Translocator protein is a marker of activated microglia in rodent models but not human neurodegenerative diseases

Microglial activation plays central roles in neuroinflammatory and neurodegenerative diseases. Positron emission tomography (PET) targeting 18 kDa Translocator Protein (TSPO) is widely used for localising inflammation in vivo, but its quantitative interpretation remains uncertain. We show that TSPO expression increases in activated microglia in mouse brain disease models but does not change in a non-human primate disease model or in common neurodegenerative and neuroinflammatory human diseases. We describe genetic divergence in the TSPO gene promoter, consistent with the hypothesis that the increase in TSPO expression in activated myeloid cells depends on the transcription factor AP1 and is unique to a subset of rodent species within the Muroidea superfamily. Finally, we identify LCP2 and TFEC as potential markers of microglial activation in humans. These data emphasise that TSPO expression in human myeloid cells is related to different phenomena than in mice, and that TSPO-PET signals in humans reflect the density of inflammatory cells rather than activation state.Published versionThe authors thank the UK MS Society for financial support (grant number: C008-16.1). DRO was funded by an MRC Clinician Scientist Award (MR/N008219/1). P.M.M. acknowledges generous support from Edmond J Safra Foundation and Lily Safra, the NIHR Senior Investigator programme and the UK Dementia Research Institute which receives its funding from DRI Ltd., funded by the UK Medical Research Council, Alzheimer’s Society, and Alzheimer’s Research UK. P.M.M. and D.R.O. thank the Imperial College Healthcare Trust-NIHR Biomedical Research Centre for infrastructure support and the Medical Research Council for support of TSPO studies (MR/N016343/1). E.A. was supported by the ALS Stichting (grant “The Dutch ALS Tissue Bank”). P.M. and B.B.T. are funded by the Swiss National Science Foundation (projects 320030_184713 and 310030_212322, respectively). S.T. was supported by an “Early Postdoc.Mobility” scholarship (P2GEP3_191446) from the Swiss National Science Foundation, a “Clinical Medicine Plus” scholarship from the Prof Dr. Max Cloëtta Foundation (Zurich, Switzerland), from the Jean et Madeleine Vachoux Foundation (Geneva, Switzerland) and from the University Hospitals of Geneva. This work was funded by NIH grants U01AG061356 (De Jager/Bennett), RF1AG057473 (De Jager/Bennett), and U01AG046152 (De Jager/Bennett) as part of the AMP-AD consortium, as well as NIH grants R01AG066831 (Menon) and U01AG072572 (De Jager/St George-Hyslop)

Modulation de la réactivité astrocytaire par ciblage de la voie JAK2-STAT3 : conséquences dans des modèles murins de la maladie d’Alzheimer

Astrocytes are emerging as key players in brain physiology. In Alzheimer’s disease (AD), astrocytes become reactive. Astrocyte reactivity (AR) is essentially characterized by morphological changes. But how the normal supportive functions of astrocytes are changed by their reactive state is unclear. Moreover, signaling cascades leading to AR are not yet determined. In this study, we aim to: 1/ demonstrate the JAK2-STAT3 pathway (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) is responsible for AR in neurodegenerative diseases ; 2/ understand the contribution of reactive astrocytes to molecular, cellular and functional alterations in AD. We already reported that the JAK2- STAT3 pathway is a central cascade for AR (Ben Haim et al., 2015). Here, we demonstrate, with new molecular tools based on viral vectors, that this pathway is necessary and sufficient to AR. Our results also show that the modulation of AR in two AD mouse models (APP/PS1dE9 and 3xTg-AD mice) influence several pathological hallmarks, but in a context-dependent manner. Overall, this work has generated new original tools to study reactive astrocytes in situ and it underlines the importance and complexity of their functions in neurodegenerative diseases.Les astrocytes sont des éléments clés de la physiologie cérébrale. Dans les maladies neurodégénératives comme la maladie d’Alzheimer (MA), les astrocytes deviennent réactifs. Cette réactivité astrocytaire (RA) est essentiellement caractérisée par des changements morphologiques. En revanche, les effets de la réactivité sur les fonctions de support des astrocytes sont mal connus. De plus, les cascades de signalisation qui conduisent à la RA restent à déterminer. Les objectifs de ce projet étaient de : 1/ démontrer que la voie JAK2-STAT3 (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) joue un rôle central dans le contrôle de la RA au cours des maladies neurodégénératives ; 2/ comprendre quelle est l’implication de la RA dans les altérations moléculaires, cellulaires et fonctionnelles observées dans la MA. Nous avons montré que la voie JAK2-STAT3 est une cascade de signalisation centrale dans la RA (Ben Haim et al., 2015). Dans ce projet, nous démontrons en utilisant de nouveaux outils moléculaires basés sur des vecteurs viraux, que cette voie est nécessaire et suffisante à la RA. Nos résultats montrent également que la modulation de la RA dans deux modèles murins de la MA (souris APP/PS1dE9 et 3xTg-AD) influence certains index pathologiques, mais de façon contexte-dépendante. L’ensemble de ce travail a permis de valider de nouveaux outils pour étudier les astrocytes réactifs in situ et souligne l’importance et la complexité de leur fonctions au cours des maladies neurodégénératives

Modulation of Astrocyte Reactivity by targeting the JAK2-STAT3 Pathway : Consequences in Alzheimer’s Disease Mouse Models

Les astrocytes sont des éléments clés de la physiologie cérébrale. Dans les maladies neurodégénératives comme la maladie d’Alzheimer (MA), les astrocytes deviennent réactifs. Cette réactivité astrocytaire (RA) est essentiellement caractérisée par des changements morphologiques. En revanche, les effets de la réactivité sur les fonctions de support des astrocytes sont mal connus. De plus, les cascades de signalisation qui conduisent à la RA restent à déterminer. Les objectifs de ce projet étaient de : 1/ démontrer que la voie JAK2-STAT3 (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) joue un rôle central dans le contrôle de la RA au cours des maladies neurodégénératives ; 2/ comprendre quelle est l’implication de la RA dans les altérations moléculaires, cellulaires et fonctionnelles observées dans la MA. Nous avons montré que la voie JAK2-STAT3 est une cascade de signalisation centrale dans la RA (Ben Haim et al., 2015). Dans ce projet, nous démontrons en utilisant de nouveaux outils moléculaires basés sur des vecteurs viraux, que cette voie est nécessaire et suffisante à la RA. Nos résultats montrent également que la modulation de la RA dans deux modèles murins de la MA (souris APP/PS1dE9 et 3xTg-AD) influence certains index pathologiques, mais de façon contexte-dépendante. L’ensemble de ce travail a permis de valider de nouveaux outils pour étudier les astrocytes réactifs in situ et souligne l’importance et la complexité de leur fonctions au cours des maladies neurodégénératives.Astrocytes are emerging as key players in brain physiology. In Alzheimer’s disease (AD), astrocytes become reactive. Astrocyte reactivity (AR) is essentially characterized by morphological changes. But how the normal supportive functions of astrocytes are changed by their reactive state is unclear. Moreover, signaling cascades leading to AR are not yet determined. In this study, we aim to: 1/ demonstrate the JAK2-STAT3 pathway (Janus Kinase 2 - Signal Transducer and Activator of Transcription 3) is responsible for AR in neurodegenerative diseases ; 2/ understand the contribution of reactive astrocytes to molecular, cellular and functional alterations in AD. We already reported that the JAK2- STAT3 pathway is a central cascade for AR (Ben Haim et al., 2015). Here, we demonstrate, with new molecular tools based on viral vectors, that this pathway is necessary and sufficient to AR. Our results also show that the modulation of AR in two AD mouse models (APP/PS1dE9 and 3xTg-AD mice) influence several pathological hallmarks, but in a context-dependent manner. Overall, this work has generated new original tools to study reactive astrocytes in situ and it underlines the importance and complexity of their functions in neurodegenerative diseases

Elusive roles for reactive astrocytes in neurodegenerative diseases

International audienceAstrocytes play crucial roles in the brain and are involved in the neuroinflammatory response. They become reactive in response to virtually all pathological situations in the brain such as axotomy, ischemia, infection, and neurodegenerative diseases (ND). Astrocyte reactivity was originally characterized by morphological changes (hypertrophy, remodeling of processes) and the overexpression of the intermediate filament glial fibrillary acidic protein (GFAP). However, it is unclear how the normal supportive functions of astrocytes are altered by their reactive state. In ND, in which neuronal dysfunction and astrocyte reactivity take place over several years or decades, the issue is even more complex and highly debated, with several conflicting reports published recently. In this review, we discuss studies addressing the contribution of reactive astrocytes to ND. We describe the molecular triggers leading to astrocyte reactivity during ND, examine how some key astrocyte functions may be enhanced or altered during the disease process, and discuss how astrocyte reactivity may globally affect ND progression. Finally we will consider the anticipated developments in this important field. With this review, we aim to show that the detailed study of reactive astrocytes may open new perspectives for ND

The complex STATes of astrocyte reactivity: How are they controlled by the JAK–STAT3 pathway?

International audienceAstrocytes play multiple important roles in brain physiology. In pathological conditions, they become reac-tive, which is characterized by morphological changes and upregulation of intermediate filament proteins. Besides these descriptive hallmarks, astrocyte reactivity involves significant transcriptional and functional changes that are far from being fully understood. Most importantly, astrocyte reactivity seems to encompass multiple states, each having a specific influence on surrounding cells and disease progression. These diverse functional states of reactivity must be regulated by subtle signaling networks. Many signaling cascades have been associated with astrocyte reactivity, but among them, the JAK-STAT3 pathway is emerging as a central regulator. In this review, we aim (i) to show that the JAK-STAT3 pathway plays a key role in the control of astrocyte reactivity, (ii) to illustrate that STAT3 is a pleiotropic molecule operating multiple functions in reactive astrocytes, and (iii) to suggest that each specific functional state of reactivity is governed by complex molecular interactions within astrocytes, which converge on STAT3. More research is needed to precisely identify the signaling networks controlling the diverse states of astro-cyte reactivity. Only then, we will be able to precisely delin-eate the therapeutic potential of reactive astrocytes in each neurological disease context.

Reactive astrocytes mediate TSPO overexpression in response to sustained CNTF exposure in the rat striatum

International audienceThe 18 kDa translocator protein (TSPO) is a classical marker of neuroinflammation targeted for in vivo molecular imaging. Microglial cells were originally thought to be the only source of TSPO overexpression but astrocytes, neurons and endothelial cells can also up-regulate TSPO depending on the pathological context. This study aims to determine the cellular origin of TSPO overexpression in a simplified model of neuroinflammation and to identify the molecular pathways involved. This is essential to better interpret TSPO molecular imaging in preclinical and clinical settings. We used lentiviral vectors (LV) to overexpress the ciliary neurotrophic factor (CNTF) in the right striatum of 2-month-old Sprague Dawley rats. A LV encoding for β-Galactosidase (LV-LacZ) was used as control. One month later, TSPO expression was measured by single-photon emission computed tomography (SPECT) imaging using [ 125 I]CLINDE. The fluorescence-activated cell sorting to radioligand-treated tissue (FACS-RTT) method was used to quantify TSPO levels in acutely sorted astrocytes, microglia, neurons and endothelial cells. A second cohort was injected with LV-CNTF and a LV encoding suppressor of cytokine signaling 3 (SOCS3), to inhibit the JAK-STAT3 pathway specifically in astrocytes. GFAP and TSPO expressions were quantified by immunofluorescence. We measured a significant increase in TSPO signal in response to CNTF by SPECT imaging. Using FACS-RTT, we observed TSPO overexpression in reactive astrocytes (+ 153 ± 62%) but also in microglia (+ 2088 ± 500%) and neurons (+ 369 ± 117%), accompanied by an increase in TSPO binding sites per cell in those three cell populations. Endothelial cells did not contribute to TSPO signal increase. Importantly, LV-SOCS3 reduced CNTF-induced astrocyte reactivity and decreased global TSPO immunoreactivity (-71% ± 30%), suggesting that TSPO overexpression is primarily mediated by reactive astrocytes. Overall, this study reveals that CNTF induces TSPO in multiple cell types in the rat striatum, through the JAK2-STAT3 pathway in astrocytes, identifying this cell type as the primary mediator of CNTF effects neuroinflammatory processes. Our results highlight the difficulty to interpret TSPO imaging in term of cellular origin without addition cellular analysis by FACS-RTT or quantitative immunostainings. Consequently, TSPO should only be used as a global marker of neuroinflammation

18 kDa Translocator Protein TSPO Is a Mediator of Astrocyte Reactivity

An increase in astrocyte reactivity has been described in Alzheimer’s disease and seems to be related to the presence of a pro-inflammatory environment. Reactive astrocytes show an increase in the density of the 18 kDa translocator protein (TSPO), but TSPO involvement in astrocyte functions remains poorly understood. The goal of this study was to better characterize the mechanisms leading to the increase in TSPO under inflammatory conditions and the associated consequences. For this purpose, the C6 astrocytic cell line was used in the presence of lipopolysaccharide (LPS) or TSPO overexpression mediated by the transfection of a plasmid encoding TSPO. The results show that nonlethal doses of LPS induced TSPO expression at mRNA and protein levels through a STAT3-dependent mechanism and increased the number of mitochondria per cell. LPS stimulated reactive oxygen species (ROS) production and decreased glucose consumption (quantified by the [18F]FDG uptake), and these effects were diminished by FEPPA, a TSPO antagonist. The transfection-mediated overexpression of TSPO induced ROS production, and this effect was blocked by FEPPA. In addition, a synergistic effect of overexpression of TSPO and LPS on ROS production was observed. These data show that the increase of TSPO in astrocytic cells is involved in the regulation of glucose metabolism and in the pro-inflammatory response. These data suggest that the overexpression of TSPO by astrocytes in Alzheimer’s disease would have rather deleterious effects by promoting the pro-inflammatory response

Low-dose brain irradiation normalizes TSPO and CLUSTERIN levels and promotes the non-amyloidogenic pathway in pre-symptomatic TgF344-AD rats

International audienceAbstract Preclinical studies have recently evaluated the impact of low-dose brain radiation therapy (LD-RT) in animal models of Alzheimer’s disease (AD) showing anti-amyloid and anti-inflammatory effects of this treatment. Its effectiveness varied, however, depending on the LD-RT protocol used and the stage when the treatment was applied. In this study, we aimed to evaluate the therapeutic potential of 10 Gy delivered in five daily fractions of 2 Gy (a protocol previously shown to induce an improvement of cognitive performances) in 9-month-old TgF344-AD rats, modeling at a pre-symptomatic stage of the disease. We showed that at an early stage, LD-RT was able to lower levels of the 18-kDa translocator protein (TSPO)-mediated neuroinflammation to normal ranges in addition to the secreted CLUSTERIN, another inflammatory protein also involved in Aβ aggregation. In addition, we demonstrated that LD-RT reduces all amyloid forms (~ − 60 to − 80%, P < 0.01 ; soluble and aggregated forms of Aβ 40 , Aβ 42 , and Aβ oligomers ). Interestingly, we showed for the first time that sAPPα levels were improved by the treatment, showing a higher activation of the non-amyloidogenic pathway, that could favor neuronal survival. The current evidence confirms the capacity of LD-RT to successfully modulate two pathological hallmarks of AD, namely amyloid and neuroinflammation, when applied before symptoms onset

Complex roles for reactive astrocytes in the triple transgenic mouse model of Alzheimer disease

In Alzheimer disease (AD), astrocytes undergo complex changes and become reactive. Theconsequences of this reaction are still unclear. To evaluate the net impact of reactive astrocytes in AD,we recently developed viral vectors targeting astrocytes that either activate or inhibit the JAK2-STAT3 pathway, a central cascade controlling astrocyte reaction.We aimed to evaluate whether reactive astrocytes contribute to Tau as well as amyloid pathologies inthe hippocampus of 3xTg-AD mice, an AD model that develops Tau hyperphosphorylation andaggregation in addition to amyloid deposition. JAK2-STAT3 pathway-mediated modulation ofreactive astrocytes in the hippocampus of 3xTg-AD mice, did not significantly influence Tauphosphorylation or amyloid processing and deposition, at early, advanced and terminal stage of thedisease. Interestingly, inhibition of the JAK2-STAT3 pathway in hippocampal astrocytes did notimprove short-term spatial memory in the Y maze but it reduced anxiety in the elevated plus maze.Our unique approach to specifically manipulate reactive astrocytes in situ show these cells may impactbehavioral outcomes without influencing Tau or amyloid pathology

Modulation of astrocyte reactivity improves functional deficits in mouse models of Alzheimer’s disease

Abstract Astrocyte reactivity and neuroinflammation are hallmarks of CNS pathological conditions such as Alzheimer’s disease. However, the specific role of reactive astrocytes is still debated. This controversy may stem from the fact that most strategies used to modulate astrocyte reactivity and explore its contribution to disease outcomes have only limited specificity. Moreover, reactive astrocytes are now emerging as heterogeneous cells and all types of astrocyte reactivity may not be controlled efficiently by such strategies. Here, we used cell type-specific approaches in vivo and identified the JAK2-STAT3 pathway, as necessary and sufficient for the induction and maintenance of astrocyte reactivity. Modulation of this cascade by viral gene transfer in mouse astrocytes efficiently controlled several morphological and molecular features of reactivity. Inhibition of this pathway in mouse models of Alzheimer’s disease improved three key pathological hallmarks by reducing amyloid deposition, improving spatial learning and restoring synaptic deficits. In conclusion, the JAK2-STAT3 cascade operates as a master regulator of astrocyte reactivity in vivo. Its inhibition offers new therapeutic opportunities for Alzheimer’s disease