How do plants feel the heat and survive?

Climate change is increasingly affecting the quality of life of organisms on Earth. More frequent, extreme, and lengthy heat waves are contributing to the sixth mass extinction of complex life forms in the Earth's history. From an anthropocentric point of view, global warming is a major threat to human health because it also compromises crop yields and food security. Thus, achieving agricultural productivity under climate change calls for closer examination of the molecular mechanisms of heat-stress resistance in model and crop plants. This requires a better understanding of the mechanisms by which plant cells can sense rising temperatures and establish effective molecular defenses, such as molecular chaperones and thermoprotective metabolites, as reviewed here, to survive extreme diurnal variations in temperature and seasonal heat waves.

Data-driven large-scale genomic analysis reveals an intricate phylogenetic and functional landscape in J-domain proteins.

The 70-kD heat shock protein (Hsp70) chaperone system is a central hub of the proteostasis network that helps maintain protein homeostasis in all organisms. The recruitment of Hsp70 to perform different and specific cellular functions is regulated by the J-domain protein (JDP) co-chaperone family carrying the small namesake J-domain, required to interact and drive the ATPase cycle of Hsp70s. Besides the J-domain, prokaryotic and eukaryotic JDPs display a staggering diversity in domain architecture, function, and cellular localization. Very little is known about the overall JDP family, despite their essential role in cellular proteostasis, development, and its link to a broad range of human diseases. In this work, we leverage the exponentially increasing number of JDP gene sequences identified across all kingdoms owing to the advancements in sequencing technology and provide a broad overview of the JDP repertoire. Using an automated classification scheme based on artificial neural networks (ANNs), we demonstrate that the sequences of J-domains carry sufficient discriminatory information to reliably recover the phylogeny, localization, and domain composition of the corresponding full-length JDP. By harnessing the interpretability of the ANNs, we find that many of the discriminatory sequence positions match residues that form the interaction interface between the J-domain and Hsp70. This reveals that key residues within the J-domains have coevolved with their obligatory Hsp70 partners to build chaperone circuits for specific functions in cells.

Paternal morphine exposure in rats reduces social play in adolescent male progeny without affecting drug-taking behavior in juvenile males or female offspring.

The ongoing opioid addiction crisis necessitates the identification of novel risk factors to improve prevention and treatment of opioid use disorder. Parental opioid exposure has recently emerged as a potential regulator of offspring vulnerability to opioid misuse, in addition to heritable genetic liability. An understudied aspect of this "missing heritability" is the developmental presentation of these cross-generational phenotypes. This is an especially relevant question in the context of inherited addiction-related phenotypes, given the prominent role of developmental processes in the etiology of psychiatric disorders. Paternal morphine self-administration was previously shown to alter the sensitivity to the reinforcing and antinociceptive properties of opioids in the next generation. Here, phenotyping was expanded to include the adolescent period, with a focus on endophenotypes related to opioid use disorders and pain. Paternal morphine exposure did not alter heroin or cocaine self-administration in male and female juvenile progeny. Further, baseline sensory reflexes related to pain were unaltered in morphine-sired adolescent rats of either sex. However, morphine-sired adolescent males exhibited a reduction in social play behavior. Our findings suggest that, in morphine-sired male offspring, paternal opioid exposure does not affect opioid intake during adolescence, suggesting that this phenotype does not emerge until later in life. Altered social behaviors in male morphine-sired adolescents indicate that the changes in drug-taking behavior in adults sired by morphine-exposed sires may be due to more complex factors not yet fully assessed.

On the evolution of chaperones and cochaperones and the expansion of proteomes across the Tree of Life.

Across the Tree of Life (ToL), the complexity of proteomes varies widely. Our systematic analysis depicts that from the simplest archaea to mammals, the total number of proteins per proteome expanded ∼200-fold. Individual proteins also became larger, and multidomain proteins expanded ∼50-fold. Apart from duplication and divergence of existing proteins, completely new proteins were born. Along the ToL, the number of different folds expanded ∼5-fold and fold combinations ∼20-fold. Proteins prone to misfolding and aggregation, such as repeat and beta-rich proteins, proliferated ∼600-fold and, accordingly, proteins predicted as aggregation-prone became 6-fold more frequent in mammalian compared with bacterial proteomes. To control the quality of these expanding proteomes, core chaperones, ranging from heat shock proteins 20 (HSP20s) that prevent aggregation to HSP60, HSP70, HSP90, and HSP100 acting as adenosine triphosphate (ATP)-fueled unfolding and refolding machines, also evolved. However, these core chaperones were already available in prokaryotes, and they comprise ∼0.3% of all genes from archaea to mammals. This challenge-roughly the same number of core chaperones supporting a massive expansion of proteomes-was met by 1) elevation of messenger RNA (mRNA) and protein abundances of the ancient generalist core chaperones in the cell, and 2) continuous emergence of new substrate-binding and nucleotide-exchange factor cochaperones that function cooperatively with core chaperones as a network.

Early life adversity promotes resilience to opioid addiction-related phenotypes in male rats and sex-specific transcriptional changes.

Experiencing some early life adversity can have an "inoculating" effect that promotes resilience in adulthood. However, the mechanisms underlying stress inoculation are unknown, and animal models are lacking. Here we used the limited bedding and nesting (LBN) model of adversity to evaluate stress inoculation of addiction-related phenotypes. In LBN, pups from postnatal days 2 to 9 and their dams were exposed to a low-resource environment. In adulthood, they were tested for addiction-like phenotypes and compared to rats raised in standard housing conditions. High levels of impulsivity are associated with substance abuse, but in males, LBN reduced impulsive choice compared to controls. LBN males also self-administered less morphine and had a lower breakpoint on a progressive ratio reinforcement schedule than controls. These effects of LBN on addiction-related behaviors were not found in females. Because the nucleus accumbens (NAc) mediates these behaviors, we tested whether LBN altered NAc physiology in drug-naïve and morphine-exposed rats. LBN reduced the frequency of spontaneous excitatory postsynaptic currents in males, but a similar effect was not observed in females. Only in males did LBN prevent a morphine-induced increase in the AMPA/NMDA ratio. RNA sequencing was performed to delineate the molecular signature in the NAc associated with LBN-derived phenotypes. LBN produced sex-specific changes in transcription, including in genes related to glutamate transmission. Collectively, these studies reveal that LBN causes a male-specific stress inoculation effect against addiction-related phenotypes. Identifying factors that promote resilience to addiction may reveal novel treatment options for patients.

Cocaine-Induced Changes in Sperm Cdkn1a Methylation Are Associated with Cocaine Resistance in Male Offspring.

Paternal environmental perturbations can influence the physiology and behavior of offspring. For example, our previous work showed reduced cocaine reinforcement in male, but not female, progeny of rat sires that self-administered cocaine. The information transfer from sire to progeny may occur through epigenetic marks in sperm, encompassing alterations in small noncoding RNAs, including microRNAs (miRNAs) and/or DNA methylation. Here, no reliable changes in miRNAs in the sperm of cocaine- relative to saline-experienced sires were identified. In contrast, 272 differentially methylated regions were observed in sperm between these groups. Two hypomethylated promoter regions in the sperm of cocaine-experienced rats were upstream of cyclin-dependent kinase inhibitor 1a (Cdkn1a). Cdkn1a mRNA also was selectively increased in the NAc of cocaine-sired male (but not female) offspring. Cocaine self-administration also enhanced Cdkn1a expression in the accumbens of cocaine-sired rats. These results suggest that changes in Cdkn1a may play a role in the reduced cocaine reinforcing efficacy observed in cocaine-sired male rats. Introducing a 90 d delay between sire self-administration and breeding reversed both cocaine resistance and the increase in accumbens Cdkn1a mRNA in male offspring, indicating that cocaine-induced epigenetic modifications are eliminated with sperm turnover. Collectively, our results indicate that cocaine self-administration produces hypomethylation of Cdkn1a in sperm and a selective increase in the expression of this gene in the NAc of male offspring, which is associated with blunted cocaine reinforcement.SIGNIFICANCE STATEMENT The relatively new field of transgenerational epigenetics explores the effects of environmental perturbations on offspring behavior and physiology. Our prior work in rats indicated that male, but not female, progeny of sires that self-administered cocaine displayed reduced cocaine reinforcement. The information transfer from sire to progeny may occur through heritable epigenetic marks in sperm, including DNA methylation. The present findings revealed two hypomethylated promoter regions upstream of the Cdkn1a gene in sire sperm. Remarkably, Cdkn1a expression was selectively decreased in offspring NAc, a brain region that regulates cocaine reinforcement.

Paternal nicotine taking elicits heritable sex-specific phenotypes that are mediated by hippocampal Satb2.

Nicotine intake, whether through tobacco smoking or e-cigarettes, remains a global health concern. An emerging preclinical literature indicates that parental nicotine exposure produces behavioral, physiological, and molecular changes in subsequent generations. However, the heritable effects of voluntary parental nicotine taking are unknown. Here, we show increased acquisition of nicotine taking in male and female offspring of sires that self-administered nicotine. In contrast, self-administration of sucrose and cocaine were unaltered in male and female offspring suggesting that the intergenerational effects of paternal nicotine taking may be reinforcer specific. Further characterization revealed memory deficits and increased anxiety-like behaviors in drug-naive male, but not female, offspring of nicotine-experienced sires. Using an unbiased, genome-wide approach, we discovered that these phenotypes were associated with decreased expression of Satb2, a transcription factor known to play important roles in synaptic plasticity and memory formation, in the hippocampus of nicotine-sired male offspring. This effect was sex-specific as no changes in Satb2 expression were found in nicotine-sired female offspring. Finally, increasing Satb2 levels in the hippocampus prevented the escalation of nicotine intake and rescued the memory deficits associated with paternal nicotine taking in male offspring. Collectively, these findings indicate that paternal nicotine taking produces heritable sex-specific molecular changes that promote addiction-like phenotypes and memory impairments in male offspring.

microRNA expression levels in the nucleus accumbens correlate with morphine-taking but not morphine-seeking behaviour in male rats.

A powerful motivation to seek opioids remains after drug cessation and intensifies during extended periods of abstinence. Unfortunately, biomarkers associated with continued drug seeking have not been described. Moreover, previous studies have focused on the effects of early abstinence with little exploration into the long-term drug-induced mechanisms that occur after extended abstinence. Here we demonstrated that 30 days (D) of forced abstinence results in a time-dependent increase in morphine seeking in a rat model of morphine self-administration (SA). We measured expression of known drug-responsive microRNAs (miRNAs) in the nucleus accumbens, an area critical for reward-related plasticity, during early or late abstinence in animals that underwent either a cue-induced relapse test or no relapse test. miRNAs are small noncoding RNAs that represent suitable biomarker candidates due to their long-lasting nature. mir-32-5p levels during early abstinence negatively correlated with active lever pressing in both cue-exposed and cue-naïve animals. mir-1298-5p positively correlated with drug SA history after a relapse test during late abstinence. When animals underwent acute abstinence with no relapse test, mir-1298-5p correlated with drug infusions and active lever pressing during SA. In late abstinence with no relapse test, mir-137-3p negatively correlated with drug infusions. Regulation of mir-32-5p target genes and significant correlation of target gene mRNA with mir-32-5p was observed after abstinence. These results indicate that lasting regulation of miRNA expression is associated with drug intake following morphine SA. In addition, we conclude that the miRNA profile undergoes regulation from early to late abstinence and miRNA expression may indicate past drug history.

H3.3 Barcoding of Nucleus Accumbens Transcriptional Activity Identifies Novel Molecular Cascades Associated with Cocaine Self-administration in Mice.

Although numerous epigenetic modifications have been associated with addiction, little work has explored the turnover of histone variants. Uniquely, the H3.3 variant incorporates stably and preferentially into chromatin independently of DNA replication at active sites of transcription and transcription factor binding. Thus, genomic regions associated with H3.3-containing nucleosomes are particularly likely to be involved in plasticity, such as following repeated cocaine exposure. A recently developed mouse line expressing a neuron-specific hemagglutinin (HA)-tagged H3.3 protein was used to track transcriptionally active sites cumulatively across 19 d of cocaine self-administration. RNA-seq and H3.3-HA ChIP-seq analyses were performed on NAcc tissue collected following cocaine or food self-administration in male mice. RNA sequencing revealed five genes upregulated in cocaine relative to food self-administering mice: Fosb, Npas4, Vgf, Nptx2, and Pmepa1, which reflect known and novel cocaine plasticity-associated genes. Subsequent ChIP-seq analysis confirmed increased H3.3 aggregation at four of these five loci, thus validating H3.3 insertion as a marker of enhanced cocaine-induced transcription. Further motif recognition analysis of the ChIP-seq data showed that cocaine-associated differential H3.3 accumulation correlated with the presence of several transcription factor binding motifs, including RBPJ1, EGR1, and SOX4, suggesting that these are potentially important regulators of molecular cascades associated with cocaine-induced neuronal plasticity. Additional ontological analysis revealed differential H3.3 accumulation mainly near genes involved in neuronal differentiation and dendrite formation. These results establish the H3.3-HA transgenic mouse line as a compelling molecular barcoding tool to identify the cumulative effects of long-term environmental perturbations, such as exposure to drugs of abuse.SIGNIFICANCE STATEMENT Histone H3.3 is a core histone variant that is stably incorporated at active sites of transcription. We used a tagged version of H3.3 expressed exclusively in neurons to delineate active transcription sites following extended cocaine self-administration in mice. This approach revealed the cumulative list of genes expressed in response to cocaine taking over the course of several weeks. We combined this technique with RNA sequencing of tissue collected from the same animals 24 h after the last cocaine exposure. Comparing these datasets provided a full picture of genes that respond to chronic cocaine exposure in NAcc neurons. These studies revealed novel transcription factors that are likely involved in cocaine-induced plasticity and addiction-like behaviors.

Rolipram treatment during consolidation ameliorates long-term object location memory in aged male mice.

Normal aging is accompanied by cognitive and memory impairments that negatively impact quality of life for the growing elderly population. Hippocampal function is most vulnerable to the deleterious effects of aging, and deficits in hippocampus-dependent memories are common amongst aged individuals. Moreover, signaling networks such as the cAMP/PKA/CREB pathway, which are critical for memory consolidation, are dampened in healthy aged subjects. Phosphodiesterase (PDE) enzymes that break down cAMP are also affected by aging, and increased break down of cAMP by PDEs may contribute to reduced activity of the cAMP/PKA/CREB signaling network in the brain of aged individuals. Here, we report that the PDE4 inhibitor rolipram administered during consolidation of hippocampus-dependent object location memory improves aged-related spatial memory deficits in aged mice.

Impaired cocaine-induced behavioral plasticity in the male offspring of cocaine-experienced sires.

Our previous work indicated that male, but not female, offspring of cocaine-experienced sires display blunted cocaine self-administration. We extended this line of investigation to examine behavioral sensitization, a commonly used model of cocaine-induced behavioral and neuronal plasticity. Results indicated that male, but not female, offspring of cocaine-taking sires showed deficits in the ability of repeated systemic cocaine injections to induce augmented locomotor activity. The reduced cocaine sensitization phenotype in male progeny was associated with changes in histone post-translational modifications, epigenetic processes that regulate gene expression by controlling the accessibility of genes to transcriptional machinery, in the nucleus accumbens of first-generation male progeny. Thus, five histone post-translational modifications were significantly altered in the male progeny of cocaine-exposed sires. In contrast, self-administration of nicotine was unaltered in male and female offspring suggesting that the intergenerational effects of paternal cocaine taking may be drug-specific. Interestingly, the reduced sensitivity to cocaine previously observed in the male offspring of cocaine-taking sires dissipated in the grand-offspring. Both male and female grand-progeny of cocaine-exposed sires showed unaltered cocaine-induced behavioral sensitization and cocaine self-administration. Taken together, these findings indicate that paternal cocaine taking produces changes in multiple cocaine addiction-related behaviors in male progeny, which do not persist beyond the first generation of offspring. Moreover, the altered sensitivity to cocaine in first-generation male progeny of cocaine-sired male offspring was associated with epigenetic modifications in the nucleus accumbens, a nucleus that plays a critical role in cocaine-associated behavioral plasticity.

Is 5 days of oral fluoroquinolone enough for acute uncomplicated pyelonephritis? The DTP randomized trial.

The treatment duration of acute uncomplicated pyelonephritis (AUP) is still under debate. As shortening treatment duration could be a means to reduce antimicrobial resistance, we aimed to establish whether 5 days of antibiotic treatment is non-inferior to 10 days in patients with AUP. We performed an open-label prospective randomized trial comparing 5 days to 10 days of fluoroquinolone treatment for AUP. The inclusion criteria were: female patients aged ≥18 years with clinical signs of urinary tract infection, fever >38 °C, and positive urinalysis. Patients were randomized to either 5 or 10 days of fluoroquinolone treatment. Outcome was cure at day 10 and day 30 after the end of treatment. One hundred patients were randomized and 12 were excluded after randomization. The mean ± standard deviation (SD) age was 31.8 ± 11 years old and the mean ± SD temperature was 38.6 ± 0.7 °C. The main bacterium involved was Escherichia coli (n = 86; 97.7%) and 3 (3.4%) patients had a positive blood culture. In the post-hoc analysis, clinical cure 10 days after the end of the treatment was 28/30 (93.3%) in the 5-day arm and 36/38 (94.7%) in the 10-day arm (p = 1.00). At day 30, the clinical cure rate was 23/23 (100%) in the 5-day arm and 20/20 (100%) in the 10-day arm (p = 1.00). The microbiological cure rate was 20/23 (87.0%) in the 5-day arm and 16/20 (80.0%) in the 10-day arm (p = 1.00). The efficacy of 5 days of fluoroquinolone treatment does not seem different from 10 days of treatment for AUP.

How data analysis affects power, reproducibility and biological insight of RNA-seq studies in complex datasets.

The sequencing of the full transcriptome (RNA-seq) has become the preferred choice for the measurement of genome-wide gene expression. Despite its widespread use, challenges remain in RNA-seq data analysis. One often-overlooked aspect is normalization. Despite the fact that a variety of factors or 'batch effects' can contribute unwanted variation to the data, commonly used RNA-seq normalization methods only correct for sequencing depth. The study of gene expression is particularly problematic when it is influenced simultaneously by a variety of biological factors in addition to the one of interest. Using examples from experimental neuroscience, we show that batch effects can dominate the signal of interest; and that the choice of normalization method affects the power and reproducibility of the results. While commonly used global normalization methods are not able to adequately normalize the data, more recently developed RNA-seq normalization can. We focus on one particular method, RUVSeq and show that it is able to increase power and biological insight of the results. Finally, we provide a tutorial outlining the implementation of RUVSeq normalization that is applicable to a broad range of studies as well as meta-analysis of publicly available data.

Syntaxin 5-dependent retrograde transport to the trans-Golgi network is required for adeno-associated virus transduction.

UNLABELLED: Intracellular transport of recombinant adeno-associated virus (AAV) is still incompletely understood. In particular, the trafficking steps preceding the release of incoming AAV particles from the endosomal system into the cytoplasm, allowing subsequent nuclear import and the initiation of gene expression, remain to be elucidated fully. Others and we previously showed that a significant proportion of viral particles are transported to the Golgi apparatus and that Golgi apparatus disruption caused by the drug brefeldin A efficiently blocks AAV serotype 2 (AAV2) transduction. However, because brefeldin A is known to exert pleiotropic effects on the entire endosomal system, the functional relevance of transport to the Golgi apparatus for AAV transduction remains to be established definitively. Here, we show that AAV2 trafficking toward the trans-Golgi network (TGN) and the Golgi apparatus correlates with transduction efficiency and relies on a nonclassical retrograde transport pathway that is independent of the retromer complex, late endosomes, and recycling endosomes. AAV2 transduction is unaffected by the knockdown of syntaxins 6 and 16, which are two major effectors in the retrograde transport of both exogenous and endogenous cargo. On the other hand, inhibition of syntaxin 5 function by small interfering RNA silencing or treatment with cyclized Retro-2 strongly decreases AAV2 transduction and transport to the Golgi apparatus. This inhibition of transduction is observed with several AAV serotypes and a number of primary and immortalized cells. Together, our data strongly suggest that syntaxin 5-mediated retrograde transport to the Golgi apparatus is a broadly conserved feature of AAV trafficking that appears to be independent of the identity of the receptors used for viral attachment. IMPORTANCE: Gene therapy constitutes a promising approach for the treatment of life-threatening conditions refractory to any other form of remedy. Adeno-associated virus (AAV) vectors are currently being evaluated for the treatment of diseases such as Duchenne muscular dystrophy, hemophilia, heart failure, Parkinson's disease, and others. Despite their promise as gene delivery vehicles, a better understanding of the biology of AAV-based vectors is necessary to improve further their efficacy. AAV vectors must reach the nucleus in order to deliver their genome, and their intracellular transport is not fully understood. Here, we dissect an important step of the intracellular journey of AAV by showing that retrograde transport of capsids to the trans-Golgi network is necessary for gene delivery. We show that the AAV trafficking route differs from that of known Golgi apparatus-targeted cargos, and we raise the possibility that this nonclassical pathway is shared by most AAV variants, regardless of their attachment receptors.

Survey of 243 ART patients having made a final disposition decision about their surplus cryopreserved embryos: the crucial role of symbolic embryo representation.

STUDY QUESTION: In couples who have chosen and confirmed the fate of surplus frozen embryos, which factors influence their decision, with a special emphasis on their symbolic representation of the embryo(s)? SUMMARY ANSWER: Embryo representation and gamete donation use significantly influence the fate of surplus cryopreserved embryos. WHAT IS KNOWN ALREADY: Previous studies report difficulties for couples to decide whether or not to continue storing their frozen embryo(s) and different factors have been already highlighted which influence their decision, including embryo conceptualization, information and support provided by the medical institution, quality of embryo(s) and life events. Little is known, however, about couples who definitely decided to stop their parental project and finalized the process of decision-making about the fate of their cryopreserved embryo(s). STUDY DESIGN, SIZE, DURATION: This prospective study was conducted over a period of 3 years (2007-2010) and included IVF/ICSI patients with surplus frozen embryos, who made a final embryo disposition decision. Among the 280 eligible IVF/ICSI patients, 247 agreed to participate in the study. According to the available options, 91 persons chose to 'stop cryopreservation', 77 chose donation to 'research' and 48 'embryo donation' to infertile couples. Furthermore, 31 participants who chose embryo donation for a parental project were refused by the center as not compatible with their mandatory medical conditions. Among them, 27 participants then selected donation to research as a new option and were included in a fourth group: 'donation to research after Refusal of Embryo Donation for parental project' or 'research-RED' (n = 27). Four participants chose 'stop cryopreservation', however, given the small number of subjects this latter group was not included in the analysis. In all, 243 participants who made a final choice concerning the fate of their cryopreserved embryos were included in this study. PARTICIPANTS/MATERIALS, SETTING, METHODS: Participants were sent a letter of invitation to a semi-structured interview of 30 min with a psychologist. Interviews were conducted separately for each partner, including a questionnaire with a common part and a specific part, according to the chosen option, and allowing a quantitative evaluation. A multivariate logistic regression model was used to assess the link between their embryo representation and their decision about their embryos' fate. MAIN RESULTS AND THE ROLE OF CHANCE: After adjustment for age, gender, gamete donation, number of children and the different embryo representations, a choice to 'stop cryopreservation' is more frequent if the embryo is represented as a child [odds ratio (OR) adjusted = 3.29, 95% confidence interval (CI) = 1.62-6.66], P = 0.0009. Representing the embryo as a project prompts patients to choose 'donation to research' [OR adjusted = 3.76, 95% CI = 1.56-9.06], P = 0.0032. Respondents are more likely to choose 'embryo donation' if they represent the embryo as a potential person [OR adjusted = 3.77, 95% CI = 1.45-9.80], P = 0.0064. Furthermore, patients who benefited from gamete donation are ∼10 times more likely to donate their embryos to another couple [OR adjusted = 10.62, 95% CI = 3.99-28.30], P < 0.0001. For more than half the participants (57%) the decision-making was easy, however, deciding to stop cryopreservation was significantly more difficult than choosing research or embryo donation (P < 0.0001). LIMITATIONS, REASONS FOR CAUTION: Socio-economic status, moral and religious affiliations are known to influence the choice of couples but analyzing these factors was not an aim of the present study. WIDER IMPLICATIONS OF THE FINDINGS: When couples definitely decide to stop their parental project, the embryo symbolic representation remains the main factor that influences the fate of their frozen embryo(s). Moreover, this representation can evolve when influenced by external events and information provided. In order to support patients who are making this difficult decision, it could be helpful to explore this symbolic representation early in the IVF/ICSI procedure, before surplus embryo freezing, as a new tool enhancing the accuracy of counseling. STUDY FUNDING/COMPETING INTERESTS: this study was supported by a grant from the 'Agence de la biomedicine (ABM)', the national regulatory ART agency, under the authority of the French Ministry of Health. The authors have no conflict of interest to declare.

Contextual fear conditioning induces differential alternative splicing.

The process of memory consolidation requires transcription and translation to form long-term memories. Significant effort has been dedicated to understanding changes in hippocampal gene expression after contextual fear conditioning. However, alternative splicing by differential transcript regulation during this time period has received less attention. Here, we use RNA-seq to determine exon-level changes in expression after contextual fear conditioning and retrieval. Our work reveals that a short variant of Homer1, Ania-3, is regulated by contextual fear conditioning. The ribosome biogenesis regulator Las1l, small nucleolar RNA Snord14e, and the RNA-binding protein Rbm3 also change specific transcript usage after fear conditioning. The changes in Ania-3 and Las1l are specific to either the new context or the context-shock association, while the changes in Rbm3 occur after context or shock only. Our analysis revealed novel transcript regulation of previously undetected changes after learning, revealing the importance of high throughput sequencing approaches in the study of gene expression changes after learning.

Enhanced anxiety in the male offspring of sires that self-administered cocaine.

We previously showed that paternal cocaine exposure reduced the reinforcing efficacy of cocaine in male offspring. Here, we sought to determine whether paternal cocaine experience could also influence anxiety levels in offspring. Male rats were allowed to self-administer cocaine (controls received saline passively) for 60 days and then were bred with naïve females. Measures of anxiety and cocaine-induced anxiogenic effects were assessed in the adult offspring. Cocaine-sired male offspring exhibited increased anxiety-like behaviors, as measured using the novelty-induced hypophagia and defensive burying tasks, relative to saline-sired males. In contrast, sire cocaine experience had no effect on anxiety-like behaviors in female offspring. When challenged with an anxiogenic (but not anorectic) dose of cocaine (2.5 mg/kg, i.p.), anxiety-like behavior was enhanced in all animals to an equal degree regardless of sire drug experience. Since anxiety and depression are often co-morbid, we also assessed measures of depressive-like behavior. Sire cocaine experience had no effect on depression-like behaviors, as measured by the forced swim task, among male offspring. In a separate group of naïve littermates, select neuronal correlates of anxiety were measured. Male offspring of cocaine-experienced sires showed increased mRNA and protein expression of corticotropin-releasing factor receptor 2 in the hippocampus. Together, these results indicate that cocaine-experienced sires produce male progeny that have increased baseline anxiety, which is unaltered by subsequent cocaine exposure.

Memory acquisition and retrieval impact different epigenetic processes that regulate gene expression.

BACKGROUND: A fundamental question in neuroscience is how memories are stored and retrieved in the brain. Long-term memory formation requires transcription, translation and epigenetic processes that control gene expression. Thus, characterizing genome-wide the transcriptional changes that occur after memory acquisition and retrieval is of broad interest and importance. Genome-wide technologies are commonly used to interrogate transcriptional changes in discovery-based approaches. Their ability to increase scientific insight beyond traditional candidate gene approaches, however, is usually hindered by batch effects and other sources of unwanted variation, which are particularly hard to control in the study of brain and behavior. RESULTS: We examined genome-wide gene expression after contextual conditioning in the mouse hippocampus, a brain region essential for learning and memory, at all the time-points in which inhibiting transcription has been shown to impair memory formation. We show that most of the variance in gene expression is not due to conditioning and that by removing unwanted variance through additional normalization we are able provide novel biological insights. In particular, we show that genes downregulated by memory acquisition and retrieval impact different functions: chromatin assembly and RNA processing, respectively. Levels of histone 2A variant H2AB are reduced only following acquisition, a finding we confirmed using quantitative proteomics. On the other hand, splicing factor Rbfox1 and NMDA receptor-dependent microRNA miR-219 are only downregulated after retrieval, accompanied by an increase in protein levels of miR-219 target CAMKIIγ. CONCLUSIONS: We provide a thorough characterization of coding and non-coding gene expression during long-term memory formation. We demonstrate that unwanted variance dominates the signal in transcriptional studies of learning and memory and introduce the removal of unwanted variance through normalization as a necessary step for the analysis of genome-wide transcriptional studies in the context of brain and behavior. We show for the first time that histone variants are downregulated after memory acquisition, and splicing factors and microRNAs after memory retrieval. Our results provide mechanistic insights into the molecular basis of cognition by highlighting the differential involvement of epigenetic mechanisms, such as histone variants and post-transcriptional RNA regulation, after acquisition and retrieval of memory.

Autorepression of yeast Hsp70 cochaperones by intramolecular interactions involving their J-domains.

The 70 kDa heat shock protein (Hsp70) chaperones control protein homeostasis in all ATP-containing cellular compartments. J-domain proteins (JDPs) coevolved with Hsp70s to trigger ATP hydrolysis and catalytically upload various substrate polypeptides in need to be structurally modified by the chaperone. Here, we measured the protein disaggregation and refolding activities of the main yeast cytosolic Hsp70, Ssa1, in the presence of its most abundant JDPs, Sis1 and Ydj1, and two swap mutants, in which the J-domains have been interchanged. The observed differences by which the four constructs differently cooperate with Ssa1 and cooperate with each other, as well as their observed intrinsic ability to bind misfolded substrates and trigger Ssa1's ATPase, indicate the presence of yet uncharacterized intramolecular dynamic interactions between the J-domains and the remaining C-terminal segments of these proteins. Taken together, the data suggest an autoregulatory role to these intramolecular interactions within both type A and B JDPs, which might have evolved to reduce energy-costly ATPase cycles by the Ssa1-4 chaperones that are the most abundant Hsp70s in the yeast cytosol.

Early resource scarcity causes cortical astrocyte enlargement and sex-specific changes in the orbitofrontal cortex transcriptome in adult rats.

Astrocyte morphology affects function, including the regulation of glutamatergic signaling. This morphology changes dynamically in response to the environment. However, how early life manipulations alter adult cortical astrocyte morphology is underexplored. Our lab uses brief postnatal resource scarcity, the limited bedding and nesting (LBN) manipulation, in rats. We previously found that LBN augments maternal behaviors and promotes later resilience to adult addiction-related behaviors, reducing impulsivity, risky decision-making, and morphine self-administration. These behaviors rely on glutamatergic transmission in the medial orbitofrontal (mOFC) and medial prefrontal (mPFC) cortex. Here we tested whether LBN changed astrocyte morphology in the mOFC and mPFC of adult rats using a novel viral approach that, unlike traditional markers, fully labels astrocytes. Prior exposure to LBN causes an increase in the surface area and volume of astrocytes in the mOFC and mPFC of adult males and females relative to control-raised rats. We next used bulk RNA sequencing of OFC tissue to assess transcriptional changes that could increase astrocyte size in LBN rats. LBN caused mainly sex-specific changes in differentially expressed genes. Pathway analysis revealed that OFC glutamatergic signaling is altered by LBN in males and females, but the gene changes in that pathway differed across sex. This may represent a convergent sex difference where glutamatergic signaling, which affects astrocyte morphology, is altered by LBN via sex-specific mechanisms. Collectively, these studies highlight that astrocytes may be an important cell type that mediates the effect of early resource scarcity on adult brain function.