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GluN3A excitatory glycine receptors control adult cortical and amygdalar circuits.

GluN3A is an atypical glycine-binding subunit of NMDA receptors (NMDARs) whose actions in the brain are mostly unknown. Here, we show that the expression of GluN3A subunits controls the excitability of mouse adult cortical and amygdalar circuits via an unusual signaling mechanism involving the formation of excitatory glycine GluN1/GluN3A receptors (eGlyRs) and their tonic activation by extracellular glycine. eGlyRs are mostly extrasynaptic and reside in specific neuronal populations, including the principal cells of the basolateral amygdala (BLA) and SST-positive interneurons (SST-INs) of the neocortex. In the BLA, tonic eGlyR currents are sensitive to fear-conditioning protocols, are subject to neuromodulation by the dopaminergic system, and control the stability of fear memories. In the neocortex, eGlyRs control the in vivo spiking of SST-INs and the behavior-dependent modulation of cortical activity. GluN3A-containing eGlyRs thus represent a novel and widespread signaling modality in the adult brain, with attributes that strikingly depart from those of conventional NMDARs.

Integrity of the circadian clock determines regularity of high-frequency and diurnal LFP rhythms within and between brain areas.

Circadian clocks control most physiological processes of many species. We specifically wanted to investigate the influence of environmental and endogenous rhythms and their interplay on electrophysiological dynamics of neuronal populations. Therefore, we measured local field potential (LFP) time series in wild-type and Cryptochrome 1 and 2 deficient (Cry1/2-/-) mice in the suprachiasmatic nucleus and the nucleus accumbens under regular light conditions and constant darkness. Using refined descriptive and statistical analyses, we systematically profiled LFP time series activity. We show that both environmental and endogenous rhythms strongly influence the rhythmicity of LFP signals and their frequency components, but also shape neuronal patterns on much smaller time scales, as neuronal activity in Cry1/2-/- mice is significantly less regular but at each time more synchronous within and between brain areas than in wild-type mice. These results show that functional circadian rhythms are integral for both circadian and non-circadian coordination of neuronal ensemble dynamics.

A modified density gradient proteomic-based method to analyze endolysosomal proteins in cardiac tissue.

The importance of lysosomes in cardiac physiology and pathology is well established, and evidence for roles in calcium signaling is emerging. We describe a label-free proteomics method suitable for small cardiac tissue biopsies based on density-separated fractionation, which allows study of endolysosomal (EL) proteins. Density gradient fractions corresponding to tissue lysate; sarcoplasmic reticulum (SR), mitochondria (Mito) (1.3 g/mL); and EL with negligible contamination from SR or Mito (1.04 g/mL) were analyzed using Western blot, enzyme activity assay, and liquid chromatography with tandem mass spectrometry (LC-MS/MS) analysis (adapted discontinuous Percoll and sucrose differential density gradient). Kyoto Encyclopedia of Genes and Genomes, Reactome, Panther, and Gene Ontology pathway analysis showed good coverage of RAB proteins and lysosomal cathepsins (including cardiac-specific cathepsin D) in the purified EL fraction. Significant EL proteins recovered included catalytic activity proteins. We thus present a comprehensive protocol and data set of guinea pig atrial EL organelle proteomics using techniques also applicable for non-cardiac tissue.

Ketone Esters Partially and Selectively Rescue Mitochondrial Bioenergetics After Acute Cervical Spinal Cord Injury in Rats: A Time-Course.

Spinal cord injury (SCI) pathology and pathophysiology can be attributed to both primary physical injury and secondary injury cascades. Secondary injury cascades involve dysregulated metabolism and energetic deficits directly linked to compromised mitochondrial bioenergetics. Rescuing mitochondrial function and reducing oxidative stress are associated with neuroprotection. In this regard, ketosis after traumatic brain injury (TBI), or after SCI, improves secondary neuropathology by decreasing oxidative stress, increasing antioxidants, reducing inflammation, and improving mitochondrial bioenergetics. Here, we follow up on our previous study and have used an exogenous ketone monoester, (R)-3-hydroxybutyl (R)-3-hydroxybutyrate (KE), as an alternative to a ketogenic diet, focusing on mitochondrial function between 1 and 14 days after injury. Starting 3 h following a cervical level 5 (C5) hemi-contusion injury, animals were fed either a standard control diet (SD) or a ketone ester diet (KED) combined with KE administered orally (OKE). We found that mitochondrial function was reduced after SCI at all times post-SCI, accompanied by reduced expression of most of the components of the electron transport chain (ETC). The KE rescued some of the bioenergetic parameters 1 day after SCI when D-β-Hydroxybutyrate (BHB) concentrations were ~2 mM. Still, most of the beneficial effects were observed 14 days after injury, with BHB concentrations reaching values of 4-6 mM. To our knowledge, this is the first report to show the beneficial effects of KE in rescuing mitochondrial function after SCI and demonstrates the suitability of KE in ameliorating the metabolic dysregulation that occurs after traumatic SCI without requiring a restrictive dietary regime.

Inhibition of adenylyl cyclase 1 by ST034307 inhibits IP3-evoked changes in sino-atrial node beat rate.

Atrial arrhythmias, such as atrial fibrillation (AF), are a major mortality risk and a leading cause of stroke. The IP3 signalling pathway has been proposed as an atrial-specific target for AF therapy, and atrial IP3 signalling has been linked to the activation of calcium sensitive adenylyl cyclases AC1 and AC8. We investigated the involvement of AC1 in the response of intact mouse atrial tissue and isolated guinea pig atrial and sino-atrial node (SAN) cells to the α-adrenoceptor agonist phenylephrine (PE) using the selective AC1 inhibitor ST034307. The maximum rate change of spontaneously beating mouse right atrial tissue exposed to PE was reduced from 14.5% to 8.2% (p = 0.005) in the presence of 1 μM ST034307, whereas the increase in tension generated in paced left atrial tissue in the presence of PE was not inhibited by ST034307 (Control = 14.2%, ST034307 = 16.3%; p > 0.05). Experiments were performed using isolated guinea pig atrial and SAN cells loaded with Fluo-5F-AM to record changes in calcium transients (CaT) generated by 10 μM PE in the presence and absence of 1 μM ST034307. ST034307 significantly reduced the beating rate of SAN cells (0.34-fold decrease; p = 0.003) but did not inhibit changes in CaT amplitude in response to PE in atrial cells. The results presented here demonstrate pharmacologically the involvement of AC1 in the downstream response of atrial pacemaker activity to α-adrenoreceptor stimulation and IP3R calcium release.

Lysosomal signalling pathways influence heart rhythm, and regulate atrial function

IP3-mediated Ca2+ release regulates atrial Ca2+ transients and pacemaker function by stimulation of adenylyl cyclases.

Inositol trisphosphate (IP3) is a Ca2+-mobilizing second messenger shown to modulate atrial muscle contraction and is thought to contribute to atrial fibrillation. Cellular pathways underlying IP3 actions in cardiac tissue remain poorly understood, and the work presented here addresses the question whether IP3-mediated Ca2+ release from the sarcoplasmic reticulum is linked to adenylyl cyclase activity including Ca2+-stimulated adenylyl cyclases (AC1 and AC8) that are selectively expressed in atria and sinoatrial node (SAN). Immunocytochemistry in guinea pig atrial myocytes identified colocalization of type 2 IP3 receptors with AC8, while AC1 was located in close vicinity. Intracellular photorelease of IP3 by UV light significantly enhanced the amplitude of the Ca2+ transient (CaT) evoked by electrical stimulation of atrial myocytes (31 ± 6% increase 60 s after photorelease, n = 16). The increase in CaT amplitude was abolished by inhibitors of adenylyl cyclases (MDL-12,330) or protein kinase A (H89), showing that cAMP signaling is required for this effect of photoreleased IP3. In mouse, spontaneously beating right atrial preparations, phenylephrine, an α-adrenoceptor agonist with effects that depend on IP3-mediated Ca2+ release, increased the maximum beating rate by 14.7 ± 0.5%, n = 10. This effect was substantially reduced by 2.5 µmol/L 2-aminoethyl diphenylborinate and abolished by a low dose of MDL-12,330, observations which are again consistent with a functional interaction between IP3 and cAMP signaling involving Ca2+ stimulation of adenylyl cyclases in the SAN pacemaker. Understanding the interaction between IP3 receptor pathways and Ca2+-stimulated adenylyl cyclases provides important insights concerning acute mechanisms for initiation of atrial arrhythmias.NEW & NOTEWORTHY This study provides evidence supporting the proposal that IP3 signaling in cardiac atria and sinoatrial node involves stimulation of Ca2+-activated adenylyl cyclases (AC1 and AC8) by IP3-evoked Ca2+ release from junctional sarcoplasmic reticulum. AC8 and IP3 receptors are shown to be located close together, while AC1 is nearby. Greater understanding of these novel aspects of the IP3 signal transduction mechanism is important for future study in atrial physiology and pathophysiology, particularly atrial fibrillation.

Bayliss Starling Prize Lecture 2023: Neuropeptide-Y being 'unsympathetic' to the broken hearted.

William Bayliss and Ernest Starling are not only famous as pioneers in cardiovascular physiology, but also responsible for the discovery of the first hormone (from the Greek 'excite or arouse'), the intestinal signalling molecule and neuropeptide secretin in 1902. Our research group focuses on neuropeptides and neuromodulators that influence cardiovascular autonomic control as potential biomarkers in disease and tractable targets for therapeutic intervention. Acute myocardial infarction (AMI) and chronic heart failure (CHF) result in high levels of cardiac sympathetic stimulation, which is a poor prognostic indicator. Although beta-blockers improve mortality in these conditions by preventing the action of the neurotransmitter noradrenaline, a substantial residual risk remains. Recently, we have identified the sympathetic co-transmitter neuropeptide-Y (NPY) as being released during AMI, leading to larger infarcts and life-threatening arrhythmia in both animal models and patients. Here, we discuss recently published data demonstrating that peripheral venous NPY levels are associated with heart failure hospitalisation and mortality after AMI, and all cause cardiovascular mortality in CHF, even when adjusting for known risk factors (including brain natriuretic peptide). We have investigated the mechanistic basis for these observations in human and rat stellate ganglia and cardiac tissue, manipulating NPY neurochemistry at the same time as using state-of-the-art imaging techniques, to establish the receptor pathways responsible for NPY signalling. We propose NPY as a new mechanistic biomarker in AMI and CHF patients and aim to determine whether specific NPY receptor blockers can prevent arrhythmia and attenuate the development of heart failure.

Compartmentalization proteomics revealed endolysosomal protein network changes in a goat model of atrial fibrillation.

Endolysosomes (EL) are known for their role in regulating both intracellular trafficking and proteostasis. EL facilitate the elimination of damaged membranes, protein aggregates, membranous organelles and play an important role in calcium signaling. The specific role of EL in cardiac atrial fibrillation (AF) is not well understood. We isolated atrial EL organelles from AF goat biopsies and conducted a comprehensive integrated omics analysis to study the EL-specific proteins and pathways. We also performed electron tomography, protein and enzyme assays on these biopsies. Our results revealed the upregulation of the AMPK pathway and the expression of EL-specific proteins that were not found in whole tissue lysates, including GAA, DYNLRB1, CLTB, SIRT3, CCT2, and muscle-specific HSPB2. We also observed structural anomalies, such as autophagic-vacuole formation, irregularly shaped mitochondria, and glycogen deposition. Our results provide molecular information suggesting EL play a role in AF disease process over extended time frames.

Activation of IP3R in atrial cardiomyocytes leads to generation of cytosolic cAMP.

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. Excessive stimulation of the inositol (1,4,5)-trisphosphate (IP3) signaling pathway has been linked to AF through abnormal calcium handling. However, little is known about the mechanisms involved in this process. We expressed the fluorescence resonance energy transfer (FRET)-based cytosolic cyclic adenosine monophosphate (cAMP) sensor EPAC-SH187 in neonatal rat atrial myocytes (NRAMs) and neonatal rat ventricular myocytes (NRVMs). In NRAMs, the addition of the α1-agonist, phenylephrine (PE, 3 µM), resulted in a FRET change of 21.20 ± 7.43%, and the addition of membrane-permeant IP3 derivative 2,3,6-tri-O-butyryl-myo-IP3(1,4,5)-hexakis(acetoxymethyl)ester (IP3-AM, 20 μM) resulted in a peak of 20.31 ± 6.74%. These FRET changes imply an increase in cAMP. Prior application of IP3 receptor (IP3R) inhibitors 2-aminoethyl diphenylborinate (2-APB, 2.5 μM) or Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to PE. Xestospongin-C (0.3 μM) significantly inhibited the change in FRET in NRAMs in response to IP3-AM. The FRET change in response to PE in NRVMs was not inhibited by 2-APB or Xestospongin-C. Finally, the localization of cAMP signals was tested by expressing the FRET-based cAMP sensor, AKAP79-CUTie, which targets the intracellular surface of the plasmalemma. We found in NRAMs that PE led to FRET change corresponding to an increase in cAMP that was inhibited by 2-APB and Xestospongin-C. These data support further investigation of the proarrhythmic nature and components of IP3-induced cAMP signaling to identify potential pharmacological targets.NEW & NOTEWORTHY This study shows that indirect activation of the IP3 pathway in atrial myocytes using phenylephrine and direct activation using IP3-AM leads to an increase in cAMP and is in part localized to the cell membrane. These changes can be pharmacologically inhibited using IP3R inhibitors. However, the cAMP rise in ventricular myocytes is independent of IP3R calcium release. Our data support further investigation into the proarrhythmic nature of IP3-induced cAMP signaling.

Lysosomal proteases and their role in signaling pathways

Lysosomes contain more than 50 acid hydrolases and are the major location for degradation of both intracellular and extracellular macromolecules. However, lysosomes also play a key role in cell signaling processes, for example, trafficking via the endosomal/lysosomal pathway, regulation of autophagy, growth factor degradation, and through their involvement in antigen presentation. In this chapter, we summarize the classification of lysosomal proteases and their roles in cell signaling, with particular focus on the cysteine, serine, and aspartic cathepsins. In addition, we summarize how disruption of these processes may be linked to specific diseases, including neurodegenerative disease, cardiovascular disease, cancer, and inflammation.

The age-dependent development of abnormal cardiac metabolism in the peroxisome proliferator-activated receptor α-knockout mouse.

BACKGROUND AND AIMS: Peroxisome proliferator-activated receptor α (PPARα) is crucial for regulating cardiac β-oxidation in the heart, liver, and kidney. Ageing can induce cardiac metabolic alterations, but the role of PPARα has not been extensively characterised. The aim of this research was to investigate the role of PPARα in the aged heart. METHODS: Hyperpolarized [1-13C]pyruvate was used to evaluate in vivo cardiac carbohydrate metabolism in fed and fasted young (3 months) and old (20-22 months) PPARα knockout (KO) mice versus controls. Cine MRI assessed cardiac structural and functional changes. Cardiac tissue analysis included qRT-PCR and Western blotting for Pparα, medium chain acyl-CoA dehydrenase (MCAD), uncoupling protein (UCP) 3, glucose transporter (GLUT) 4 and PDH kinase (PDK) 1,2, and 4 expression. RESULTS: PPARα-KO hearts from both young and old mice showed significantly reduced Pparα mRNA and a 58-59 % decrease in MCAD protein levels compared to controls. Cardiac PDH flux was similar in young control and PPARα-KO mice but 96 % higher in old PPARα-KO mice. Differences between genotypes were consistent in fed and fasted states, with reduced PDH flux when fasted. Increased PDH flux was accompanied by a 179 % rise in myocardial GLUT4 protein. No differences in PDK 1, 2, or 4 protein levels were observed between fed groups, indicating the increased PDH flux in aged PPARα-KO mice was not due to changes in PDH phosphorylation. CONCLUSIONS: Aged PPARα-KO mice demonstrated higher cardiac PDH flux compared to controls, facilitated by increased myocardial GLUT4 protein levels, leading to enhanced glucose uptake and glycolysis.

Hypothesis-driven genome-wide association studies provide novel insights into genetics of reading disabilities.

Reading Disability (RD) is often characterized by difficulties in the phonology of the language. While the molecular mechanisms underlying it are largely undetermined, loci are being revealed by genome-wide association studies (GWAS). In a previous GWAS for word reading (Price, 2020), we observed that top single-nucleotide polymorphisms (SNPs) were located near to or in genes involved in neuronal migration/axon guidance (NM/AG) or loci implicated in autism spectrum disorder (ASD). A prominent theory of RD etiology posits that it involves disturbed neuronal migration, while potential links between RD-ASD have not been extensively investigated. To improve power to identify associated loci, we up-weighted variants involved in NM/AG or ASD, separately, and performed a new Hypothesis-Driven (HD)-GWAS. The approach was applied to a Toronto RD sample and a meta-analysis of the GenLang Consortium. For the Toronto sample (n = 624), no SNPs reached significance; however, by gene-set analysis, the joint contribution of ASD-related genes passed the threshold (p~1.45 × 10-2, threshold = 2.5 × 10-2). For the GenLang Cohort (n = 26,558), SNPs in DOCK7 and CDH4 showed significant association for the NM/AG hypothesis (sFDR q = 1.02 × 10-2). To make the GenLang dataset more similar to Toronto, we repeated the analysis restricting to samples selected for reading/language deficits (n = 4152). In this GenLang selected subset, we found significant association for a locus intergenic between BTG3-C21orf91 for both hypotheses (sFDR q 

Correlation between the number of interstitial neurons of the white matter and number of neurons within cortical layers: Histological analyses in postnatal macaque.

We have examined the number and distribution of NeuN-immunoreactive cortical white matter interstitial cells (WMICs) and compared them to the neurons in layers 1-6 across the overlying cortex in coronal sections from postnatal macaques. The data have been gathered from over 300 selected regions at gyral crowns, at sulci, and at linear regions of the cortex where we also determined cortical layer thicknesses: standard thicknesses and tangential thicknesses. Cortical thicknesses and cell numbers showed variability according to gyral, linear, or sulcal regions. In spite of these variations, our standardized cell numbers in layers 1 to 6b and interstitial cells underlying layer 6b-white matter boundary have shown a consistent correlation between the number of WMICs and the number of layer 5 and 6a cortical neurons on all cortical regions studied: for each WMIC, there are on the order of five cortical neurons in layer 5 and approximately three cortical neurons in layer 6a, irrespective of the origins of the selected cortical area or whether they are from gyral, linear, or sulcal regions. We propose that the number of interstitial neurons in the postnatal macaque cortex is correlated to the density of neurons within layers 5 and 6a and, from a clinical perspective, the change in density or distribution of interstitial neurons in schizophrenia or epilepsy may in fact be linked to the number of layers 5 and 6a neurons.

Utrophin correlates with disease severity in Duchenne muscular dystrophy.

This month in Med, the description of an unusually severely affected DMD patient suffering from a large deletion in the dystrophin gene confirms that absence of utrophin worsens the dystrophy and supports the concept that utrophin upregulation ameliorates the pathology. This study may guide the development of dystrophin-based gene therapies.

Early deficits in an in vitro striatal microcircuit model carrying the Parkinson's GBA-N370S mutation.

Understanding medium spiny neuron (MSN) physiology is essential to understand motor impairments in Parkinson's disease (PD) given the architecture of the basal ganglia. Here, we developed a custom three-chambered microfluidic platform and established a cortico-striato-nigral microcircuit partially recapitulating the striatal presynaptic landscape in vitro using induced pluripotent stem cell (iPSC)-derived neurons. We found that, cortical glutamatergic projections facilitated MSN synaptic activity, and dopaminergic transmission enhanced maturation of MSNs in vitro. Replacement of wild-type iPSC-derived dopamine neurons (iPSC-DaNs) in the striatal microcircuit with those carrying the PD-related GBA-N370S mutation led to a depolarisation of resting membrane potential and an increase in rheobase in iPSC-MSNs, as well as a reduction in both voltage-gated sodium and potassium currents. Such deficits were resolved in late microcircuit cultures, and could be reversed in younger cultures with antagonism of protein kinase A activity in iPSC-MSNs. Taken together, our results highlight the unique utility of modelling striatal neurons in a modular physiological circuit to reveal mechanistic insights into GBA1 mutations in PD.

Histone transcription regulator Slm9 is required for cytoophidium biogenesis.

The cytoophidium, a subcellular structure composed of CTP synthase, can be observed during the division of Schizosaccharomyces pombe. Cytoophidium formation changes periodically with the cell cycle of yeast cells. Here, we find that histone chaperone Slm9 is required for the integrity of cytoophidia in fission yeast. When the slm9 gene is knocked out, we observe that morphological characteristics, the abundance of cytoophidia and the division of the yeast cells are significantly affected. Fragmented cytoophidia occur in slm9 mutant cells, a phenomenon rarely observed in wild-type cells. Our study reveals a potential link between a chromosomal regulatory factor and cytoophidium biogenesis.

eCRUIS captures RNA-protein interaction in vitro and in vivo.

As an information bridge between DNA and protein, RNA regulates cellular processes and gene expression in various ways. From its synthesis to degradation, RNA is associated with a range of RNA-binding proteins. Therefore, it is necessary to develop innovative methods to study the interaction between RNA and proteins. Previously, we developed an RNA-centric method, called CRISPR-based RNA-United Interacting System (CRUIS), to capture RNA-protein interaction in cells. On this basis, here we develop an enhanced CRUIS (eCRUIS) by combining the power of dCas13d and the engineered promiscuous ligase TurboID. The current version allows us to rapidly label RNA-binding proteins on the target RNA within 30 minutes, potentially for in vivo use. By introducing bait-assay with exogenous RNA, we confirm that eCRUIS can effectively label RNA-binding proteins on bait RNA in a short time. eCRUIS provides a broader range of in vitro and in vivo applications for studying RNA-protein interactions.

The Impact of Developmental and Metabolic Cues on Cytoophidium Formation.

The cytoophidium, composed mainly of CTP synthase (CTPS), is a newly discovered dynamic filamentous structure in various organisms such as archaea, bacteria, and humans. These filamentous structures represent a fascinating example of intracellular compartmentation and dynamic regulation of metabolic enzymes. Currently, cytoophidia have been proven to be tightly regulated and highly dynamic, responding rapidly to developmental and metabolic cues and playing a critical role in maintaining cellular homeostasis. In this review, we would like to discuss in detail the characteristics, mechanisms, functions, and potential applications of this conservative but promising organelle.

Differential Cytoophidium Assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe.

The de novo synthesis of cytidine 5'-triphosphate (CTP) is catalyzed by the enzyme CTP synthase (CTPS), which is known to form cytoophidia across all three domains of life. In this study, we use the budding yeast Saccharomyces cerevisiae and the fission yeast Schizosaccharomyces pombe as model organisms to compare cytoophidium assembly under external environmental and intracellular CTPS alterations. We observe that under low and high temperature conditions, cytoophidia in fission yeast gradually disassemble, while cytoophidia in budding yeast remain unaffected. The effect of pH changes on cytoophidia maintenance in the two yeast species is different. When cultured in the yeast-saturated cultured medium, cytoophidia in fission yeast disassemble, while cytoophidia in budding yeast gradually form. Overexpression of CTPS results in the presence and maintenance of cytoophidia in both yeast species from the log phase to the stationary phase. In summary, our results demonstrate differential cytoophidium assembly between Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most studied yeast species.