Extracellular ATP/adenosine dynamics in the brain and its role in health and disease

Extracellular ATP and adenosine are neuromodulators that regulate numerous neuronal functions in the brain. Neuronal activity and brain insults such as ischemic and traumatic injury upregulate these neuromodulators, which exert their effects by activating purinergic receptors. In addition, extracellular ATP/adenosine signaling plays a pivotal role in the pathogenesis of neurological diseases. Virtually every cell type in the brain contributes to the elevation of ATP/adenosine, and various mechanisms underlying this increase have been proposed. Extracellular adenosine is thought to be mainly produced via the degradation of extracellular ATP. However, adenosine is also released from neurons and glia in the brain. Therefore, the regulation of extracellular ATP/adenosine in physiological and pathophysiological conditions is likely far more complex than previously thought. To elucidate the complex mechanisms that regulate extracellular ATP/adenosine levels, accurate methods of assessing their spatiotemporal dynamics are needed. Several novel techniques for acquiring spatiotemporal information on extracellular ATP/adenosine, including fluorescent sensors, have been developed and have started to reveal the mechanisms underlying the release, uptake and degradation of ATP/adenosine. Here, we review methods for analyzing extracellular ATP/adenosine dynamics as well as the current state of knowledge on the spatiotemporal dynamics of ATP/adenosine in the brain. We focus on the mechanisms used by neurons and glia to cooperatively produce the activity-dependent increase in ATP/adenosine and its physiological and pathophysiological significance in the brain

The astrocytic TRPA1 channel mediates an intrinsic protective response to vascular cognitive impairment via LIF production

認知症に対する新たな生体防御機構の発見 --アストロサイトのTRPA1活性化が、LIF産生を介して白質傷害や認知機能障害を防ぐ--. 京都大学プレスリリース. 2023-07-24.Vascular cognitive impairment (VCI) refers to cognitive alterations caused by vascular disease, which is associated with various types of dementia. Because chronic cerebral hypoperfusion (CCH) induces VCI, we used bilateral common carotid artery stenosis (BCAS) mice as a CCH-induced VCI model. Transient receptor potential ankyrin 1 (TRPA1), the most redox-sensitive TRP channel, is functionally expressed in the brain. Here, we investigated the pathophysiological role of TRPA1 in CCH-induced VCI. During early-stage CCH, cognitive impairment and white matter injury were induced by BCAS in TRPA1-knockout but not wild-type mice. TRPA1 stimulation with cinnamaldehyde ameliorated BCAS-induced outcomes. RNA sequencing analysis revealed that BCAS increased leukemia inhibitory factor (LIF) in astrocytes. Moreover, hydrogen peroxide-treated TRPA1-stimulated primary astrocyte cultures expressed LIF, and culture medium derived from these cells promoted oligodendrocyte precursor cell myelination. Overall, TRPA1 in astrocytes prevents CCH-induced VCI through LIF production. Therefore, TRPA1 stimulation may be a promising therapeutic approach for VCI

Upregulation of P2Y2 receptors by retinoids in normal human epidermal keratinocytes

Retinoids, vitamin A derivatives, are important regulators of the growth and differentiation of skin cells. Although retinoids are therapeutically used for several skin ailments, little is known about their effects on P2 receptors, known to be involved in various functions in the skin. DNA array analysis showed that treatment of normal human epidermal keratinocytes (NHEKs) with all-trans-retinoic acid (ATRA), an agonist to RAR (retinoic acid receptor), enhanced the expression of mRNA for the P2Y2 receptor, a metabotropic P2 receptor that is known to be involved in the proliferation of the epidermis. The expression of other P2 receptors in NHEKs was not affected by ATRA. ATRA increased the mRNA for the P2Y2 receptor in a concentration-dependent fashion (1 nM to 1 μM). Am80, a synthesized agonist to RAR, showed a similar enhancement, whereas 9-cis-retinoic acid (9-cisRA), an agonist to RXR (retinoid X receptor), enhanced P2Y2 gene expression to a lesser extent. Ca2+ imaging analysis showed that ATRA also increased the function of P2Y2 receptors in NHEKs. Retinoids are known to enhance the turnover of the epidermis by increasing both proliferation and terminal differentiation. The DNA microarray analysis also revealed that ATRA upregulates various genes involved in the differentiation of NHEKs. Our present results suggest that retinoids, at least in part, exert their proliferative effects by upregulating P2Y2 receptors in NHEKs. This effect of retinoids may be closely related to their therapeutic effect against various ailments or aging events in skins such as over-keratinization, pigmentation and re-modeling

Astrocytic dysfunction induced by ABCA1 deficiency causes optic neuropathy

Astrocyte abnormalities have received great attention for their association with various diseases in the brain but not so much in the eye. Recent independent genome-wide association studies of glaucoma, optic neuropathy characterized by retinal ganglion cell (RGC) degeneration, and vision loss found that single-nucleotide polymorphisms near the ABCA1 locus were common risk factors. Here, we show that Abca1 loss in retinal astrocytes causes glaucoma-like optic neuropathy in aged mice. ABCA1 was highly expressed in retinal astrocytes in mice. Thus, we generated macroglia-specific Abca1-deficient mice (Glia-KO) and found that aged Glia-KO mice had RGC degeneration and ocular dysfunction without affected intraocular pressure, a conventional risk factor for glaucoma. Single-cell RNA sequencing revealed that Abca1 deficiency in aged Glia-KO mice caused astrocyte-triggered inflammation and increased the susceptibility of certain RGC clusters to excitotoxicity. Together, astrocytes play a pivotal role in eye diseases, and loss of ABCA1 in astrocytes causes glaucoma-like neuropathy

Direct Observation of ATP-Induced Conformational Changes in Single P2X4 Receptors

The ATP-gated P2X4 receptor is a cation channel, which is important in various pathophysiological events. The architecture of the P2X4 receptor in the activated state and how to change its structure in response to ATP binding are not fully understood. Here, we analyze the architecture and ATP-induced structural changes in P2X4 receptors using fast-scanning atomic force microscopy (AFM). AFM images of the membrane-dissociated and membrane-inserted forms of P2X4 receptors and a functional analysis revealed that P2X4 receptors have an upward orientation on mica but lean to one side. Time-lapse imaging of the ATP-induced structural changes in P2X4 receptors revealed two different forms of activated structures under 0 Ca2+ conditions, namely a trimer structure and a pore dilation-like tripartite structure. A dye uptake measurement demonstrated that ATP-activated P2X4 receptors display pore dilation in the absence of Ca2+. With Ca2+, the P2X4 receptors exhibited only a disengaged trimer and no dye uptake was observed. Thus our data provide a new insight into ATP-induced structural changes in P2X4 receptors that correlate with pore dynamics

The Astrocyte-Targeted Therapy by Bushi for the Neuropathic Pain in Mice

BACKGROUND: There is accumulating evidence that the activation of spinal glial cells, especially microglia, is a key event in the pathogenesis of neuropathic pain. However, the inhibition of microglial activation is often ineffective, especially for long-lasting persistent neuropathic pain. So far, neuropathic pain remains largely intractable and a new therapeutic strategy for the pain is still required. METHODS/PRINCIPAL FINDINGS: Using Seltzer model mice, we investigated the temporal aspect of two types of neuropathic pain behaviors, i.e., thermal hyperalgesia and mechanical allodynia, as well as that of morphological changes in spinal microglia and astrocytes by immunohistochemical studies. Firstly, we analyzed the pattern of progression in the pain behaviors, and found that the pain consisted of an "early induction phase" and subsequent "late maintenance phase". We next analyzed the temporal changes in spinal glial cells, and found that the induction and the maintenance phase of pain were associated with the activation of microglia and astrocytes, respectively. When Bushi, a Japanese herbal medicine often used for several types of persistent pain, was administered chronically, it inhibited the maintenance phase of pain without affecting the induction phase, which was in accordance with the inhibition of astrocytic activation in the spinal cord. These analgesic effects and the inhibition of astrocytic activation by Bushi were mimicked by the intrathecal injection of fluorocitrate, an inhibitor of astrocytic activation. Finally, we tested the direct effect of Bushi on astrocytic activation, and found that Bushi suppressed the IL-1β- or IL-18-evoked ERK1/2-phosphorylation in cultured astrocytes but not the ATP-evoked p38- and ERK1/2-phosphorylation in microglia in vitro. CONCLUSIONS: Our results indicated that the activation of spinal astrocytes was responsible for the late maintenance phase of neuropathic pain in the Seltzer model mice and, therefore, the inhibition of astrocytic activation by Bushi could be a useful therapeutic strategy for treating neuropathic pain

Regulation of cell-to-cell communication mediated by astrocytic ATP in the CNS

It has become apparent that glial cells, especially astrocytes, not merely supportive but are integrative, being able to receive inputs, assimilate information and send instructive chemical signals to other neighboring cells including neurons. At first, the excitatory neurotransmitter glutamate was found to be a major extracellular messenger that mediates these communications because it can be released from astrocytes in a Ca2+-dependent manner, diffused, and can stimulate extra-synaptic glutamate receptors in adjacent neurons, leading to a dynamic modification of synaptic transmission. However, recently extracellular ATP has come into the limelight as an important extracellular messenger for these communications. Astrocytes express various neurotransmitter receptors including P2 receptors, release ATP in response to various stimuli and respond to extracellular ATP to cause various physiological responses. The intercellular communication “Ca2+ wave” in astrocytes was found to be mainly mediated by the release of ATP and the activation of P2 receptors, suggesting that ATP is a dominant “gliotransmitter” between astrocytes. Because neurons also express various P2 receptors and synapses are surrounded by astrocytes, astrocytic ATP could affect neuronal activities and even dynamically regulate synaptic transmission in adjacent neurons as if forming a “tripartite synapse” In this review, we summarize the role of astrocytic ATP, as compared with glutamate, in gliotransmission and synaptic transmission in neighboring cells, mainly focusing on the hippocampus. Dynamic communication between astrocytes and neurons mediated by ATP would be a key event in the processing or integration of information in the CNS

ATP receptors in pain sensation: Involvement of spinal microglia and P2X4 receptors

There is abundant evidence that extracellular ATP and other nucleotides have an important role in pain signaling at both the periphery and in the CNS. At first, it was thought that ATP was simply involved in acute pain, since ATP is released from damaged cells and excites directly primary sensory neurons by activating their receptors. However, neither blocking P2X/Y receptors pharmacologically nor suppressing the expression of P2X/Y receptors molecularly in sensory neurons or in the spinal cord had an effect on acute physiological pain. The focus of attention now is on the possibility that endogenous ATP and its receptor system might be activated in pathological pain states, particularly in neuropathic pain. Neuropathic pain is often a consequence of nerve injury through surgery, bone compression, diabetes or infection. This type of pain can be so severe that even light touching can be intensely painful; unfortunately, this state is generally resistant to currently available treatments. An important advance in our understanding of the mechanisms involved in neuropathic pain has been made by a recent work demonstrating the crucial role of ATP receptors (i.e., P2X3 and P2X4 receptors). In this review, we summarize the role of ATP receptors, particularly the P2X4 receptor, in neuropathic pain. The expression of P2X4 receptors in the spinal cord is enhanced in spinal microglia after peripheral nerve injury, and blocking pharmacologically and suppressing molecularly P2X4 receptors produce a reduction of the neuropathic pain behaviour. Understanding the key roles of ATP receptors including P2X4 receptors may lead to new strategies for the management of neuropathic pain