Synaptic BMAL1 phosphorylation controls circadian hippocampal plasticity.

The time of day strongly influences adaptive behaviors like long-term memory, but the correlating synaptic and molecular mechanisms remain unclear. The circadian clock comprises a canonical transcription-translation feedback loop (TTFL) strictly dependent on the BMAL1 transcription factor. We report that BMAL1 rhythmically localizes to hippocampal synapses in a manner dependent on its phosphorylation at Ser42 [pBMAL1(S42)]. pBMAL1(S42) regulates the autophosphorylation of synaptic CaMKIIα and circadian rhythms of CaMKIIα-dependent molecular interactions and LTP but not global rest/activity behavior. Therefore, our results suggest a model in which repurposing of the clock protein BMAL1 to synapses locally gates the circadian timing of plasticity.

Defining diurnal fluctuations in mouse choroid plexus and CSF at high molecular, spatial, and temporal resolution.

Transmission and secretion of signals via the choroid plexus (ChP) brain barrier can modulate brain states via regulation of cerebrospinal fluid (CSF) composition. Here, we developed a platform to analyze diurnal variations in male mouse ChP and CSF. Ribosome profiling of ChP epithelial cells revealed diurnal translatome differences in metabolic machinery, secreted proteins, and barrier components. Using ChP and CSF metabolomics and blood-CSF barrier analyses, we observed diurnal changes in metabolites and cellular junctions. We then focused on transthyretin (TTR), a diurnally regulated thyroid hormone chaperone secreted by the ChP. Diurnal variation in ChP TTR depended on Bmal1 clock gene expression. We achieved real-time tracking of CSF-TTR in awake TtrmNeonGreen mice via multi-day intracerebroventricular fiber photometry. Diurnal changes in ChP and CSF TTR levels correlated with CSF thyroid hormone levels. These datasets highlight an integrated platform for investigating diurnal control of brain states by the ChP and CSF.

Keeping an eye on circadian time in clinical research and medicine.

BACKGROUND: Daily rhythms are observed in humans and almost all other organisms. Most of these observed rhythms reflect both underlying endogenous circadian rhythms and evoked responses from behaviours such as sleep/wake, eating/fasting, rest/activity, posture changes and exercise. For many research and clinical purposes, it is important to understand the contribution of the endogenous circadian component to these observed rhythms. CONTENT: The goal of this manuscript is to provide guidance on best practices in measuring metrics of endogenous circadian rhythms in humans and promote the inclusion of circadian rhythms assessments in studies of health and disease. Circadian rhythms affect all aspects of physiology. By specifying minimal experimental conditions for studies, we aim to improve the quality, reliability and interpretability of research into circadian and daily (i.e., time-of-day) rhythms and facilitate the interpretation of clinical and translational findings within the context of human circadian rhythms. We describe protocols, variables and analyses commonly used for studying human daily rhythms, including how to assess the relative contributions of the endogenous circadian system and other daily patterns in behaviours or the environment. We conclude with recommendations for protocols, variables, analyses, definitions and examples of circadian terminology. CONCLUSION: Although circadian rhythms and daily effects on health outcomes can be challenging to distinguish in practice, this distinction may be important in many clinical settings. Identifying and targeting the appropriate underlying (patho)physiology is a medical goal. This review provides methods for identifying circadian effects to aid in the interpretation of published work and the inclusion of circadian factors in clinical research and practice.

Persistent CO2 reactivity deficits are associated with neurological dysfunction up to one year after repetitive mild closed head injury in adolescent mice.

Cerebrovascular reactivity (CVR) deficits in adolescents with concussion may persist after resolution of neurological symptoms. Whether or not CVR deficits predict long term neurological function is unknown. We used adolescent mice closed head injury (CHI) models (54 g, 107 cm or 117 cm drop height), followed by blood oxygenation level dependent (BOLD)-functional MRI with CO2 challenge to assess CVR and brain connectivity. At one week, 3HD 107 cm mice showed delayed BOLD responses (p = 0.0074), normal striatal connectivity, and an impaired respiratory rate response to CO2 challenge (p = 0.0061 in ΔRmax). The 107 cm group developed rotarod deficits at 6 months (p = 0.02) and altered post-CO2 brain connectivity (3-fold increase in striatum to motor cortex correlation coefficient) by one year, but resolved their CVR and respiratory rate impairments, and did not develop cognitive or circadian activity deficits. In contrast, the 117 cm group had persistent CVR (delay time: p = 0.016; washout time: p = 0.039) and circadian activity deficits (free-running period: 23.7 hr in sham vs 23.9 hr in 3HD; amplitude: 0.15 in sham vs 0.2 in 3HD; peak activity: 18 in sham vs 21 in 3HD) at one year. Persistent CVR deficits after concussion may portend long-term neurological dysfunction. Further studies are warranted to determine the utility of CVR to predict chronic neurological outcome after mild traumatic brain injury.

Modified Precordial Lead R-Wave Deflection Interval Predicts Left- and Right-Sided Idiopathic Outflow Tract Ventricular Arrhythmias.

OBJECTIVES: This study evaluated if modifying electrocardiographic (ECG) precordial leads to a higher intercostal position improved the accuracy of outflow tract ventricular arrhythmia (OTVA) localization. BACKGROUND: Precordial ECG prediction algorithms that use a standard lead configuration localize OTVA with variable accuracy. METHODS: Patients who underwent OTVA ablation were prospectively enrolled to have a standard and modified (high) precordial ECG. R- and S-wave amplitudes and intervals were measured to develop an algorithm that differentiated the right ventricular outflow tract (RVOT) and the left ventricular outflow tract (LVOT) with high accuracy-the modified lead R-wave deflection interval (RWDI). This interval was defined from the earliest QRS onset (using all modified leads) to the lead with longest R-wave deflection. The RWDI was compared with all other ECG algorithms. RESULTS: A total of 50 patients (38 women; mean age 51 ± 17 years) had successful catheter ablation for OTVA (RVOT 60%, LVOT 40%). The modified lead RWDI was significantly shorter in the RVOT group (18.5 ms, interquartile range 25th to 75th percentile [IQR25-75]: 0 to 29.5 ms) compared with the LVOT group (67.5 ms, IQR25-75: 56.5 to 77 ms; p < 0.05). Using a RWDI ≤40 ms to predict an RVOT focus, the sensitivity and specificity of the modified lead RWDI were 100% and 95%, respectively; the area under the receiver-operating characteristic curve was 0.96. This was superior to all previously developed algorithms. In a computed tomography analysis (n = 50), the modified leads were significantly closer to the outflow tracts compared with the standard precordial leads. CONCLUSIONS: The modified lead RWDI is a simple, easily interpretable algorithm that can potentially differentiate a right- or left-sided origin of OTVA with high accuracy.

Position Statement on the Management of Cardiac Electrophysiology and Cardiac Implantable Electronic Devices in Australia During the COVID-19 Pandemic: A Living Document.

The COVID-19 pandemic poses a significant stress on health resources in Australia. The Heart Rhythm Council of the Cardiac Society of Australia and New Zealand aims to provide a framework for efficient resource utilisation balanced with competing risks when appropriately treating patients with cardiac arrhythmias. This document provides practical recommendations for the electrophysiology (EP) and cardiac implantable electronic devices (CIED) services in Australia. The document will be updated regularly as new evidence and knowledge is gained with time.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Assessment of ablation catheter contact on valve annulus: Implications on accessory pathway ablation.

BACKGROUND: Catheter-tissue contact force is an important factor influencing lesion size and efficacy and thereby potential for arrhythmia recurrence following accessory pathway (AP) radiofrequency ablation. We aim to evaluate adequacy and perception of catheter contact on the tricuspid and mitral annuli. METHODS: Data were collected from 42 patients undergoing catheter ablation. Operators were blinded to contact force information and reported perceived contact (poor, moderate, or good) while positioning the catheter at four tricuspid annular sites (12, 9, 6 and 4 o'clock positions; abbreviated as TA12, TA9, TA6 and TA4) and three mitral annular sites (3, 5 and 7 o'clock positions; abbreviated as MA3, MA5 and MA7) through long vascular sheaths. RESULTS: The highest and lowest mean contact forces were obtained at MA7 (13.3 ±â€¯1.7 g) and TA12 (3.6 g ±â€¯1.3 g) respectively. Mean contact force on tricuspid annulus (6.1 g ±â€¯0.9 g) was lower than mitral annulus (9.8 ±â€¯0.9 g) locations (p = 0.0036), with greater proportion of sites with <10 g contact force (81.7% vs 60.4%; p = 0.0075). Perceived contact had no impact on measured mean contact force for both mitral and tricuspid annular positions (p = 0.959 and 0.671 respectively). There was correlation of both impedance and atrial electrogram amplitude with contact force, though insufficient to be clinically applicable. CONCLUSION: A high proportion of annular catheter applications have low contact force despite being performed with long vascular sheaths in the hands of experienced operators. In addition, there was no impact of operator perceived contact force on actual measured contact force. This may carry implications for success of AP ablation.

Deletion of Neuronal GLT-1 in Mice Reveals Its Role in Synaptic Glutamate Homeostasis and Mitochondrial Function.

The glutamate transporter GLT-1 is highly expressed in astrocytes but also in neurons, primarily in axon terminals. We generated a conditional neuronal GLT-1 KO using synapsin 1-Cre (synGLT-1 KO) to elucidate the metabolic functions of GLT-1 expressed in neurons, here focusing on the cerebral cortex. Both synaptosomal uptake studies and electron microscopic immunocytochemistry demonstrated knockdown of GLT-1 in the cerebral cortex in the synGLT-1 KO mice. Aspartate content was significantly reduced in cerebral cortical extracts as well as synaptosomes from cerebral cortex of synGLT-1 KO compared with control littermates. 13C-Labeling of tricarboxylic acid cycle intermediates originating from metabolism of [U-13C]-glutamate was significantly reduced in synGLT-1 KO synaptosomes. The decreased aspartate content was due to diminished entry of glutamate into the tricarboxylic acid cycle. Pyruvate recycling, a pathway necessary for full glutamate oxidation, was also decreased. ATP production was significantly increased, despite unaltered oxygen consumption, in isolated mitochondria from the synGLT-1 KO. The density of mitochondria in axon terminals and perisynaptic astrocytes was increased in the synGLT-1 KO. Intramitochondrial cristae density of synGLT-1 KO mice was increased, suggesting increased mitochondrial efficiency, perhaps in compensation for reduced access to glutamate. SynGLT-1 KO synaptosomes exhibited an elevated oxygen consumption rate when stimulated with veratridine, despite a lower baseline oxygen consumption rate in the presence of glucose. GLT-1 expressed in neurons appears to be required to provide glutamate to synaptic mitochondria and is linked to neuronal energy metabolism and mitochondrial function.SIGNIFICANCE STATEMENT All synaptic transmitters need to be cleared from the extracellular space after release, and transporters are used to clear glutamate released from excitatory synapses. GLT-1 is the major glutamate transporter, and most GLT-1 is expressed in astrocytes. Only 5%-10% is expressed in neurons, primarily in axon terminals. The function of GLT-1 in axon terminals remains unknown. Here, we used a conditional KO approach to investigate the significance of the expression of GLT-1 in neurons. We found multiple abnormalities of mitochondrial function, suggesting impairment of glutamate utilization by synaptic mitochondria in the neuronal GLT-1 KO. These data suggest that GLT-1 expressed in axon terminals may be important in maintaining energy metabolism and biosynthetic activities mediated by presynaptic mitochondria.

Mechanisms of sleep and circadian ontogeny through the lens of neurodevelopmental disorders.

Sleep is a mysterious, developmentally regulated behavior fundamental for cognition in both adults and developing animals. A large number of studies offer a substantive body of evidence that demonstrates that the ontogeny of sleep architecture parallels brain development. Sleep deprivation impairs the consolidation of learned tasks into long-term memories and likely links sleep to the neural mechanisms underlying memory and its physiological roots in brain plasticity. Consistent with this notion is the alarming frequency of sleep and circadian rhythm dysfunction in children with neurodevelopmental disorders (NDDs). While the mechanisms underlying sleep dysfunction in most NDDs still remains poorly understood, here we will review several sentinel examples of monogenetic NDDs with both well-established connections to synaptic dysfunction and evidence of sleep or circadian dysfunction: Tuberous Sclerosis Complex, Fragile X Syndrome, and Angelman Syndrome. We suggest that the coincident maturation of sleep with synaptic physiology is one of the core reasons for the commonplace disruption of sleep in NDDs and argue that disorders with well-defined molecular genetics can provide a unique lens for understanding and unraveling the molecular correlates that link the development of sleep and circadian rhythms to health and disease.

Proteomics, Post-translational Modifications, and Integrative Analyses Reveal Molecular Heterogeneity within Medulloblastoma Subgroups.

There is a pressing need to identify therapeutic targets in tumors with low mutation rates such as the malignant pediatric brain tumor medulloblastoma. To address this challenge, we quantitatively profiled global proteomes and phospho-proteomes of 45 medulloblastoma samples. Integrated analyses revealed that tumors with similar RNA expression vary extensively at the post-transcriptional and post-translational levels. We identified distinct pathways associated with two subsets of SHH tumors, and found post-translational modifications of MYC that are associated with poor outcomes in group 3 tumors. We found kinases associated with subtypes and showed that inhibiting PRKDC sensitizes MYC-driven cells to radiation. Our study shows that proteomics enables a more comprehensive, functional readout, providing a foundation for future therapeutic strategies.

mGluR5 Modulation of Behavioral and Epileptic Phenotypes in a Mouse Model of Tuberous Sclerosis Complex.

Drugs targeting metabotropic glutamate receptor 5 (mGluR5) have therapeutic potential in autism spectrum disorders (ASD), including tuberous sclerosis complex (TSC). The question whether inhibition or potentiation of mGluR5 could be beneficial depends, among other factors, on the specific indication. To facilitate the development of mGluR5 treatment strategies, we tested the therapeutic utility of mGluR5 negative and positive allosteric modulators (an mGluR5 NAM and PAM) for TSC, using a mutant mouse model with neuronal loss of Tsc2 that demonstrates disease-related phenotypes, including behavioral symptoms of ASD and epilepsy. This model uniquely enables the in vivo characterization and rescue of the electrographic seizures associated with TSC. We demonstrate that inhibition of mGluR5 corrects hyperactivity, seizures, and elevated de novo synaptic protein synthesis. Conversely, positive allosteric modulation of mGluR5 results in the exacerbation of hyperactivity and epileptic phenotypes. The data suggest a meaningful therapeutic potential for mGluR5 NAMs in TSC, which warrants clinical exploration and the continued development of mGluR5 therapies.

Aberrant Proteostasis of BMAL1 Underlies Circadian Abnormalities in a Paradigmatic mTOR-opathy.

Tuberous sclerosis complex (TSC) is a neurodevelopmental disorder characterized by mutations in either the TSC1 or TSC2 genes, whose products form a critical inhibitor of the mechanistic target of rapamycin (mTOR). Loss of TSC1/2 gene function renders an mTOR-overactivated state. Clinically, TSC manifests with epilepsy, intellectual disability, autism, and sleep dysfunction. Here, we report that mouse models of TSC have abnormal circadian rhythms. We show that mTOR regulates the proteostasis of the core clock protein BMAL1, affecting its translation, degradation, and subcellular localization. This results in elevated levels of BMAL1 and a dysfunctional clock that displays abnormal timekeeping under constant conditions and exaggerated responses to phase resetting. Genetically lowering the dose of BMAL1 rescues circadian behavioral phenotypes in TSC mouse models. These findings indicate that BMAL1 deregulation is a feature of the mTOR-activated state and suggest a molecular mechanism for mitigating circadian phenotypes in a neurodevelopmental disorder.

Neural Circuitry of Wakefulness and Sleep.

Sleep remains one of the most mysterious yet ubiquitous animal behaviors. We review current perspectives on the neural systems that regulate sleep/wake states in mammals and the circadian mechanisms that control their timing. We also outline key models for the regulation of rapid eye movement (REM) sleep and non-REM sleep, how mutual inhibition between specific pathways gives rise to these distinct states, and how dysfunction in these circuits can give rise to sleep disorders.

New Insights Into an Old Arrhythmia: High-Resolution Mapping Demonstrates Conduction and Substrate Variability in Right Atrial Macro-Re-Entrant Tachycardia.

OBJECTIVES: Using high-resolution 3-dimensional (3D) mapping, the aim of this study was to further characterize right atrial macro-re-entrant tachycardias and answer unresolved questions in the understanding of this arrhythmia. BACKGROUND: Despite advances in understanding of the mechanisms of right atrial macro-re-entrant tachycardias, many questions lack definitive answers. The advent of high-resolution 3D mapping provides an opportunity to gain further insights into the nature of these common circuits. METHODS: A total of 25 patients with right atrial macro-re-entrant tachycardia were studied. High-resolution 3D mapping (Rhythmia mapping system, Boston Scientific, Natick, Massachusetts) was performed. Regional voltage and conduction velocity were determined. Maps were analyzed to characterize wave front propagation patterns in all atrial regions. The relationship between substrate and conduction was evaluated. RESULTS: A total of 42 right atrial macro-re-entrant circuits were observed. The most common location of the posterior line of block was the posteromedial right atrium (73%). This line of block continued superiorly into the superior vena cava, taking an oblique course to finish on the anterior superior vena cava aspect in 73%. Conduction delay at the crista terminalis was less common (23%). Conduction slowing or block was seen at the limbus of the fossa ovalis (73%) and Eustachian ridge (77%). Highly variable and localized areas of slow conduction were also observed in the inferior septum (45%), superior septum (27%), anterosuperior right atrium (23%), and lateral right atrium (23%). Localized conduction slowing was seen in the cavotricuspid isthmus in 50% of patients, but there was no generalized conduction slowing in this isthmus. The voltage in regions of slow conduction was significantly lower compared with areas of normal conduction velocity (p < 0.001). Conduction channels were observed in 55% of patients. CONCLUSIONS: High-resolution 3D mapping has provided new insights into the nature of right atrial macro-re-entrant tachycardias. Variable regions of abnormal atrial substrate were associated with conduction slowing and block. Individual variation in propagation patterns was observed in association with this variable substrate. (Mapping of Atrial Arrhythmias Using High Spatial Resolution Mapping Catheters and the Rhythmia Mapping System; ACTRN12615000544572).