Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia.

COVID-19 is a disease with unique characteristics that include lung thrombosis1, frequent diarrhoea2, abnormal activation of the inflammatory response3 and rapid deterioration of lung function consistent with alveolar oedema4. The pathological substrate for these findings remains unknown. Here we show that the lungs of patients with COVID-19 contain infected pneumocytes with abnormal morphology and frequent multinucleation. The generation of these syncytia results from activation of the SARS-CoV-2 spike protein at the cell plasma membrane level. On the basis of these observations, we performed two high-content microscopy-based screenings with more than 3,000 approved drugs to search for inhibitors of spike-driven syncytia. We converged on the identification of 83 drugs that inhibited spike-mediated cell fusion, several of which belonged to defined pharmacological classes. We focused our attention on effective drugs that also protected against virus replication and associated cytopathicity. One of the most effective molecules was the antihelminthic drug niclosamide, which markedly blunted calcium oscillations and membrane conductance in spike-expressing cells by suppressing the activity of TMEM16F (also known as anoctamin 6), a calcium-activated ion channel and scramblase that is responsible for exposure of phosphatidylserine on the cell surface. These findings suggest a potential mechanism for COVID-19 disease pathogenesis and support the repurposing of niclosamide for therapy.

Distance-dependent gradient in NMDAR-driven spine calcium signals along tapering dendrites.

Neurons receive a multitude of synaptic inputs along their dendritic arbor, but how this highly heterogeneous population of synaptic compartments is spatially organized remains unclear. By measuring N-methyl-d-aspartic acid receptor (NMDAR)-driven calcium responses in single spines, we provide a spatial map of synaptic calcium signals along dendritic arbors of hippocampal neurons and relate this to measures of synapse structure. We find that quantal NMDAR calcium signals increase in amplitude as they approach a thinning dendritic tip end. Based on a compartmental model of spine calcium dynamics, we propose that this biased distribution in calcium signals is governed by a gradual, distance-dependent decline in spine size, which we visualized using serial block-face scanning electron microscopy. Our data describe a cell-autonomous feature of principal neurons, where tapering dendrites show an inverse distribution of spine size and NMDAR-driven calcium signals along dendritic trees, with important implications for synaptic plasticity rules and spine function.

Transcription factor LIM homeobox 7 (Lhx7) maintains subtype identity of cholinergic interneurons in the mammalian striatum.

The generation and maintenance of a plethora of neuronal subtypes is essential for normal brain function. Nevertheless, little is known about the molecular mechanisms that maintain the defining characteristics of neurons following their initial postmitotic specification. Using conditional gene ablation in mice, we demonstrate here that the homeodomain protein LIM homeobox (Lhx)7 is essential for maintaining the morphological and molecular characteristics of cholinergic interneurons of the striatum. Lhx7-depleted cholinergic interneurons extinguish expression of several subtype-specific markers, including choline acetyl transferase and Isl1, and are respecified into Lhx6-expressing mature GABAergic interneurons. Additional expression studies support a model where Lhx7 controls the choice between cholinergic or GABAergic identity by gating a cross inhibitory regulation between Isl1 and Lhx6. By demonstrating that the switch between alternative striatal interneuron fates depends on persistent activity of a single transcription factor, we provide evidence that the intrinsic plasticity of mammalian forebrain neuronal subtypes is maintained after the initial specification and lineage commitment and possibly throughout life.

The LIM homeodomain protein Lhx6 regulates maturation of interneurons and network excitability in the mammalian cortex.

Deletion of LIM homeodomain transcription factor-encoding Lhx6 gene in mice results in defective tangential migration of cortical interneurons and failure of differentiation of the somatostatin (Sst)- and parvalbumin (Pva)-expressing subtypes. Here, we characterize a novel hypomorphic allele of Lhx6 and demonstrate that reduced activity of this locus leads to widespread differentiation defects in Sst(+) interneurons, but relatively minor and localized changes in Pva(+) interneurons. The reduction in the number of Sst-expressing cells was not associated with a loss of interneurons, because the migration and number of Lhx6-expressing interneurons and expression of characteristic molecular markers, such as calretinin or Neuropeptide Y, were not affected in Lhx6 hypomorphic mice. Consistent with a selective deficit in the differentiation of Sst(+) interneurons in the CA1 subfield of the hippocampus, we observed reduced expression of metabotropic Glutamate Receptor 1 in the stratum oriens and characteristic changes in dendritic inhibition, but normal inhibitory input onto the somatic compartment of CA1 pyramidal cells. Moreover, Lhx6 hypomorphs show behavioral, histological, and electroencephalographic signs of recurrent seizure activity, starting from early adulthood. These results demonstrate that Lhx6 plays an important role in the maturation of cortical interneurons and the formation of inhibitory circuits in the mammalian cortex.

Excitatory and inhibitory synapses show a tight subcellular correlation that weakens over development.

Neurons receive correlated levels of excitation and inhibition, a feature that is important for proper brain function. However, how this relationship between excitatory and inhibitory inputs is established during the dynamic period of circuit wiring remains unexplored. Using multiple techniques, including in utero electroporation, electron microscopy, and electrophysiology, we reveal a tight correlation in the distribution of excitatory and inhibitory synapses along the dendrites of developing CA1 hippocampal neurons. This correlation was present within short dendritic stretches (<20 µm) and, surprisingly, was most pronounced during early development, sharply declining with maturity. The tight matching between excitation and inhibition was unexpected, as inhibitory synapses lacked an active zone when formed and exhibited compromised evoked release. We propose that inhibitory synapses form as a stabilizing scaffold to counterbalance growing excitation levels. This relationship diminishes over time, suggesting a critical role for a subcellular balance in early neuronal function and circuit formation.

Synaptic plasticity, memory and the hippocampus: a neural network approach to causality.

Two facts about the hippocampus have been common currency among neuroscientists for several decades. First, lesions of the hippocampus in humans prevent the acquisition of new episodic memories; second, activity-dependent synaptic plasticity is a prominent feature of hippocampal synapses. Given this background, the hypothesis that hippocampus-dependent memory is mediated, at least in part, by hippocampal synaptic plasticity has seemed as cogent in theory as it has been difficult to prove in practice. Here we argue that the recent development of transgenic molecular devices will encourage a shift from mechanistic investigations of synaptic plasticity in single neurons towards an analysis of how networks of neurons encode and represent memory, and we suggest ways in which this might be achieved. In the process, the hypothesis that synaptic plasticity is necessary and sufficient for information storage in the brain may finally be validated.

Stochastic yet biased expression of multiple Dscam splice variants by individual cells.

The Drosophila melanogaster gene Dscam is essential for axon guidance and has 38,016 possible alternative splice forms. This diversity can potentially be used to distinguish cells. We analyzed the Dscam mRNA isoforms expressed by different cell types and individual cells. The choice of splice variants expressed is regulated both spatially and temporally. Different subtypes of photoreceptors express broad yet distinctive spectra of Dscam isoforms. Single-cell RT-PCR documented that individual cells express several different Dscam isoforms and allowed an estimation of the diversity that is present. For example, we estimate that each R3/R4 photoreceptor cell expresses 14-50 distinct mRNAs chosen from the spectrum of thousands of splice variants distinctive of its cell type. Thus, the Dscam repertoire of each cell is different from those of its neighbors, providing a potential mechanism for generating unique cell identity in the nervous system and elsewhere.

Simulation of the Influence of the Radial Graded Porosity Distribution on Elastic Modulus of γ/ß Phase Ti-Based Alloy Foams for Bone Implant.

This research aims to examine how a radial graded porosity distribution affects the elastic modulus by conducting simulations on Ti-based alloy foams with face-centered cubic and body-centered cubic crystal structures. Four types of foams were analyzed; commercially pure-Ti, Ti-13Ta-6Mn (TTM), Ti-13Ta-(TT) and Ti-13Ta-6Sn (TTS), (all in at.%). Four radial graded porosity distribution configurations were modeled and simulated using the finite element analysis (FEA). The radial graded porosity distribution configurations were generated using a Material Designer (Ansys) with a pore range of 200 to 600 µm. These radial graded porosity distributions had average porosity values of 0, 20, 30 and 40%. The consolidated samples that were obtained through a powder metallurgy technique in two step samples were synthesized using a powder metallurgy technique, with the elastic moduli values of the aforementioned Ti based alloys being measured by ultrasound using ~110, ~69, ~61 and ~65 GPa, respectively. The results showed that the modulus decreased as a function of porosity level in all simulated materials. The TTM, TT and TTS foams, with average porosities of 20, 30 and 40%, exhibited an modulus smaller than 30 GPa, which is a requirement to be used as a biomaterial in human bones. The TT foams showed the lowest modulus when compared to the other foams. Finally, certain theoretical models were used to obtain the modulus, the best being; the Gibson-Ashby model (α = 1 and n = 2.5) for the cp-Ti foams and Knudsen-Spriggs model (b = 3.06) for the TTM, TT and TTS foams.

Aberrant axon initial segment plasticity and intrinsic excitability of ALS hiPSC motor neurons.

Dysregulated neuronal excitability is a hallmark of amyotrophic lateral sclerosis (ALS). We sought to investigate how functional changes to the axon initial segment (AIS), the site of action potential generation, could impact neuronal excitability in ALS human induced pluripotent stem cell (hiPSC) motor neurons. We find that early TDP-43 and C9orf72 hiPSC motor neurons show an increase in the length of the AIS and impaired activity-dependent AIS plasticity that is linked to abnormal homeostatic regulation of neuronal activity and intrinsic hyperexcitability. In turn, these hyperactive neurons drive increased spontaneous myofiber contractions of in vitro hiPSC motor units. In contrast, late hiPSC and postmortem ALS motor neurons show AIS shortening, and hiPSC motor neurons progress to hypoexcitability. At a molecular level, aberrant expression of the AIS master scaffolding protein ankyrin-G and AIS-specific voltage-gated sodium channels mirror these dynamic changes in AIS function and excitability. Our results point toward the AIS as an important site of dysfunction in ALS motor neurons.

Multi-synaptic boutons are a feature of CA1 hippocampal connections in the stratum oriens.

Excitatory synapses are typically described as single synaptic boutons (SSBs), where one presynaptic bouton contacts a single postsynaptic spine. Using serial section block-face scanning electron microscopy, we found that this textbook definition of the synapse does not fully apply to the CA1 region of the hippocampus. Roughly half of all excitatory synapses in the stratum oriens involved multi-synaptic boutons (MSBs), where a single presynaptic bouton containing multiple active zones contacted many postsynaptic spines (from 2 to 7) on the basal dendrites of different cells. The fraction of MSBs increased during development (from postnatal day 22 [P22] to P100) and decreased with distance from the soma. Curiously, synaptic properties such as active zone (AZ) or postsynaptic density (PSD) size exhibited less within-MSB variation when compared with neighboring SSBs, features that were confirmed by super-resolution light microscopy. Computer simulations suggest that these properties favor synchronous activity in CA1 networks.

Sociodemographic and Clinical Factors Associated with Treatment Outcomes for Drug-resistant Tuberculosis.

Drug-resistant tuberculosis (DR-TB) continues to be a serious public health problem. The objective of this study was to evaluate the sociodemographic, radiological, clinical, and outcome characteristics and assess the determinants of unfavorable outcomes in DR-TB. The descriptive-analytical study was carried out in a reference outpatient clinic in Rio de Janeiro, Brazil, among DR-TB cases that received treatment between February 2016 and October 2020, using descriptive statistics, χ2 test, and logistic regression multivariate. Of the 148 cases, 12.2% were resistant to rifampicin, 12.2% were resistant to isoniazid, 18.2% were polyresistant, 56.1% multidrug resistant, and 1.3% were extensively drug resistant. Most of the patients were men, aged up to 44 years, with brown or black skin, having up to 8 years of schooling, unemployed or working in the informal economy, and of low income. Presenting with acquired resistance or positive sputum smear microscopy in the diagnosis, taking more than four drugs, and being unemployed were associated with unfavorable outcomes. Having no income or acquired resistance doubled the chances of unfavorable outcomes. There was a high proportion of unfavorable outcomes, thereby highlighting the need to concentrate efforts on planning and executing public policies that include the severity of DR-TB and its risk factors.

Activity-Dependent Plasticity of Axo-axonic Synapses at the Axon Initial Segment.

The activity-dependent rules that govern the wiring of GABAergic interneurons are not well understood. Chandelier cells (ChCs) are a type of GABAergic interneuron that control pyramidal cell output through axo-axonic synapses that target the axon initial segment. In vivo imaging of ChCs during development uncovered a narrow window (P12-P18) over which axons arborized and formed connections. We found that increases in the activity of either pyramidal cells or individual ChCs during this temporal window result in a reversible decrease in axo-axonic connections. Voltage imaging of GABAergic transmission at the axon initial segment (AIS) showed that axo-axonic synapses were depolarizing during this period. Identical manipulations of network activity in older mice (P40-P46), when ChC synapses are inhibitory, resulted instead in an increase in axo-axonic synapses. We propose that the direction of ChC synaptic plasticity follows homeostatic rules that depend on the polarity of axo-axonic synapses.

The variable transmembrane domain of Drosophila N-cadherin regulates adhesive activity.

Drosophila N-cadherin (CadN) is an evolutionarily conserved classic cadherin which has a large, complex extracellular domain and a catenin-binding cytoplasmic domain. The CadN locus contains three modules of alternative exons (7a/b, 13a/b, and 18a/b) and undergoes alternative splicing to generate multiple isoforms. Using quantitative transcript analyses and green fluorescent protein-based cell sorting, we found that during development CadN alternative splicing is regulated in a temporal but not cell-type-specific fashion. In particular, exon 18b is predominantly expressed during early developmental stages, while exon 18a is prevalent at the late developmental and adult stages. All CadN isoforms share the same molecular architecture but have different sequences in their extracellular and transmembrane domains, suggesting functional diversity. In vitro quantitative cell aggregation assays revealed that all CadN isoforms mediate homophilic interactions, but the isoforms encoded by exon 18b have a higher adhesive activity than those by its alternative, 18a. Domain-swapping experiments further revealed that the different sequences in the transmembrane domains of isoforms are responsible for their differential adhesive activities. CadN alternative splicing might provide a novel mechanism to fine-tune its adhesive activity at different developmental stages or to restrict the use of high-affinity 18b-type isoforms at the adult stage.

Transplantation of Chemogenetically Engineered Cortical Interneuron Progenitors into Early Postnatal Mouse Brains.

Neuronal development is regulated by a complex combination of environmental and genetic factors. Assessing the relative contribution of each component is a complicated task, which is particularly difficult in regards to the development of γ-aminobutyric acid (GABA)ergic cortical interneurons (CIs). CIs are the main inhibitory neurons in the cerebral cortex, and they play key roles in neuronal networks, by regulating both the activity of individual pyramidal neurons, as well as the oscillatory behavior of neuronal ensembles. They are generated in transient embryonic structures (medial and caudal ganglionic eminences - MGE and CGE) that are very difficult to efficiently target using in utero electroporation approaches. Interneuron progenitors migrate long distances during normal embryonic development, before they integrate in the cortical circuit. This remarkable ability to disperse and integrate into a developing network can be hijacked by transplanting embryonic interneuron precursors into early post-natal host cortices. Here, we present a protocol that allows genetic modification of embryonic interneuron progenitors using focal ex vivo electroporation. These engineered interneuron precursors are then transplanted into early post-natal host cortices, where they will mature into easily identifiable CIs. This protocol allows the use of multiple genetically encoded tools, or the ability to regulate the expression of specific genes in interneuron progenitors, in order to investigate the impact of either genetic or environmental variables on the maturation and integration of CIs.

Endogenous calcium buffers regulate fast exocytosis in the synaptic terminal of retinal bipolar cells.

Calcium-triggered exocytosis at the synapse is suppressed by addition of calcium chelators, but the effects of endogenous Ca(2+) buffers have not been tested. We find that 80% of Ca(2+) binding sites in the synaptic terminal of retinal bipolar cells were associated with mobile molecules that suppressed activation of Ca(2+)-sensitive K(+) channels with an efficiency equivalent to approximately 1.2 mM BAPTA. Removing these buffers caused a 30-fold increase in the number of vesicles released by Ca(2+) tail currents lasting approximately 0.5 ms and a 2-fold increase in the rapidly releasable pool of vesicles (RRP). The effects of BAPTA and EGTA indicate that vesicles comprising the RRP were docked at variable distances from Ca(2+) channels. We propose that endogenous Ca(2+) buffers regulate the size of the RRP by suppressing the release of vesicles toward the periphery of the active zone.

Analysis of Dscam diversity in regulating axon guidance in Drosophila mushroom bodies.

Dscam is an immunoglobulin (Ig) superfamily member that regulates axon guidance and targeting in Drosophila. Alternative splicing potentially generates 38,016 isoforms differing in their extracellular Ig and transmembrane domains. We demonstrate that Dscam mediates the sorting of axons in the developing mushroom body (MB). This correlates with the precise spatiotemporal pattern of Dscam protein expression. We demonstrate that MB neurons express different arrays of Dscam isoforms and that single MB neurons express multiple isoforms. Two different Dscam isoforms differing in their extracellular domains introduced as transgenes into single mutant cells partially rescued the mutant phenotype. Expression of one isoform of Dscam in a cohort of MB neurons induced dominant phenotypes, while expression of a single isoform in a single cell did not. We propose that different extracellular domains of Dscam share a common function and that differences in isoforms expressed on the surface of neighboring axons influence interactions between them.

Homeostatic Regulation of Interneuron Apoptosis During Cortical Development.

The mammalian cortex consists of two main neuronal types: the principal excitatory pyramidal neurons (PNs) and the inhibitory interneurons (INs). The interplay between these two neuronal populations - which drive excitation and inhibition (E/I balance), respectively - is crucial for controlling the overall activity in the brain. A number of neurological and psychiatric disorders have been associated with changes in E/I balance. It is not surprising, therefore, that neural networks employ several different mechanisms to maintain their firing rates at a stable level, collectively referred as homeostatic forms of plasticity. Here, we share our views on how the size of IN populations may provide an early homeostatic checkpoint for controlling brain activity. In a recent paper published in Cell Reports, we demonstrate that the extent of IN apoptosis during a critical early postnatal period is plastic, cell type specific, and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that a critical interplay between the physiological state of the network and its cellular units fine-tunes the size of IN populations with the aim of stabilizing network activity.

A Distance-Dependent Distribution of Presynaptic Boutons Tunes Frequency-Dependent Dendritic Integration.

How presynaptic inputs and neurotransmitter release dynamics are distributed along a dendritic tree is not well established. Here, we show that presynaptic boutons that form onto basal dendrites of CA1 pyramidal neurons display a decrease in active zone (AZ) size with distance from the soma, resulting in a distance-dependent increase in short-term facilitation. Our findings suggest that the spatial distribution of short-term facilitation serves to compensate for the electrotonic attenuation of subthreshold distal inputs during repeated stimulation and fine-tunes the preferred input frequency of dendritic domains.

Modulation of Apoptosis Controls Inhibitory Interneuron Number in the Cortex.

Cortical networks are composed of excitatory projection neurons and inhibitory interneurons. Finding the right balance between the two is important for controlling overall cortical excitation and network dynamics. However, it is unclear how the correct number of cortical interneurons (CIs) is established in the mammalian forebrain. CIs are generated in excess from basal forebrain progenitors, and their final numbers are adjusted via an intrinsically determined program of apoptosis that takes place during an early postnatal window. Here, we provide evidence that the extent of CI apoptosis during this critical period is plastic and cell-type specific and can be reduced in a cell-autonomous manner by acute increases in neuronal activity. We propose that the physiological state of the emerging neural network controls the activity levels of local CIs to modulate their numbers in a homeostatic manner.

Permethrin induces histological and cytological changes in the midgut of the predatory bug, Podisus nigrispinus.

Insecticides used in the agriculture and forestry have side effects on non-target organisms used as natural enemies. This study evaluated the histopathology and cytotoxicity of permethrin on the midgut of the non-target predatory bug, Podisus nigrispinus (Heteroptera: Pentatomidae) used in the biological control of pest insects. The toxicity and survival of this insect were determined using six concentrations of permethrin via ingestion. Histological and ultraestutural changes of the midgut of P. nigrispinus were analyzed after exposure to permethrin. The insecticide caused toxicity in P. nigrispinus with LC50 = 0.46 µg L-1 and survival of 47% after 72 h of exposure. The histological changes in the midgut were irregularly bordered epithelium, cytoplasmic vacuolization and apocrine secretions in the lumen after 6 h following exposure to the insecticide. Cytotoxic effects such as granules and vacuoles secreted into the lumen, presence of autophagosomes, and dilatation of infolds of the basal plasma membrane were observed in the three regions of the midgut. Cells of the midgut in apoptosis occurred after 12 h of exposure. Permethrin causes toxic effects, inhibits survival, and produces changes in the histology and cytology of the midgut in P. nigrispinus, suggesting that the cell stress induced by this insecticide can disrupt physiological processes such as digestion, compromising the potential of the predator as a biological control agent of pests. The low selectivity of permethrin to a non-target organism such as the predatory bug, P. nigrispinus indicates that the associated use of this insecticide in biological control should be better evaluated.