HDAC4 governs a transcriptional program essential for synaptic plasticity and memory.

Neuronal activity influences genes involved in circuit development and information processing. However, the molecular basis of this process remains poorly understood. We found that HDAC4, a histone deacetylase that shuttles between the nucleus and cytoplasm, controls a transcriptional program essential for synaptic plasticity and memory. The nuclear import of HDAC4 and its association with chromatin is negatively regulated by NMDA receptors. In the nucleus, HDAC4 represses genes encoding constituents of central synapses, thereby affecting synaptic architecture and strength. Furthermore, we show that a truncated form of HDAC4 encoded by an allele associated with mental retardation is a gain-of-function nuclear repressor that abolishes transcription and synaptic transmission despite the loss of the deacetylase domain. Accordingly, mice carrying a mutant that mimics this allele exhibit deficits in neurotransmission, spatial learning, and memory. These studies elucidate a mechanism of experience-dependent plasticity and define the biological role of HDAC4 in the brain.

Synaptotagmin-11 mediates a vesicle trafficking pathway that is essential for development and synaptic plasticity.

Synaptotagmin-11 (Syt11) is a Synaptotagmin isoform that lacks an apparent ability to bind calcium, phospholipids, or SNARE proteins. While human genetic studies have linked mutations in the Syt11 gene to schizophrenia and Parkinson's disease, the localization or physiological role of Syt11 remain unclear. We found that in neurons, Syt11 resides on abundant vesicles that differ from synaptic vesicles and resemble trafficking endosomes. These vesicles recycle via the plasma membrane in an activity-dependent manner, but their exocytosis is slow and desynchronized. Constitutive knockout mice lacking Syt11 died shortly after birth, suggesting Syt11-mediated membrane transport is required for survival. In contrast, selective ablation of Syt11 in excitatory forebrain neurons using a conditional knockout did not affect life span but impaired synaptic plasticity and memory. Syt11-deficient neurons displayed normal secretion of fast neurotransmitters and peptides but exhibited a reduction of long-term synaptic potentiation. Hence, Syt11 is an essential component of a neuronal vesicular trafficking pathway that differs from the well-characterized synaptic vesicle trafficking pathway but is also essential for life.

Activity-dependent IGF-1 exocytosis is controlled by the Ca(2+)-sensor synaptotagmin-10.

Synaptotagmins Syt1, Syt2, Syt7, and Syt9 act as Ca(2+)-sensors for synaptic and neuroendocrine exocytosis, but the function of other synaptotagmins remains unknown. Here, we show that olfactory bulb neurons secrete IGF-1 by an activity-dependent pathway of exocytosis, and that Syt10 functions as the Ca(2+)-sensor that triggers IGF-1 exocytosis in these neurons. Deletion of Syt10 impaired activity-dependent IGF-1 secretion in olfactory bulb neurons, resulting in smaller neurons and an overall decrease in synapse numbers. Exogenous IGF-1 completely reversed the Syt10 knockout phenotype. Syt10 colocalized with IGF-1 in somatodendritic vesicles of olfactory bulb neurons, and Ca(2+)-binding to Syt10 caused these vesicles to undergo exocytosis, thereby secreting IGF-1. Thus, Syt10 controls a previously unrecognized pathway of Ca(2+)-dependent exocytosis that is spatially and temporally distinct from Ca(2+)-dependent synaptic vesicle exocytosis controlled by Syt1. Our findings thereby reveal that two different synaptotagmins can regulate functionally distinct Ca(2+)-dependent membrane fusion reactions in the same neuron.

A genetically targeted reporter for PET imaging of deep neuronal circuits in mammalian brains.

Positron emission tomography (PET) allows biomolecular tracking but PET monitoring of brain networks has been hampered by a lack of suitable reporters. Here, we take advantage of bacterial dihydrofolate reductase, ecDHFR, and its unique antagonist, TMP, to facilitate in vivo imaging in the brain. Peripheral administration of radiofluorinated and fluorescent TMP analogs enabled PET and intravital microscopy, respectively, of neuronal ecDHFR expression in mice. This technique can be used to the visualize neuronal circuit activity elicited by chemogenetic manipulation in the mouse hippocampus. Notably, ecDHFR-PET allows mapping of neuronal projections in non-human primate brains, demonstrating the applicability of ecDHFR-based tracking technologies for network monitoring. Finally, we demonstrate the utility of TMP analogs for PET studies of turnover and self-assembly of proteins tagged with ecDHFR mutants. These results establish opportunities for a broad spectrum of previously unattainable PET analyses of mammalian brain circuits at the molecular level.

HYBRiD: hydrogel-reinforced DISCO for clearing mammalian bodies.

The recent development of solvent- and polymer-based brain-clearing techniques has advanced our ability to visualize the mammalian nervous system in three dimensions. However, it remains challenging to image the mammalian body en bloc. Here we developed HYBRiD (hydrogel-based reinforcement of three-dimensional imaging solvent-cleared organs (DISCO)), by recombining components of organic- and polymer-based clearing pipelines. We achieved high transparency and protein retention, as well as compatibility with direct fluorescent imaging and immunostaining in cleared mammalian bodies. Using parvalbumin- and somatostatin-Cre models, we demonstrated the utility of HYBRiD for whole-body imaging of genetically encoded fluorescent reporters without antibody enhancement of signals in newborn and juvenile mice. Using K18-hACE2 transgenic mice, HYBRiD enabled perfusion-free clearing and visualization of SARS-CoV-2 infection in a whole mouse chest, revealing macroscopic and microscopic features of viral pathology in the same sample. HYBRiD offers a simple and universal solution to visualize large heterogeneous body parts or entire animals for basic and translational research.

Different requirements of functional telomeres in neural stem cells and terminally differentiated neurons.

Telomeres have been studied extensively in peripheral tissues, but their relevance in the nervous system remains poorly understood. Here, we examine the roles of telomeres at distinct stages of murine brain development by using lineage-specific genetic ablation of TRF2, an essential component of the shelterin complex that protects chromosome ends from the DNA damage response machinery. We found that functional telomeres are required for embryonic and adult neurogenesis, but their uncapping has surprisingly no detectable consequences on terminally differentiated neurons. Conditional knockout of TRF2 in post-mitotic immature neurons had virtually no detectable effect on circuit assembly, neuronal gene expression, and the behavior of adult animals despite triggering massive end-to-end chromosome fusions across the brain. These results suggest that telomeres are dispensable in terminally differentiated neurons and provide mechanistic insight into cognitive abnormalities associated with aberrant telomere length in humans.

Activity-Induced Cortical Glutamatergic Neuron Nascent Proteins.

Neuronal activity initiates signaling cascades that culminate in diverse outcomes including structural and functional neuronal plasticity, and metabolic changes. While studies have revealed activity-dependent neuronal cell type-specific transcriptional changes, unbiased quantitative analysis of cell-specific activity-induced dynamics in newly synthesized proteins (NSPs) synthesis in vivo has been complicated by cellular heterogeneity and a relatively low abundance of NSPs within the proteome in the brain. Here we combined targeted expression of mutant MetRS (methionine tRNA synthetase) in genetically defined cortical glutamatergic neurons with tight temporal control of treatment with the noncanonical amino acid, azidonorleucine, to biotinylate NSPs within a short period after pharmacologically induced seizure in male and female mice. By purifying peptides tagged with heavy or light biotin-alkynes and using direct tandem mass spectrometry detection of biotinylated peptides, we quantified activity-induced changes in cortical glutamatergic neuron NSPs. Seizure triggered significant changes in ∼300 NSPs, 33% of which were decreased by seizure. Proteins mediating excitatory and inhibitory synaptic plasticity, including SynGAP1, Pak3, GEPH1, Copine-6, and collybistin, and DNA and chromatin remodeling proteins, including Rad21, Smarca2, and Ddb1, are differentially synthesized in response to activity. Proteins likely to play homeostatic roles in response to activity, such as regulators of proteastasis, intracellular ion control, and cytoskeleton remodeling proteins, are activity induced. Conversely, seizure decreased newly synthetized NCAM, among others, suggesting that seizure induced degradation. Overall, we identified quantitative changes in the activity-induced nascent proteome from genetically defined cortical glutamatergic neurons as a strategy to discover downstream mediators of neuronal plasticity and generate hypotheses regarding their function.SIGNIFICANCE STATEMENT Activity-induced neuronal and synaptic plasticity are mediated by changes in the protein landscape, including changes in the activity-induced newly synthesized proteins; however, identifying neuronal cell type-specific nascent proteome dynamics in the intact brain has been technically challenging. We conducted an unbiased proteomic screen from which we identified significant activity-induced changes in ∼300 newly synthesized proteins in genetically defined cortical glutamatergic neurons within 20 h after pharmacologically induced seizure. Bioinformatic analysis of the dynamic nascent proteome indicates that the newly synthesized proteins play diverse roles in excitatory and inhibitory synaptic plasticity, chromatin remodeling, homeostatic mechanisms, and proteasomal and metabolic functions, extending our understanding of the diversity of plasticity mechanisms.

Direct Homogeneous Synthesis of Compounds with Two O Atoms and Long-Chain Hydrocarbons from CO and H2: Co-Ru/N-methylpyrrolidone Catalyst.

The homogeneous acetic acid synthesis-type Ru-Co-Li/N-methylpyrrolidone catalyst for CO and H2 transformations has been studied at moderately high pressures. For 1CO:2H2, low acetic acid selectivity has been observed, along with remarkable methyl acetate selectivity, the absence of aldehydes and ethyl acetate and sharp deviations from the Anderson-Schultz-Flory distribution for both alcaohols and long-chain hydrocarbons. For 1CO:1H2 and slightly elevated pressure, acetic acid selectivity slightly increased, notable ethyl acetate formation was detected, and both long-chain hydrocarbons and alcohols disappeared. Hypotheses are discussed about the direct parallel formation of all observed product groups (hydrocarbons, alcohols, esters, and acetic acid) and hydrocarbon chain growth limitations according to the formed Ru-Co cluster size in the presence of the aforementioned catalytic system.

Methyl and Ethyl Ethers of Glycerol as Potential Green Low-Melting Technical Fluids.

The study is dedicated to the consideration of lower alkyl ethers of glycerol as potential components of low-melting technical fluids (e.g., heat transfer fluids, hydraulic fluids, aircraft de-icing fluids, etc.). Four isomeric mixtures of glycerol ethers (GMME-monomethyl; GDME-dimethyl; GMEE-monoethyl; GDEE-diethyl) were synthesized from epichlorohydrin and methanol/ethanol in the presence of sodium and subjected to detailed characterization as pure compounds and as aqueous solutions (30-90 vol%). The temperature and concentration dependencies of density, viscosity, cloud point, boiling range, specific heat capacity, thermal conductivity, and rubber swelling were obtained. On the basis of the data obtained, a comparison was made between the aqueous solutions of glycerol ethers and of other common bases for low-melting liquids (glycerol, ethylene glycol, and propylene glycol). Pure glycerol ethers could potentially be used as technical fluids in a very wide temperature range-from -114 to 150 °C. It was further demonstrated that in low temperature applications (e.g., in low-temperature chiller systems) the glycerol-ether-based aqueous heat transfer fluids could provide enhanced efficiency when compared to the glycerol- or propylene-glycol-based ones due to their lower viscosities and favorable environmental properties.

In Situ-Generated, Dispersed Cu Catalysts for the Catalytic Hydrogenolysis of Glycerol.

The present study is dedicated to the experimental verification of a concept for the hydrogenolysis of glycerol over in situ-generated Cu dispersed particles (Cu-DP). The Cu-DP were generated by in situ reduction of a precursor salt (Cu(OAc)2, CuSO4, CuCl2) in the presence of KOH and were active in glycerol conversion under hydrogen (T = 200-220 °C, p(H2) = 1-4 MPa), where 1,2-propylene glycol (PG) and lactic acid (LA) were detected to be the main products. The influence of the reaction conditions (temperature, hydrogen pressure, reaction time, catalyst-to-feed ratio and the KOH/Cu ratio) on the yields of the products is described. It was shown that the selectivity between the PG and LA could be tuned by changing p(H2) or by the KOH amount, i.e., higher yields of LA corresponded to lower p(H2) and higher alkalinity of the reaction media. The activity of the in situ-generated Cu-DP was found to be comparable to that of an industrial Cu-Cr2O3 catalyst. The Cu-DP catalysts were characterized by XRD, XPS, HRTEM and SEM. During the reaction, the catalyst evolved by the sintering and recrystallization of the separate Cu-DP; the crystallite sizes after 1 and 15 h reaction times amounted to 35 and 49 nm, respectively.

Correction: LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

[This corrects the article DOI: 10.1371/journal.pbio.1002516.].

Bio-Based Solvents and Gasoline Components From Renewable 2,3-Butanediol and 1,2-Propanediol: Synthesis and Characterization.

In this study approaches for chemical conversions of the renewable compounds 1,2-propanediol (1,2-PD) and 2,3-butanediol (2,3-BD) that yield the corresponding cyclic ketals and glycol ethers have been investigated experimentally. The characterization of the obtained products as potential green solvents and gasoline components is discussed. Cyclic ketals have been obtained by the direct reaction of the diols with lower aliphatic ketones (1,2-PD + acetone → 2,2,4-trimethyl-1,3-dioxolane (TMD) and 2,3-BD + butanone-2 → 2-ethyl-2,4,5-trimethyl-1,3-dioxolane (ETMD)), for which the ΔH0r, ΔS0r and ΔG0r values have been estimated experimentally. The monoethers of diols could be obtained through either hydrogenolysis of the pure ketals or from the ketone and the diol via reductive alkylation. In the both reactions, the cyclic ketals (TMD and ETMD) have been hydrogenated in nearly quantitative yields to the corresponding isopropoxypropanols (IPP) and 3-sec-butoxy-2-butanol (SBB) under mild conditions (T = 120-140 °C, p(H2) = 40 bar) with high selectivity (>93%). Four products (TMD, ETMD, IPP and SBB) have been characterized as far as their physical properties are concerned (density, melting/boiling points, viscosity, calorific value, evaporation rate, Antoine equation coefficients), as well as their solvent ones (Kamlet-Taft solvatochromic parameters, miscibility, and polymer solubilization). In the investigation of gasoline blending properties, TMD, ETMD, IPP and SBB have shown remarkable antiknock performance with blending antiknock indices of 95.2, 92.7, 99.2 and 99.7 points, respectively.

LKB1 Regulates Mitochondria-Dependent Presynaptic Calcium Clearance and Neurotransmitter Release Properties at Excitatory Synapses along Cortical Axons.

Individual synapses vary significantly in their neurotransmitter release properties, which underlie complex information processing in neural circuits. Presynaptic Ca2+ homeostasis plays a critical role in specifying neurotransmitter release properties, but the mechanisms regulating synapse-specific Ca2+ homeostasis in the mammalian brain are still poorly understood. Using electrophysiology and genetically encoded Ca2+ sensors targeted to the mitochondrial matrix or to presynaptic boutons of cortical pyramidal neurons, we demonstrate that the presence or absence of mitochondria at presynaptic boutons dictates neurotransmitter release properties through Mitochondrial Calcium Uniporter (MCU)-dependent Ca2+ clearance. We demonstrate that the serine/threonine kinase LKB1 regulates MCU expression, mitochondria-dependent Ca2+ clearance, and thereby, presynaptic release properties. Re-establishment of MCU-dependent mitochondrial Ca2+ uptake at glutamatergic synapses rescues the altered neurotransmitter release properties characterizing LKB1-null cortical axons. Our results provide novel insights into the cellular and molecular mechanisms whereby mitochondria control neurotransmitter release properties in a bouton-specific way through presynaptic Ca2+ clearance.

Inducible control of gene expression with destabilized Cre.

Acute manipulation of gene and protein function in the brain is essential for understanding the mechanisms of nervous system development, plasticity and information processing. Here we describe a technique based on a destabilized Cre recombinase (DD-Cre) whose activity is controlled by the antibiotic trimethoprim (TMP). We show that DD-Cre triggers rapid TMP-dependent recombination of loxP-flanked ('floxed') alleles in mouse neurons in vivo and validate the use of this system for neurobehavioral research.

Epigenetic therapy for Friedreich ataxia.

OBJECTIVE: To investigate whether a histone deacetylase inhibitor (HDACi) would be effective in an in vitro model for the neurodegenerative disease Friedreich ataxia (FRDA) and to evaluate safety and surrogate markers of efficacy in a phase I clinical trial in patients. METHODS: We used a human FRDA neuronal cell model, derived from patient induced pluripotent stem cells, to determine the efficacy of a 2-aminobenzamide HDACi (109) as a modulator of FXN gene expression and chromatin histone modifications. FRDA patients were dosed in 4 cohorts, ranging from 30mg/day to 240mg/day of the formulated drug product of HDACi 109, RG2833. Patients were monitored for adverse effects as well as for increases in FXN mRNA, frataxin protein, and chromatin modification in blood cells. RESULTS: In the neuronal cell model, HDACi 109/RG2833 increases FXN mRNA levels and frataxin protein, with concomitant changes in the epigenetic state of the gene. Chromatin signatures indicate that histone H3 lysine 9 is a key residue for gene silencing through methylation and reactivation through acetylation, mediated by the HDACi. Drug treatment in FRDA patients demonstrated increased FXN mRNA and H3 lysine 9 acetylation in peripheral blood mononuclear cells. No safety issues were encountered. INTERPRETATION: Drug exposure inducing epigenetic changes in neurons in vitro is comparable to the exposure required in patients to see epigenetic changes in circulating lymphoid cells and increases in gene expression. These findings provide a proof of concept for the development of an epigenetic therapy for this fatal neurological disease.

Nr4a1 regulates cell-specific transcriptional programs in inhibitory GABAergic interneurons.

The patterns of synaptic connectivity and physiological properties of diverse neuron types are shaped by distinct gene sets. Our study demonstrates that, in the mouse forebrain, the transcriptional profiles of inhibitory GABAergic interneurons are regulated by Nr4a1, an orphan nuclear receptor whose expression is transiently induced by sensory experiences and is required for normal learning. Nr4a1 exerts contrasting effects on the local axonal wiring of parvalbumin- and somatostatin-positive interneurons, which innervate different subcellular domains of their postsynaptic partners. The loss of Nr4a1 activity in these interneurons results in bidirectional, cell-type-specific transcriptional switches across multiple gene families, including those involved in surface adhesion and repulsion. Our findings reveal that combinatorial synaptic organizing codes are surprisingly flexible and highlight a mechanism by which inducible transcription factors can influence neural circuit structure and function.

Synaptic architecture of a memory engram in the mouse hippocampus.

Memory engrams are formed through experience-dependent remodeling of neural circuits, but their detailed architectures have remained unresolved. Using 3D electron microscopy, we performed nanoscale reconstructions of the hippocampal CA3-CA1 pathway following chemogenetic labeling of cellular ensembles with a remote history of correlated excitation during associative learning. Projection neurons involved in memory acquisition expanded their connectomes via multi-synaptic boutons without altering the numbers and spatial arrangements of individual axonal terminals and dendritic spines. This expansion was driven by presynaptic activity elicited by specific negative valence stimuli, regardless of the co-activation state of postsynaptic partners. The rewiring of initial ensembles representing an engram coincided with local, input-specific changes in the shapes and organelle composition of glutamatergic synapses, reflecting their weights and potential for further modifications. Our findings challenge the view that the connectivity among neuronal substrates of memory traces is governed by Hebbian mechanisms, and offer a structural basis for representational drifts.

Phosphorylation of pyruvate dehydrogenase inversely associates with neuronal activity.

For decades, the expression of immediate early genes (IEGs) such as FOS has been the most widely used molecular marker representing neuronal activation. However, to date, there is no equivalent surrogate available for the decrease of neuronal activity. Here, we developed an optogenetic-based biochemical screen in which population neural activities can be controlled by light with single action potential precision, followed by unbiased phosphoproteomic profiling. We identified that the phosphorylation of pyruvate dehydrogenase (pPDH) inversely correlated with the intensity of action potential firing in primary neurons. In in vivo mouse models, monoclonal antibody-based pPDH immunostaining detected activity decreases across the brain, which were induced by a wide range of factors including general anesthesia, chemogenetic inhibition, sensory experiences, and natural behaviors. Thus, as an inverse activity marker (IAM) in vivo, pPDH can be used together with IEGs or other cell-type markers to profile and identify bi-directional neural dynamics induced by experiences or behaviors.

Push-and-pull regulation of the fusion pore by synaptotagmin-7.

In chromaffin cells, Ca(2+) binding to synaptotagmin-1 and -7 triggers exocytosis by promoting fusion pore opening and fusion pore expansion. Synaptotagmins contain two C2 domains that both bind Ca(2+) and contribute to exocytosis; however, it remains unknown whether the C2 domains act similarly or differentially to promote opening and expansion of fusion pores. Here, we use patch amperometry measurements in WT and synaptotagmin-7-mutant chromaffin cells to analyze the role of Ca(2+) binding to the two synaptotagmin-7 C2 domains in exocytosis. We show that, surprisingly, Ca(2+) binding to the C2A domain suffices to trigger fusion pore opening but that the resulting fusion pores are unstable and collapse, causing a dramatic increase in kiss-and-run fusion events. Thus, synaptotagmin-7 controls fusion pore dynamics during exocytosis via a push-and-pull mechanism in which Ca(2+) binding to both C2 domains promotes fusion pore opening, but the C2B domain is selectively essential for continuous expansion of an otherwise unstable fusion pore.

PET Waste Recycling into BTX Fraction Using In Situ Obtained Nickel Phosphide.

The annual production of plastic waste is a serious ecological problem as it causes substantial pollution of the environment. Polyethylene terephthalate, a material usually found in disposable plastic bottles, is one of the most popular material used for packaging in the world. In this paper, it is proposed to recycle polyethylene terephthalate waste bottles into benzene-toluene-xylene fraction using a heterogeneous nickel phosphide catalyst formed in situ during the polyethylene terephthalate recycling process. The catalyst obtained was characterized using powder X-ray diffraction, high-resolution transmission electron microscopy, and X-ray photoelectron spectroscopy techniques. The catalyst was shown to contain a Ni2P phase. Its activity was studied in a temperature range of 250-400 °C and a H2 pressure range of 5-9 MPa. The highest selectivity for benzene-toluene-xylene fraction was 93% at quantitative conversion.