FM1-43 Dye Memorizes Piezo1 Activation in the Trigeminal Nociceptive System Implicated in Migraine Pain.

It has been proposed that mechanosensitive Piezo1 channels trigger migraine pain in trigeminal nociceptive neurons, but the mechanosensitivity of satellite glial cells (SGCs) supporting neuronal sensitization has not been tested before. Moreover, tools to monitor previous Piezo1 activation are not available. Therefore, by using live calcium imaging with Fluo-4 AM and labeling with FM1-43 dye, we explored a new strategy to identify Piezo channels' activity in mouse trigeminal neurons, SGCs, and isolated meninges. The specific Piezo1 agonist Yoda1 induced calcium transients in both neurons and SGCs, suggesting the functional expression of Piezo1 channels in both types of cells. In Piezo1-transfected HEK cells, FM1-43 produced only a transient fluorescent response, whereas co-application with Yoda1 provided higher transient signals and a remarkable long-lasting FM1-43 'tail response'. A similar Piezo1-related FM1-43 trapping was observed in neurons and SGCs. The non-specific Piezo channel blocker, Gadolinium, inhibited the transient peak, confirming the involvement of Piezo1 receptors. Finally, FM1-43 labeling demonstrated previous activity in meningeal tissues 3.5 h after Yoda1 washout. Our data indicated that trigeminal neurons and SGCs express functional Piezo channels, and their activation provides sustained labeling with FM1-43. This long-lasting labelling can be used to monitor the ongoing and previous activation of Piezo1 channels in the trigeminal nociceptive system, which is implicated in migraine pain.

Neuron-astrocyte transmitophagy is altered in Alzheimer's disease.

Under physiological conditions in vivo astrocytes internalize and degrade neuronal mitochondria in a process called transmitophagy. Mitophagy is widely reported to be impaired in neurodegeneration but it is unknown whether and how transmitophagy is altered in Alzheimer's disease (AD). Here we report that the internalization of neuronal mitochondria is significantly increased in astrocytes isolated from AD mouse brains. We also demonstrate that the degradation of neuronal mitochondria by astrocytes is increased in AD mice at the age of 6 months onwards. Furthermore, we demonstrate for the first time a similar phenomenon between human neurons and AD astrocytes, and in murine hippocampi in vivo. The results suggest the involvement of S100a4 in impaired mitochondrial transfer between neurons and AD astrocytes together with significant increases in the mitophagy regulator and reactive oxygen species in aged AD astrocytes. These findings demonstrate altered neuron-supporting functions of AD astrocytes and provide a starting point for studying the molecular mechanisms of transmitophagy in AD.

Microglial amyloid beta clearance is driven by PIEZO1 channels.

BACKGROUND: Microglia are the endogenous immune cells of the brain and act as sensors of pathology to maintain brain homeostasis and eliminate potential threats. In Alzheimer's disease (AD), toxic amyloid beta (Aß) accumulates in the brain and forms stiff plaques. In late-onset AD accounting for 95% of all cases, this is thought to be due to reduced clearance of Aß. Human genome-wide association studies and animal models suggest that reduced clearance results from aberrant function of microglia. While the impact of neurochemical pathways on microglia had been broadly studied, mechanical receptors regulating microglial functions remain largely unexplored. METHODS: Here we showed that a mechanotransduction ion channel, PIEZO1, is expressed and functional in human and mouse microglia. We used a small molecule agonist, Yoda1, to study how activation of PIEZO1 affects AD-related functions in human induced pluripotent stem cell (iPSC)-derived microglia-like cells (iMGL) under controlled laboratory experiments. Cell survival, metabolism, phagocytosis and lysosomal activity were assessed using real-time functional assays. To evaluate the effect of activation of PIEZO1 in vivo, 5-month-old 5xFAD male mice were infused daily with Yoda1 for two weeks through intracranial cannulas. Microglial Iba1 expression and Aß pathology were quantified with immunohistochemistry and confocal microscopy. Published human and mouse AD datasets were used for in-depth analysis of PIEZO1 gene expression and related pathways in microglial subpopulations. RESULTS: We show that PIEZO1 orchestrates Aß clearance by enhancing microglial survival, phagocytosis, and lysosomal activity. Aß inhibited PIEZO1-mediated calcium transients, whereas activation of PIEZO1 with a selective agonist, Yoda1, improved microglial phagocytosis resulting in Aß clearance both in human and mouse models of AD. Moreover, PIEZO1 expression was associated with a unique microglial transcriptional phenotype in AD as indicated by assessment of cellular metabolism, and human and mouse single-cell datasets. CONCLUSION: These results indicate that the compromised function of microglia in AD could be improved by controlled activation of PIEZO1 channels resulting in alleviated Aß burden. Pharmacological regulation of these mechanoreceptors in microglia could represent a novel therapeutic paradigm for AD.

Functional Characterization of Mechanosensitive Piezo1 Channels in Trigeminal and Somatic Nerves in a Neuron-on-Chip Model.

Mechanosensitive ion channels, Piezo1 and 2, are activated by pressure and involved in diverse physiological functions, including senses of touch and pain, proprioception and many more. Understanding their function is important for elucidating the mechanosensitive mechanisms of a range of human diseases. Recently, Piezo channels were suggested to be contributors to migraine pain generation. Migraine is typically characterized by allodynia and mechanical hyperalgesia associated with the activation and sensitization of trigeminal ganglion (TG) nerve fibers. Notably, migraine specific medicines are ineffective for other types of pain, suggesting a distinct underlying mechanism. To address, in a straightforward manner, the specificity of the mechanosensitivity of trigeminal vs. somatic nerves, we compared the activity of Piezo1 channels in mouse TG neurons vs. dorsal root ganglia (DRG) neurons. We assessed the functional expression of Piezo1 receptors using a conventional live calcium imaging setup equipped with a multibarrel application system and utilizing a microfluidic chip-based setup. Surprisingly, the TG neurons, despite higher expression of the Piezo1 gene, were less responsive to Piezo1 agonist Yoda1 than the DRG neurons. This difference was more prominent in the chip-based setup, suggesting that certain limitations of the conventional approach, such as turbulence, can be overcome by utilizing microfluidic devices with laminar solution flow.

Urban air particulate matter induces mitochondrial dysfunction in human olfactory mucosal cells.

BACKGROUND: The adverse effects of air pollutants including particulate matter (PM) on the central nervous system is increasingly reported by epidemiological, animal and post-mortem studies in the last decade. Oxidative stress and inflammation are key consequences of exposure to PM although little is known of the exact mechanism. The association of PM exposure with deteriorating brain health is speculated to be driven by PM entry via the olfactory system. How air pollutants affect this key entry site remains elusive. In this study, we investigated effects of urban size-segregated PM on a novel cellular model: primary human olfactory mucosal (hOM) cells. RESULTS: Metabolic activity was reduced following 24-h exposure to PM without evident signs of toxicity. Results from cytometric bead array suggested a mild inflammatory response to PM exposure. We observed increased oxidative stress and caspase-3/7 activity as well as perturbed mitochondrial membrane potential in PM-exposed cells. Mitochondrial dysfunction was further verified by a decrease in mitochondria-dependent respiration. Transient suppression of the mitochondria-targeted gene, neuronal pentraxin 1 (NPTX1), was carried out, after being identified to be up-regulated in PM2.5-1 treated cells via RNA sequencing. Suppression of NPTX1 in cells exposed to PM did not restore mitochondrial defects resulting from PM exposure. In contrast, PM-induced adverse effects were magnified in the absence of NPTX1, indicating a critical role of this protein in protection against PM effects in hOM cells. CONCLUSION: Key mitochondrial functions were perturbed by urban PM exposure in a physiologically relevant cellular model via a mechanism involving NPTX1. In addition, inflammatory response and early signs of apoptosis accompanied mitochondrial dysfunction during exposure to PM. Findings from this study contribute to increased understanding of harmful PM effects on human health and may provide information to support mitigation strategies targeted at air pollution.

The Emerging Role of Mechanosensitive Piezo Channels in Migraine Pain.

Recently discovered mechanosensitive Piezo channels emerged as the main molecular detectors of mechanical forces. The functions of Piezo channels range from detection of touch and pain, to control of the plastic changes in different organs. Recent studies suggested the role of Piezo channels in migraine pain, which is supposed to originate from the trigeminovascular nociceptive system in meninges. Interestingly, migraine pain is associated with such phenomenon as mechanical hypersensitivity, suggesting enhanced mechanotransduction. In the current review, we present the data that propose the implication of Piezo channels in migraine pain, which has a distinctive pulsatile character. These data include: (i) distribution of Piezo channels in the key elements of the trigeminovascular nociceptive system; (ii) the prolonged functional activity of Piezo channels in meningeal afferents providing a mechanistical basis for mechanotransduction in nociceptive nerve terminals; (iii) potential activation of Piezo channels by shear stress and pulsating blood flow; and (iv) modulation of these channels by emerging chemical agonists and modulators, including pro-nociceptive compounds. Achievements in this quickly expanding field should open a new road for efficient control of Piezo-related diseases including migraine and chronic pain.

Modulating Mitochondrial DNA Heteroplasmy with Mitochondrially Targeted Endonucleases.

Mitochondria, mainly known as energy factories of eukaryotic cells, also exert several additional signaling and metabolic functions and are today recognized as major cellular biosynthetic and signaling hubs. Mitochondria possess their own genome (mitochondrial DNA-mtDNA), that encodes proteins essential for oxidative phosphorylation, and mutations in it are an important contributor to human disease. The mtDNA mutations often exist in heteroplasmic conditions, with both healthy and mutant versions of the mtDNA residing in patients' cells and the level of mutant mtDNA may vary between different tissues and organs and affect the clinical outcome of the disease. Thus, shifting the ratio between healthy and mutant mtDNA in patients' cells provides an intriguing therapeutic option for mtDNA diseases. In this review we describe current strategies for modulating mitochondrial heteroplasmy levels with engineered endonucleases including mitochondrially targeted TALENs and Zinc finger nucleases (ZFNs) and discuss their therapeutic potential. These gene therapy tools could in the future provide therapeutic help both for patients with mitochondrial disease as well as in preventing the transfer of pathogenic mtDNA mutations from a mother to her offspring.

The role of the meningeal lymphatic system in local meningeal inflammation and trigeminal nociception.

A system of lymphatic vessels has been recently characterized in the meninges, with a postulated role in 'cleaning' the brain via cerebral fluid drainage. As meninges are the origin site of migraine pain, we hypothesized that malfunctioning of the lymphatic system should affect the local trigeminal nociception. To test this hypothesis, we studied nociceptive and inflammatory mechanisms in the hemiskull preparations (containing the meninges) of K14-VEGFR3-Ig (K14) mice lacking the meningeal lymphatic system. We recorded the spiking activity of meningeal afferents and estimated the local mast cells population, calcitonin gene-related peptide (CGRP) and cytokine levels as well as the dural trigeminal innervation in freshly-isolated hemiskull preparations from K14-VEGFR3-Ig (K14) or wild type C57BL/6 mice (WT). Spiking activity data have been confirmed in an acquired model of meningeal lymphatic dysfunction (AAV-mVEGFR3(1-4)Ig induced lymphatic ablation). We found that levels of the pro-inflammatory cytokine IL12-p70 and CGRP, implicated in migraine, were reduced in the meninges of K14 mice, while the levels of the mast cell activator MCP-1 were increased. The other migraine-related pro-inflammatory cytokines (basal and stimulated), did not differ between the two genotypes. The patterns of trigeminal innervation in meninges remained unchanged and we did not observe alterations in basal or ATP-induced nociceptive firing in the meningeal afferents associated with meningeal lymphatic dysfunction. In summary, the lack of meningeal lymphatic system is associated with a new balance between pro- and anti-migraine mediators but does not directly trigger meningeal nociceptive state.

Modeling a Nociceptive Neuro-Immune Synapse Activated by ATP and 5-HT in Meninges: Novel Clues on Transduction of Chemical Signals Into Persistent or Rhythmic Neuronal Firing.

Extracellular ATP and serotonin (5-HT) are powerful triggers of nociceptive firing in the meninges, a process supporting headache and whose cellular mechanisms are incompletely understood. The current study aimed to develop, with the neurosimulator NEURON, a novel approach to explore in silico the molecular determinants of the long-lasting, pulsatile nature of migraine attacks. The present model included ATP and 5-HT release, ATP diffusion and hydrolysis, 5-HT uptake, differential activation of ATP P2X or 5-HT3 receptors, and receptor subtype-specific desensitization. The model also tested the role of branched meningeal fibers with multiple release sites. Spike generation and propagation were simulated using variable contribution by potassium and sodium channels in a multi-compartment fiber environment. Multiple factors appeared important to ensure prolonged nociceptive firing potentially relevant to long-lasting pain. Crucial roles were observed in: (i) co-expression of ATP P2X2 and P2X3 receptor subunits; (ii) intrinsic activation/inactivation properties of sodium Nav1.8 channels; and (iii) temporal and spatial distribution of ATP/5-HT release sites along the branches of trigeminal nerve fibers. Based on these factors we could obtain either persistent activation of nociceptive firing or its periodic bursting mimicking the pulsating nature of pain. In summary, our model proposes a novel tool for the exploration of peripheral nociception to test the contribution of clinically relevant factors to headache including migraine pain.

Mechanosensitive meningeal nociception via Piezo channels: Implications for pulsatile pain in migraine?

BACKGROUND: Recent discovery of mechanosensitive Piezo receptors in trigeminal ganglia suggested the novel molecular candidate for generation of migraine pain. However, the contribution of Piezo channels in migraine pathology was not tested yet. Therefore, in this study, we explored a potential involvement of Piezo channels in peripheral trigeminal nociception implicated in generation of migraine pain. METHODS: We used immunohistochemistry, calcium imaging, calcitonin gene related peptide (CGRP) release assay and electrophysiology in mouse and rat isolated trigeminal neurons and rat hemiskulls to study action of various stimulants of Piezo receptors on migraine-related peripheral nociception. RESULTS: We found that essential (35%) fraction of isolated rat trigeminal neurons responded to chemical Piezo1 agonist Yoda1 and about a half of Yoda1 positive neurons responded to hypo-osmotic solution (HOS) and a quarter to mechanical stimulation by focused ultrasound (US). In ex vivo hemiskull preparation, Yoda1 and HOS largely activated persistent nociceptive firing in meningeal branches of trigeminal nerve. By using our novel cluster analysis of pain spikes, we demonstrated that 42% of fibers responded to Piezo1 agonist and 20% of trigeminal fibers were activated by Yoda1 and by capsaicin, suggesting expression of Piezo receptors in TRPV1 positive peptidergic nociceptive nerve fibers. Consistent with this, Yoda1 promoted the release of the key migraine mediator CGRP from hemiskull preparation. CONCLUSION: Taken together, our data suggest the involvement of mechanosensitive Piezo receptors, in particular, Piezo1 subtype in peripheral trigeminal nociception, which provides a new view on mechanotransduction in migraine pathology and suggests novel molecular targets for anti-migraine medicine.

Endoscopic Transsphenoidal Approach in Treatment of Germinomas of the Chiasmosellar Region.

INTRODUCTION: Germinogenic central nervous system (CNS) tumors represent a heterogeneous group of tumors, constituting approximately 0.4% of all primary brain tumors. Removal of the tumor has no prognostic value. In "pure" primary germinomas of the CNS, the alpha-fetoprotein and human chorionic gonadotropin levels are within normal limits, and no specific biochemical tumor markers currently exist for this tumor type, making histological verification crucial for the choice of treatment tactics. When the tumor is located in the chiasmosellar region, one of the possible verification methods is endoscopic endonasal transsphenoidal biopsy. OBJECTIVE: The main objective of the study is to demonstrate the feasibility and safety of endoscopic transsphenoidal approach for histological verification of primary germinomas of the CNS with chiasmosellar localization. MATERIALS AND METHODS: The current study includes 13 patients with "pure" germinomas of the chiasmosellar region who underwent endoscopic endonasal surgical interventions with subsequent treatment according to the "Germinoma 2008" protocol. RESULTS: The extent of surgical intervention ranged from biopsy (4) to partial (5) and total (4) removal of the tumor. In all cases, histological verification of the diagnosis was achieved and none of the patients presented with cerebrospinal fluid leaks and/or meningitis in the postoperative period, allowing to evaluate endoscopic intervention in our patient series as safe and effective. Two out of 13 patients were lost to follow-up. CONCLUSION: The endoscopic endonasal approach for histological verification and removal of chiasmosellar region germinomas is safe, and in some cases, less traumatic for the patient than transcranial and transventricular approaches.

Parasympathetic Cholinergic and Neuropeptide Mechanisms of Migraine.

CONTEXT: Migraine mechanisms remain largely uncovered for various reasons including a very high complexity of the neurophysiological mechanisms implicated in this disorder and a plethora of endogenous biologically active compounds involved in the pathological process. The functional role of parasympathetic innervation of meninges and cholinergic mechanisms of migraine are among little explored issues despite multiple evidence indirectly indicating the role of acetylcholine (ACh) and its analogues in migraine and other types of headache. In the current short review, we discuss morphological, functional, and clinical issues related to the role of ACh and its analogues such as carbachol and nicotine in this most common neurological disorder. EVIDENCE ACQUISITION: In the present work, studies published from 1953 to 2016 were investigated. Literature was searched with following keywords: acetylcholine (ACh), carbachol, nicotine, parasympathetic, mast cells, vasoactive intestinal polypeptide (VIP), and pituitary adenylate cyclase-activating polypeptide (PACAP). RESULTS: Parasympathetic fibers originated from SPG and trigeminal nerves can interact at the level of meninges which is considered to be the origin site of migraine pain. Here, in dura mater, ACh, VIP, and PACAP released by parasympathetic afferents can both affect mast cells provoking its degranulation and additional release of neurotransmitters, or they can directly affect trigeminal nerves inducing nociception. CONCLUSIONS: In summary, cholinergic mechanisms in migraine and other types of headache remain little elucidated and future studies should clarify the role of parasympathetic nerves and molecular mechanisms of cholinergic modulation within the nociceptive system.

Cholinergic Nociceptive Mechanisms in Rat Meninges and Trigeminal Ganglia: Potential Implications for Migraine Pain.

BACKGROUND: Parasympathetic innervation of meninges and ability of carbachol, acetylcholine (ACh) receptor (AChR) agonist, to induce headaches suggests contribution of cholinergic mechanisms to primary headaches. However, neurochemical mechanisms of cholinergic regulation of peripheral nociception in meninges, origin place for headache, are almost unknown. METHODS: Using electrophysiology, calcium imaging, immunohistochemistry, and staining of meningeal mast cells, we studied effects of cholinergic agents on peripheral nociception in rat hemiskulls and isolated trigeminal neurons. RESULTS: Both ACh and carbachol significantly increased nociceptive firing in peripheral terminals of meningeal trigeminal nerves recorded by local suction electrode. Strong nociceptive firing was also induced by nicotine, implying essential role of nicotinic AChRs in control of excitability of trigeminal nerve endings. Nociceptive firing induced by carbachol was reduced by muscarinic antagonist atropine, whereas the action of nicotine was prevented by the nicotinic blocker d-tubocurarine but was insensitive to the TRPA1 antagonist HC-300033. Carbachol but not nicotine induced massive degranulation of meningeal mast cells known to release multiple pro-nociceptive mediators. Enzymes terminating ACh action, acetylcholinesterase (AChE) and butyrylcholinesterase, were revealed in perivascular meningeal nerves. The inhibitor of AChE neostigmine did not change the firing per se but induced nociceptive activity, sensitive to d-tubocurarine, after pretreatment of meninges with the migraine mediator CGRP. This observation suggested the pro-nociceptive action of endogenous ACh in meninges. Both nicotine and carbachol induced intracellular Ca2+ transients in trigeminal neurons partially overlapping with expression of capsaicin-sensitive TRPV1 receptors. CONCLUSION: Trigeminal nerve terminals in meninges, as well as dural mast cells and trigeminal ganglion neurons express a repertoire of pro-nociceptive nicotinic and muscarinic AChRs, which could be activated by the ACh released from parasympathetic nerves. These receptors represent a potential target for novel therapeutic interventions in trigeminal pain and probably in migraine.