Microglial P2X4 receptors are essential for spinal neurons hyperexcitability and tactile allodynia in male and female neuropathic mice.

In neuropathic pain, recent evidence has highlighted a sex-dependent role of the P2X4 receptor in spinal microglia in the development of tactile allodynia following nerve injury. Here, using internalization-defective P2X4mCherryIN knockin mice (P2X4KI), we demonstrate that increased cell surface expression of P2X4 induces hypersensitivity to mechanical stimulations and hyperexcitability in spinal cord neurons of both male and female naive mice. During neuropathy, both wild-type (WT) and P2X4KI mice of both sexes develop tactile allodynia accompanied by spinal neuron hyperexcitability. These responses are selectively associated with P2X4, as they are absent in global P2X4KO or myeloid-specific P2X4KO mice. We show that P2X4 is de novo expressed in reactive microglia in neuropathic WT and P2X4KI mice of both sexes and that tactile allodynia is relieved by pharmacological blockade of P2X4 or TrkB. These results show that the upregulation of P2X4 in microglia is crucial for neuropathic pain, regardless of sex.

Microglial P2X4 receptors promote ApoE degradation and contribute to memory deficits in Alzheimer's disease.

Numerous evidences support that microglia contributes to the progression of Alzheimer's disease. P2X4 receptors are ATP-gated channels with high calcium permeability, which are de novo expressed in a subset of reactive microglia associated with various pathological contexts, contributing to microglial functions. P2X4 receptors are mainly localized in lysosomes and trafficking to the plasma membrane is tightly regulated. Here, we investigated the role of P2X4 in the context of Alzheimer's disease (AD). Using proteomics, we identified Apolipoprotein E (ApoE) as a specific P2X4 interacting protein. We found that P2X4 regulates lysosomal cathepsin B (CatB) activity promoting ApoE degradation; P2rX4 deletion results in higher amounts of intracellular and secreted ApoE in both bone-marrow-derived macrophage (BMDM) and microglia from APPswe/PSEN1dE9 brain. In both human AD brain and APP/PS1 mice, P2X4 and ApoE are almost exclusively expressed in plaque-associated microglia. In 12-month-old APP/PS1 mice, genetic deletion of P2rX4 reverses topographical and spatial memory impairment and reduces amount of soluble small aggregates of Aß1-42 peptide, while no obvious alteration of plaque-associated microglia characteristics is observed. Our results support that microglial P2X4 promotes lysosomal ApoE degradation, indirectly altering Aß peptide clearance, which in turn might promotes synaptic dysfunctions and cognitive deficits. Our findings uncover a specific interplay between purinergic signaling, microglial ApoE, soluble Aß (sAß) species and cognitive deficits associated with AD.

Comparative analysis of transcriptome remodeling in plaque-associated and plaque-distant microglia during amyloid-ß pathology progression in mice.

BACKGROUND: Research in recent years firmly established that microglial cells play an important role in the pathogenesis of Alzheimer's disease (AD). In parallel, a series of studies showed that, under both homeostatic and pathological conditions, microglia are a heterogeneous cell population. In AD, amyloid-ß (Aß) plaque-associated microglia (PAM) display a clearly distinct phenotype compared to plaque-distant microglia (PCM), suggesting that these two microglia subtypes likely differently contribute to disease progression. So far, molecular characterization of PAM was performed indirectly using single cell RNA sequencing (scRNA-seq) approaches or based on markers that are supposedly up-regulated in this microglia subpopulation. METHODS: In this study based on a well-characterized AD mouse model, we combined cell-specific laser capture microdissection and RNA-seq analysis to i) identify, without preconceived notions of the molecular and/or functional changes that would affect these cells, the genes and gene networks that are dysregulated in PAM or PCM at three critical stages of the disease, and ii) to investigate the potential contribution of both plaque-associated and plaque-distant microglia. RESULTS: First, we established that our approach allows selective isolation of microglia, while preserving spatial information and preventing transcriptome changes induced by classical purification approaches. Then, we identified, in PAM and PCM subpopulations, networks of co-deregulated genes and analyzed their potential functional roles in AD. Finally, we investigated the dynamics of microglia transcriptomic remodeling at early, intermediate and late stages of the disease and validated select findings in postmortem human AD brain. CONCLUSIONS: Our comprehensive study provides useful transcriptomic information regarding the respective contribution of PAM and PCM across the Aß pathology progression. It highlights specific pathways that would require further study to decipher their roles across disease progression. It demonstrates that the proximity of microglia to Aß-plaques dramatically alters the microglial transcriptome and reveals that these changes can have both positive and negative impacts on the surrounding cells. These opposing effects may be driven by local microglia heterogeneity also demonstrated by this study. Our approach leads to molecularly define the less well studied plaque-distant microglia. We show that plaque-distant microglia are not bystanders of the disease, although the transcriptomic changes are far less striking compared to what is observed in plaque-associated microglia. In particular, our results suggest they may be involved in Aß oligomer detection and in Aß-plaque initiation, with increased contribution as the disease progresses.

Analysis of CX3CR1 haplodeficiency in male and female APPswe/PSEN1dE9 mice along Alzheimer disease progression.

Microglia, the resident immune cells of the brain, have recently emerged as key players in Alzheimer Disease (AD) pathogenesis, but their roles in AD remain largely elusive and require further investigation. Microglia functions are readily altered when isolated from their brain environment, and microglia reporter mice thus represent valuable tools to study the contribution of these cells to neurodegenerative diseases such as AD. The CX3CR1+/eGFP mice is one of the most popular microglia reporter mice, and has been used in numerous studies to investigate in vivo microglial functions, including in the context of AD research. However, until now, the impact of CX3CR1 haplodeficiency on the typical features of Alzheimer Disease has not been studied in depth. To fill this gap, we generated APPswe/PSEN1dE9:CX3CR1+/eGFP mice and analyzed these mice for Alzheimer's like pathology and neuroinflammation hallmarks. More specifically, using robust multifactorial statistical and multivariate analyses, we investigated the impact of CX3CR1 deficiency in both males and females, at three typical stages of the pathology progression: at early stage when Amyloid-ß (Aß) deposition just starts, at intermediate stage during Aß accumulation phase and at more advanced stages when Aß plaque number stabilizes. We found that CX3CR1 haplodeficiency had little impact on the progression of the pathology in the APPswe/PSEN1dE9 model and demonstrated that the APPswe/PSEN1dE9:CX3CR1+/eGFP line is a relevant and useful model to study the role of microglia in Alzheimer Disease. In addition, although Aß plaques density is higher in females compared to age-matched males, we show that their glial reaction, inflammation status and memory deficits are not different.

P2X-GCaMPs as Versatile Tools for Imaging Extracellular ATP Signaling.

ATP is an extracellular signaling molecule involved in numerous physiological and pathologic processes. However, in situ characterization of the spatiotemporal dynamic of extracellular ATP is still challenging because of the lack of sensor with appropriate specificity, sensitivity, and kinetics. Here, we report the development of biosensors based on the fusion of cation permeable ATP receptors (P2X) to genetically encoded calcium sensors [genetically encoded calcium indicator (GECI)]. By combining the features of P2X receptors with the high signal-to-noise ratio of GECIs, we generated ultrasensitive green and red fluorescent sniffers that detect nanomolar ATP concentrations in situ and also enable the tracking of P2X receptor activity. We provide the proof of concept that these sensors can dynamically track ATP release evoked by depolarization in mouse neurons or by extracellular hypotonicity. Targeting these P2X-based biosensors to diverse cell types should advance our knowledge of extracellular ATP dynamics in vivo.

A P2rx7 Passenger Mutation Affects the Vitality and Function of T cells in Congenic Mice.

Among laboratory mouse strains many genes are differentially expressed in the same cell population. As consequence, gene targeting in 129-derived embryonic stem cells (ESCs) and backcrossing the modified mice onto the C57BL/6 background can introduce passenger mutations in the close proximity of the targeted gene. Here, we demonstrate that several transgenic mice carry a P2rx7 passenger mutation that affects the function of T cells. By the example of P2rx4tm1Rass we demonstrate that P2X4ko T cells express higher levels of P2X7 and are more sensitive toward the P2X7 activators ATP and NAD+, rendering these cells more vulnerable toward NAD-induced cell death (NICD) compared with wild type (WT). The enhanced NICD sensitivity confounded functional assays e.g. cytokine production and cell migration. Our results need to be considered when working with P2rx4tm1Rass mice or other 129-based transgenic strains that target P2rx7 neighboring genes.

Procedures for Culturing and Genetically Manipulating Murine Hippocampal Postnatal Neurons.

Neuronal hippocampal cultures are simple and valuable models for studying neuronal function. While embryonic cultures are widely used for different applications, mouse postnatal cultures are still challenging, lack reproducibility and/or exhibit inappropriate neuronal activity. Yet, postnatal cultures have major advantages such as allowing genotyping of pups before culture and reducing the number of experimental animals. Herein we describe a simple and fast protocol for culturing and genetically manipulating hippocampal neurons from P0 to P3 mice. This protocol provides reproducible cultures exhibiting a consistent neuronal development, normal excitatory over inhibitory neuronal ratio and a physiological neuronal activity. We also describe simple and efficient procedures for genetic manipulation of neurons using transfection reagent or lentiviral particles. Overall, this method provides a detailed and validated protocol allowing to explore cellular mechanisms and neuronal activity in postnatal hippocampal neurons in culture.

Multimeric Ionotropic Purinoceptor Detection by Protein Cross-Linking.

P2X receptor subunits (P2X1 to P2X7) assemble to form trimeric homomers or heteromers. Here, we describe the use of protein cross-linking to study the composition of P2X receptor complexes. This simple protocol is useful for determining the stoichiometry of P2X heteromeric receptors as well as for assessing the effect of point mutation, truncation, or concatenation on the quaternary architecture of these receptors.

Multimeric Purinoceptor Detection by Bioluminescence Resonance Energy Transfer.

Assays based on bioluminescence resonance energy transfer (BRET) provide a sensitive and simple method to study protein-protein interactions in live cells. Here we describe a protocol using BRET technique to investigate potential interactions between P2X subunits. This approach combined with bimolecular fluorescence complementation (BiFC) can also be employed to determine the stoichiometry of heteromeric P2X receptors.

Generation and Characterization of Specific Monoclonal Antibodies and Nanobodies Directed Against the ATP-Gated Channel P2X4.

The P2X4 channel is involved in different physiological and pathological conditions and functions in the nervous system. Despite the existence of several mouse models for which the expression of the gene was manipulated, there is still little information on the expression of the protein at the cellular level. In particular, supposedly specific available antibodies have often proved to recognize unrelated proteins in P2X4-deficient mice. Here, we used an in vivo DNA vaccine approach to generate a series of monoclonal antibodies and nanobodies specific for human, mouse, and rat P2X4 channels. We further characterized these antibodies and show that they solely recognize the native form of the proteins both in biochemical and cytometric applications. Some of these antibodies prove to specifically recognize P2X4 channels by immunostaining in brain or sensory ganglia slices, as well as at the cellular and subcellular levels. Due to their clonality, these different antibodies should represent versatile tools for further characterizing the cellular functions of P2X4 in the nervous system as well as at the periphery.

Microglia Reactivity: Heterogeneous Pathological Phenotypes.

A century ago, Pío del Río-Hortega discovered that microglial cells are endowed with remarkable dynamic and plastic capabilities. The real-time plasticity of microglia could be revealed, however, only during the last 15 years with the development of new transgenic animal models and new molecular and functional analysis methods. Phenotyping microglia in situ with these new tools sealed the fate of the classical two state model of "resting" microglia in physiological conditions and "activated" microglia in pathological conditions. Our current view on functional behavior of microglia takes into account the exquisite reactivity of these immune cells to changes occurring in the CNS in both physiological and pathological conditions. We briefly review here the results and methods that have uncovered the dynamics and versatility of microglial reactivity.

Blocking α2δ-1 Subunit Reduces Bladder Hypersensitivity and Inflammation in a Cystitis Mouse Model by Decreasing NF-kB Pathway Activation.

Bladder pain is frequently associated with bladder inflammation, as in conditions like interstitial cystitis (IC), for which current analgesic therapies have limited efficacy. The antinociceptive effect of alpha-2-delta (α2δ) ligands on inflammation-associated visceral pain like that experienced in cystitis has been poorly investigated. To investigate the effect of pregabalin (PGB), an α2δ ligand, we evaluated its impact on mechanical hyperalgesia in a mouse model of cystitis induced by cyclophosphamide (CYP). We further studied its effect on inflammation and NF-kB pathway activation. Acute cystitis was induced by intraperitoneal injection of 150 mg kg-1 of CYP in C57Bl/6J male mice. PGB was subcutaneously injected (30 mg kg-1) 3 h after CYP injection. The effect of PGB on CYP-induced mechanical referred hyperalgesia (abdominal Von Frey test), inflammation (organ weight, cytokine production, α2δ subunit level, NF-kB pathway activation) were assessed 1 h after its injection. In parallel, its effect on cytokine production, α2δ subunit level and NF-kB pathway activation was assessed in vitro on peritoneal exudate cells (PECs) stimulated with LPS. PGB treatment decreased mechanical referred hyperalgesia. Interestingly, it had an anti-inflammatory effect in the cystitis model by reducing pro-inflammatory cytokine production. PGB also inhibited NF-kB pathway activation in the cystitis model and in macrophages stimulated with LPS, in which it blocked the increase in intracellular calcium. This study shows the efficacy of PGB in hypersensitivity and inflammation associated with cystitis. It is therefore of great interest in assessing the benefit of α2δ ligands in patients suffering from cystitis.

P2X4 receptor controls microglia activation and favors remyelination in autoimmune encephalitis.

Microglia survey the brain microenvironment for signals of injury or infection and are essential for the initiation and resolution of pathogen- or tissue damage-induced inflammation. Understanding the mechanism of microglia responses during pathology is hence vital to promote regenerative responses. Here, we analyzed the role of purinergic receptor P2X4 (P2X4R) in microglia/macrophages during autoimmune inflammation. Blockade of P2X4R signaling exacerbated clinical signs in the experimental autoimmune encephalomyelitis (EAE) model and also favored microglia activation to a pro-inflammatory phenotype and inhibited myelin phagocytosis. Moreover, P2X4R blockade in microglia halted oligodendrocyte differentiation in vitro and remyelination after lysolecithin-induced demyelination. Conversely, potentiation of P2X4R signaling by the allosteric modulator ivermectin (IVM) favored a switch in microglia to an anti-inflammatory phenotype, potentiated myelin phagocytosis, promoted the remyelination response, and ameliorated clinical signs of EAE Our results provide evidence that P2X4Rs modulate microglia/macrophage inflammatory responses and identify IVM as a potential candidate among currently used drugs to promote the repair of myelin damage.

The microglial reaction signature revealed by RNAseq from individual mice.

Microglial cells have a double life as the immune cells of the brain in times of stress but have also specific physiological functions in homeostatic conditions. In pathological contexts, microglia undergo a phenotypic switch called "reaction" that promotes the initiation and the propagation of neuro-inflammation. Reaction is complex, molecularly heterogeneous and still poorly characterized, leading to the concept that microglial reactivity might be too diverse to be molecularly defined. However, it remains unknown whether reactive microglia from different pathological contexts share a common molecular signature. Using improved flow cytometry and RNAseq approaches we studied, with higher statistical power, the remodeling of microglia transcriptome in a mouse model of sepsis. Through bioinformatic comparison of our results with published datasets, we defined the microglial reactome as a set of genes discriminating reactive from homeostatic microglia. Ultimately, we identified a subset of 86 genes deregulated in both acute and neurodegenerative conditions. Our data provide a new comprehensive resource that includes functional analysis and specific molecular markers of microglial reaction which represent new tools for its unambiguous characterization.

Sensory neuronal P2RX4 receptors controls BDNF signaling in inflammatory pain.

Chronic inflammatory and neuropathic pains are major public health concerns. Potential therapeutic targets include the ATP-gated purinergic receptors (P2RX) that contribute to these pathological types of pain in several different cell types. The purinergic receptors P2RX2 and P2RX3 are expressed by a specific subset of dorsal root ganglion neurons and directly shape pain processing by primary afferents. In contrast the P2RX4 and P2RX7 are mostly expressed in myeloid cells, where activation of these receptors triggers the release of various pro-inflammatory molecules. Here, we demonstrate that P2RX4 also controls calcium influx in mouse dorsal root ganglion neurons. P2RX4 is up-regulated in pain-processing neurons during long lasting peripheral inflammation and it co-localizes with Brain-Derived Neurotrophic Factor (BDNF). In the dorsal horn of the spinal cord, BDNF-dependent signaling pathways, phosphorylation of Erk1/2 and of the GluN1 subunit as well as the down regulation of the co-transporter KCC2, which are triggered by peripheral inflammation are impaired in P2RX4-deficient mice. Our results suggest that P2RX4, expressed by sensory neurons, controls neuronal BDNF release that contributes to hyper-excitability during chronic inflammatory pain and establish P2RX4 in sensory neurons as a new potential therapeutic target to treat hyperexcitability during chronic inflammatory pain.

Purinergic signaling in epilepsy.

Until recently, analysis of the mechanisms underlying epilepsy was centered on neuron dysfunctions. Accordingly, most of the available pharmacological treatments aim at reducing neuronal excitation or at potentiating neuronal inhibition. These therapeutic options can lead to obvious secondary effects, and, moreover, seizures cannot be controlled by any known medication in one-third of the patients. A purely neurocentric view of brain functions and dysfunctions has been seriously questioned during the past 2 decades because of the accumulation of experimental data showing the functional importance of reciprocal interactions between glial cells and neurons. In the case of epilepsy, our current knowledge of the human disease and analysis of animal models clearly favor the involvement of astrocytes and microglial cells during the progression of the disease, including at very early stages, opening the way to the identification of new therapeutic targets. Purinergic signaling is a fundamental feature of neuron-glia interactions, and increasing evidence indicates that modifications of this pathway contribute to the functional remodeling of the epileptic brain. This Review discusses the recent experimental results indicating the roles of astrocytic and microglial P2X and P2Y receptors in epilepsy. © 2016 Wiley Periodicals, Inc.

Evidence for Status Epilepticus and Pro-Inflammatory Changes after Intranasal Kainic Acid Administration in Mice.

Kainic acid (KA) is routinely used to elicit status epilepticus (SE) and epileptogenesis. Among the available KA administration protocols, intranasal instillation (IN) remains understudied. Dosages of KA were instilled IN in mice. Racine Scale and Video-EEG were used to assess and quantify SE onset. Time spent in SE and spike activity was quantified for each animal and confirmed by power spectrum analysis. Immunohistochemistry and qPCR were performed to define brain inflammation occurring after SE, including activated microglial phenotypes. Long term video-EEG recording was also performed. Titration of IN KA showed that a dose of 30 mg/kg was associated with low mortality while eliciting SE. IN KA provoked at least one behavioral and electrographic SE in the majority of the mice (>90%). Behavioral and EEG SE were accompanied by a rapid and persistent microglial-astrocytic cell activation and hippocampal neurodegeneration. Specifically, microglial modifications involved both pro- (M1) and anti-inflammatory (M2) genes. Our initial long-term video-EEG exploration conducted using a small cohort of mice indicated the appearance of spike activity or SE. Our study demonstrated that induction of SE is attainable using IN KA in mice. Typical pro-inflammatory brain changes were observed in this model after SE, supporting disease pathophysiology. Our results are in favor of the further development of IN KA as a means to study seizure disorders. A possibility for tailoring this model to drug testing or to study mechanisms of disease is offered.

Spatiotemporal pattern of action potential firing in developing inner hair cells of the mouse cochlea.

Inner hair cells (IHCs) are the primary transducer for sound encoding in the cochlea. In contrast to the graded receptor potential of adult IHCs, immature hair cells fire spontaneous calcium action potentials during the first postnatal week. This spiking activity has been proposed to shape the tonotopic map along the ascending auditory pathway. Using perforated patch-clamp recordings, we show that developing IHCs fire spontaneous bursts of action potentials and that this pattern is indistinguishable along the basoapical gradient of the developing cochlea. In both apical and basal IHCs, the spiking behavior undergoes developmental changes, where the bursts of action potential tend to occur at a regular time interval and have a similar length toward the end of the first postnatal week. Although disruption of purinergic signaling does not interfere with the action potential firing pattern, pharmacological ablation of the α9α10 nicotinic receptor elicits an increase in the discharge rate. We therefore suggest that in addition to carrying place information to the ascending auditory nuclei, the IHCs firing pattern controlled by the α9α10 receptor conveys a temporal signature of the cochlear development.

Optical control of an ion channel gate.

The powerful optogenetic pharmacology method allows the optical control of neuronal activity by photoswitchable ligands tethered to channels and receptors. However, this approach is technically demanding, as it requires the design of pharmacologically active ligands. The development of versatile technologies therefore represents a challenging issue. Here, we present optogating, a method in which the gating machinery of an ATP-activated P2X channel was reprogrammed to respond to light. We found that channels covalently modified by azobenzene-containing reagents at the transmembrane segments could be reversibly turned on and off by light, without the need of ATP, thus revealing an agonist-independent, light-induced gating mechanism. We demonstrate photocontrol of neuronal activity by a light-gated, ATP-insensitive P2X receptor, providing an original tool devoid of endogenous sensitivity to delineate P2X signaling in normal and pathological states. These findings open new avenues to specifically activate other ion channels independently of their natural stimulus.

Involvement of P2X4 receptors in hippocampal microglial activation after status epilepticus.

Within the central nervous system, functions of the ATP-gated receptor-channel P2X4 (P2X4R) are still poorly understood, yet P2X4R activation in neurons and microglia coincides with high or pathological neuronal activities. In this study, we investigated the potential involvement of P2X4R in microglial functions in a model of kainate (KA)-induced status epilepticus (SE). We found that SE was associated with an induction of P2X4R expression in the hippocampus, mostly localized in activated microglial cells. In P2X4R-deficient mice, behavioral responses during KA-induced SE were unaltered. However, 48h post SE specific features of microglial activation, such as cell recruitment and upregulation of voltage-dependent potassium channels were impaired in P2X4R-deficient mice, whereas the expression and function of other microglial purinergic receptors remained unaffected. Consistent with the role of P2X4R in activity-dependent degenerative processes, the CA1 area was partially protected from SE-induced neuronal death in P2X4R-deficient mice compared with wild-type animals. Our findings demonstrate that P2X4Rs are brought into play during neuronal hyperexcitability and that they control specific aspects of microglial activation. Our results also suggest that P2X4Rs contribute to excitotoxic damages by regulating microglial activation.