extracellular matrix components to support axonal growth and repair, while others inhibit neuronal repair and regeneration. This chapter will introduce some of the cellular and molecular components involved in astrocyte responses induced by inflammatory mediators or pathogens during neuroinflammation or neuroinfectious diseases

Regulation of Microglial Functions by Purinergic Mechanisms in the Healthy and Diseased CNS

Microglial cells, the resident macrophages of the central nervous system (CNS), exist in a process-bearing, ramified/surveying phenotype under resting conditions. Upon activation by cell-damaging factors, they get transformed into an amoeboid phenotype releasing various cell products including pro-inflammatory cytokines, chemokines, proteases, reactive oxygen/nitrogen species, and the excytotoxic ATP and glutamate. In addition, they engulf pathogenic bacteria or cell debris and phagocytose them. However, already resting/surveying microglia have a number of important physiological functions in the CNS; for example, they shield small disruptions of the blood–brain barrier by their processes, dynamically interact with synaptic structures, and clear surplus synapses during development. In neurodegenerative illnesses, they aggravate the original disease by a microglia-based compulsory neuroinflammatory reaction. Therefore, the blockade of this reaction improves the outcome of Alzheimer’s Disease, Parkinson’s Disease, multiple sclerosis, amyotrophic lateral sclerosis, etc. The function of microglia is regulated by a whole array of purinergic receptors classified as P2Y12, P2Y6, P2Y4, P2X4, P2X7, A2A, and A3, as targets of endogenous ATP, ADP, or adenosine. ATP is sequentially degraded by the ecto-nucleotidases and 5′-nucleotidase enzymes to the almost inactive inosine as an end product. The appropriate selective agonists/antagonists for purinergic receptors as well as the respective enzyme inhibitors may profoundly interfere with microglial functions and reconstitute the homeostasis of the CNS disturbed by neuroinflammation

Identifying Key Regulators of the Immune/Inflammatory Response for Developing Anticonvulsive and Antiepileptogenic Approaches

Epilepsy therapy is based on drugs that treat the symptoms, seizures, rather than the disease. There are no treatments for modifying the course of the disease, improving prognosis. Another unmet need is the absence of biomarkers able to identify patients at risk of developing epilepsy or with a progressive disease. Among the potential pathways for attaining these unmet needs, we focused on neuroinflammation since it is a pathological process occurring in experimental epileptogenesis and in human epilepsy. Interleukin-1β (IL-1β)/IL-1Receptor type 1 (IL-1R1) and High Mobility Group Box 1 (HMGB1)/Toll-like Receptor 4 (TLR4) axes are key initiators of neuroinflammation following epileptogenic injuries. Experimental and clinical evidence showed that endogenous anti-inflammatory mechanisms are not efficiently activated in epileptogenic tissue. Among these mechanisms, we focused on specialized mediators driving the process of “resolution of inflammation”. We demonstrated that induction of pro-resolving response is delayed during epileptogenesis compared to activation of neuroinflammation. Based on this, we studied the antiepileptogenic and disease-modifying effects of two pharmacological strategies aimed at (1) anticipating and boosting resolution of neuroinflammation by enhancing pro-resolving mechanisms and (2) blocking the neuroinflammatory response with a combined anti-inflammatory treatment targeting IL-1β/IL-1R1 and HMGB1/TLR4 pathways. (1) The pro-resolving treatment decreased neuroinflammation, ameliorated cognitive deficits and reduced spontaneous seizure frequency and duration; (2) the combined anti-inflammatory treatment prevented disease progression. The role of HMGB1 and its isoforms was investigated in epileptogenesis and drug-resistance. In two animal models of epileptogenesis the pathologic disulfide HMGB1 isoform progressively increased in blood before epilepsy onset, and prospectively identified animals that developed the disease. Moreover, the combined anti-inflammatory approach prevented disease progression and the increase in HMGB1 isoforms in blood during epileptogenesis. In conclusion, pro-resolving and anti-inflammatory treatments have disease-modifying effects and HMGB1 isoforms are potential mechanistic biomarkers for epileptogenesis, underlying the need of validation in prospective clinical studies

Alterations of High-Energy Brain Metabolites Across Multiple Neurodegenerative Disorders

Brain energy metabolism is vital for many cellular processes including homeostasis and thus disturbances to metabolism can be the cause or consequence of neurodegeneration. Metabolic discrepancies have been hypothesized to be involved but less frequently demonstrated to be manipulated by acute and chronic neurodegenerative disorders such as stroke, traumatic brain injury, and epilepsy. I hypothesized that cerebral energy levels are decreased in my model systems for Alzheimer\u27s disease, Autism, Down syndrome, and HIV-1 dementia and that dietary treatments could enhance energy reserves and protect against neurodegenerative disease. For rodent studies, I utlilized a high-energy head focused microwave irradiation system to kill animals but most importantly to snap-inactivate all cerebral enzymes, including those that contribute to the rapid degradation of high-energy phosphate compounds. I found that energy levels are diminished in a high-cholesterol diet model for Alzheimer\u27s disease in rabbit, a trisomic mouse model for Down syndrome (Ts65Dn), and following administration of Tat to primary mouse cortical cultures as a model for HIV-1 dementia. My experiments also examined the extent to which protection is provided by creatine supplementation and the ketogenic diet in models of HIV-1 dementia and epilepsy, respectively. Creatine bioenergetically protected against Tat-induced decreases in cellular levels of ATP, Tat-induced mitochondrial hypopolarization, and Tat-induced mitochondrial permeability transition pore opening. My calorie restricted ketogenic diet studies demonstrated this diet\u27s ability to protect against chemically induced seizures. As well, I observed a coordinated upregulation of all differentially regulated transcripts encoding energy metabolism enzymes, increased numbers of mitochondrial profiles, and ultimately augmented high-energy phosphate levels in seizure naïve rats. My studies demonstrate compromised brain energy levels in the aforementioned neurodegenerative disorders and that dietary treatments such as creatine supplementation for HIV-1 dementia and the ketogenic diet for epilepsy, may protect cerebral function by enhancing neuroenergetics

Imaging Neuroinflammation in Progressive Multiple Sclerosis

Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system CNS), where inflammation and neurodegeneration lead to irreversible neuronal damage. In MS, a dysfunctional immune system causes auto‐reactive lymphocytes to migrate into CNS where they initiate an inflammatory cascade leading to focal demyelination, axonal degeneration and neuronal loss. One of the hallmarks of neuronal injury and neuroinflammation is the activation of microglia. Activated microglia are found not only in the focal inflammatory lesions, but also diffusely in the normal‐appearing white matter (NAWM), especially in progressive MS. The purine base, adenosine is a ubiquitous neuromodulator in the CNS and also participates in the regulation of inflammation. The effect of adenosine mediated via adenosine A2A receptors has been linked to microglial activation, whereas modulating A2A receptors may exert neuroprotective effects. In the majority of patients, MS presents with a relapsing disease course, later advancing to a progressive phase characterised by a worsening, irreversible disability. Disease modifying treatments can reduce the severity and progression in relapsing MS, but no efficient treatment exists for progressive MS. The aim of this research was to investigate the prevalence of adenosine A2A receptors and activated microglia in progressive MS by using in vivo positron emission tomography (PET) imaging and [11C]TMSX and [11C](R)‐PK11195 radioligands. Magnetic resonance imaging (MRI) with diffusion tensor imaging (DTI) was performed to evaluate structural brain damage. Non‐invasive input function methods were also developed for the analyses of [11C]TMSX PET data. Finally, histopathological correlates of [11C](R)‐PK11195 radioligand binding related to chronic MS lesions were investigated in post‐mortem samples of progressive MS brain using autoradiography and immunohistochemistry. [11C]TMSX binding to A2A receptors was increased in NAWM of secondary progressive MS (SPMS) patients when compared to healthy controls, and this correlated to more severe atrophy in MRI and white matter disintegration (reduced fractional anisotropy, FA) in DTI. The non‐invasive input function methods appeared as feasible options for brain [11C]TMSX images obviating arterial blood sampling. [11C](R)‐PK11195 uptake was increased in the NAWM of SPMS patients when compared to patients with relapsing MS and healthy controls. Higher [11C](R)‐PK11195 binding in NAWM and total perilesional area of T1 hypointense lesions was associated with more severe clinical disability, increased brain atrophy, higher lesion load and reduced FA in NAWM in the MS patients. In autoradiography, increased perilesional [11C](R)‐PK11195 uptake was associated with increased microglial activation identified using immunohistochemistry. In conclusion, brain [11C]TMSX PET imaging holds promise in the evaluation of diffuse neuroinflammation in progressive MS. Being a marker of microglial activation, [11C](R)‐ PK11195 PET imaging could possibly be used as a surrogate biomarker in the evaluation of the neuroinflammatory burden and clinical disease severity in progressive MS.Siirretty Doriast