Pannexin-1 opening in neuronal edema causes cell death but also leads to protection via increased microglia contacts.

Neuronal swelling during cytotoxic edema is triggered by Na+ and Cl- entry and is Ca2+ independent. However, the causes of neuronal death during swelling are unknown. Here, we investigate the role of large-conductance Pannexin-1 (Panx1) channels in neuronal death during cytotoxic edema. Panx1 channel inhibitors reduce and delay neuronal death in swelling triggered by voltage-gated Na+ entry with veratridine. Neuronal swelling causes downstream production of reactive oxygen species (ROS) that opens Panx1 channels. We confirm that ROS activates Panx1 currents with whole-cell electrophysiology and find scavenging ROS is neuroprotective. Panx1 opening and subsequent ATP release attract microglial processes to contact swelling neurons. Depleting microglia using the CSF1 receptor antagonist PLX3397 or blocking P2Y12 receptors exacerbates neuronal death, suggesting that the Panx1-ATP-dependent microglia contacts are neuroprotective. We conclude that cytotoxic edema triggers oxidative stress in neurons that opens Panx1 to trigger death but also initiates neuroprotective feedback mediated by microglia contacts.

hiPSC-derived GRN-deficient astrocytes delay spiking activity of developing neurons.

Frontotemporal dementia (FTD) refers to a group of neurodegenerative disorders that are characterized by pathology predominantly localized to the frontal and temporal lobes. Approximately 40% of FTD cases are familial, and up to 20% of these are caused by heterozygous loss of function mutations in the gene encoding for progranulin (PGRN), GRN. The mechanisms by which loss of PGRN leads to FTD remain incompletely understood. While astrocytes and microglia have long been linked to the neuropathology of FTD due to mutations in GRN (FTD-GRN), a primary mechanistic role of these supporting cells have not been thoroughly addressed. In contrast, mutations in MAPT, another leading cause of familial FTD, greatly alters astrocyte gene expression leading to subsequent non-cell autonomous effects on neurons, suggesting similar mechanisms may be present in FTD-GRN. Here, we utilized human induced pluripotent stem cell (hiPSC)-derived neural tissue carrying a homozygous GRN R493X-/- knock-in mutation to investigate in vitro whether GRN mutant astrocytes have a non-cell autonomous effect on neurons. Using microelectrode array (MEA) analysis, we demonstrate that the development of spiking activity of neurons cultured with GRN R493X-/- astrocytes was significantly delayed compared to cultures with WT astrocytes. Histological analysis of synaptic markers in these cultures showed an increase in GABAergic synaptic markers and a decrease in glutamatergic synaptic markers during this period when activity was delayed. We also demonstrate that this effect may be due in-part to soluble factors. Overall, this work represents one of the first studies investigating astrocyte-induced neuronal pathology in GRN mutant hiPSCs, and supports the hypothesis of astrocyte involvement in the early pathophysiology of FTD.

KCC2 drives chloride microdomain formation in dendritic blebbing.

Intracellular chloride ion concentration ([Cl-]i) homeostasis is critical for excitatory/inhibitory balance and volume regulation in neurons. We quantitatively map spatiotemporal dendritic [Cl-]i dynamics during N-methyl-d-aspartate (NMDA) excitotoxicity to determine how Cl- changes contribute to localized dendritic swelling (blebbing) in stroke-like conditions. Whole-cell patch clamp electrophysiology combined with simultaneous fluorescence lifetime imaging (FLIM) of the Cl- dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE; MQAE-FLIM) reliably report resting and dynamic [Cl-]i shifts in dendrites. NMDA application generates spatially restricted and persistent high [Cl-]i subdomains at dendritic blebs in a process that requires Ca2+ influx and the subsequent opening of small-conductance Ca2+-activated K+ (SK) channels. We propose sustained and localized K+ efflux increased extracellular K+ concentrations ([K+]o) sufficiently at discrete regions to reverse K+-Cl- cotransporter (KCC2) transport and trigger synaptic swelling. Together, our data establish a mechanism for KCC2 to generate pathological [Cl-]i microdomains in blebbing with relevance for multiple neurological disorders.

Simultaneous imaging of redox states in dystrophic neurites and microglia at Aß plaques indicate lysosome accumulation not microglia correlate with increased oxidative stress.

The inter-relationship between microglia dynamics and oxidative stress (Ox-stress) in dystrophic neurites (DNs) at Alzheimer's Disease (AD) plaques may contribute to the pathological changes in neurons. We developed new in vivo imaging strategies to combine EGFP expression in microglia with neuronal expression of genetically encoded ratiometric redox sensors (rogRFP2 or roGFP1), and immunohistochemistry to investigate how microglia influence Ox-stress at amyloid plaques in 5xFAD AD mice. By simultaneously imaging microglia morphology and neuronal Ox-stress over time in vivo and in fixed brains we found that microglia preferentially enwrapped DNs exhibiting the greatest degree of Ox-stress. After microglia were partially depleted with the CSF1 receptor antagonist PLX3397, Ox-stress in DNs increased in a manner that was inversely correlated to the extent of coverage of the adjacent Aß plaques by the remaining microglia. These data suggest that microglia do not create Ox-stress at Aß plaques but instead create protective barriers around Aß plaques possibly reducing the spread of Aß. Intracranial injection of Aß was sufficient to induce neuronal Ox-stress suggesting it to be the initial trigger of Ox-stress generation. Although Ox-stress is increased in DNs, neuronal survival is enhanced following microglia depletion indicating complex and multifactorial roles of microglia with both neurotoxic and neuroprotective components. Increased Ox-stress of DNs was correlated with higher LAMP1 and ubiquitin immunoreactivity supporting proposed mechanistic links between lysosomal accumulation in DNs and their intrinsic generation of Ox-stress. Our results suggest protective as well as neurotoxic roles for microglia at plaques and that the generation of Ox-stress of DNs could intrinsically be generated via lysosomal disruption rather than by microglia. In Brief: Simultaneous imaging of microglia and neuronal Ox-stress revealed a double-edged role for microglia in 5xFAD mice. Plaque associated microglia were attracted to and enwrapped Aß plaques as well as the most highly oxidized DNs. After partial depletion of microglia, DNs were larger with greater levels of Ox-stress. Despite increased Ox-stress after microglia removal neuronal survival improved. Greater Ox-stress was correlated with increased levels of LAMP1 and ubiquitin thereby linking lysosome accumulation and Ox-stress in DNs.

An in vitro bioengineered model of the human arterial neurovascular unit to study neurodegenerative diseases.

INTRODUCTION: The neurovascular unit (NVU) - the interaction between the neurons and the cerebrovasculature - is increasingly important to interrogate through human-based experimental models. Although advanced models of cerebral capillaries have been developed in the last decade, there is currently no in vitro 3-dimensional (3D) perfusible model of the human cortical arterial NVU. METHOD: We used a tissue-engineering technique to develop a scaffold-directed, perfusible, 3D human NVU that is cultured in native-like flow conditions that mimics the anatomy and physiology of cortical penetrating arteries. RESULTS: This system, composed of primary human vascular cells (endothelial cells, smooth muscle cells and astrocytes) and induced pluripotent stem cell (iPSC) derived neurons, demonstrates a physiological multilayer organization of the involved cell types. It reproduces key characteristics of cortical neurons and astrocytes and enables formation of a selective and functional endothelial barrier. We provide proof-of-principle data showing that this in vitro human arterial NVU may be suitable to study neurovascular components of neurodegenerative diseases such as Alzheimer's disease (AD), as endogenously produced phosphorylated tau and beta-amyloid accumulate in the model over time. Finally, neuronal and glial fluid biomarkers relevant to neurodegenerative diseases are measurable in our arterial NVU model. CONCLUSION: This model is a suitable research tool to investigate arterial NVU functions in healthy and disease states. Further, the design of the platform allows culture under native-like flow conditions for extended periods of time and yields sufficient tissue and media for downstream immunohistochemistry and biochemistry analyses.

Microglial metabolic flexibility supports immune surveillance of the brain parenchyma.

Microglia are highly motile cells that continuously monitor the brain environment and respond to damage-associated cues. While glucose is the main energy substrate used by neurons in the brain, the nutrients metabolized by microglia to support surveillance of the parenchyma remain unexplored. Here, we use fluorescence lifetime imaging of intracellular NAD(P)H and time-lapse two-photon imaging of microglial dynamics in vivo and in situ, to show unique aspects of the microglial metabolic signature in the brain. Microglia are metabolically flexible and can rapidly adapt to consume glutamine as an alternative metabolic fuel in the absence of glucose. During insulin-induced hypoglycemia in vivo or in aglycemia in acute brain slices, glutaminolysis supports the maintenance of microglial process motility and damage-sensing functions. This metabolic shift sustains mitochondrial metabolism and requires mTOR-dependent signaling. This remarkable plasticity allows microglia to maintain their critical surveillance and phagocytic roles, even after brain neuroenergetic homeostasis is compromised.

Suppression of Presynaptic Glutamate Release by Postsynaptic Metabotropic NMDA Receptor Signalling to Pannexin-1.

The impact of pannexin-1 (Panx1) channels on synaptic transmission is poorly understood. Here, we show that selective block of Panx1 in single postsynaptic hippocampal CA1 neurons from male rat or mouse brain slices causes intermittent, seconds long increases in the frequency of sEPSC following Schaffer collateral stimulation. The increase in sEPSC frequency occurred without an effect on evoked neurotransmission. Consistent with a presynaptic origin of the augmented glutamate release, the increased sEPSC frequency was prevented by bath-applied EGTA-AM or TTX. Manipulation of a previously described metabotropic NMDAR pathway (i.e., by preventing ligand binding to NMDARs with competitive antagonists or blocking downstream Src kinase) also increased sEPSC frequency similar to that seen when Panx1 was blocked. This facilitated glutamate release was absent in transient receptor potential vanilloid 1 (TRPV1) KO mice and prevented by the TRPV1 antagonist, capsazepine, suggesting it required presynaptic TRPV1. We show presynaptic expression of TRPV1 by immunoelectron microscopy and link TRPV1 to Panx1 because Panx1 block increases tissue levels of the endovanilloid, anandamide. Together, these findings demonstrate an unexpected role for metabotropic NMDARs and postsynaptic Panx1 in suppression of facilitated glutamate neurotransmission.SIGNIFICANCE STATEMENT The postsynaptic ion and metabolite channel, pannexin-1, is regulated by metabotropic NMDAR signaling through Src kinase. This pathway suppresses facilitated release of presynaptic glutamate during synaptic activity by regulating tissue levels of the transient receptor potential vanilloid 1 agonist anandamide.

Green fluorescent protein emission obscures metabolic fluorescent lifetime imaging of NAD(P)H.

Autofluorescence of endogenous molecules can provide valuable insights in both basic research and clinical applications. One such technique is fluorescence lifetime imaging (FLIM) of NAD(P)H, which serves as a correlate of glycolysis and electron transport chain rates in metabolically active tissue. A powerful advantage of NAD(P)H-FLIM is the ability to measure cell-specific metabolism within heterogeneous tissues. Cell-type specific identification is most commonly achieved with directed green fluorescent protein (GFP) expression. However, we demonstrate that NAD(P)H-FLIM should not be analyzed in GFP-expressing cells, as GFP molecules themselves emit photons in the blue spectrum with short fluorescence lifetimes when imaged using two-photon excitation at 750 nm. This is substantially different from the reported GFP emission wavelength and lifetime after two-photon excitation at 910 nm. These blue GFP photons are indistinguishable from free NAD(P)H by both emission spectra and fluorescence lifetime. Therefore, NAD(P)H-FLIM in GFP-expressing cells will lead to incorrect interpretations of metabolic rates, and thus, these techniques should not be combined.

Regulation of pannexin channels in the central nervous system by Src family kinases.

Pannexins form single membrane channels that regulate the passage of ions, small molecules and metabolites between the intra- and extracellular compartments. In the central nervous system, these channels are integrated into numerous signaling cascades that shape brain physiology and pathology. Post-translational modification of pannexins is complex, with phosphorylation emerging as a prominent form of functional regulation. While much is still not known regarding the specific kinases and modified amino acids, recent reports support a role for Src family tyrosine kinases (SFK) in regulating pannexin channel activity. This review outlines the current evidence supporting SFK-dependent pannexin phosphorylation in the CNS and examines the importance of these modifications in the healthy and diseased brain.

Rap2 and TNIK control Plexin-dependent tiled synaptic innervation in C. elegans.

During development, neurons form synapses with their fate-determined targets. While we begin to elucidate the mechanisms by which extracellular ligand-receptor interactions enhance synapse specificity by inhibiting synaptogenesis, our knowledge about their intracellular mechanisms remains limited. Here we show that Rap2 GTPase (rap-2) and its effector, TNIK (mig-15), act genetically downstream of Plexin (plx-1) to restrict presynaptic assembly and to form tiled synaptic innervation in C. elegans. Both constitutively GTP- and GDP-forms of rap-2 mutants exhibit synaptic tiling defects as plx-1 mutants, suggesting that cycling of the RAP-2 nucleotide state is critical for synapse inhibition. Consistently, PLX-1 suppresses local RAP-2 activity. Excessive ectopic synapse formation in mig-15 mutants causes a severe synaptic tiling defect. Conversely, overexpression of mig-15 strongly inhibited synapse formation, suggesting that mig-15 is a negative regulator of synapse formation. These results reveal that subcellular regulation of small GTPase activity by Plexin shapes proper synapse patterning in vivo.

Site-Specific Regulation of P2X7 Receptor Function in Microglia Gates Morphine Analgesic Tolerance.

Tolerance to the analgesic effects of opioids is a major problem in chronic pain management. Microglia are implicated in opioid tolerance, but the core mechanisms regulating their response to opioids remain obscure. By selectively ablating microglia in the spinal cord using a saporin-conjugated antibody to Mac1, we demonstrate a causal role for microglia in the development, but not maintenance, of morphine tolerance in male rats. Increased P2X7 receptor (P2X7R) activity is a cardinal feature of microglial activation, and in this study we found that morphine potentiates P2X7R-mediated Ca2+ responses in resident spinal microglia acutely isolated from morphine tolerant rats. The increased P2X7R function was blocked in cultured microglia by PP2, a Src family protein tyrosine kinase inhibitor. We identified Src family kinase activation mediated by µ-receptors as a key mechanistic step required for morphine potentiation of P2X7R function. Furthermore, we show by site-directed mutagenesis that tyrosine (Y382-384) within the P2X7R C-terminus is differentially modulated by repeated morphine treatment and has no bearing on normal P2X7R function. Intrathecal administration of a palmitoylated peptide corresponding to the Y382-384 site suppressed morphine-induced microglial reactivity and preserved the antinociceptive effects of morphine in male rats. Thus, site-specific regulation of P2X7R function mediated by Y382-384 is a novel cellular determinant of the microglial response to morphine that critically underlies the development of morphine analgesic tolerance.SIGNIFICANCE STATEMENT Controlling pain is one of the most difficult challenges in medicine and its management is a requirement of a large diversity of illnesses. Although morphine and other opioids offer dramatic and impressive relief of pain, their impact is truncated by loss of efficacy (analgesic tolerance). Understanding why this occurs and how to prevent it are of critical importance in improving pain therapies. We uncovered a novel site (Y382-384) within the P2X7 receptor that can be targeted to blunt the development of morphine analgesic tolerance, without affecting normal P2X7 receptor function. Our findings provide a critical missing mechanistic piece, site-specific modulation by Y382-384, that unifies P2X7R function to the activation of spinal microglia and the development of morphine tolerance.

Metabotropic NMDA receptor signaling couples Src family kinases to pannexin-1 during excitotoxicity.

Overactivation of neuronal N-methyl-D-aspartate receptors (NMDARs) causes excitotoxicity and is necessary for neuronal death. In the classical view, these ligand-gated Ca(2+)-permeable ionotropic receptors require co-agonists and membrane depolarization for activation. We report that NMDARs signal during ligand binding without activation of their ion conduction pore. Pharmacological pore block with MK-801, physiological pore block with Mg(2+) or a Ca(2+)-impermeable NMDAR variant prevented NMDAR currents, but did not block excitotoxic dendritic blebbing and secondary currents induced by exogenous NMDA. NMDARs, Src kinase and Panx1 form a signaling complex, and activation of Panx1 required phosphorylation at Y308. Disruption of this NMDAR-Src-Panx1 signaling complex in vitro or in vivo by administration of an interfering peptide either before or 2 h after ischemia or stroke was neuroprotective. Our observations provide insights into a new signaling modality of NMDARs that has broad-reaching implications for brain physiology and pathology.

Ionotropic receptors and ion channels in ischemic neuronal death and dysfunction.

Loss of energy supply to neurons during stroke induces a rapid loss of membrane potential that is called the anoxic depolarization. Anoxic depolarizations result in tremendous physiological stress on the neurons because of the dysregulation of ionic fluxes and the loss of ATP to drive ion pumps that maintain electrochemical gradients. In this review, we present an overview of some of the ionotropic receptors and ion channels that are thought to contribute to the anoxic depolarization of neurons and subsequently, to cell death. The ionotropic receptors for glutamate and ATP that function as ligand-gated cation channels are critical in the death and dysfunction of neurons. Interestingly, two of these receptors (P2X7 and NMDAR) have been shown to couple to the pannexin-1 (Panx1) ion channel. We also discuss the important roles of transient receptor potential (TRP) channels and acid-sensing ion channels (ASICs) in responses to ischemia. The central challenge that emerges from our current understanding of the anoxic depolarization is the need to elucidate the mechanistic and temporal interrelations of these ion channels to fully appreciate their impact on neurons during stroke.

Anoxia-induced NMDA receptor activation opens pannexin channels via Src family kinases.

Anoxic depolarization of pyramidal neurons results from a large inward current that is activated, in part, by excessive glutamate release during exposure to anoxia/ischemia. Pannexin-1 (Panx1) channels can be activated both by ischemia and NMDA receptors (NMDARs), but the mechanisms of Panx1 activation are unknown. We used whole-cell recordings to show that pharmacological inhibition or conditional genetic deletion of Panx1 strongly attenuates the anoxic depolarization of CA1 pyramidal neurons in acute brain slices from rats and mice. Anoxia or exogenous NMDA activated Src family kinases (SFKs), as measured by increased phosphorylation of SFKs at Y416. The SFK inhibitor PP2 prevented Src activation and Panx1 opening during anoxia. A newly developed interfering peptide that targets the SFK consensus-like sequence of Panx1 (Y308) attenuated the anoxic depolarization (AD) without affecting SFK activation. Importantly, the NMDAR antagonists, D-APV and R-CPP, attenuated AD currents carried by Panx1, and the combined application of D-APV and (10)panx (a Panx1 blocker) inhibited AD currents to the same extent as either blocker alone. We conclude that activation of NMDARs during anoxia/ischemia recruits SFKs to open Panx1, leading to sustained neuronal depolarizations.