Shadow imaging for panoptical visualization of brain tissue in vivo.

Progress in neuroscience research hinges on technical advances in visualizing living brain tissue with high fidelity and facility. Current neuroanatomical imaging approaches either require tissue fixation (electron microscopy), do not have cellular resolution (magnetic resonance imaging) or only give a fragmented view (fluorescence microscopy). Here, we show how regular light microscopy together with fluorescence labeling of the interstitial fluid in the extracellular space provide comprehensive optical access in real-time to the anatomical complexity and dynamics of living brain tissue at submicron scale. Using several common fluorescence microscopy modalities (confocal, light-sheet and 2-photon microscopy) in mouse organotypic and acute brain slices and the intact mouse brain in vivo, we demonstrate the value of this straightforward 'shadow imaging' approach by revealing neurons, microglia, tumor cells and blood capillaries together with their complete anatomical tissue contexts. In addition, we provide quantifications of perivascular spaces and the volume fraction of the extracellular space of brain tissue in vivo.

Another win for mimetic peptides in stroke: Disruption of TRPM2-NMDAR signaling.

Glutamate excitotoxicity during ischemia triggers an intracellular signaling avalanche leading to cell death, yet blocking NMDA receptors directly in human stroke trials failed. In this issue of Neuron, Zong et al. (2022) disrupt downstream NMDAR-TRPM2 coupling to improve stroke outcomes, supporting intracellular NMDAR signaling as an alternate therapeutic target.

Limiting RyR2 Open Time Prevents Alzheimer's Disease-Related Neuronal Hyperactivity and Memory Loss but Not ß-Amyloid Accumulation.

Neuronal hyperactivity is an early primary dysfunction in Alzheimer's disease (AD) in humans and animal models, but effective neuronal hyperactivity-directed anti-AD therapeutic agents are lacking. Here we define a previously unknown mode of ryanodine receptor 2 (RyR2) control of neuronal hyperactivity and AD progression. We show that a single RyR2 point mutation, E4872Q, which reduces RyR2 open time, prevents hyperexcitability, hyperactivity, memory impairment, neuronal cell death, and dendritic spine loss in a severe early-onset AD mouse model (5xFAD). The RyR2-E4872Q mutation upregulates hippocampal CA1-pyramidal cell A-type K+ current, a well-known neuronal excitability control that is downregulated in AD. Pharmacologically limiting RyR2 open time with the R-carvedilol enantiomer (but not racemic carvedilol) prevents and rescues neuronal hyperactivity, memory impairment, and neuron loss even in late stages of AD. These AD-related deficits are prevented even with continued ß-amyloid accumulation. Thus, limiting RyR2 open time may be a hyperactivity-directed, non-ß-amyloid-targeted anti-AD strategy.

Author Correction: Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

Stress gates an astrocytic energy reservoir to impair synaptic plasticity.

Astrocytes support the energy demands of synaptic transmission and plasticity. Enduring changes in synaptic efficacy are highly sensitive to stress, yet whether changes to astrocyte bioenergetic control of synapses contributes to stress-impaired plasticity is unclear. Here we show in mice that stress constrains the shuttling of glucose and lactate through astrocyte networks, creating a barrier for neuronal access to an astrocytic energy reservoir in the hippocampus and neocortex, compromising long-term potentiation. Impairing astrocytic delivery of energy substrates by reducing astrocyte gap junction coupling with dominant negative connexin 43 or by disrupting lactate efflux was sufficient to mimic the effects of stress on long-term potentiation. Furthermore, direct restoration of the astrocyte lactate supply alone rescued stress-impaired synaptic plasticity, which was blocked by inhibiting neural lactate uptake. This gating of synaptic plasticity in stress by astrocytic metabolic networks indicates a broader role of astrocyte bioenergetics in determining how experience-dependent information is controlled.

A novel motif in the proximal C-terminus of Pannexin 1 regulates cell surface localization.

The Pannexin 1 (Panx1) ion and metabolite channel is expressed in a wide variety of cells where it regulates a number of cell behaviours including proliferation and differentiation. Panx1 is expressed on the cell surface as well as intracellular membranes. Previous work suggests that a region within the proximal Panx1 C-terminus (Panx1CT) regulates cell surface localization. Here we report the discovery of a putative leucine-rich repeat (LRR) motif in the proximal Panx1CT necessary for Panx1 cell surface expression in HEK293T cells. Deletion of the putative LRR motif results in significant loss of Panx1 cell surface distribution. Outcomes of complementary cell surface oligomerization and glycosylation state analyses were consistent with reduced cell surface expression of Panx1 LRR deletion mutants. Of note, the oligomerization analysis revealed the presence of putative dimers and trimers of Panx1 at the cell surface. Expression of Panx1 increased HEK293T cell growth and reduced doubling time, while expression of a Panx1 LRR deletion mutant (highly conserved segment) did not reproduce this effect. In summary, here we discovered the presence of a putative LRR motif in the Panx1CT that impacts on Panx1 cell surface localization. Overall these findings provide new insights into the molecular mechanisms underlying C-terminal regulation of Panx1 trafficking and raise potential new lines of investigation with respect to Panx1 oligomerization and glycosylation.

Interaction Between Pannexin 1 and Caveolin-1 in Smooth Muscle Can Regulate Blood Pressure.

Objective- Sympathetic nerve innervation of vascular smooth muscle cells (VSMCs) is a major regulator of arteriolar vasoconstriction, vascular resistance, and blood pressure. Importantly, α-adrenergic receptor stimulation, which uniquely couples with Panx1 (pannexin 1) channel-mediated ATP release in resistance arteries, also requires localization to membrane caveolae. Here, we test whether localization of Panx1 to Cav1 (caveolin-1) promotes channel function (stimulus-dependent ATP release and adrenergic vasoconstriction) and is important for blood pressure homeostasis. Approach and Results- We use in vitro VSMC culture models, ex vivo resistance arteries, and a novel inducible VSMC-specific Cav1 knockout mouse to probe interactions between Panx1 and Cav1. We report that Panx1 and Cav1 colocalized on the VSMC plasma membrane of resistance arteries near sympathetic nerves in an adrenergic stimulus-dependent manner. Genetic deletion of Cav1 significantly blunts adrenergic-stimulated ATP release and vasoconstriction, with no direct influence on endothelium-dependent vasodilation or cardiac function. A significant reduction in mean arterial pressure (total=4 mm Hg; night=7 mm Hg) occurred in mice deficient for VSMC Cav1. These animals were resistant to further blood pressure lowering using a Panx1 peptide inhibitor Px1IL2P, which targets an intracellular loop region necessary for channel function. Conclusions- Translocalization of Panx1 to Cav1-enriched caveolae in VSMCs augments the release of purinergic stimuli necessary for proper adrenergic-mediated vasoconstriction and blood pressure homeostasis.

The prolyl isomerase FKBP25 regulates microtubule polymerization impacting cell cycle progression and genomic stability.

FK506 binding proteins (FKBPs) catalyze the interconversion of cis-trans proline conformers in proteins. Importantly, FK506 drugs have anti-cancer and neuroprotective properties, but the effectors and mechanisms underpinning these properties are not well understood because the cellular function(s) of most FKBP proteins are unclear. FKBP25 is a nuclear prolyl isomerase that interacts directly with nucleic acids and is associated with several DNA/RNA binding proteins. Here, we show the catalytic FKBP domain binds microtubules (MTs) directly to promote their polymerization and stabilize the MT network. Furthermore, FKBP25 associates with the mitotic spindle and regulates entry into mitosis. This interaction is important for mitotic spindle dynamics, as we observe increased chromosome instability in FKBP25 knockdown cells. Finally, we provide evidence that FKBP25 association with chromatin is cell-cycle regulated by Protein Kinase C phosphorylation. This disrupts FKBP25-DNA contacts during mitosis while maintaining its interaction with the spindle apparatus. Collectively, these data support a model where FKBP25 association with chromatin and MTs is carefully choreographed to ensure faithful genome duplication. Additionally, they highlight that FKBP25 is a MT-associated FK506 receptor and potential therapeutic target in MT-associated diseases.

Transcriptional and post-translational regulation of pannexins.

Pannexins are a 3-membered family of proteins that form large pore ion and metabolite channels in vertebrates. The impact of pannexins on vertebrate biology is intricately tied to where and when they are expressed, and how they are modified, once produced. The purpose of this review is therefore to outline our current understanding of transcriptional and post-translational regulation of pannexins. First, we briefly summarize their discovery and characteristics. Next, we describe several aspects of transcriptional regulation, including cell and tissue-specific expression, dynamic expression over development and disease, as well as new insights into the underlying molecular machinery involved. Following this, we delve into the role of post-translational modifications in the regulation of trafficking and channel properties, highlighting important work on glycosylation, phosphorylation, S-nitrosylation and proteolytic cleavage. Embedded throughout, we also highlight important knowledge gaps and avenues of future research. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.

Regulation of Pannexin 1 Surface Expression by Extracellular ATP: Potential Implications for Nervous System Function in Health and Disease.

Pannexin 1 (Panx1) channels are widely recognized for their role in ATP release, and as follows, their function is closely tied to that of ATP-activated P2X7 purinergic receptors (P2X7Rs). Our recent work has shown that extracellular ATP induces clustering of Panx1 with P2X7Rs and their subsequent internalization through a non-canonical cholesterol-dependent mechanism. In other words, we have demonstrated that extracellular ATP levels can regulate the cell surface expression of Panx1. Here we discuss two situations in which we hypothesize that ATP modulation of Panx1 surface expression could be relevant for central nervous system function. The first scenario involves the development of new neurons in the ventricular zone. We propose that ATP-induced Panx1 endocytosis could play an important role in regulating the balance of cell proliferation, survival, and differentiation within this neurogenic niche in the healthy brain. The second scenario relates to the spinal cord, in which we posit that an impairment of ATP-induced Panx1 endocytosis could contribute to pathological neuroplasticity. Together, the discussion of these hypotheses serves to highlight important outstanding questions regarding the interplay between extracellular ATP, Panx1, and P2X7Rs in the nervous system in health and disease.

P2X7 receptor cross-talk regulates ATP-induced pannexin 1 internalization.

In the nervous system, extracellular ATP levels transiently increase in physiological and pathophysiological circumstances, effecting key signalling pathways in plasticity and inflammation through purinergic receptors. Pannexin 1 (Panx1) forms ion- and metabolite-permeable channels that mediate ATP release and are particularly enriched in the nervous system. Our recent study demonstrated that elevation of extracellular ATP triggers Panx1 internalization in a concentration- and time-dependent manner. Notably, this effect was sensitive to inhibition of ionotropic P2X7 purinergic receptors (P2X7Rs). Here, we report our novel findings from the detailed investigation of the mechanism underlying P2X7R-Panx1 cross-talk in ATP-stimulated internalization. We demonstrate that extracellular ATP triggers and is required for the clustering of P2X7Rs and Panx1 on Neuro2a cells through an extracellular physical interaction with the Panx1 first extracellular loop (EL1). Importantly, disruption of P2X7R-Panx1 clustering by mutation of tryptophan 74 within the Panx1 EL1 inhibits Panx1 internalization. Notably, P2X7R-Panx1 clustering and internalization are independent of P2X7R-associated intracellular signalling pathways (Ca2+ influx and Src activation). Further analysis revealed that cholesterol is required for ATP-stimulated P2X7R-Panx1 clustering at the cell periphery. Taken together, our data suggest that extracellular ATP induces and is required for Panx1 EL1-mediated, cholesterol-dependent P2X7R-Panx1 clustering and endocytosis. These findings have important implications for understanding the role of Panx1 in the nervous system and provide important new insights into Panx1-P2X7R cross-talk.

ATP stimulates pannexin 1 internalization to endosomal compartments.

The ubiquitous pannexin 1 (Panx1) ion- and metabolite-permeable channel mediates the release of ATP, a potent signalling molecule. In the present study, we provide striking evidence that ATP, in turn, stimulates internalization of Panx1 to intracellular membranes. These findings hold important implications for understanding the regulation of Panx1 when extracellular ATP is elevated. In the nervous system, this includes phenomena such as synaptic plasticity, pain, precursor cell development and stroke; outside of the nervous system, this includes things like skeletal and smooth muscle activity and inflammation. Within 15 min, ATP led to significant Panx1-EGFP internalization. In a series of experiments, we determined that hydrolysable ATP is the most potent stimulator of Panx1 internalization. We identified two possible mechanisms for Panx1 internalization, including activation of ionotropic purinergic (P2X) receptors and involvement of a putative ATP-sensitive residue in the first extracellular loop of Panx1 (Trp(74)). Internalization was cholesterol-dependent, but clathrin, caveolin and dynamin independent. Detailed analysis of Panx1 at specific endosome sub-compartments confirmed that Panx1 is expressed in endosome membranes of the classical degradation pathway under basal conditions and that elevation of ATP levels diverts a sub-population to recycling endosomes. This is the first report detailing endosome localization of Panx1 under basal conditions and the potential for ATP regulation of its surface expression. Given the ubiquitous expression profile of Panx1 and the importance of ATP signalling, these findings are of critical importance for understanding the role of Panx1 in health and disease.

Powerful partnership: crosstalk between pannexin 1 and the cytoskeleton.

The roles of pannexin 1 (Panx1) large-pore ion and metabolite channels are becoming recognized in many physiological and pathophysiological scenarios. Recent evidence has tightly linked Panx1 trafficking and function to the cytoskeleton, a multi-component network that provides critical structural support, transportation, and scaffolding functions in all cell types. Here we review early work demonstrating the mechanosensitive activation of Panx1 channels, and expand on more recent evidence directly linking Panx1 to the cytoskeleton. Further, we examine the reciprocal regulation between Panx1 and the cytoskeleton, and discuss the involvement of Panx1 in cytoskeletal-regulated cell behaviors. Finally, we identify important gaps in the current knowledge surrounding this emerging Panx1-cytoskeleton relationship.

Pore positioning: current concepts in Pannexin channel trafficking.

Pannexins (Panxs) are a multifaceted family of ion and metabolite channels that play key roles in a number of physiological and pathophysiological settings. These single membrane large-pore channels exhibit a variety of tissue, cell type, and subcellular distributions. The lifecycles of Panxs are complex, yet must be understood to accurately target these proteins for future therapeutic use. Here we review the basics of Panx function and localization, and then analyze the recent advances in knowledge regarding Panx trafficking. We examine several intrinsic features of Panxs including specific post-translational modifications, the divergent C-termini, and oligomerization, all of which contribute to Panx anterograde transport pathways. Further, we examine the potential influence of extrinsic factors, such as protein-protein interactions, on Panx trafficking. Finally, we highlight what is currently known with respect to Panx internalization and retrograde transport, and present new data illustrating Panx1 internalization following an activating stimulus.

Analysis of a pannexin 2-pannexin 1 chimeric protein supports divergent roles for pannexin C-termini in cellular localization.

Pannexins (Panxs) are a three-member family of large pore ion channels permeable to ions and small molecules. Recent elegant work has demonstrated that the Panx1 C-terminus plays an important role in channel trafficking. Panx2, another family member, has a longer and highly dissimilar C-terminus. Interestingly, Panx1 is readily found at the plasma membrane, while Panx2 is mainly present on intracellular membranes. Here we used overlap-extension cloning to create the first chimeric Panx, consisting of Panx2 with the Panx1 C-terminus (Panx2(Panx1CT)), to determine whether the Panx1 C-terminus influences the trafficking of Panx2. We are the first to observe a high level of co-localization between Panx2 and the endolysosomal enriched mannose-6-phosphate receptor. Interestingly this distinct localization of Panx2 is altered by the presence of the Panx1 C-terminus. These novel observations support previous data indicating the importance of the C-terminus in the control of Panx trafficking, and highlight the complexity of molecular signals involved.