Impairment of hippocampal astrocyte-mediated striatal dopamine release and locomotion in Alzheimer's disease.

BACKGROUND: Clinical and translational research has identified deficits in the dopaminergic neurotransmission in the striatum in Alzheimer's disease (AD) and this could be related to the pathophysiology of psychiatric symptoms appearing even at early stages of the pathology. HYPOTHESIS: We hypothesized that AD pathology in the hippocampus may influence dopaminergic neurotransmission even in the absence of AD-related lesion in the mesostriatal circuit. METHODS: We chemogenetically manipulated the activity of hippocampal neurons and astrocytes in wild-type and hemizygous TgF344-AD (Tg) rats, an animal model of AD pathology. We assessed the brain-wide functional output of this manipulation using in vivo Single Photon Emission Computed Tomography to measure cerebral blood flow and D2/3 receptor binding, in response to acute (3 mg kg-1 i.p.) and chronic (0.015 mg/ml in drinking water, 28 days) stimulation of neurons or astrocytes with clozapine N-oxide. We also assessed the effects of the chronic chemogenetic manipulations on D2 receptor density, low or high aggregated forms of amyloid Aß40 and Aß42, astrocytes and microglial reactivity, and the capacity of astrocytes and microglia to surround and phagocytize Aß both locally and in the striatum. RESULTS: We showed that acute and chronic neuronal and astrocytic stimulation induces widespread effects on the brain regional activation pattern, notably with an inhibition of striatal activation. In the Tg rats, both these effects were blunted. Chemogenetic stimulation in the hippocampus increased microglial density and its capacity to limit AD pathology, whereas these effects were absent in the striatum perhaps as a consequence of the altered connectivity between the hippocampus and the striatum. CONCLUSIONS: Our work suggests that hippocampal AD pathology may alter mesostriatal signalling and induce widespread alterations of brain activity. Neuronal and astrocytic activation may induce a protective, Aß-limiting phenotype of microglia, which surrounds Aß plaques and limits Αß concentration more efficiently.

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.

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.

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

The 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 ß-Galactosidase (LV-LacZ) was used as control. One month later, TSPO expression was measured by single-photon emission computed tomography (SPECT) imaging using [125I]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.

The 18 kDa translocator protein is associated with microglia in the hippocampus of non-demented elderly subjects.

Increase in the brain expression of the 18 kDa translocator protein (TSPO) is considered as a marker of neuroinflammation in the context of brain diseases, such as Alzheimer's disease (AD). However, in non-demented subjects with Alzheimer's neuropathology, TSPO accumulation in hippocampus subdivisions has not been fully characterized. To determine if TSPO is associated with the presence of amyloid ß plaques and/or phosphorylated Tau accumulation, we analyzed hippocampal sections using immunohistochemistry of 14 non-demented subjects with positive staining for Aß and/or phosphorylated Tau. TSPO expression was heterogenous with higher accumulation in the CA2/3 and subiculum subfields of the hippocampus. Its distribution closely resembled that of the microglial IBA1 marker and of the Aß42 amyloid form. In addition, positive correlations were observed between TSPO and IBA1 densities in CA4, CA2/3 and the subiculum but not with either the astrocyte GFAP marker or the AD-type Aß and Tau markers. This study sustains the hypothesis that TSPO is mainly associated with microglia and in Aß42-rich subdivisions in the hippocampus of non-demented elderly individuals.

Fluorescence-Activated Cell Sorting-Radioligand Treated Tissue (FACS-RTT) to Determine the Cellular Origin of Radioactive Signal.

Glial cells probably have a considerable implication in the pathophysiology of neurodegenerative disorders, such as Alzheimer's disease (AD). Their alterations are perhaps associated with a pro-inflammatory state. The TgF344-AD rat strain has been designed to express human APP and human PS1ΔE9 genes, encoding for amyloid proteins Aß-40 and Aß-42 and displays amyloid pathology and cognitive deficits with aging. The TgF344-AD rat model is used in this study to evaluate the cellular origin of the 18 kDa translocator protein (TSPO, a marker of glial cell activation) binding, and the 5HT2A-receptor (5HT2AR) serotonin receptor levels that are possibly disrupted in AD. The technique presented here is Fluorescence-Activated Cell Sorting to Radioligand Treated Tissue (FACS-RTT), a quantitative cell-type-specific technique complementary to in vivo PET or SPECT or ex vivo/in vitro autoradiography techniques. It quantifies the same radiolabeled tracer used prior for imaging, using a γ counter after cytometry cell sorting. This allows determining the cellular origin of the radiolabeled protein with high cellular specificity and sensitivity. For example, studies with FACS-RTT showed that (i) the increase in TSPO binding was associated with microglia in a rat model of lipopolysaccharide (LPS)-induced neuroinflammation, (ii) an increase in TSPO binding at 12- and 18-months was associated with astrocytes first, and then microglia in the TgF344-AD rats compared to wild type (WT) rats, and (iii) the striatal density of 5HT2AR decreases in astrocytes at 18 months in the same rat AD model. Interestingly, this technique can be extended to virtually all radiotracers.