miR-7 controls glutamatergic transmission and neuronal connectivity in a Cdr1as-dependent manner.

The circular RNA (circRNA) Cdr1as is conserved across mammals and highly expressed in neurons, where it directly interacts with microRNA miR-7. However, the biological function of this interaction is unknown. Here, using primary cortical murine neurons, we demonstrate that stimulating neurons by sustained depolarization rapidly induces two-fold transcriptional upregulation of Cdr1as and strong post-transcriptional stabilization of miR-7. Cdr1as loss causes doubling of glutamate release from stimulated synapses and increased frequency and duration of local neuronal bursts. Moreover, the periodicity of neuronal networks increases, and synchronicity is impaired. Strikingly, these effects are reverted by sustained expression of miR-7, which also clears Cdr1as molecules from neuronal projections. Consistently, without Cdr1as, transcriptomic changes caused by miR-7 overexpression are stronger (including miR-7-targets downregulation) and enriched in secretion/synaptic plasticity pathways. Altogether, our results suggest that in cortical neurons Cdr1as buffers miR-7 activity to control glutamatergic excitatory transmission and neuronal connectivity important for long-lasting synaptic adaptations.

Uncoupling endosomal CLC chloride/proton exchange causes severe neurodegeneration.

CLC chloride/proton exchangers may support acidification of endolysosomes and raise their luminal Cl- concentration. Disruption of endosomal ClC-3 causes severe neurodegeneration. To assess the importance of ClC-3 Cl- /H+ exchange, we now generate Clcn3unc/unc mice in which ClC-3 is converted into a Cl- channel. Unlike Clcn3-/- mice, Clcn3unc/unc mice appear normal owing to compensation by ClC-4 with which ClC-3 forms heteromers. ClC-4 protein levels are strongly reduced in Clcn3-/- , but not in Clcn3unc/unc mice because ClC-3unc binds and stabilizes ClC-4 like wild-type ClC-3. Although mice lacking ClC-4 appear healthy, its absence in Clcn3unc/unc /Clcn4-/- mice entails even stronger neurodegeneration than observed in Clcn3-/- mice. A fraction of ClC-3 is found on synaptic vesicles, but miniature postsynaptic currents and synaptic vesicle acidification are not affected in Clcn3unc/unc or Clcn3-/- mice before neurodegeneration sets in. Both, Cl- /H+ -exchange activity and the stabilizing effect on ClC-4, are central to the biological function of ClC-3.

Proton electrochemical gradient: Driving and regulating neurotransmitter uptake.

Accumulation of neurotransmitters in the lumen of synaptic vesicles (SVs) relies on the activity of the vacuolar-type H+ -ATPase. This pump drives protons into the lumen, generating a proton electrochemical gradient (ΔµH+ ) across the membrane. Recent work has demonstrated that the balance between the chemical (ΔpH) and electrical (ΔΨ) components of ΔµH+ is regulated differently by some distinct vesicle types. As different neurotransmitter transporters use ΔpH and ΔΨ with different relative efficiencies, regulation of this gradient balance has the potential to influence neurotransmitter uptake. Nevertheless, the underlying mechanisms responsible for this regulation remain poorly understood. In this review, we provide an overview of current neurotransmitter uptake models, with a particular emphasis on the distinct roles of the electrical and chemical gradients and current hypotheses for regulatory mechanisms.

KV 10.1 opposes activity-dependent increase in Ca²âº influx into the presynaptic terminal of the parallel fibre-Purkinje cell synapse.

KEY POINTS: Voltage-gated KV 10.1 potassium channels are widely expressed in the mammalian brain but their function remains poorly understood. We report that KV 10.1 is enriched in the presynaptic terminals and does not take part in somatic action potentials. In parallel fibre synapses in the cerebellar cortex, we find that KV 10.1 regulates Ca(2+) influx and neurotransmitter release during repetitive high-frequency activity. Our results describe the physiological role of mammalian KV 10.1 for the first time and help understand the fine-tuning of synaptic transmission. The voltage-gated potassium channel KV 10.1 (Eag1) is widely expressed in the mammalian brain, but its physiological function is not yet understood. Previous studies revealed highest expression levels in hippocampus and cerebellum and suggested a synaptic localization of the channel. The distinct activation kinetics of KV 10.1 indicate a role during repetitive activity of the cell. Here, we confirm the synaptic localization of KV 10.1 both biochemically and functionally and that the channel is sufficiently fast at physiological temperature to take part in repolarization of the action potential (AP). We studied the role of the channel in cerebellar physiology using patch clamp and two-photon Ca(2+) imaging in KV 10.1-deficient and wild-type mice. The excitability and action potential waveform recorded at granule cell somata was unchanged, while Ca(2+) influx into axonal boutons was enhanced in mutants in response to stimulation with three APs, but not after a single AP. Furthermore, mutants exhibited a frequency-dependent increase in facilitation at the parallel fibre-Purkinje cell synapse at high firing rates. We propose that KV 10.1 acts as a modulator of local AP shape specifically during high-frequency burst firing when other potassium channels suffer cumulative inactivation.

Differential functional consequences of GRIN2A mutations associated with schizophrenia and neurodevelopmental disorders.

Human genetic studies have revealed rare missense and protein-truncating variants in GRIN2A, encoding for the GluN2A subunit of the NMDA receptors, that confer significant risk for schizophrenia (SCZ). Mutations in GRIN2A are also associated with epilepsy and developmental delay/intellectual disability (DD/ID). However, it remains enigmatic how alterations to the same protein can result in diverse clinical phenotypes. Here, we performed functional characterization of human GluN1/GluN2A heteromeric NMDA receptors that contain SCZ-linked GluN2A variants, and compared them to NMDA receptors with GluN2A variants associated with epilepsy or DD/ID. Our findings demonstrate that SCZ-associated GRIN2A variants were predominantly loss-of-function (LoF), whereas epilepsy and DD/ID-associated variants resulted in both gain- and loss-of-function phenotypes. We additionally show that M653I and S809R, LoF GRIN2A variants associated with DD/ID, exert a dominant-negative effect when co-expressed with a wild-type GluN2A, whereas E58Ter and Y698C, SCZ-linked LoF variants, and A727T, an epilepsy-linked LoF variant, do not. These data offer a potential mechanism by which SCZ/epilepsy and DD/ID-linked variants can cause different effects on receptor function and therefore result in divergent pathological outcomes.

Molecular mechanisms of schizophrenia: Insights from human genetics.

Schizophrenia is a debilitating psychiatric disorder that affects millions of people worldwide; however, its etiology is poorly understood at the molecular and neurobiological levels. A particularly important advance in recent years is the discovery of rare genetic variants associated with a greatly increased risk of developing schizophrenia. These primarily loss-of-function variants are found in genes that overlap with those implicated by common variants and are involved in the regulation of glutamate signaling, synaptic function, DNA transcription, and chromatin remodeling. Animal models harboring mutations in these large-effect schizophrenia risk genes show promise in providing additional insights into the molecular mechanisms of the disease.

Mouse mutants in schizophrenia risk genes GRIN2A and AKAP11 show EEG abnormalities in common with schizophrenia patients.

Schizophrenia is a heterogeneous psychiatric disorder with a strong genetic basis, whose etiology and pathophysiology remain poorly understood. Exome sequencing studies have uncovered rare, loss-of-function variants that greatly increase risk of schizophrenia [1], including loss-of-function mutations in GRIN2A (aka GluN2A or NR2A, encoding the NMDA receptor subunit 2A) and AKAP11 (A-Kinase Anchoring Protein 11). AKAP11 and GRIN2A mutations are also associated with bipolar disorder [2], and epilepsy and developmental delay/intellectual disability [1, 3, 4], respectively. Accessible in both humans and rodents, electroencephalogram (EEG) recordings offer a window into brain activity and display abnormal features in schizophrenia patients. Does loss of Grin2a or Akap11 in mice also result in EEG abnormalities? We monitored EEG in heterozygous and homozygous knockout Grin2a and Akap11 mutant mice compared with their wild-type littermates, at 3- and 6-months of age, across the sleep/wake cycle and during auditory stimulation protocols. Grin2a and Akap11 mutants exhibited increased resting gamma power, attenuated auditory steady-state responses (ASSR) at gamma frequencies, and reduced responses to unexpected auditory stimuli during mismatch negativity (MMN) tests. Sleep spindle density was reduced in a gene dose-dependent manner in Akap11 mutants, whereas Grin2a mutants showed increased sleep spindle density. The EEG phenotypes of Grin2a and Akap11 mutant mice show a variety of abnormal features that overlap considerably with human schizophrenia patients, reflecting systems-level changes caused by Grin2a and Akap11 deficiency. These neurophysiologic findings further substantiate Grin2a and Akap11 mutants as genetic models of schizophrenia and identify potential biomarkers for stratification of schizophrenia patients.

Brain-region-specific changes in neurons and glia and dysregulation of dopamine signaling in Grin2a mutant mice.

A genetically valid animal model could transform our understanding of schizophrenia (SCZ) disease mechanisms. Rare heterozygous loss-of-function (LoF) mutations in GRIN2A, encoding a subunit of the NMDA receptor, greatly increase the risk of SCZ. By transcriptomic, proteomic, and behavioral analyses, we report that heterozygous Grin2a mutant mice show (1) large-scale gene expression changes across multiple brain regions and in neuronal (excitatory and inhibitory) and non-neuronal cells (astrocytes and oligodendrocytes), (2) evidence of hypoactivity in the prefrontal cortex (PFC) and hyperactivity in the hippocampus and striatum, (3) an elevated dopamine signaling in the striatum and hypersensitivity to amphetamine-induced hyperlocomotion (AIH), (4) altered cholesterol biosynthesis in astrocytes, (5) a reduction in glutamatergic receptor signaling proteins in the synapse, and (6) an aberrant locomotor pattern opposite of that induced by antipsychotic drugs. These findings reveal potential pathophysiologic mechanisms, provide support for both the "hypo-glutamate" and "hyper-dopamine" hypotheses of SCZ, and underscore the utility of Grin2a-deficient mice as a genetic model of SCZ.

Single synapse glutamate imaging reveals multiple levels of release mode regulation in mammalian synapses.

Mammalian central synapses exhibit vast heterogeneity in signaling strength. To understand the extent of this diversity, how it is achieved, and its functional implications, characterization of a large number of individual synapses is required. Using glutamate imaging, we characterized the evoked release probability and spontaneous release frequency of over 24,000 individual synapses. We found striking variability and no correlation between action potential-evoked and spontaneous synaptic release strength, suggesting distinct regulatory mechanisms. Subpixel localization of individual evoked and spontaneous release events reveals tight spatial regulation of evoked release and enhanced spontaneous release outside of evoked release region. Using on-stage post hoc immune-labeling of vesicle-associated proteins, Ca2+-sensing proteins, and soluble presynaptic proteins we were able to show that distinct molecular ensembles are associated with evoked and spontaneous modes of synaptic release.

Maternal and fetal effects of COVID-19 virus on a complicated triplet pregnancy: a case report.

BACKGROUND: Coronavirus disease 2019 (COVID-19), the global pandemic that has spread throughout the world, is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Given the limited scientific evidence on the manifestations and potential impact of this virus on pregnancy, we decided to report this case. CASE PRESENTATION: The patient was a 38 year-old Iranian woman with a triplet pregnancy and a history of primary infertility, as well as hypothyroidism and gestational diabetes. She was hospitalized at 29 weeks and 2 days gestational age due to elevated liver enzymes, and finally, based on a probable diagnosis of gestational cholestasis, she was treated with ursodeoxycholic acid. On the first day of hospitalization, sonography was performed, which showed that biophysical scores and amniotic fluid were normal in all three fetuses, with normal Doppler findings in two fetuses and increased umbilical artery resistance (pulsatility index [PI] > 95%) in one fetus. On day 4 of hospitalization, she developed fever, cough and myalgia, and her COVID-19 test was positive. Despite mild maternal symptoms, exacerbated placental insufficiency occurred in two of the fetuses leading to the rapid development of absent umbilical artery end-diastolic flow. Finally, 6 days later, the patient underwent cesarean section due to rapid exacerbation of placental insufficiency and declining biophysical score in two of the fetuses. Nasopharyngeal swab COVID-19 tests were negative for the first and third babies and positive for the second baby. The first and third babies died 3 and 13 days after birth, respectively, due to collapsed white lung and sepsis. The second baby was discharged in good general condition. The mother was discharged 3 days after cesarean section. She had no fever at the time of discharge and was also in good general condition. CONCLUSIONS: This was a complicated triplet pregnancy, in which, after maternal infection with COVID-19, despite mild maternal symptoms, exacerbated placental insufficiency occurred in two of the fetuses, and the third fetus had a positive COVID-19 test after birth. Therefore, in cases of pregnancy with COVID-19 infection, in addition to managing the mother, it seems that physicians would be wise to also give special attention to the possibility of acute placental insufficiency and subsequent fetal hypoxia, and also the probability of vertical transmission.

Clathrin coat controls synaptic vesicle acidification by blocking vacuolar ATPase activity.

Newly-formed synaptic vesicles (SVs) are rapidly acidified by vacuolar adenosine triphosphatases (vATPases), generating a proton electrochemical gradient that drives neurotransmitter loading. Clathrin-mediated endocytosis is needed for the formation of new SVs, yet it is unclear when endocytosed vesicles acidify and refill at the synapse. Here, we isolated clathrin-coated vesicles (CCVs) from mouse brain to measure their acidification directly at the single vesicle level. We observed that the ATP-induced acidification of CCVs was strikingly reduced in comparison to SVs. Remarkably, when the coat was removed from CCVs, uncoated vesicles regained ATP-dependent acidification, demonstrating that CCVs contain the functional vATPase, yet its function is inhibited by the clathrin coat. Considering the known structures of the vATPase and clathrin coat, we propose a model in which the formation of the coat surrounds the vATPase and blocks its activity. Such inhibition is likely fundamental for the proper timing of SV refilling.

Imaging Activity-Dependent Signaling Dynamics at the Neuronal Synapse Using FRET-Based Biosensors.

In this chapter, we introduce the combined use of FRET-based biosensors and synaptic markers as an effective tool for studying intracellular signaling pathways in small synaptic terminals of neuronal cells. The approach is based on the unmixing of excitation/emission spectral fingerprints of a FRET donor and acceptor pair, as well as a lipophilic styryl dye, FM1-43, loaded into presynaptic terminals. The destaining of FM1-43 during evoked release provides a map to guide the sampling of fluorescence for FRET analysis. In the example presented here, we measure the temporal dynamics of cAMP at the presynaptic terminal using an intramolecular CFP/YFP-based FRET sensor. However, this methodology can be applied to investigate the spatial and temporal regulation of a variety of signaling processes, as well as dynamic changes in protein-protein interaction.

Serotonin Transporter Associated Protein Complexes Are Enriched in Synaptic Vesicle Proteins and Proteins Involved in Energy Metabolism and Ion Homeostasis.

The serotonin transporter (SERT) mediates Na+-dependent high-affinity serotonin uptake and plays a key role in regulating extracellular serotonin concentration in the brain and periphery. To gain novel insight into SERT regulation, we conducted a comprehensive proteomics screen to identify components of SERT-associated protein complexes in the brain by employing three independent approaches. In vivo SERT complexes were purified from rat brain using an immobilized high-affinity SERT ligand, amino-methyl citalopram. This approach was combined with GST pulldown and yeast two-hybrid screens using N- and C-terminal cytoplasmic transporter domains as bait. Potential SERT associated proteins detected by at least two of the interaction methods were subjected to gene ontology analysis resulting in the identification of functional protein clusters that are enriched in SERT complexes. Prominent clusters include synaptic vesicle proteins, as well as proteins involved in energy metabolism and ion homeostasis. Using subcellular fractionation and electron microscopy we provide further evidence that SERT is indeed associated with synaptic vesicle fractions, and colocalizes with small vesicular structures in axons and axon terminals. We also show that SERT is found in close proximity to mitochondrial membranes in both, hippocampal and neocortical regions. We propose a model of the SERT interactome, in which SERT is distributed between different subcellular compartments through dynamic interactions with site-specific protein complexes. Finally, our protein interaction data suggest novel hypotheses for the regulation of SERT activity and trafficking, which ultimately impact on serotonergic neurotransmission and serotonin dependent brain functions.

Single-vesicle imaging reveals different transport mechanisms between glutamatergic and GABAergic vesicles.

Synaptic transmission is mediated by the release of neurotransmitters, which involves exo-endocytotic cycling of synaptic vesicles. To maintain synaptic function, synaptic vesicles are refilled with thousands of neurotransmitter molecules within seconds after endocytosis, using the energy provided by an electrochemical proton gradient. However, it is unclear how transmitter molecules carrying different net charges can be efficiently sequestered while maintaining charge neutrality and osmotic balance. We used single-vesicle imaging to monitor pH and electrical gradients and directly showed different uptake mechanisms for glutamate and γ-aminobutyric acid (GABA) operating in parallel. In contrast to glutamate, GABA was exchanged for protons, with no other ions participating in the transport cycle. Thus, only a few components are needed to guarantee reliable vesicle filling with different neurotransmitters.

Conferral of allostery to Thermus sp. GH5 methylglyoxal synthase by a single mutation.

There is huge number of oligomeric proteins that show allosteric behaviour as soon as their allosteric effector is provided. Thermus sp. GH5 methylglyoxal synthase is also a homohexameric protein, which displays cooperative behaviour when phosphate concentration increases. Previous studies on this enzyme have indicated that binding of phosphate leads to formation of specific interactions which makes the enzyme capable of displaying allosteric behaviour when its substrate is bound. In this study, it has been shown that a single mutation, independent of phosphate, provides the requirements for showing such cooperative behaviour. However, it is proposed that the allosteric mechanism triggered by phosphate is different from that applied by the mutation. These findings point towards the fact that allostery can be acquired, modulated or eliminated by any alteration in structure and/or dynamics of the proteins and all proteins are potentially capable of showing cooperative behaviour as soon as their prerequisites for this phenomenon are provided by the allosteric effector.

Membrane fusion intermediates via directional and full assembly of the SNARE complex.

Cellular membrane fusion is thought to proceed through intermediates including docking of apposed lipid bilayers, merging of proximal leaflets to form a hemifusion diaphragm, and fusion pore opening. A membrane-bridging four-helix complex of soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) mediates fusion. However, how assembly of the SNARE complex generates docking and other fusion intermediates is unknown. Using a cell-free reaction, we identified intermediates visually and then arrested the SNARE fusion machinery when fusion was about to begin. Partial and directional assembly of SNAREs tightly docked bilayers, but efficient fusion and an extended form of hemifusion required assembly beyond the core complex to the membrane-connecting linkers. We propose that straining of lipids at the edges of an extended docking zone initiates fusion.