Inter- and transgenerational inheritance of behavioral phenotypes

Adult animal behaviors are determined by complex and dynamic changes in gene expression in different brain regions and are influenced by life experiences and environmental exposures. These stimuli affect gene expression through intricate mechanisms of regulation that largely implicate epigenetic factors, such as, DNA methylation, histone post-translational modifications, and non-coding RNAs (ncRNAs). Through these molecular pathways, some of the behavioral phenotypes associated with life experiences can be stably transmitted to descendants, sometimes across several generations. Rodent studies indicate that parental stressful and traumatic experiences can lead to behavioral despair, risk-taking behaviors, altered sociability and atypical responses to stressful stimuli in the offspring, whereas parental environmental enrichment has been associated with improved cognition and stress resilience in the offspring. Similar observations have been made in humans; children and grandchildren of genocide survivors show increased psychopathology and emotional disturbances. At the molecular level, changes in germline ncRNAs have been identified as likely vectors of transmission in rodents. The mechanisms linking behavioral stimuli to the germline, and factors responsible for these changes and their persistence across generations remain, however, largely unidentified

Neural functions of calcineurin in synaptic plasticity and memory

Major brain functions depend on neuronal processes that favor the plasticity of neuronal circuits while at the same time maintaining their stability. The mechanisms that regulate brain plasticity are complex and engage multiple cascades of molecular components that modulate synaptic efficacy. Protein kinases (PKs) and phosphatases (PPs) are among the most important of these components that act as positive and negative regulators of neuronal signaling and plasticity, respectively. In these cascades, the PP protein phosphatase 2B or calcineurin (CaN) is of particular interest because it is the only Ca(2+)-activated PP in the brain and a major regulator of key proteins essential for synaptic transmission and neuronal excitability. This review describes the primary properties of CaN and illustrates its functions and modes of action by focusing on several representative targets, in particular glutamate receptors, striatal enriched protein phosphatase (STEP), and neuromodulin (GAP43), and their functional significance for synaptic plasticity and memory

High Efficiency RNA Extraction From Sperm Cells Using Guanidinium Thiocyanate Supplemented With Tris(2-Carboxyethyl)Phosphine

The extraction of high-quality ribonucleic acid (RNA) from tissues and cells is a key step in many biological assays. Guanidinium thiocyanate-phenol-chloroform (AGPC) is a widely used and efficient method to obtain pure RNA from most tissues and cells. However, it is not efficient with some cells like sperm cells because they are resistant to chaotropic lysis solutions containing guanidinium thiocyanate such as Buffer RLT+ and Trizol. Here, we show that disulfide bonds are responsible for the chemical resistance of sperm cells to RNA extraction reagents. We show that while β-mercaptoethanol (βME) can increase sperm lysis in Buffer RLT+, it has no effect in Trizol and leaves sperm cells intact. We measured the reduction of disulfide bonds in 2,2′-dithiodipyridine (DTDP) and observed that βME has a pH-dependent activity in chaotropic solutions, suggesting that pH is a limiting factor. We identified tris(2-carboxyethyl)phosphine (TCEP) as an efficient lysis enhancer of AGPC solutions that can retain reducing activity even at acidic pH. Trizol supplemented with TCEP allows the complete and rapid lysis of sperm cells, increasing RNA yield by 100-fold and resulting in RNA with optimal quality for reverse transcription and polymerase chain reaction. Our findings highlight the importance of efficient cell lysis and extraction of various macromolecules for bulk and single-cell assays, and can be applied to other lysis-resistant cells and vesicles, thereby optimizing the amount of required starting material and animals

Inheritable Effect of Unpredictable Maternal Separation on Behavioral Responses in Mice

The long-term impact of early stress on behavior and emotions is well documented in humans, and can be modeled in experimental animals. In mice, maternal separation during early postnatal development induces poor and disorganized maternal care, and results in behavioral deficits that persist through adulthood. Here, we examined the long-term effect of unpredictable maternal separation combined with maternal stress on behavior and its transmissibility. We report that unpredictable maternal separation from birth to postnatal day 14 in C57Bl/6J mice has mild behavioral effects in the animals when adult, but that its combination with maternal stress exacerbates this effect. Further, the behavioral deficits are transmitted to the following generation through females, an effect that is independent of maternal care and is not affected by cross-fostering. The combined manipulation does not alter basic components of the hypothalamic–pituitary–adrenal axis but decreases the expression of the corticotropin releasing factor receptor 2 (CRFR2) in several nuclei of the amygdala and the hypothalamus in the brain of maternal-separated females. These results suggest a non-genomic mode of transmission of the impact of early stress in mice

Protein Phosphatase 1 (PP1) Is a Post-Translational Regulator of the Mammalian Circadian Clock

Circadian clocks coordinate the timing of important biological processes. Interconnected transcriptional and post-translational feedback loops based on a set of clock genes generate and maintain these rhythms with a period of about 24 hours. Many clock proteins undergo circadian cycles of post-translational modifications. Among these modifications, protein phosphorylation plays an important role in regulating activity, stability and intracellular localization of clock components. Several protein kinases were characterized as regulators of the circadian clock. However, the function of protein phosphatases, which balance phosphorylation events, in the mammalian clock mechanism is less well understood. Here, we identify protein phosphatase 1 (PP1) as regulator of period and light-induced resetting of the mammalian circadian clock. Down-regulation of PP1 activity in cells by RNA interference and in vivo by expression of a specific inhibitor in the brain of mice tended to lengthen circadian period. Moreover, reduction of PP1 activity in the brain altered light-mediated clock resetting behavior in mice, enhancing the phase shifts in either direction. At the molecular level, diminished PP1 activity increased nuclear accumulation of the clock component PER2 in neurons. Hence, PP1, may reduce PER2 phosphorylation thereby influencing nuclear localization of this protein. This may at least partially influence period and phase shifting properties of the mammalian circadian clock

Single-Cell Multiomics Techniques: From Conception to Applications

Recent advances in methods for single-cell analyses and barcoding strategies have led to considerable progress in research. The development of multiplexed assays offers the possibility to conduct parallel analyses of multiple factors and processes for comprehensive characterization of cellular and molecular states in health and disease. These technologies have expanded extremely rapidly in the past years and constantly evolve and provide better specificity, precision and resolution. This review summarizes recent progress in single-cell multiomics approaches, and focuses, in particular, on the most innovative techniques that integrate genome, epigenome and transcriptome profiling. It describes the methodologies, discusses their advantages and limitations, and explains how they have been applied to studies on cell heterogeneity and differentiation, and epigenetic reprogramming. Keywords: chromatin accessibility; epigenomics; genomics; multiomics; single-cell; transcriptomic

Influence of Early Stress on Social Abilities and Serotonergic Functions across Generations in Mice

Exposure to adverse environments during early development is a known risk factor for several psychiatric conditions including antisocial behavior and personality disorders. Here, we induced social anxiety and altered social recognition memory in adult mice using unpredictable maternal separation and maternal stress during early postnatal life. We show that these social defects are not only pronounced in the animals directly subjected to stress, but are also transmitted to their offspring across two generations. The defects are associated with impaired serotonergic signaling, in particular, reduced 5HT1A receptor expression in the dorsal raphe nucleus, and increased serotonin level in a dorsal raphe projection area. These findings underscore the susceptibility of social behaviors and serotonergic pathways to early stress, and the persistence of their perturbation across generations

Prion protein and Aβ-related synaptic toxicity impairment

Alzheimer's disease (AD), the most common neurodegenerative disorder, goes along with extracellular amyloid-β (Aβ) deposits. The cognitive decline observed during AD progression correlates with damaged spines, dendrites and synapses in hippocampus and cortex. Numerous studies have shown that Aβ oligomers, both synthetic and derived from cultures and AD brains, potently impair synaptic structure and functions. The cellular prion protein (PrPC) was proposed to mediate this effect. We report that ablation or overexpression of PrPC had no effect on the impairment of hippocampal synaptic plasticity in a transgenic model of AD. These findings challenge the role of PrPC as a mediator of Aβ toxicity

Dynamics of transcriptional programs and chromatin accessibility in mouse spermatogonial cells from early postnatal to adult life

In mammals, spermatogonial cells (SCs) are undifferentiated male germ cells in testis quiescent until birth that self-renew and differentiate to produce spermatogenic cells and functional sperm across life. The transcriptome of SCs is highly dynamic and timely regulated during postnatal development. We examined if such dynamics involves changes in chromatin organization by profiling the transcriptome and chromatin accessibility in SCs from early postnatal stages to adulthood in mice using RNA-seq and ATAC-seq. By integrating transcriptomic and epigenomic features, we show that SCs undergo massive chromatin remodeling during postnatal development that correlates with distinct gene expression profiles and transcription factors (TF) motif enrichment. We identify genomic regions with significantly different chromatin accessibility in adult SCs that are marked by histone modifications associated with enhancers and promoters. Some of the regions with increased accessibility correspond to transposable element subtypes enriched in multiple TFs motifs and close to differentially expressed genes. Our results underscore the dynamics of chromatin organization in developing germ cells and the involvement of the regulatory genome

Using mice from different breeding sites fails to improve replicability of results from single-laboratory studies

Theoretical and empirical evidence indicates that low external validity due to rigorous standardization of study populations is a cause of poor replicability in animal research. Here we report a multi-laboratory study aimed at investigating whether heterogenization of study populations by using animals from different breeding sites increases the replicability of results from single-laboratory studies. We used male C57BL/6J mice from six different breeding sites to test a standardized against a heterogenized (HET) study design in six independent replicate test laboratories. For the standardized design, each laboratory ordered mice from a single breeding site (each laboratory from a different one), while for the HET design, each laboratory ordered proportionate numbers of mice from the five remaining breeding sites. To test our hypothesis, we assessed 14 outcome variables, including body weight, behavioral measures obtained from a single session on an elevated plus maze, and clinical blood parameters. Both breeding site and test laboratory affected variation in outcome variables, but the effect of test laboratory was more pronounced for most outcome variables. Moreover, heterogenization of study populations by breeding site (HET) did not reduce variation in outcome variables between test laboratories, which was most likely due to the fact that breeding site had only little effect on variation in outcome variables, thereby limiting the scope for HET to reduce between-lab variation. We conclude that heterogenization of study populations by breeding site has limited capacity for improving the replicability of results from single-laboratory animal studies