TSPO: kaleidoscopic 18-kDa amid biochemical pharmacology, control and targeting of mitochondria

The 18-kDa translocator protein (TSPO) localizes in the outer mitochondrial membrane (OMM) of cells and is readily up-regulated under various pathological conditions such as cancer, inflammation, mechanical lesions and neurological diseases. Able to bind with high affinity synthetic and endogenous ligands, its core biochemical function resides in the translocation of cholesterol into the mitochondria influencing the subsequent steps of (neuro-)steroid synthesis and systemic endocrine regulation. Over the years, however, TSPO has also been linked to core cellular processes such as apoptosis and autophagy. It interacts and forms complexes with other mitochondrial proteins such as the voltage-dependent anion channel (VDAC) via which signalling and regulatory transduction of these core cellular events may be influenced. Despite nearly 40 years of study, the precise functional role of TSPO beyond cholesterol trafficking remains elusive even though the recent breakthroughs on its high-resolution crystal structure and contribution to quality-control signalling of mitochondria. All this along with a captivating pharmacological profile provides novel opportunities to investigate and understand the significance of this highly conserved protein as well as contribute the development of specific therapeutics as presented and discussed in the present review

Radiopharmaceuticals for PET imaging of neuroinflammation - Les radiopharmaceutiques pour l’imagerie TEP de la neuroinflammation

Abstract Recently, accumulating evidence has revealed that neuroinflammation appears to be the cornerstone of many neurological diseases including stroke, multiple sclerosis, Alzheimer's disease or Parkinson's disease. Neuroinflammation causes neuronal damages by activation of numerous cells and molecular mediators in diseases involving the inflammatory process. In this article, we focus on noninvasive molecular imaging of radioligands that target inflammatory cells and molecules involved in neuroinflammation. PET is in fact one of the most promising imaging techniques to visualize and quantify neuroinflammation in vivo. We have also summarized the potential neuroinflammation imaging targets and corresponding PET radioligands. Résumé Des données scientifiques récentes et de plus en plus nombreuses ont mis en évidence le rôle central joué par le processus de neuroinflammation dans la physiopathologie de nombreuses maladies neurologiques, telles que l’accident vasculaire cérébral, la sclérose en plaques, la maladie d’Alzheimer ou encore la maladie de Parkinson. Dans ces maladies impliquant le processus inflammatoire, la neuro-inflammation cause en effet des dommages neuronaux par activation de nombreuses cellules et médiateurs moléculaires. L’imagerie par tomographie par émission de positons (TEP) apparaît comme une approche prometteuse pour visualiser et quantifier in vivo la neuro-inflammation de façon non invasive, grâce en particulier au développement de radioligands ciblant spécifiquement diverses molécules impliquées dans cette réaction inflammatoire cérébrale. Dans cette revue sont présentés les cibles moléculaires potentielles pour l’imagerie TEP de la neuro-inflammation ainsi que les médicaments radiopharmaceutiques correspondants

Imagerie moléculaire de la neuroinflammation dans la maladie de Parkinson (étude préclinique dans un modèle animal de rat)

Bien que les mécanismes moléculaires précis à l origine de la neurodégénérescence dopaminergique ne soient pas encore totalement connus, un ensemble de preuves épidémiologiques, cliniques et expérimentales indiquent que la neuroinflammation peut avoir un rôle important dans la pathogenèse de la MP. L étude des liens spatio-temporels entre la neuroinflammation et la neurodégénérescence au cours de la MP pourrait améliorer la compréhension du mécanisme physiopathologique et aussi l'accessibilité à un diagnostic précoce et/ou à de nouvelles approches thérapeutiques anti-inflammatoires. Le développement actuel des méthodes non invasives d'imagerie moléculaire permettant la surveillance directe du processus de neuroinflammation devrait être utile à cet effet. La cible moléculaire de choix dans ce domaine est la protéine de 18 kDa translocateur (TSPO), biomarqueur sensible associée à la neuroinflammation, qui est surexprimé dans les microglies activées. Dans le travail présenté ici nous avons réalisé l'évaluation longitudinale des deux mécanismes physiopathologiques en parallèle avec les modifications de la fonction dopaminergique à plusieurs points au cours du temps après lésion à la 6-OHDA chez le rat, modèle qui imite un stade précoce de la MP. Après l'administration unilatérale, intra-striatale de la 6-OHDA, nous avons quantifié l'évolution temporelle de la TSPO, de l immunoréactivité TH et du DAT dans le striatum et la SNC de 3 à 56 jours post-lésion (jpl). L augmentation de la liaison des ligands de la TSPO utilisés, c-à-d [3H]-PK11195 et [125I]-CLINDE, a été observée dans le striatum lésé à 3, 7 et 14 jpl, suivie d'un retour progressif à un niveau basal à 56 jpl. Le profil de liaison dans la SNC a montré une augmentation progressive de la fixation qui débute à 3 jpl, avec un pic à 14 jpl, et diminue progressivement jusqu'à ce que 56 jpl. Dans ce modèle de rongeur de la MP, les processus neuroinflammatoire et neurodégénératif surviennent de façon concomitante. La présence transitoire de l'activation microgliale pourrait être impliquée dans l apparition et l'installation durable de la perte neuronale dopaminergique. Cette étude confirme donc le lien entre la neuroinflammation et de la neurodégénérescence et met aussi l'accent sur l'intérêt du CLINDE comme traceur potentiel de la neuroinflammation in-vivo en fournissant des informations précieuses pour le diagnostic précoce et le suivi longitudinal de la progression de la maladie, avec des applications potentielles chez l'homme. En effet, la détection précoce de la neuroinflammation, de façon antérieure à une perte neuronale cliniquement significative, pourrait devenir un enjeu majeur dans la prise en charge pré-symptomatique de la MP. Dans ce sens, nous mettons en évidence l existence d'une fenêtre thérapeutique, survenant juste après la lésion, qui peut être proposé pour l'introduction de traitements anti-inflammatoires qui viseraient à ralentir le processus neurodégénératif. La poursuite de l exploration des relations entre la neuroinflammation et la neurodégénéréscence in-vivo dans le même modèle animal avec la méthode d imagerie micro-TEP, transposable à l homme, en utilisant en parallèle le [18F]-DPA714 pour la TSPO et le [18F]-LBT999 pour le DAT est en cours.Although the precise molecular mechanisms causing the dopaminergic neurodegeneration are still not totally understood, a body of epidemiological, clinical and experimental evidence indicates that neuroinflammation may have an important role in the pathogenesis of PD. Study of spatio-temporal links between neuroinflammation and neurodegeneration during the course of PD would improve understanding of the physiopathological mechanism and also accessibility to early diagnosis and/or new antiinflammatory therapeutic approaches. The current development of non-invasive molecular imaging methods allowing direct monitoring of the neuroinflammation process should be valuable for this purpose. The molecular target of choice in this field is the 18 kDa translocator protein (TSPO), a sensitive biomarker associated with neuroinflammation, which is over-expressed in activated microglia. In the study presented here we achieved the longitudinal evaluation of both physiopayhological mechanisms in parallel with the modifications of dopaminergic function at several time-points after 6-OHDA lesion in the rat that mimics an early stage of PD. After unilateral intra-striatal 6-OHDA administration, we quantified the temporal evolution of the TSPO, TH immunoreactivity and DAT in the striatum and the SNc from 3 to 56 days post-lesion (dpl). Increased binding of TSPO ligands used, i.e. [3H]PK11195 and [125I]CLINDE, was observed in the lesioned striatum at 3, 7 and 14 dpl, followed by a progressive return to the basal level at 56 dpl. The binding profile in the SNc showed progressive binding beginning at 3 dpl, peaking at 14 dpl, and progressively decreasing until 56 dpl. In this rodent model of PD, the neuroinflammatory and neurodegenerative processes occurred concomitantly. The transitory occurrence of microglial activation could be involved in the advent and the lasting installation of dopaminergic neuron loss. This study supports the link between neuroinflammation and neurodegeneration and emphasizes the interest of CLINDE as potent in vivo tracer of neuroinflammation by providing valuable information for early diagnosis and longitudinal follow-up of disease progression, with potential applications to human patients. Indeed, early detection of neuroinflammation, prior to a clinically significant loss of neurons, could become a major issue in the management of pre-symptomatic PD. To support this idea, we demonstrate the existence of a therapeutic window, occurring just after the lesion, which may be proposed for the introduction of anti-inflammatory treatments that aimed to slow the neurodegenerative process. Further exploration of the relationship between neuroinflammation and neurodegeneration in vivo in the same animal model with the method of micro-PET imaging, transposable to humans, using in parallel the [18F]-DPA714 for TSPO and [18F]-LBT999 for DAT is pending.TOURS-Bibl.électronique (372610011) / SudocSudocFranceF

Insights into the Cyclooxygenase Pathway in a Progressive Rat Model of Parkinson’s Disease Induced by Prostaglandin J2: Protection with Ibuprofen

Parkinson’s disease (PD) is a neurodegenerative disease with aging as a major risk factor. Its defining symptoms are motor deficits that are primarily associated with dopaminergic neuronal loss in the substantia nigra pars compacta (SNpc) in the midbrain. Post-mortem PD brains exhibit abnormal intraneuronal inclusions of α-synuclein and ubiquitinated protein aggregates known as Lewy bodies, a hallmark of PD pathology. Currently, there is no validated biomarker for PD. Especially the early stage of PD is difficult to detect as the pathology develops progressively. While symptom-managing treatments are available, there is no neuroprotective treatment as of yet, mainly due to the unknown cause of PD. Animal models of PD have greatly expanded our knowledge on different aspects of PD. However, each has challenges on closely mimicking PD’s major pathological conditions, especially the progressive nature of the pathology. Among the existing PD animal models, a shared feature is neuroinflammation, which has been increasingly implicated in the development of PD by findings from genome-wide association studies, PD-risk genes, environmental risk factors and post-mortem studies. Transition of acute neuroinflammation to the chronic state is critical in the development and worsening of neurodegeneration associated with inflammation. The cyclooxygenase (COX) pathway is a major contributing factor to the development of PD. Firstly, COX-2 is upregulated in the substantia nigra of PD patients. Secondly, existing genetic and toxin models of PD indicate the involvement of the COX pathway. Thirdly, epidemiological studies report the effect of non-steroid-anti-inflammatory drugs (NSAIDs) in lowering PD risk. However, NSAIDs are not an ideal treatment against PD due to their serious side effects. Prostaglandins are products of the COX pathway but their profile and role in the progression of PD pathology are not fully understood. As endogenous products of neuroinflammation, prostaglandins may be highly relevant to the etiology of PD. The overall goal of my studies was to gain insights into how prostaglandins contribute to PD neurodegeneration in vivo. Goal 1: Establish a rat model of neuroinflammation displaying parkinsonian-like pathology induced by the highly neurotoxic prostaglandin J2 (PGJ2), and characterize the impact of PGJ2 on key factors of the PGD2/J2 signaling pathway (Chapter 2). We found that unilateral nigral PGJ2-microinfusions induced progressive PD-like pathology in the rats. PGJ2-treated rats exhibited dose-dependent and progressive dopaminergic neuronal loss in the SNpc, correlated motor deficits, and gliosis involving microglia and astrocytes. Ibuprofen, an NSAID, prevented most of the PD-like pathology shown in PGJ2-treated rats. We explored the effect of PGJ2-treatment on COX-2, lipocalin-type PGD2 synthase (L-PGDS), PGD2/J2 receptor 2 (DP2), and 15-hydroxyprostaglandin dehydrogenase (15-PGDH), a PG-deactivating enzyme. We found that COX-2, L-PGDS, and 15-PGDH levels increased significantly in PGJ2-treated rats compared to controls in the SNpc DA neurons. DP2 receptors were found predominantly expressed on dopaminergic neurons. The levels of COX-2 and L-PGDS co-localization in microglia were increased upon PGJ2-treatment. However, 15-PGDH levels were did not differ among all rat groups, while high levels of 15-PGDH were detected in SNpc oligodendrocytes. Goal 2: Assess chronic neuroinflammation in the PGJ2-induced rat model in vivo with micro-PET imaging (Chapter 3). We performed µPET imaging with the translocator protein (TSPO) radioligand [11C]PK11195 at two different time points (weeks four and eight), post DMSO and PGJ2-injections. Higher [11C]PK11195 uptake was observed in rats that received two PGJ2-injections compared to controls (DMSO-treated) at both time points, indicating chronic glial activation. Conclusion: our studies establish that the PGJ2-induced rat model recapitulates the progressive nature of PD pathology albeit in a relatively short term. Chronic neuroinflammation exhibited in the PGJ2 rat model of PD was assessed with PET imaging in vivo. Studies with the PGJ2‑induced rat model of PD have potential to identify and optimize treatments against neurotoxic inflammation, and to evaluate novel PET radiotracers for neuroinflammation. Our studies with the PGJ2-induced rat model of PD, strongly support that therapeutic targets downstream of cyclooxygenases, such as DP2 receptors and L-PGDS, have potential against PD pathology

PET Agents in Dementia:An Overview

This article presents an overview of imaging agents for PET that have been applied for research and diagnostic purposes in patients affected by dementia. Classified by the target which the agents visualize, seven groups of tracers can be distinguished, namely radiopharmaceuticals for: (1) Misfolded proteins (beta-amyloid, tau, alpha-synuclein), (2) Neuroinflammation (overexpression of translocator protein), (3) Elements of the cholinergic system, (4) Elements of monoamine neurotransmitter systems, (5) Synaptic density, (6) Cerebral energy metabolism (glucose transport/ hexokinase), and (7) Various other proteins. This last category contains proteins involved in mechanisms underlying neuroinflammation or cognitive impairment, which may also be potential therapeutic targets. Many receptors belong to this category: AMPA, cannabinoid, colony stimulating factor 1, metabotropic glutamate receptor 1 and 5 (mGluR1, mGluR5), opioid (kappa, mu), purinergic (P2X7, P2Y12), sigma-1, sigma-2, receptor for advanced glycation endproducts, and triggering receptor expressed on myeloid cells-1, besides several enzymes: cyclooxygenase-1 and 2 (COX-1, COX-2), phosphodiesterase-5 and 10 (PDE5, PDE10), and tropomyosin receptor kinase. Significant advances in neuroimaging have been made in the last 15 years. The use of 2-[F-18]-fluoro-2-deoxy-D-glucose (FDG) for quantification of regional cerebral glucose metabolism is well-established. Three tracers for beta-amyloid plaques have been approved by the Food and Drug Administration and European Medicines Agency. Several tracers for tau neurofibrillary tangles are already applied in clinical research. Since many novel agents are in the preclinical or experimental stage of development, further advances in nuclear medicine imaging can be expected in the near future. PET studies with established tracers and tracers for novel targets may result in early diagnosis and better classification of neurodegenerative disorders and in accurate monitoring of therapy trials which involve these targets. PET data have prognostic value and may be used to assess the response of the human brain to interventions, or to select the appropriate treatment strategy for an individual patient. (C) 2021 The Authors. Published by Elsevier Inc

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

Synthesis and biological evalutation of novel N,N-dialkyl-2-arylindol-3-ylglyoxylamide TSPO ligands.

A series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamide derivatives was design, synthesized and tested as TSPO ligands. The SARs of these compounds were rationalized in light of the pharmacophore/topological model of TSPO binding site made up of three lipophilic pockets (L1, L3 and L4) and a H-bond donor group (H1). The aim of this work is the design and the synthesis of a novel series of N,N-dialkyl-2-phenylindol-3-ylglyoxylamide featuring suitable substituentes on the indole nucleus for good pharmacokinetic parameters. The affinity towards TSPO of all the new compounds was tested by binding assays performed on rat kidney membranes. For those compounds which showed a Ki value in the sub-nanomolar range, the ability to induce functional effects trough the TSPO was also tested. In particular we carried out experiments on cell growth in metabolic stress conditions. The effects of some compounds (the most promising ones in term of affinity) were evaluated by proliferation MTS assay and crystal violet staining. The obtained results showed an increase in cell growth after 48 hours of treatment with compounds, in respect to the control, suggesting their neuroprotective action

Kinetic modelling of [(11)C]PBR28 for 18 kDa translocator protein PET data:A validation study of vascular modelling in the brain using XBD173 and tissue analysis

The 18 kDa translocator protein (TSPO) is a marker of microglia activation in the central nervous system and represents the main target of radiotracers for the in vivo quantification of neuroinflammation with positron emission tomography (PET). TSPO PET is methodologically challenging given the heterogeneous distribution of TSPO in blood and brain. Our previous studies with the TSPO tracers [11C]PBR28 and [11C]PK11195 demonstrated that a model accounting for TSPO binding to the endothelium improves the quantification of PET data. Here, we performed a validation of the kinetic model with the additional endothelial compartment through a displacement study. Seven subjects with schizophrenia, all high-affinity binders, underwent two [11C]PBR28 PET scans before and after oral administration of 90 mg of the TSPO ligand XBD173. The addition of the endothelial component provided a signal compartmentalization much more consistent with the underlying biology, as only in this model, the blocking study produced the expected reduction in the tracer concentration of the specific tissue compartment, whereas the non-displaceable compartment remained unchanged. In addition, we also studied TSPO expression in vessels using 3D reconstructions of histological data of frontal lobe and cerebellum, demonstrating that TSPO positive vessels account for 30% of the vascular volume in cortical and white matter