A_2AR upregulation in the temporal cortex of patients with frontotemporal dementia (FTLD-tau) carrying P301L mutations. (A) Western blot analysis of tau phosphorylation at S396 and A_2AR expression in the cortex of FTLD-tau patients and age-matched controls (CTRL). Data revealed an expected tau hyperphosphorylation at S396 in the cortex of FTLD-tau patients as well as a significant increase of A_2AR expression. *P < 0.05 versus Control using Student’s t-test. n = 3 per group. Putamen sample was used as a positive control for A_2AR immunoreactivity. (B) Immunohistochemical detection of A_2AR in the temporal cortex from the same FTLD-tau patients and age-matched controls. Staining intensity was first measured in individual A_2AR⁺ cells (red) detected in controls (n = 1616 cells) and FTLD-tau patients (n = 2153 cells) from 9–11 region of interests per individual representing n = 29–30 images per group overall. In line with biochemical data, quantitative immunohistochemical evaluation indicated an upsurge of cellular A_2AR expression in the cortex of FTLD-tau patients (i). In addition, in FTLD-tau patients, A_2AR staining intensity was compared between neurons exhibiting tau pathology i.e. AT8-positive (green, indicated by an arrow; n = 370) and AT8-negative cells (indicated by an asterisk; n = 1818) from 9–11 region of interests per individual representing 30 images per group overall. Analysis revealed a higher A_2AR staining intensity in AT8-positive neurons as compared to AT8-negative cells (ii). DAPI (blue) represents cell nuclei. ^***P < 0.001 versus Control using Student’s t-test. Results are expressed as mean ± SEM. Scale bar = 20 µm.

Conditional (Tet-Off) transgenic mouse model of neuronal A_2AR overexpression. (A) Conditional overexpression of A_2AR in neurons is achieved by crossing of CaMKII-tTA mice, expressing the transactivator protein tTA, under the control of a neuronal forebrain promoter (CaMKII) with the TRE-A_2AR strain, in which murine A_2AR is under the control of a Tet-responsive element. A_2AR expression is elicited in CaMKII-expressing neurons by the binding of the tTA protein to the TRE promoter. Transgene expression is maintained off from mating until offspring weaning (P28) by doxycycline (0.2 mg/ml in drinking water) to avoid potential perinatal effects linked to early A_2AR overexpression. (B) Representative western blots of A_2AR in the hippocampus of double CaMKII-tTA/TRE-A_2AR (A_2AR mice) and littermate controls (WT, wild-type). In absence of doxycycline, at P28 (P28 w/o Dox, left), double transgenic A_2AR animals exhibited receptor immunoreactivity while its level remained undetectable in the hippocampus of wild-type animals. Doxycycline treatment from mating to P28 (P28 w/ Dox, middle) abolished A_2A overexpression. Doxycycline removal from P28 promoted hippocampal A_2AR overexpression in the latter animals as exemplified in 6 month-old animals i.e. 5 months after doxycycline removal (right). (C) A_2AR immunostaining by immunohistochemistry under the same experimental conditions showing expression of the receptor in animals treated (middle) or not with doxycycline (left) as well as receptor re-expression following doxycycline withdrawal (right). Upper panels represent immunostainings at the level of the striatum and lower panels at the level of the hippocampus and cortex. Scale bar = 1 mm. (D) Co-immunostainings with A_2AR (red) and either neuronal (NeuN), microglial (Iba1) or astrocytic (GFAP and S100β) markers (green) showing the neuronal-specificity of A_2AR overexpression in CaMKII-tTA/TRE-A_2AR mice. DAPI (blue) represents cell nuclei. Scale bar = 20 µm. (E) Co-immunostainings between A_2AR (red), NeuN (as marker of mature neurons, white) and doublecortin (DCX, as marker of immature neurons, green) in CaMKII-tTA/TRE-A_2AR mice (A_2AR). A_2AR was not expressed in immature neurons. Scale bar = 100 µm. (F) Averaged time course of field excitatory postsynaptic potentials (fEPSP) after perfusion with SCH58261 (50 nM) for 30 min on hippocampal slices from wild-type and double CaMKII-tTA/TRE-A_2AR transgenic mice (*P < 0.05, n = 5 per group). A_2AR blockade significantly inhibited fEPSPs in double transgenic mice suggesting a gain of function of A_2AR upon their overexpression, whereby A_2AR exerts a tonic control on basal synaptic transmission, a phenomenon that is not observed in wild-type animals.

Neuronal overexpression of A_2AR favours spatial memory deficits in THY-Tau22 transgenic mice. Effects of neuronal overexpression of A_2AR on spontaneous activity, anxiety-like behaviour, spatial learning and memory of THY-Tau22 mice. (A and B) No change of either spontaneous locomotion or velocity was observed using actimetry. (C) Anxiety-like behaviour evaluated using elevated plus maze. Double transgenic mice overexpressing A_2AR performed as wild-type controls. As expected, tau transgenic mice spent more time in the open arms, a change similarly observed in triple tau/A_2AR transgenic mice. ^***P < 0.001 versus wild-type mice using one-way ANOVA followed by Tukey’s post hoc test. (D) Evaluation of the spatial learning using the Barnes maze task revealed that all groups of animals learned the position of the escape box in a time-dependent manner during the four days of training. (E) During the probe test, while displaying a preference, A_2AR mice spent significantly less time in the target quadrant (T) than wild-type controls. At the early age tested (5–6 months old), tau transgenic mice did not exhibit significant memory impairments with a strong preference for the target quadrant. In contrast, triple tau/A_2AR mice did not show preference for the target quadrant (T) over the other quadrants (O), supporting significant spatial memory deficits. ^$P < 0.05 versus wild-type mice; °P < 0.05, °°P < 0.01, °°°P < 0.001 versus Target quadrant using one-way ANOVA followed by Tukey’s post hoc test. (F) In agreement, the latency to reach the target hole was significantly increased for tau/A_2AR mice as compared to the other experimental groups. °°P < 0.01 versus wild-type mice using one-way ANOVA followed by Tukey’s post hoc test. n = 7–22 per group. Results are expressed as mean ± SEM.

Impact of neuronal A_2AR overexpression on hippocampal tau pathology. Human tau expression, phosphorylation and aggregation in the hippocampus of triple transgenic mice (tau/A_2AR) versus tau transgenic controls were evaluated by immunohistochemistry, bidimensional electrophoresis (2D) and western blots. (A) Co-immunostainings with A_2AR (red) and human tau (TauE1E2 antibody, human total tau, green) in the CA1 and dentate gyrus (DG) regions of triple tau/A_2AR transgenic mice. Neurons expressing human tau transgene (arrows) were found to overexpress A_2AR. DAPI (blue) represents cell nuclei. Scale bar = 50 µm. (B) 2D profile of total human tau (Cter antibody) in triple tau/A_2AR mice and littermate tau controls, shows an increase of tau isovariants in the acidic range of PI (arrow). (C) Quantification of tau phosphorylation at T181, S199, S212/T214 (AT100), S262, S396 and S404 epitopes, as well as dephosphorylated tau (tau-1) in triple tau/A_2AR animals and littermates tau controls. Analysis revealed tau hyperphosphorylation in tau/A_2AR mice signed by increased pS396 and reduced tau-1 (dephosphorylated tau). ^#P < 0.05, ^##P < 0.01 versus tau mice using Student’s t-test. n = 6–7 per group. (D) Conformational tau immunostaining using MC1 antibody in triple tau/A_2AR animals and littermates tau controls revealed no difference between groups. n = 5–11 per group. Scale bar = 500 µm. Results are expressed as mean ± SEM.

Neuronal overexpression of A_2AR promotes the upregulation of a microglial transcriptomic signature in the hippocampus of tau transgenic mice. RNA sequencing analysis of the hippocampus of wild-type, double A_2AR, tau and triple tau/A_2AR animals at the age of 6 months (n = 4 per genotype). (A) Volcano plot showing the 505 genes differentially regulated genes between tau/A_2AR mice and tau mice. Red dots represent significantly dysregulated genes with log2 fold change > 0.32 and adjusted P-value < 0.05. Sixty-four genes were found significantly upregulated (right) and 441 significantly downregulated (left). (B) Functional annotation of the 64 upregulated genes in the triple tau/A_2AR mice versus tau was performed with DAVID for GOTERM_Biological Process and showed a significant association with immune system processes, innate immune response and phagocytosis engulfment. (C) Known and predicted protein interaction (STRING) of the genes belonging to the significant GO term processes shown in C. (D) Heat map representing the cellular enrichment of each upregulated gene based on a transcriptome database of purified populations of neurons, astrocytes, oligodendrocyte precursor cells (OPC), newly formed oligodendrocytes (NFO), myelinating oligodendrocytes (MO), microglia and endothelial cells (). Relative cellular enrichment of each gene is given as the percentage of highest expression. Expression of 54 out of the 64 genes upregulated was knowledgeable in the database. Among these 54 genes, 33 were particularly enriched in microglial cells, contrasting with the lack of neuronal enrichment. (E) Cell number and cell morphology of Iba1-immunolabeled microglia (green) were analysed in confocal images using custom-written ImageJ plugins. A representative confocal image, the 3D reconstruction, visualization of spanned volume and cell skeleton derived from one representative cell in the confocal image are shown. (F and G) Quantification of microglia cell number and the morphological parameters ramification index, spanned volume and total tree length of cell skeleton revealed no difference between the mouse groups in the CA1 (F) or dentate gyrus (G) regions. n = 5–6 mice per genotype. Results are expressed as mean ± SEM. Scale bar = 20 μm.

Neuronal overexpression of A_2AR in tau mice is associated with upregulation of C1q and hippocampal atrophy. (A) Independent, quantitative PCR analysis of C1qa, C1qb and C1qc, the three genes encoding for functional heterotrimeric C1q protein. ^#P < 0.05 versus tau mice using one-way ANOVA followed by Tukey’s post hoc test. n = 6–14 per group. (B). Representative images of anti-C1q immunohistochemistry and related quantification showing an upregulation of C1q immunoreactivity in the hippocampus of tau/A_2AR mice, with a particular intense signal in the dentate gyrus (DG, arrow). Scale bar = 1 mm. ^###P < 0.001 versus tau mice using one-way ANOVA followed by Tukey’s post hoc test. n = 3–11 per group. (C) Morphometric analysis of dentate gyrus thickness in the hippocampus of all experimental groups ^#P < 0.05, ^##P < 0.01 versus tau mice using one-way ANOVA followed by Tukey’s post hoc test. n = 5–11 per group.

C1q upregulation in the temporal cortex of patients with frontotemporal dementia (FTLD-tau) carrying P301L mutations. Western blot analysis of C1q levels in the cortex of FTLD-tau patients and age-matched controls (CTRL) revealing a significant increase in tauopathic patients. ^**P < 0.01 versus Control using Student’s t-test. n = 3 per group.

Neuronal overexpression of A_2AR in tau mice is associated with synaptic loss. (A) Characteristic immunofluorescence for inhibitory and excitatory presynaptic markers VGAT (green) and VGLUT1 (red) in the dentate gyrus from wild-type, A_2AR, tau and tau/A_2AR mice, at low (scale bar = 50 µm) and higher magnification (inset, scale bar = 10 µm). A marked decrease in VGLUT1 but not VGAT immunoreactivity was observed in the molecular layer of the dentate gyrus of tau/A2AR mice. The total number of VGLUT1 synapses per area was decreased in tau/A_2AR mice in the molecular layer (B) but not in the hilus (D). VGAT synapses remained unaffected in the molecular layer (C). ^#P < 0.05 versus tau mice using one-way ANOVA followed by Tukey’s post hoc test, n = 14–16 images from four mice per group. Results are expressed as mean ± SEM.