Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model

Therapeutic approaches providing effective medication for Alzheimer’s disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms, while a pharmacological treatment that can reverse synaptic and memory deficits in AD mice was thus far not identified. Repurposing food and drug administration (FDA)-approved drugs for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. The sphingosine-1 phosphate analog fingolimod (FTY720) was approved recently for treatment of multiple sclerosis patients. Here, we addressed whether fingolimod rescues AD-related synaptic deficits and memory dysfunction in an amyloid precursor protein/presenilin-1 (APP/PS1) AD mouse model when medication starts after onset of symptoms (at five months). Male mice received intraperitoneal injections of fingolimod for one to two months starting at five to six months. This treatment rescued spine density as well as long-term potentiation in hippocampal cornu ammonis-1 (CA1) pyramidal neurons, that were both impaired in untreated APP/PS1 animals at six to seven months of age. Immunohistochemical analysis with markers of microgliosis (ionized calcium-binding adapter molecule 1; Iba1) and astrogliosis (glial fibrillary acid protein; GFAP) revealed that our fingolimod treatment regime strongly down regulated neuroinflammation in the hippocampus and neocortex of this AD model. These effects were accompanied by a moderate reduction of Aβ accumulation in hippocampus and neocortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology that is believed to underlie memory deficits in AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes

Anti-Inflammatory Treatment with FTY720 Starting after Onset of Symptoms Reverses Synaptic Deficits in an AD Mouse Model

Therapeutic approaches providing effective medication for Alzheimer’s disease (AD) patients after disease onset are urgently needed. Previous studies in AD mouse models suggested that physical exercise or changed lifestyle can delay AD-related synaptic and memory dysfunctions when treatment started in juvenile animals long before onset of disease symptoms, while a pharmacological treatment that can reverse synaptic and memory deficits in AD mice was thus far not identified. Repurposing food and drug administration (FDA)-approved drugs for treatment of AD is a promising way to reduce the time to bring such medication into clinical practice. The sphingosine-1 phosphate analog fingolimod (FTY720) was approved recently for treatment of multiple sclerosis patients. Here, we addressed whether fingolimod rescues AD-related synaptic deficits and memory dysfunction in an amyloid precursor protein/presenilin-1 (APP/PS1) AD mouse model when medication starts after onset of symptoms (at five months). Male mice received intraperitoneal injections of fingolimod for one to two months starting at five to six months. This treatment rescued spine density as well as long-term potentiation in hippocampal cornu ammonis-1 (CA1) pyramidal neurons, that were both impaired in untreated APP/PS1 animals at six to seven months of age. Immunohistochemical analysis with markers of microgliosis (ionized calcium-binding adapter molecule 1; Iba1) and astrogliosis (glial fibrillary acid protein; GFAP) revealed that our fingolimod treatment regime strongly down regulated neuroinflammation in the hippocampus and neocortex of this AD model. These effects were accompanied by a moderate reduction of Aβ accumulation in hippocampus and neocortex. Our results suggest that fingolimod, when applied after onset of disease symptoms in an APP/PS1 mouse model, rescues synaptic pathology that is believed to underlie memory deficits in AD mice, and that this beneficial effect is mediated via anti-neuroinflammatory actions of the drug on microglia and astrocytes

Modulations of ASIC3 channels by endogenous lipids and temperature

Les canaux ioniques ASIC3 (Acid-Sensing Ion Channel 3) sont des canaux sodiques excitateurs majoritairement exprimés dans le système nerveux périphérique. Les variations extracellulaires du pH sont considérées comme le principal signal activateur des ASIC3, qui sont essentiellement vus comme des senseurs de protons. Ces dernières années, il a été démontré que la lysophosphatidylcholine (LPC) et l'acide arachidonique (AA), qui sont des lipides endogènes, activent les canaux ASIC3 à pH physiologique en l'absence d'acidification extracellulaire.Durant ma thèse, j'ai participé à une étude mettant en évidence des taux importants de LPC16:0 dans les liquides synoviaux de patients souffrants de diverses pathologies articulaires douloureuses, incluant l'osthéoartrose. Ces taux de LPC16:0 ont été corrélés avec les scores de douleur dans la cohorte de patients ostéoarthritiques, et des injections intra-articulaires de ce lipides chez l'animal induisent un comportement douloureux chronique associé à des comorbidités anxio-dépressives. Les animaux invalidés pour le canal ASIC3, ou co-injectés avec un inhibiteur d'ASIC3 sont protégés de ces effets.J'ai pu montrer in vitro que l'application de LPC16:0 est capable d'activer spécifiquement le canal ASIC3 exprimé en système hétérologue, mais également les canaux ASIC3 natifs des neurones sensoriels et nociceptifs issus des ganglions rachidiens (DRG) de souris en culture primaire. De plus, les courants natifs induits par la LPC16:0 dans les neurones DRG sont associés à une dépolarisation membranaire dépendante d'ASIC3. Grace à ces travaux, nous pouvons émettre l'hypothèse que la LPC16:0 intra-articulaire active les canaux ASIC3 des neurones DRG innervant l'articulation, générant une dépolarisation qui conduit au développement d'un état douloureux chronique, au moins chez la souris et potentiellement chez l'homme.Mes travaux de thèse m'ont également permis d'identifier deux nouveaux lipides endogènes capables de moduler positivement les canaux ASIC3. Il s'agit du lysoPAF et du PAF, deux éther-lipides analogues structuraux de la LPC. Ils sont non seulement capables de potentialiser les courants ASIC3 induits par l'acidité, mais aussi d'activer les canaux en l'absence d'acidification extracellulaire et sont indépendants du récepteur au PAF. Ces effets sont comparables à ceux de la LPC et il semble que les trois lipides agissent sur le canal ASIC3 via un mécanisme d'action commun. Mes résultats suggèrent plutôt un effet direct sur le canal, ou dans son environnement proche, plutôt qu'un effet lié à des déformations membranaires par les lipides. J'ai pu démontrer que le lysoPAF est capable d'activer les canaux ASIC3 natifs dans les neurones DRG et d'induire une dépolarisation membranaire concomitante, suggérant que ce lipide pourrait, comme la LPC, avoir des effets proalgiques.J'ai également pu montrer durant ma thèse que les canaux ASIC3 sont sensibles aux froid. Les basses températures ayant pour effet augmenter l'amplitude des courants induit par l'acidification et de ralentir les cinétiques d'inactivation. Cependant, la délétion d'ASIC3 ne semble pas altérer la sensibilité au froid des neurones DRG en culture primaire.Pour conclure, mes travaux de thèse ont permis de faire mettre en évidence la LPC16:0 comme facteur déclencheur des douleurs articulaires, via l'activation d'ASIC3. De plus, j'ai pu identifier deux nouveaux modulateurs lipidiques endogènes du canal ASIC3, le PAF et le lysoPAF, ce qui ouvre de nouvelles perceptives quant au(x) rôle(s) de ces lipides dans la douleur. La modulation des canaux ASIC3 par différents stimuli tels que l'acidité, les lipides et le froid, renforcent l'idée que ce camanl se comporte comme un détecteur de coincidence.Acid-Sensing Ion Channel 3 (ASIC3) are excitatory sodium channels predominantly expressed in the peripheral nervous system. Extracellular pH changes are considered the main activating signal for ASIC3, which are essentially seen as proton sensors. In recent years, it has been shown that lysophosphatidylcholine (LPC) and arachidonic acid (AA), which are endogenous lipids, activate ASIC3 channels at physiological pH in the absence of extracellular acidification.During my thesis, I participated in a study that demonstrated significant levels of LPC16:0 in synovial fluids of patients suffering from various painful joint pathologies, including osteoarthritis. These LPC16:0 levels were correlated with pain scores in the osteoarthritic cohort, and intra-articular injections of this lipid in animals induced chronic pain behaviour associated with anxiety-depressive comorbidities. Animals invalidated for the ASIC3 channel, or co-injected with an ASIC3 inhibitor are protected from these effects.I was able to show in vitro that the application of LPC16:0 is able to specifically activate the heterologously expressed ASIC3 channel, but also the native ASIC3 channels of sensory and nociceptive neurons from dorsal root ganglia (DRG) of primary cultured mice. Furthermore, native LPC16:0-induced currents in DRG neurons are associated with ASIC3-dependent membrane depolarisation. Based on this work, we can hypothesise that intra-articular LPC16:0 activates ASIC3 channels in DRG neurons innervating the joint, generating a depolarisation that leads to the development of a chronic pain state, at least in mice and potentially in humans.My thesis work also allowed me to identify two new endogenous lipids capable of positively modulating ASIC3 channels. These are lysoPAF and PAF, two ether lipids that are structural analogues of LPC. They are not only able to potentiate acid-induced ASIC3 currents, but also to activate the channels in the absence of extracellular acidification and are independent of the PAF receptor. These effects are comparable to those of LPC and it appears that all three lipids act on the ASIC3 channel via a common mechanism of action. My results suggest a direct effect on the channel, or in its immediate environment, rather than an effect related to membrane deformations by the lipids. I was able to demonstrate that lysoPAF is able to activate native ASIC3 channels in DRG neurons and to induce a concomitant membrane depolarisation, suggesting that this lipid could, like LPC, have pro-algesic effects.I also showed during my thesis that ASIC3 channels are cold sensitive. Low temperatures increase the amplitude of acidification-induced currents and slow down the inactivation kinetics. However, deletion of ASIC3 does not appear to alter the cold sensitivity of DRG neurons in primary culture.In conclusion, my thesis work allowed me to identify LPC16:0 as a trigger for joint pain, via ASIC3 activation. In addition, I was able to identify two new endogenous lipid modulators of the ASIC3 channel, PAF and lysoPAF, which opens new insights into the role(s) of these lipids in pain. The modulation of ASIC3 channels by different stimuli such as acidity, lipids and cold, reinforce the idea that this camanl behaves as a coincidence detector

Single Subcutaneous Injection of Lysophosphatidyl-Choline Evokes ASIC3-Dependent Increases of Spinal Dorsal Horn Neuron Activity

International audienceLysophosphatidyl-choline (LPC), a member of the phospholipid family, is an emerging player in pain. It is known to modulate different pain-related ion channels, including Acid-Sensing Ion Channel 3 (ASIC3), a cationic channel mainly expressed in peripheral sensory neurons. LPC potentiates ASIC3 current evoked by mild acidifications, but can also activate the channel at physiological pH. Very recently, LPC has been associated to chronic pain in patients suffering from fibromyalgia or osteoarthritis. Accordingly, repetitive injections of LPC within mouse muscle or joint generate both persistent pain-like and anxiety-like behaviors in an ASIC3-dependent manner. LPC has also been reported to generate acute pain behaviors when injected intraplantarly in rodents. Here, we explore the mechanism of action of a single cutaneous injection of LPC by studying its effects on spinal dorsal horn neurons. We combine pharmacological, molecular and functional approaches including in vitro patch clamp recordings and in vivo recordings of spinal neuronal activity. We show that a single cutaneous injection of LPC exclusively affects the nociceptive pathway, inducing an ASIC3-dependent sensitization of nociceptive fibers that leads to hyperexcitabilities of both high threshold (HT) and wide dynamic range (WDR) spinal neurons. ASIC3 is involved in LPC-induced increase of WDR neuron’s windup as well as in WDR and HT neuron’s mechanical hypersensitivity, and it participates, together with TRPV1, to HT neuron’s thermal hypersensitivity. The nociceptive input induced by a single LPC cutaneous rather induces short-term sensitization, contrary to previously described injections in muscle and joint. If the effects of peripheral LPC on nociceptive pathways appear to mainly depend on peripheral ASIC3 channels, their consequences on pain may also depend on the tissue injected. Our findings contribute to a better understanding of the nociceptive signaling pathway activated by peripheral LPC via ASIC3 channels, which is an important step regarding the ASIC3-dependent roles of this phospholipid in acute and chronic pain conditions

Dual contribution of ASIC1a channels in the spinal processing of pain information by deep projection neurons revealed by computational modeling.

Dorsal horn of the spinal cord is an important crossroad of pain neuraxis, especially for the neuronal plasticity mechanisms that can lead to chronic pain states. Windup is a well-known spinal pain facilitation process initially described several decades ago, but its exact mechanism is still not fully understood. Here, we combine both ex vivo and in vivo electrophysiological recordings of rat spinal neurons with computational modeling to demonstrate a role for ASIC1a-containing channels in the windup process. Spinal application of the ASIC1a inhibitory venom peptides mambalgin-1 and psalmotoxin-1 (PcTx1) significantly reduces the ability of deep wide dynamic range (WDR) neurons to develop windup in vivo. All deep WDR-like neurons recorded from spinal slices exhibit an ASIC current with biophysical and pharmacological characteristics consistent with functional expression of ASIC1a homomeric channels. A computational model of WDR neuron supplemented with different ASIC1a channel parameters accurately reproduces the experimental data, further supporting a positive contribution of these channels to windup. It also predicts a calcium-dependent windup decrease for elevated ASIC conductances, a phenomenon that was experimentally validated using the Texas coral snake ASIC-activating toxin (MitTx) and calcium-activated potassium channel inhibitory peptides (apamin and iberiotoxin). This study supports a dual contribution to windup of calcium permeable ASIC1a channels in deep laminae projecting neurons, promoting it upon moderate channel activity, but ultimately leading to calcium-dependent windup inhibition associated to potassium channels when activity increases

Effect of spinal application of mambalgin-1 and PcTx1 on WDR neuron activities evoked by Aβ and Aδ fibers.

A, Typical recordings obtained following stimulation of a WDR neuron receptive field by 10 repetitive brushings (non-noxious stimulations), before (control) and after a 10-min application of mambalgin-1 (30μM) at the spinal cord level. B, The total number of AP evoked during the brushing protocol described in A are compared before (control 1 and 2, which represent two brushing experiments that were performed consecutively at 10 min intervals) and after applications of either mambalgin-1 or PcTx1 30μM (n = 11–12, pC, Typical recordings showing the activity of a WDR neuron during a windup protocol (16 repetitive electrical stimulations at 1Hz). Only the recordings obtained at stimulation 1, 5, 10 and 15 are represented. The vertical dashed lines represent the time ranges where the activity of the WDR is considered to be evoked by Aδ or C fibres. D, Total number of AP evoked by Aδ during windup protocols before (control) after applications of either mambalgin-1 or PcTx1 (n = 13–14, *, p (PDF)</p

Computational model of WDR neuron with ASIC1a channel parameters and a synaptic cleft acidification system.

A, Schematic representation of the computational model used, with a WDR projection neuron receiving directly a 20-synapse connection from both Aδ-fibers and C-fibers. B, Simulation curve showing the evolution of synaptic pH as a function of time during windup-inducing stimulations. C, Simulation windup curves obtained without (control, no ASIC, black dots) and with moderate homomeric ASIC1a conductances (g is 0.01nS, 0.05nS, 0.1nS and 0.2nS, green squares). D, Simulation windup curves obtained in control condition (no ASIC, black dots) and with high homomeric ASIC1a conductances (0.2nS, 1nS and 1.4nS, full squares). E, Simulation windup curves obtained without (control, no ASIC, black dots) and with moderate heteromeric ASIC1a conductances g is 0.4nS, 0.8nS, 1.5nS and 3nS, green squares). F, Simulation windup curves obtained in control condition (no ASIC, black dots) and with high heteromeric ASIC1a conductances (3nS and 15nS, full squares). Compared to control, gradually adding homomeric or heteromeric ASIC1a channels first increases windup, then decreases it. Removing the calcium conductance in ASIC parameters (same conductances, open squares) suppresses the inhibitory effect of high ASIC conductances and strongly potentiates windup.</p

The ASIC1a activator MitTx inhibits windup <i>in vivo</i>.

A-B, Spinal applications of MitTx (2nd) dose dependently and reversibly inhibits windup (n = 5–9; panel A: *ppvs. MitTx 10−6 M, #p##p####pvs. MitTx 5.10−7 M, &p&&p&&&p&&&&pvs. MitTx 10−7 M, $$p$$$pvs. MitTx 5.10−8 M; panel B: *ppst), respectively; Mixed-effect analyses followed by Dunnet’s multiple comparison tests). C, Simulation of the effect of MitTx on windup (Control: no ASIC conductance; with ASIC: with a native homomeric ASIC1a conductance of 0.2nS; with ASIC+MitTx: sustained full activation (without inactivation) of ASIC1a with a same conductance of 0.2nS). Note that because the ASIC channel is constitutively and fully activated, the outcome of this simulation does not depend on the pH and dynamics of the channel, and is therefore the same for all ASIC types (homomeric, heteromeric). D-E, The inhibitory effect of MitTx 5.10-7M is abolished by spinal pre- and co-application of the KCa blockers apamin and iberiotoxin (D, 10-6M each). Removing these two blockers restored the windup inhibition induced by MitTx (application sequence: control / Apamin+IberioTx / Apamin+IberioTx+MitTx / MitTx). As a comparison, data already presented in B and showing the effect of MitTx 5.10-7M applied alone, i.e., without pre-application of KCa blockers, are also represented on the bargraph in E (n = 5–9; panel D: **ppp<0.001, one-way ANOVA followed by a Dunnet’s multiple comparison test).</p

Characterization of a native ASIC1a-type current in deep projection neurons from laminae V.

A, Typical voltage-clamp recordings of deep projection neurons (laminae V) obtained from spinal cord slices following extracellular acidifications from pH7.3 to pH6.6. Extracellular applications of the ASIC1a inhibitory peptides PcTx1 (30nM) or mambalgin-1 (1μM) both decrease the amplitude of native pH6.6-induced currents. B, Inactivation kinetics of native pH6.6-induced currents recorded in spinal neurons (native) are compared to those of ASIC1a homomeric currents form HEK293 transfected cells (form pH7.4 to 6.6). Inactivation rates (τ) were obtained by fitting current inactivation decays with a mono-exponential (n = 23 and 20 for spinal neurons and ASIC1a-HEK293 cells, respectively, p = 0.2399, unpaired t-test). C, Peak amplitude of the native ASIC current recorded in neurons following four consecutive pH6.6 extracellular acidifications (from 7.3 to 6.6 every 60s). Amplitudes are normalized to the first pH6.6-evoked current (n = 14, no significant tachyphylaxis process with p = 0.1882, one-way ANOVA test followed by a Dunnet’s post hoc test). Inset shows the same experiment performed on ASIC1a homomeric current recorded in HEK293 cells (from 7.4 to 6.6 every 60s, n = 20, significant tachyphylaxis process with ***pppost hoc test). D, Statistical analysis of both PcTx1 (30nM) and mambalgin-1 (1μM) effects on the native pH6.6-induced current amplitudes recorded as in A (n = 9 and 5, respectively, ****p<0.0001, paired Student t-test).</p

Effects of PcTx1 and mambalgin-1 on ASIC1a homomeric and heteromeric channels.

A, Typical ASIC1a, ASIC1a/2a and ASIC1a/2b currents recorded at -50 mV from HEK293 transfected cells following extracellular acidification to pH5.0. B, Statistical analysis of the current densities recorded in A. Both the transient (tr) and sustained (sst) currents were measured, and the ratio sst/tr is represented on the bargraph on the bottom right (n = 8–25, One-way ANOVA with pC-D, Effects of PcTx1 (30 nM or 300 nM) and mambalgin-1 (1μM) on pH-evoked ASIC1a (pH7.4 to pH6.0), ASIC1a/2a (pH7.4 to pH6.0) and ASIC1a/2b (pH8.0 to pH6.0) currents (n = 5–11).</p