Biphasic Npas4 expression promotes inhibitory plasticity and suppression of fear memory consolidation in mice.

Long-term memories are believed to be encoded by unique transcriptional signatures in the brain. The expression of immediate early genes (IEG) promotes structural and molecular changes required for memory consolidation. Recent evidence has shown that the brain is equipped with mechanisms that not only promote, but actively constrict memory formation. However, it remains unknown whether IEG expression may play a role in memory suppression. Here we uncovered a novel function of the IEG neuronal PAS domain protein 4 (Npas4), as an inducible memory suppressor gene of highly salient aversive experiences. Using a contextual fear conditioning paradigm, we found that low stimulus salience leads to monophasic Npas4 expression, while highly salient learning induces a biphasic expression of Npas4 in the hippocampus. The later phase requires N-methyl-D-aspartate (NMDA) receptor activity and is independent of dopaminergic neurotransmission. Our in vivo pharmacological and genetic manipulation experiments suggested that the later phase of Npas4 expression restricts the consolidation of a fear memory and promote behavioral flexibility, by facilitating fear extinction and the contextual specificity of fear responses. Moreover, immunofluorescence and electrophysiological analysis revealed a concomitant increase in synaptic input from cholecystokinin (CCK)-expressing interneurons. Our results demonstrate how salient experiences evoke unique temporal patterns of IEG expression that fine-tune memory consolidation. Moreover, our study provides evidence for inducible gene expression associated with memory suppression as a possible mechanism to balance the consolidation of highly salient memories, and thereby to evade the formation of maladaptive behavior.

Towards a Light-mediated Gene Therapy for the Eye using Caged Ethinylestradiol and the Inducible Cre/lox System.

Increasingly, retinal pathologies are being treated with virus-mediated gene therapies. To be able to target viral transgene expression specifically to the pathological regions of the retina with light, we established an in vivo photoactivated gene expression paradigm for retinal tissue. Based on the inducible Cre/lox system, we discovered that ethinylestradiol is a suitable alternative to Tamoxifen as ethinylestradiol is more amenable to modification with photosensitive protecting compounds, i.e., "caging." Identification of ethinylestradiol as a ligand for the mutated human estradiol receptor was supported by in silico binding studies showing the reduced binding of caged ethinylestradiol. Caged ethinylestradiol was injected into the eyes of double transgenic GFAP-CreERT2 mice with a Cre-dependent tdTomato reporter transgene followed by irradiation with light of 450 nm. Photoactivation significantly increased retinal tdTomato expression compared to controls. We thus demonstrated a first step towards the development of a targeted, light-mediated gene therapy for the eyes.

In Vivo Optical Interrogation of Neuronal Responses to Genetic, Cell Type-Specific Silencing.

We established a low background, Cre-dependent version of the inducible Tet-On system for fast, cell type-specific transgene expression in vivo Coexpression of a constitutive, Cre-dependent fluorescent marker selectively allowed single-cell analyses before and after inducible, Tet-dependent transgene expression. Here, we used this method for precise, acute manipulation of neuronal activity in the living brain. The goal was to study neuronal network homeostasis at cellular resolution. Single induction of the potassium channel Kir2.1 produced cell type-specific silencing within hours that lasted for at least 3 d. Longitudinal in vivo imaging of spontaneous calcium transients and neuronal morphology demonstrated that prolonged silencing did not alter spine densities or synaptic input strength. Furthermore, selective induction of Kir2.1 in parvalbumin interneurons increased the activity of surrounding neurons in a distance-dependent manner. This high-resolution, inducible interference and interval imaging of individual cells (high I5, HighFive) method thus allows visualizing temporally precise, genetic perturbations of defined cells.SIGNIFICANCE STATEMENT Gene function is studied by KO or overexpression of a specific gene followed by analyses of phenotypic changes. However, being able to predict and analyze exactly those cells in which genetic manipulation will occur is not possible. We combined two prominent transgene overexpression methods to fluorescently highlight the targeted cells appropriately before cell type-specific transgene induction. By inducing a potassium channel that decreases neuronal firing, we investigated how neuronal networks in the living mouse brain possibly compensate swift changes in cellular activities. Unlike in vitro, known compensatory homeostatic mechanisms, such as changes in synapses, were not observed in vivo Overall, we demonstrated with our method rapid genetic manipulation and analysis of neuronal activities as well as precision transgene expression.

High-content analysis of neuronal morphology in human iPSC-derived neurons.

We present a high-content analysis (HCA) protocol for monitoring the outgrowth capacity of human neurons derived from induced pluripotent stem cells (iPSCs). We describe steps to perform HCA imaging, followed by quantifying the morphology of dendrites and axons within a high-throughput system to evaluate neurons obtained through various differentiation approaches. This protocol can be used to screen for modulators of neuronal morphogenesis or neurotoxicity. The approach can be applied to patient-derived iPSCs to identify patient-specific defects and possible therapeutic strategies. For complete details on the use and execution of this protocol, please refer to Zink et al. (2020) and Inak et al. (2021). The protocol can be used in combination with Zink et al. (2022).

Single-dose ethanol intoxication causes acute and lasting neuronal changes in the brain.

Alcohol intoxication at early ages is a risk factor for the development of addictive behavior. To uncover neuronal molecular correlates of acute ethanol intoxication, we used stable-isotope-labeled mice combined with quantitative mass spectrometry to screen more than 2,000 hippocampal proteins, of which 72 changed synaptic abundance up to twofold after ethanol exposure. Among those were mitochondrial proteins and proteins important for neuronal morphology, including MAP6 and ankyrin-G. Based on these candidate proteins, we found acute and lasting molecular, cellular, and behavioral changes following a single intoxication in alcohol-naïve mice. Immunofluorescence analysis revealed a shortening of axon initial segments. Longitudinal two-photon in vivo imaging showed increased synaptic dynamics and mitochondrial trafficking in axons. Knockdown of mitochondrial trafficking in dopaminergic neurons abolished conditioned alcohol preference in Drosophila flies. This study introduces mitochondrial trafficking as a process implicated in reward learning and highlights the potential of high-resolution proteomics to identify cellular mechanisms relevant for addictive behavior.

Hypothesis: protein and RNA attributes are continuously optimized over time.

BACKGROUND: Little is known why proteins and RNAs exhibit half-lives varying over several magnitudes. Despite many efforts, a conclusive link between half-lives and gene function could not be established suggesting that other determinants may influence these molecular attributes. RESULTS: Here, I find that with increasing gene age there is a gradual and significant increase of protein and RNA half-lives, protein structure, and other molecular attributes that tend to affect protein abundance. These observations are accommodated in a hypothesis which posits that new genes at 'birth' are not optimized and thus their products exhibit low half-lives and less structure but continuous mutagenesis eventually improves these attributes. Thus, the protein and RNA products of the oldest genes obtained their high degrees of stability and structure only after billions of years while the products of younger genes had less time to be optimized and are therefore less stable and structured. Because more stable proteins with lower turnover require less transcription to maintain the same level of abundance, reduced transcription-associated mutagenesis (TAM) would fixate the changes by increasing gene conservation. CONCLUSIONS: Consequently, the currently observed diversity of molecular attributes is a snapshot of gene products being at different stages along their temporal path of optimization.

Improving electrical properties of iPSC-cardiomyocytes by enhancing Cx43 expression.

The therapeutic potential of induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs) is limited by immature functional features including low impulse propagation and reduced cell excitability. Key players regulating electrical activity are voltage-gated Na+ channels (Nav1.5) and gap junctions built from connexin-43 (Cx43). Here we tested the hypothesis that enhanced Cx43 expression increases intercellular coupling and influences excitability by modulating Nav1.5. Using transgenic approaches, Cx43 and Nav1.5 localization and cell coupling were studied by confocal imaging. Nav1.5 currents and action potentials (APs) were measured using the patch-clamp technique. Enhanced sarcolemmal Cx43 expression significantly improved intercellular coupling and accelerated dye transfer kinetics. Furthermore, Cx43 modulated Nav1.5 function leading to significantly higher current and enhanced AP upstroke velocities, thereby improving electrical activity as measured by microelectrode arrays. These findings suggest a mechanistic link between cell coupling and excitability controlled by Cx43 expression in iPSC-CMs. Therefore, we propose Cx43 as novel molecular target for improving electrical properties of iPSC-CMs to match the functional properties of native myocytes.

Synthesis and Characterization of Photoactivatable Doxycycline Analogues Bearing Two-Photon-Sensitive Photoremovable Groups Suitable for Light-Induced Gene Expression.

We report the synthesis and photolytic properties of caged 9-aminodoxycycline derivatives modified with 2-{4'-bis-[2-(2methoxyethoxy)ethyl]-4-nitrobiphenyl-3-yl}prop-1-oxy (EANBP) and PEG7-ylated (7-diethylamino-2-oxo-2H-chromen-4-yl)methyl (PEG7-DEACM) groups. 9-Aminodoxycycline is a tetracycline analogue capable of activating transcription through the inducible TetOn transgene expression system and can be regioselectively coupled to two-photon-sensitive photo-removable protecting groups by carbamoylation. The EANBP-based caged 9-aminodoxycycline showed complex photochemical reactions but did release 10 % of 9-aminodoxycycline. However, 9-(PEG7-DEACMamino)doxycycline exhibited excellent photolysis efficiency at 405 nm with quantitative release of 9-aminodoxycycline and a 0.21 uncaging quantum yield. Thanks to the good two-photon sensitivity of the DEACM chromophore, 9-aminodoxycycline release by two-photon photolysis is possible, with calculated action cross-sections of up to 4.0 GM at 740 nm. Therefore, 9-(PEG7-DEACMamino)doxycycline represents a very attractive tool for the development of a light-induced gene expression method in living cells.

A Dual SILAC Proteomic Labeling Strategy for Quantifying Constitutive and Cell-Cell Induced Protein Secretion.

Recent evidence suggests that the extracellular protein milieu is much more complex than previously assumed as various secretome analyses from different cell types described the release of hundreds to thousands of proteins. The extracellular function of many of these proteins has yet to be determined particularly in the context of three-dimensional tissues with abundant cell-cell contacts. Toward this goal, we developed a strategy of dual SILAC labeling astrocytic cultures for in silico exclusion of unlabeled proteins from serum or neurons used for stimulation. For constitutive secretion, this strategy allowed the precise quantification of the extra-to-intracellular protein ratio of more than 2000 identified proteins. Ratios covered 4 orders of magnitude indicating that the intracellular vs extracellular contributions of different proteins can be variable. Functionally, the secretome of labeled forebrain astrocytic cultures specifically changed within hours after adding unlabeled, "physiological" forebrain neurons. "Nonphysiological" cerebellar hindbrain neurons, however, elicited a different, highly repulsive secretory response. Our data also suggest a significant association of constitutive secretion with the classical secretion pathway and regulated secretion with unconventional pathways. We conclude that quantitative proteomics can help to elucidate general principles of cellular secretion and provide functional insight into the abundant extracellular presence of proteins.

Axon-carrying dendrites convey privileged synaptic input in hippocampal neurons.

Neuronal processing is classically conceptualized as dendritic input, somatic integration, and axonal output. The axon initial segment, the proposed site of action potential generation, usually emanates directly from the soma. However, we found that axons of hippocampal pyramidal cells frequently derive from a basal dendrite rather than from the soma. This morphology is particularly enriched in central CA1, the principal hippocampal output area. Multiphoton glutamate uncaging revealed that input onto the axon-carrying dendrites (AcDs) was more efficient in eliciting action potential output than input onto regular basal dendrites. First, synaptic input onto AcDs generates action potentials with lower activation thresholds compared with regular dendrites. Second, AcDs are intrinsically more excitable, generating dendritic spikes with higher probability and greater strength. Thus, axon-carrying dendrites constitute a privileged channel for excitatory synaptic input in a subset of cortical pyramidal cells.

Modification of purified proteins with photochemical protection compounds for high-resolution photoactivation of protein function in vitro and in vivo.

Specific and targeted photoactivation of protein function inside cells, tissues, or whole organisms can be achieved with reversible inhibition of proteins by conjugation with photolabile protection compounds ("caging"). In vitro caging of proteins is thought to cause sterical or functional hindrance of amino acid side chains that are important for protein activity. Following the modification, the caged protein is introduced into the biological system and high-resolution irradiation ensures specific release of protein function in the desired areas. Here, I describe the entire caging procedure and highlight a few of the caveats of photoactivation in living cells.

A molecular correlate of ocular dominance columns in the developing mammalian visual cortex.

Ocular dominance (OD) columns, alternating regions of left and right eye input in the visual cortex of higher mammals, have long been thought to develop from an initially intermingled state by an activity-dependent process. While indirect evidence points to potential alternative mechanisms based on molecular cues, direct proof for a molecular difference between left- and right eye columns is missing. Here, we show that heat shock protein 90 alpha (Hsp90α) is expressed in a clustered fashion in the developing cat visual cortex. Clusters of Hsp90α-positive cells are in register with OD columns of the ipsilateral eye as early as postnatal day 16, when OD columns have just appeared. Importantly, a periodic, clustered expression of Hsp90α is already present weeks before OD columns have started to form, suggesting that molecular differences between future left and right eye OD columns may contribute to the segregated termination of eye specific afferents in the developing visual cortex.

Systems-wide proteomic analysis in mammalian cells reveals conserved, functional protein turnover.

The turnover of each protein in the mammalian proteome is a functionally important characteristic. Here, we employed high-resolution mass spectrometry to quantify protein dynamics in nondividing mammalian cells. The ratio of externally supplied versus endogenous amino acids to de novo protein synthesis was about 17:1. Using subsaturating SILAC labeling, we obtained accurate turnover rates of 4106 proteins in HeLa and 3528 proteins in C2C12 cells. Comparison of these human and mouse cell lines revealed a highly significant turnover correlation of protein orthologs and thus high species conservation. Functionally, we observed statistically significant trends for the turnover of phosphoproteins and gene ontology categories that showed extensive covariation between mouse and human. Likewise, the members of some protein complexes, such as the proteasome, have highly similar turnover rates. The high species conservation and the low complex variances thus imply great regulatory fine-tuning of protein turnover.

Doxycycline-dependent photoactivated gene expression in eukaryotic systems.

High spatial and temporal resolution of conditional gene expression is typically difficult to achieve in whole tissues or organisms. We synthesized two reversibly inhibited, photoactivatable ('caged') doxycycline derivatives with different membrane permeabilities for precise spatial and temporal light-controlled activation of transgenes based on the 'Tet-on' system. After incubation with caged doxycycline or caged cyanodoxycycline, we induced gene expression by local irradiation with UV light or by two-photon uncaging in diverse biological systems, including mouse organotypic brain cultures, developing mouse embryos and Xenopus laevis tadpoles. The amount of UV light needed for induction was harmless as we detected no signs of toxicity. This method allows high-resolution conditional transgene expression at different spatial scales, ranging from single cells to entire complex organisms.

Silencing and un-silencing of tetracycline-controlled genes in neurons.

To identify the underlying reason for the controversial performance of tetracycline (Tet)-controlled regulated gene expression in mammalian neurons, we investigated each of the three components that comprise the Tet inducible systems, namely tetracyclines as inducers, tetracycline-transactivator (tTA) and reverse tTA (rtTA), and tTA-responsive promoters (P(tets)). We have discovered that stably integrated P(tet) becomes functionally silenced in the majority of neurons when it is inactive during development. P(tet) silencing can be avoided when it is either not integrated in the genome or stably-integrated with basal activity. Moreover, long-term, high transactivator levels in neurons can often overcome integration-induced P(tet) gene silencing, possibly by inducing promoter accessibility.

Bidirectional activity-dependent morphological plasticity in hippocampal neurons.

Dendritic spines on pyramidal neurons receive the vast majority of excitatory input and are considered electrobiochemical processing units, integrating and compartmentalizing synaptic input. Following synaptic plasticity, spines can undergo morphological plasticity, which possibly forms the structural basis for long-term changes in neuronal circuitry. Here, we demonstrate that spines on CA1 pyramidal neurons from organotypic slice cultures show bidirectional activity-dependent morphological plasticity. Using two-photon time-lapse microscopy, we observed that low-frequency stimulation induced NMDA receptor-dependent spine retractions, whereas theta burst stimulation led to the formation of new spines. Moreover, without stimulation the number of spine retractions was on the same order of magnitude as the stimulus-induced spine gain or loss. Finally, we found that the ability of neurons to eliminate spines in an activity-dependent manner decreased with developmental age. Taken together, our data show that hippocampal neurons can undergo bidirectional morphological plasticity; spines are formed and eliminated in an activity-dependent way.