Background
Formation of toxic amyloid-β (Aβ) species and their accumulation in plaques are key hallmarks in the pathogenesis of Alzheimer’s disease (AD) but it has now been recognized that, far ahead of plaques formation, soluble Aβ oligomers (Aβo) are the pathology-triggering species [
1]. More specifically, Aβo manage a progressive loss of synaptic connectivity leading to neurodegeneration. Astrocytes are important safeguards of synaptic function and it becomes increasingly evident that these cells accomplish a more important role in brain function than previously thought. The loss of synapses may reflect functional downfall of astrocytes. These cells possess receptors and signaling machinery for all known neurotransmitters thus sensing neuronal activity [
2]. They also actively secrete gliotransmitters such as ATP, glutamate, D-serine hence modulating activity of neuronal receptors [
3,
4]. Consequently, through their involvement in the tripartite synapse, they both sense and modulate synaptic output [
5].
Unlike neurons, astrocytes are electrically non-excitable cells but they are equipped with numerous channels, receptors or exchangers triggering Ca
2+ signals. Thus, astrocytic excitability is based on highly spatiotemporally coordinated fluctuations of intracellular Ca
2+ concentration relying on plasmalemmal and intracellular channels [
6]. Important progress has been made in studying astrocytes branches Ca
2+ signaling since they are the primary sites for interactions with neurons [
7‐
9]. Direct monitoring of Ca
2+ dynamics in the processes of adult mouse hippocampal astrocytes has revealed intense local and compartmentalized activity that is dissociated from activity in the cell body [
8]. However, until recently, it was difficult to specifically explore this astrocytic calcium activity since channels and receptors involved in astrocytic calcium signaling were commonly expressed in neurons. However, among the receptors involved in astrocytic calcium signaling, transient receptor potential A1 (TRPA1) channel seems to be specifically expressed in astrocytes and absent from neurons within hippocampus
stratum radiatum [
10,
11]. The discovery of TRPA1 channel as an important mediator of Ca
2+ entry restricted to astrocytes in the mouse hippocampus provided new opportunity to explore astrocyte signaling in relation to neuron-astrocyte interaction particularly in case of synaptic dysregulation.
In late transgenic AD mouse models, i.e. in phases associated with plaques formation, it has been shown that astrocytic calcium activity dramatically increases, becomes synchronous nearby Aβ plaques and coordinates calcium signals at long distance [
12]. Within somatosensory cortex, this astrocyte hyperactivity around plaques is mediated by purinergic signaling [
13]. Surprisingly, astrocytes behavior and reactions in early phases of AD remained largely undefined despite their probable involvement in the progressive loss of synaptic connectivity and in the complex and critical cellular phase of AD [
14]. We thus chose to study the impact of soluble Aβ oligomers on astrocytic function at the onset of early defensive cellular phase well before astrogliosis or inflammatory processes.
In this work, we monitored astrocytic calcium activity within the CA1 stratum radiatum region of the mouse hippocampus both at the astrocytic population level and at a single cell level, focusing in the astrocytic arbor. We characterized spontaneous Ca2+ signaling properties at these two related levels and showed that Aβo exposition induced at once a global and a local Ca2+ hyperactivity. We showed that this hyperactivity was independent of neuronal activity and was totally restored to physiological level by blocking TRPA1 channels. This TRPA1 channels-dependent influence of Aβo on astrocyte activity consequently impacted neighboring CA1 neurons, increasing glutamatergic spontaneous activity. In an AD mouse model, we showed that astrocyte hyperactivity was an early phenomenon setting up at the onset of Aβ production, being also related to neuronal hyperactivity and preceding TRPA1 channel overexpression. Overall, our data provide a novel mechanism for the understanding of early toxicity of soluble Aβo species.
Methods
Slice preparation
Coronal hippocampal slices (ranging from 300 to 350 μm thick) were prepared from Swiss mice (postnatal day 17–23; Janvier, Le Genest St Isle, France) or APP/PS1–21 transgenic mice [
15] (postnatal day 19–28) together with control littermates. Mice were killed by decerebration and decapitated. The brain was rapidly removed and cut in ice-cold cutting ACSF containing (in mM): 2.5 KCl, 7 MgCl
2, 0.5 CaCl
2, 1.2 NaH
2PO
4, 25 NaHCO
3, 11 D-glucose and 234 sucrose bubbled with 95% O
2 and 5% CO
2, with a vibratome VT1200S (Leica, Wetzlar, Germany). Slices containing the hippocampus were placed in ACSF containing (in mM): 126 NaCl, 2.5 KCl, 1.2 MgCl
2, 2.5 CaCl
2, 1.2 NaH
2PO
4 bubbled with 95% O
2 and 5% CO
2 and supplemented with 1 mM sodium pyruvate at room temperature for a recovery period.
Slices bulk loading with Fluo-4 AM.
Briefly, 350 μm coronal slices were loaded with the calcium indicator dye Fluo-4 by immersion for 45 min at 35 °C in a bath containing 5 μM Fluo-4 AM (Life Technologies), 0.01% Pluronic acid F-127 (Molecular Probes), 0.005% Cremophor EL (Sigma-Aldrich) and 0.05% DMSO (dimethyl sulfoxide, Sigma-Aldrich) in ACSF. The loading chamber was continuously oxygenated with 95% O
2 and 5% CO
2. Slices were then transferred into dye-free ACSF for at least 45 min prior to experiments. Mainly live astrocyte took up the fluorescent dye with these conditions [
16,
17].
Single-astrocyte dye loading with Fluo-4
Coronal 300 μm slices were transferred to a chamber allowing constant perfusion with ACSF at room temperature, bubbled with 95% O
2 and 5% CO
2 on the stage of an upright compound microscope (Eclipse E600 FN, Nikon, Paris, France) equipped with a water immersion 60× objective (NA 1.0) and an infrared-differential interference contrast optics with CCD camera (Optronis VX45, Kehl, Germany). Glass pipettes 8–11 MΩ (Harvard apparatus) were filled with intracellular solution containing (in mM): 105 K-gluconate, 30 KCl, 10 phosphocreatine, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Tris, 0.2 Fluo-4 pentapotassium salt (Life Technologies), adjusted to pH 7.2 with KOH. Signals were amplified by Axopatch 200B, sampled by a Digidata 1440A interface and recorded with pClamp8 software (Molecular Devices, Foster City, USA). Astrocytes were identified based on morphological, localization in the
stratum radiatum and negative resting potential (between −70 and −80 mV). Input resistance was calculated by measuring current in response to a 10 mV pulse with 80 ms duration, near the end of the voltage command. Only passive astrocytes showing linear
I/V relationship and low input resistance (~ 50 MΩ) were kept for dye loading. After achieving whole-cell configuration, access resistance was constantly monitored, and astrocytes were excluded from this study when this parameter varied >20% throughout the experiment. To allow sufficient diffusion of the dye and avoid astrocyte dialysis, the time in whole-cell configuration was limited to less than 5 min. Then, the patch pipette was carefully withdrawn to allow the astrocyte to recover. In order to maximize the diffusion of the dye into the astrocytic processes, we waited at least 15 min before calcium imaging [
8,
18].
Calcium imaging
Bulk or single-astrocyte loaded slices were placed in a constantly perfused chamber on the stage of an upright compound microscope (Eclipse E600 FN, Nikon, Paris, France) equipped with a water immersion 40× (NA 0.8) or 60× (NA 1.0) objective and a confocal head (confocal C1 head, Nikon, Paris, France). Excitation was achieved with light at 488 nm and emission was filtered with a 515 ± 15 nm filter. Images were acquired with EZ-C1 software (Nikon, Paris, France) at 1.2 s intervals in a single confocal plane over a period of 5 min.
Bulk loading calcium imaging data analysis
Prior to analysis, raw images were stabilized (when needed if slight
x-y drift occurred during recordings, z drifts were excluded) using ImageJ plugin Template Matching.
CalSignal software was used to measured intracellular Ca
2+ activity, analyzing the fluorescence signal F within each region of interest (ROI) corresponding to the cell body area of each astrocyte [
19]. F
0 was calculated for each ROI on the recording period. Based on the ΔF/F
0 ratios, significant fluorescence variations were detected and a Ca
2+ event was defined as a significant and continuous signal increase larger than a fixed threshold followed by a significant and continuous signal decrease larger than the same threshold. Thus, cells were defined as active when fluorescence increased ≥2 standard deviations relative to baseline fluorescence. After peak detection, each Ca
2+ transients were visually checked by the operator. The theoretical Poisson distribution was calculated by the method of least squares approximating λ until it most closely fits the observed frequency distribution.
Single astrocyte loading data analysis
Ca
2+ transients were measured in two-dimensional images, in individual subregions matching the shape of the astrocyte structure. Manually selected ROIs (~1 μm
2) were placed along astrocytic processes lying in the focal plane [
8] and a ROI was also selected in the soma if accessible. Prior to analysis, raw images were stabilized (when needed if slight
x-y drift occurred during recordings, z drifts were excluded) using ImageJ plugins Template Matching and filtered with 3D Hybrid Median Filter [
7].
CalSignal software was used to measure intracellular Ca
2+ activity, analyzing the fluorescence signal F within each ROI. As described above, significant changes in fluorescence were detected on the basis of the calculated ΔF/F
0 ratios. Each Ca
2+ transients within ROI were visually checked by the operator and reported in a raster plot in order to discriminate focal activities from expanded ones (as described by [
8]). At the end of each recording, z-stacks (0.5 μm steps) were performed to obtain tri-dimensional projections of astrocyte territories revealed by Fluo-4 loading. Images were then filtered with 3D Hybrid Median Filter plugin in ImageJ.
Electrophysiological recordings
Whole-cell recordings were made from the somata of visually identified CA1 pyramidal neurons. Patch pipettes (4–6 MΩ) were filled with an internal solution containing (in mM): 105 K-gluconate, 30 KCl, 10 phosphocreatine, 10 HEPES, 4 ATP-Mg, 0.3 GTP-Tris, 0.3 EGTA, adjusted to pH 7.2 with KOH. Spontaneous excitatory post-synaptic currents (sEPSCs) were collected at a membrane holding potential of −65 mV which is close to the reverse potential of GABA. All recordings were done at room temperature (22–24 °C) and only a single neuron was studied per slice. sEPSCs and their kinetics were analyzed in 5-min epochs within the time frame of the recordings. Each epoch was compared to the initial 5-min recording and sEPSCs frequencies were normalized to this initial value. Access resistance was constantly monitored and recordings were excluded from this study when this parameter varied >20% throughout the experiment. Recordings were analyzed using the Clampfit module of the pClamp8 software (Molecular Devices, Foster City, USA) with a threshold at −20 pA to exclude miniature EPSCs.
Aβ oligomerization, monomer purification and drug application
Recombinant Aβ
1–42 peptide (Bachem) was resuspended in 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) to 1 mM until complete resuspension as described previously [
20]. Following HFIP evaporation, Aβ oligomers were prepared by diluting Aβ to 1 mM in DMSO, then to 100 μM in ice-cold ACSF with immediate vortexing and bath sonication, and then incubated at 4 °C for 24 h with mild agitation.
When appropriate, the Aβ monomer is purified on a C18 column (SPE-Chromabond-HRX C18 ec, 200 μl, 5 mg, Macherey-Nagel, France). The column was equilibrated with 0.1% trifluoroacetic acid (TFA) in water. Immediately after dilution in DMSO, the Aβ sample was loaded and the column was washed three times with 0.1% TFA. Then, a gradient of acetonitrile from 30 to 60% was applied (Additional file
1). Fractions (0.1 ml) were collected. The elution profile was determined by measuring the absorbance at 275 nm. The peak fraction was collected and the concentration of peptide was determined by absorbance at 275 nm using ɛ
275 nm = 1400 M
−1 cm
−1. The peptide is then stored at −80 °C.
Aβo, Aβm, tetrodotoxin (TTX; Latoxan, Valence, France), Ca2+-free solution (ACSF - 0 Ca2+ - 1 mM EGTA) and HC 030031 (Sigma-Aldrich) were bath applied at the appropriate concentration during 5 min before and during calcium imaging or electrophysiological recordings. Minocycline hydrochloride (Sigma-Aldrich) was bath applied during 15 min before and during calcium imaging recording.
Immunohistochemistry
Mice were deeply anesthetized with 10% chloral hydrate and perfused intracardially with 10 ml 0.9% NaCl followed by 35 ml 4% paraformaldehyde in 0.1 M PBS, pH 7.3. Brains were rapidly removed, post-fixed overnight at 4 °C in 4% paraformaldehyde, immersed in 20% sucrose in 0.1 M PBS, pH 7.5 overnight, frozen in cooled (−35 °C) isopentane and stored at −30 °C. Serial frontal sections (30 μm thick) were cut with a cryostat microtome (HM 500 M, Microm, Francheville, France). Sections were blocked by incubation with 3% bovine serum albumin in TBS-Tween-Triton (TBSTT) (0.1 M Tris Base, 0.15 M NaCl, 0.1% Tween, 0.1% Triton X-100) for 30 min (dilution/blocking buffer). Tissue sections were then incubated overnight at 4 °C with either an anti-NeuN antibody (AbCys, France, mouse monoclonal; 1:500), anti-GFAP antibody (Molecular Probes, USA, mouse monoclonal; 1:1000), anti-TRPA1 antibody (Novus, USA, rabbit polyclonal; 1:100) or anti-Iba-1 antibody (Wako, USA, rabbit polyclonal; 1:500). Tissue sections were washed in TBSTT and incubated for 2 h at room temperature with Cyanin 3- (Jackson Immuno Research Laboratories, USA; 1:1000) or Alexa 488-conjugated secondary antibodies (Life Tecchnology, USA; 1:1000). Sections were washed in TBSTT and mounted in Dako fluorescent mounting medium (Dako, USA).
When appropriate, amyloid-β deposits were stained using Thioflavine S [
21]. Sections were re-hydrated in TBS buffer (0.1 M Tris Base, 0.15 M NaCl), incubated in filtered 1% aqueous Thioflavine S (Sigma, France) for 8 min at room temperature, in the dark and washed several times in TBS buffer.
Image acquisition
Sections were examined with a Zeiss LSM 710 confocal laser scanning microscope with a Plan Apochromat 20× objective (NA 0.8) or an oil immersion Plan Neofluor 40× objective (NA 1.3) and translating platform with motorized crossed roller stages. When appropriate, mosaics were acquired for each channel separately with “Zen” software, in a 12-bit format, using the tile scan function. For TRPA1 and GFAP co-staining, sections were also acquired with a Zeiss Airyscan module with an oil immersion Plan Apochromat 63× objective (NA 1.46) to improve lateral resolution (~140 nm) and signal-to-noise ratios. For illustration, images were merged with ImageJ software.
Immunoblotting
Dissected hippocampi from 1-month old APP/PS1–21 mice were homogenized in cold buffer containing 0.32 M sucrose and 10 mM HEPES, pH 7.4. Samples were maintained at 4 °C during all steps of the experiments. Homogenates were cleared at 1000 x g for 10 min to remove nuclei and large debris. Samples in loading buffer were boiled for 10 min and equal amounts of proteins (20 μg, quantified by micro-BCA assay (Pierce) in duplicate extracts) were resolved on a 4–20% gradient Bis-Tris polyacrylamide precast stain free gels (Bio-Rad) in denaturing conditions. Proteins were transferred to a polyvinylidene difluoride membrane (Millipore) for 30 min at 4 °C. Membranes were blocked with 3% dry milk in Tris-Buffered Saline (TBS: 10 mM Tris, 150 mM NaCl, pH 7.4) containing 0.1% Tween for 1 h at room temperature. Membranes were probed with anti-TRPA1 antibody (Novus, USA; 1:2000) and anti-GFAP antibody (Dako, USA, rabbit polyclonal; 1:100000) diluted in 3% dry milk in 0.1% Tween TBS overnight at 4 °C. Membranes were washed in 0.2% Tween TBS and probed with HRP-conjugated anti-rabbit IgG (Fab’) (Interchim, France; 1:40,000) antibody for 45 min at room temperature. After washes, specific proteins were visualized with an enhanced chemiluminescence ECL Detection System (Bio-Rad) and the chemidoc system (Bio-Rad). Chemiluminescence signals were normalized to protein loading signals acquired using Stain-free pre-cast gels (Bio-Rad).
Statistical analysis
Data were analyzed using R (the R Project for Statistical Computing) [
22]. Comparisons between two groups were conducted with the two-tailed Mann-Whitney test. Kruskal-Wallis test followed by Pairwise comparison using Wilcoxon rank sum test was used when needed for multiple comparisons. Proportions of hyperactive/active astrocyte and focal/expanded activities were compared with χ
2-test. Data were expressed as mean ± SEM accompanied by distribution of experimental points. Graphic significance levels were *,
p < 0.05; **,
p < 0.01 and ***,
p < 0.001.
Discussion
In this study, we investigated the contribution of astrocytes in early Aβo toxicity by studying calcium signaling in different parts of the whole astrocyte territory. We found that astroglia is a frontline target of Aβo exhibiting a global and local Ca2+ hyperactivity that involves TRPA1 channels. This TRPA1 channel-dependent astrocytic Ca2+ hyperactivity exerts regulatory influences on synaptic function and is linked to the glutamatergic synapse hyperactivity recorded in CA1 neurons. Concurrently to these acute Aβo-induced effects, astrocytes in young APP/PS1–21 mice hippocampus elicit a similar pattern of calcium hyperactivity in close relationship with the setting up of a precocious neuronal hyperactivity that are both reversed when TRPA1 channel is blocked. Moreover, the TRPA1 channel is gradually overexpressed at the onset of Aβ production in this AD mouse model.
Intracellular Ca
2+ transients are considered as the primary signal by which astrocytes interact with each other and with neighboring neurons. Ca
2+ has been extensively studied within the astrocytic cell body and thick branches. More recently, local Ca
2+ dynamics in distal fine processes has been investigated emphasizing a highly compartmentalized signaling, interconnected with physiological transmission at neighboring synapses [
8,
9]. Compartmentalization of astrocytic Ca
2+ dynamics needs to be attentively considered in order to understand how astrocytes may contribute to brain information processing [
8]. We thus chose to study both levels of information (i.e. global population signaling and local microdomain signaling) combining bulk loading and single cell astrocyte loading. Genetically encoded Ca
2+ indicators (GECIs) have been recently used to study Ca
2+ signals in distal thin processes [
9]. Alternatively, patch pipette loading give access to the whole territory of a single astrocyte and, currently, Fluo-4 is far more sensitive than GECIs therefore enabling to track smaller signals [
8]. Accordingly, we observed a similar and even better diffusion of Fluo-4 in a single astrocyte compared to SR101. We first characterized the physiological calcium activity of mouse CA1
stratum radiatum astrocytes and showed that this activity is fully autonomous, i.e. independent of neuronal activity, both at the astrocytic population level and at the microdomain level. This is in agreement with data obtained in mouse CA1 hippocampus [
30,
33] but not with astrocyte behavior in the dentate gyrus where expanded Ca
2+ events were partly dependent on neuronal activity [
8]. Interestingly, external Ca
2+ entry is the main source of Ca
2+ within thin processes whereas it only partly contributes to somatic signaling. This discrepancy between Ca
2+ sources in astrocyte soma and distal processes has already been described in brain slices [
34]. This might be supported by the subcellular location of calcium stores that are concentrated in the cell body and thick processes but are almost absent from thin processes [
23].
A central element of the pathogenesis of AD is the progressive accumulation of Aβo species, ultimately resulting in the formation of plaques. Yet, small soluble Aβ oligomers are sufficient to induce several features of the AD phenotype [
1]. The paths by which Aβo leads to neurodegeneration are probably multifactorial but all converge all towards synaptic dysfunction. The major challenge of AD research is to understand the complex cellular reaction underlying the long prodromal phase of AD [
14]. Astrocytes are an integral part of synaptic transmission and are therefore critical for the establishment and maintenance of neuronal health [
5]. They contribute to neuronal dysfunction by being proinflammatory [
35] but also play a protective role, e.g. through the release of gliotransmitters [
36] and Aβ clearance [
37,
38]. It is therefore of major importance to distinguish the beneficial from the deleterious impact of Aβo on astrocyte function. Aβo has already been involved as a direct effector on astrocytes in primary cultures [
39], in hippocampal slices [
40] and in vivo [
12] but here we showed a peculiar rapid action on compartmentalized calcium activity, activating a membrane Ca
2+ permeable channel. Indeed, Aβo application triggers global Ca
2+ hyperactivity in CA1 hippocampus astroglial population together with an intensification of the compartmentalized Ca
2+ activity in the astrocytic processes and a spatial extension of the size of the expanded Ca
2+ events within microdomains. These effects are specific to oligomeric forms of Aβ since application of the monomeric form in the same conditions had no impact on astrocytic calcium activity. We reported that the effect of Aβo on astrocyte excitability is fully independent of neuronal activity since TTX application does not prevent the Aβo effect neither on the global hyperactivity nor on the compartmentalized hyperactivity in processes. Concurrently, microglia activation does not participate in this astrocytic hyperactivity, at least in the time scale studied, whereas longer applications of Aβ activate microglia together with astroglia [
24]. Thus, astrocytes seem to express a distinctive precocious detector involved at the onset of Aβo appearance. Removal of external Ca
2+ largely inhibits Aβo-induced astrocyte hyperactivity at the population level while it has no effect in physiological conditions. Removal of external Ca
2+ also inhibits the majority of the compartmentalized Aβo-induced hyperactivity. Thus, transmembrane Ca
2+ entry carries most of the Aβo-induced hyperactivity. Remarkably, we showed that both global and compartmentalized hyperactivities are driven by TRPA1-dependent Ca
2+ entry since HC 030031, a specific TRPA1 channel inhibitor [
29], strongly abolishes Aβo-induced astrocyte hyperexcitability and totally restores the spatiotemporal properties of Ca
2+ events back to a physiological level.
The TRPA1 channel is a Ca
2+ permeable non-selective cation channel initially known to be expressed in primary afferent nociceptive neurons [
29]. In mouse CA1 hippocampus, TRPA1 channels are found to be preferentially expressed in astrocytes [
10]. However, their involvement in physiological astrocytic Ca
2+ signaling is highly debated. It has been shown that TRPA1 channels contribute to maintain basal Ca
2+ levels and regulate ~20% of spontaneous Ca
2+ signals within astrocyte branches [
11] but in the end, poorly take part in basal astrocytic Ca
2+ signaling [
30,
33]. Our data assume that TRPA1 channels are only slightly involved in the astrocytic Ca
2+ signaling in physiological conditions. However, we highlighted they are quickly and largely involved in case of Aβo presence. The absence of an obvious involvement of these channels in astrocyte physiological Ca
2+ signaling is startling since we evidenced a TRPA1 channel expression in thick and in adjacent thin astrocytic processes. Thus, TRPA1 channels might only behave as an “aggression sensor”. Indeed, TRPA1channel gating is particularly regulated by numerous electrophilic activators - such as reactive oxygen species, reactive nitrogen species or oxidized lipids - and also functions as a mechanosensor [
27]. Hence, TRPA1 channels might be directly targeted by Aβo or might be secondarily activated through an Aβo-induced oxidative stress and/or through its mechanosensor properties if Aβo binds to the astrocytic cholesterol-rich plasma membrane [
14].
Strikingly, in young APP/PS1–21 mice (~3–4 weeks), we observed a similar pattern of astrocytic hyperactivity starting at the beginning of Aβ overproduction in the hippocampus, long before its aggregation into plaques. These early repercussions in young APP/PS1–21 mice were restricted to the frequency of astrocytic Ca
2+ events in either the astrocytic population and microdomains of astrocytic processes with fewer impacts on the proportion of active cells or microdomains. This suggests a gradual impact of surrounding Aβ on astrocyte signaling, increasing the frequency of compartmentalized Ca
2+ events and, to a lesser extent, the proportion of active territories within the astrocytic processes. These impacts on astrocytic processes go along with a noteworthy redistribution of the frequency of Ca
2+ events within the astrocytic population. Interestingly, blockade of TRPA1 channels with HC 030031 abolished the astrocyte Ca
2+ hyperactivity. Overall, TRPA1 channel signaling seems to be at the frontline in mediating these Aβo progressive effects in early stages of AD. Data obtained in an advanced AD transgenic model showed an astrocyte network hyperactivity in cortical areas close to Aβ plaques and an involvement of metabotropic purinergic signaling in this astrocyte hyperactivity [
13]. This suggests a differential evolution of astrocyte engagement in AD pathogenesis depending on the stage, the structure and the physiopathological state of the astrocyte. Likewise, it has been reported that the TRPA1 channels’ protein level was increased in hippocampal astrocytes of 8 month-old APP/PS1 mice at where it mediated inflammation through astrocyte activation [
41]. Here, we showed that TRPA1 channel expression in hippocampus is increased much earlier, as soon as 1 month of age, in a more aggressive AD mouse model (APP/PS1–21). These data point towards a TRPA1 channel contribution in early stages of pathophysiology, that is as soon as the Aβo level increases and long before the setting up of astrogliosis or inflammatory mechanisms.
Numerous laboratory studies in the past decade have shown that Aβo impairs synaptic function and synaptic structure [
42]. However, how soluble Aβo initiates these effects remains to be determined. Each astrocyte deploys many fine processes to contact up to 140,000 synapses in the CA1 region [
43]. As we highlighted an intense and early effect of Aβo on astrocyte Ca
2+ activity within processes, we assessed the link with spontaneous neuronal activity. Indeed Aβo is also known to enhance spontaneous neuronal excitability in CA1 [
32,
44,
45]. Consistently, we showed here that Aβo induces a rapid and strong increase of spontaneous EPSCs frequency in CA1 neurons. Strikingly, blocking TRPA1 channels totally prevents this Aβo neuronal impact. Yet, when we blocked neuronal activity with TTX, we did not affect the Aβo-induced astrocyte hyperactivity which would be partly the case if a neuronal TRPA1 was involved. This precocious Aβo impact thus seems to trigger a one-way communication from astrocyte to neuron related to TRPA1 activation. This TRPA1-dependent neuronal hyperactivity was similarly observed in APP/PS1–21 mice at the onset of Aβ overproduction testifying its physiopathological relevance in the AD initiation process. It has been shown that Aβ can increase astrocytic release of glutamate to the extrasynaptic space resulting in the activation of extrasynaptic NMDARs and the disruption of neuronal signaling [
40,
46,
47]. Besides, astrocytes can regulate synaptic and extrasynaptic neurotransmitter concentrations, such as glutamate, in a Ca
2+-dependent manner e.g. via vesicular release, bidirectional transport or hemichannel opening [
48]. We will further decipher pathways implemented by the Aβ-induced TRPA1-mediated Ca
2+ entry that consequently affect neuronal transmission.
It has been demonstrated that soluble Aβo can affect astrocyte signaling properties in various ways in mouse hippocampal CA1 astrocytes [
40,
49,
50]. To some extent, the involvement of TRPA1 channels superimposed to these effects, directly affecting local synaptic function in a distinctive precocious manner. This actor might thus contribute to the complex cellular phase of AD, upstream of symptomatic neurodegeneration [
14].