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There are more than 55,000 variable number tandem repeats (VNTRs) in the human genome, notable for both their striking polymorphism and mutability. Despite their role in human evolution and genomic variation, they have yet to be studied collectively and in detail, partially owing to their large size, variability, and predominant location in noncoding regions. Here, we examine 467 VNTRs that are human-specific expansions, unique to one location in the genome, and not associated with retrotransposons. We leverage publicly available long-read genomes, including from the Human Genome Structural Variant Consortium, to ascertain the exact nucleotide composition of these VNTRs and compare their composition of alleles. We then confirm repeat unit composition in more than 3000 short-read samples from the 1000 Genomes Project. Our analysis reveals that these VNTRs contain highly structured repeat motif organization, modified by frequent deletion and duplication events. Although overall VNTR compositions tend to remain similar between 1000 Genomes Project superpopulations, we describe a notable exception with substantial differences in repeat composition (in PCBP3), as well as several VNTRs that are significantly different in length between superpopulations (in ART1, PROP1, DYNC2I1, and LOC102723906). We also observe that most of these VNTRs are expanded in archaic human genomes, yet remain stable in length between single generations. Collectively, our findings indicate that repeat motif variability, repeat composition, and repeat length are all informative modalities to consider when characterizing VNTRs and their contribution to genomic variation.

Tandem repeats are proposed to contribute to human-specific traits, and more than 40 tandem repeat expansions are known to cause neurological disease. Here, we characterize a human-specific 69 bp variable number tandem repeat (VNTR) in the last intron of WDR7, which exhibits striking variability in both copy number and nucleotide composition, as revealed by long-read sequencing. In addition, greater repeat copy number is significantly enriched in three independent cohorts of individuals with sporadic amyotrophic lateral sclerosis (ALS). Each unit of the repeat forms a stem-loop structure with the potential to produce microRNAs, and the repeat RNA can aggregate when expressed in cells. We leveraged its remarkable sequence variability to align the repeat in 288 samples and uncover its mechanism of expansion. We found that the repeat expands in the 3′-5′ direction, in groups of repeat units divisible by two. The expansion patterns we observed were consistent with duplication events, and a replication error called template switching. We also observed that the VNTR is expanded in both Denisovan and Neanderthal genomes but is fixed at one copy or fewer in non-human primates. Evaluating the repeat in 1000 Genomes Project samples reveals that some repeat segments are solely present or absent in certain geographic populations. The large size of the repeat unit in this VNTR, along with our multiplexed sequencing strategy, provides an unprecedented opportunity to study mechanisms of repeat expansion, and a framework for evaluating the roles of VNTRs in human evolution and disease.

Gene knockdown using short hairpin RNAs (shRNAs) is a promising strategy for targeting dominant mutations; however, delivering too much shRNA can disrupt the processing of endogenous microRNAs (miRNAs) and lead to toxicity. Here, we sought to understand the effect that excessive shRNAs have on muscle miRNAs by treating mice with recombinant adeno-associated viral vectors (rAAVs) that produce shRNAs with 19-nt or 21-nt stem sequences. Small RNA sequencing of their muscle and liver tissues revealed that shRNA expression was highest in the heart, where mice experienced substantial cardiomyopathy when shRNAs accumulated to 51.2% ± 13.7% of total small RNAs. With the same treatment, shRNAs in other muscle tissues reached only 12.1% ± 5.0% of total small RNAs. Regardless of treatment, the predominant heart miRNAs remained relatively stable across samples. Instead, the lower-expressed miR-451, one of the few miRNAs processed independently of Dicer, changed in relation to shRNA level and toxicity. Our data suggest that a protective mechanism exists in cardiac tissue for maintaining the levels of most miRNAs in response to shRNA delivery, in contrast with what has been shown in the liver. Quantifying miRNA profiles after excessive shRNA delivery illuminates the host response to rAAV-shRNA, allowing for safer and more robust therapeutic gene knockdown., Graphical Abstract

Paternal stress exposure is known to impact the development of stress-related behaviors in offspring. Previous work has highlighted the importance of sperm mediated factors, such as RNAs, in transmitting the effects of parental stress. However, a key unanswered question is whether mothers’ behavior could drive or modulate the transmission of paternal stress effects on offspring development. Here we investigate how chronic variable stress in Balb/C mice influences the sex-specific development of anxiety- and depression-like neural and behavioral development in offspring. Moreover, we examined how stressed fathers influenced mate maternal investment towards their offspring and how this may modulate the transmission of paternal stress effects on offspring. We show that paternal stress leads to sex-specific effects on offspring behavior. Males that are chronically stressed sire female offspring that show increased anxiety and depression-like behaviors. However, male offspring of stressed fathers show reductions in anxiety- and depression-behaviors and are generally more exploratory. Moreover, we show that females mated with stressed males gain less weight during pregnancy and provide less care towards their offspring which additionally influenced offspring development. These data indicate that paternal stress can influence offspring development directly and indirectly via changes in mothers, with implications for divergent development between male and female offspring.

Alzheimer’s disease is the most common neurodegenerative disease, characterized by dementia and premature death. Early-onset familial Alzheimer’s disease is caused in part by pathogenic variants in presenilin 1 (PSEN1) and presenilin 2 (PSEN2), and alternative splicing of these two genes has been implicated in both familial and sporadic Alzheimer’s disease. Here, we leveraged targeted isoform-sequencing to characterize thousands of complete PSEN1 and PSEN2 transcripts in the prefrontal cortex of individuals with sporadic Alzheimer’s disease, familial Alzheimer’s disease (carrying PSEN1 and PSEN2 variants), and controls. Our results reveal alternative splicing patterns of PSEN2 specific to sporadic Alzheimer’s disease, including a human-specific cryptic exon present in intron 9 of PSEN2 as well as a 77 bp intron retention product before exon 6 that are both significantly elevated in sporadic Alzheimer’s disease samples, alongside a significantly lower percentage of canonical full-length PSEN2 transcripts versus familial Alzheimer’s disease samples and controls. Both alternatively spliced products are predicted to generate a prematurely truncated PSEN2 protein and were corroborated in an independent cerebellum RNA-sequencing dataset. In addition, our data in PSEN variant carriers is consistent with the hypothesis that PSEN1 and PSEN2 variants need to produce full-length but variant proteins to contribute to the onset of Alzheimer’s disease, although intriguingly there were far fewer full-length transcripts carrying pathogenic alleles versus wild-type alleles in PSEN2 variant carriers. Finally, we identify frequent RNA editing at Alu elements present in an extended 3′ untranslated region in PSEN2. Overall, this work expands the understanding of PSEN1 and PSEN2 variants in Alzheimer’s disease, shows that transcript differences in PSEN2 may play a role in sporadic Alzheimer’s disease, and suggests novel mechanisms of Alzheimer’s disease pathogenesis.

There are over 55,000 variable number tandem repeats (VNTRs) in the human genome, notable for both their striking polymorphism and mutability. Despite their role in human evolution and genomic variation, they have yet to be studied collectively and in detail, partially due to their large size, variability, and predominant location in non-coding regions. Here, we examine 467 VNTRs that are human-specific expansions, unique to one location in the genome, and not associated with retrotransposons. We leverage publicly available long-read genomes – including from the Human Genome Structural Variant Consortium – to ascertain the exact nucleotide composition of these VNTRs, and compare their composition of alleles. We then confirm repeat unit composition in over 3000 short-read samples from the 1000 Genomes Project. Our analysis reveals that these VNTRs contain remarkably structured repeat motif organization, modified by frequent deletion and duplication events. While overall VNTR compositions tend to remain similar between 1000 Genomes Project super-populations, we describe a notable exception with substantial differences in repeat composition (in PCBP3), as well as several VNTRs that are significantly different in length between super-populations (in ART1, PROP1, WDR60, and LOC102723906). We also observe that most of these VNTRs are expanded in archaic human genomes, yet remain stable in length between single generations. Collectively, our findings indicate that repeat motif variability, repeat composition, and repeat length are all informative modalities to consider when characterizing VNTRs and their contribution to genomic variation.

Half of all human transcripts are thought to be regulated by microRNAs. Therefore, quantifying microRNA expression can reveal underlying mechanisms in disease states and provide therapeutic targets and biomarkers. Here, we detail how to accurately quantify microRNAs. Briefly, this method describes isolating microRNAs, ligating them to adaptors suitable for high-throughput sequencing, amplifying the final products, and preparing a sample library. Then, we explain how to align the obtained sequencing reads to microRNA hairpins, and quantify, normalize, and calculate their differential expression. Versatile and robust, this combined experimental workflow and bioinformatic analysis enables users to begin with tissue extraction and finish with microRNA quantification.

Prenatal exposure to polycyclic aromatic hydrocarbons (PAH) has been associated with sustained effects on the brain and behavior in offspring. However, the mechanisms have yet to be determined. We hypothesized that prenatal exposure to ambient PAH in mice would be associated with impaired neurocognition, increased anxiety, altered cortical expression of Bdnf and Grin2b, and greater DNA methylation of Bdnf. Our results indicated that during open-field testing, prenatal PAH–exposed offspring spent more time immobile and less time exploring. Females produced more fecal boli. Offspring prenatally exposed to PAH displayed modest reductions in overall exploration of objects. Further, prenatal PAH exposure was associated with lower cortical expression of Grin2b and Bdnf in males and greater Bdnf IV promoter methylation. Epigenetic differences within the Bdnf IV promoter correlated with Bdnf gene expression but not with the observed behavioral outcomes, suggesting that additional targets may account for these PAH-associated effects.

This chapter reviews endocrine, behavioral, and reproductive outcomes of the ketogenic diet (KD) in rodent mouse and rat models. KD treatment can result in conditions seen in metabolic syndrome, including dyslipidemia and inflammation. Females raised on low protein KDs will experience delayed puberty onset. Results presented suggest an increase in ketones and a decrease in spatial memory as percent protein drops. Postpubertal female pups fed KD experienced similar cognitive decline to KD-fed dams, despite consuming only normal rodent chow since weaning. This finding is consistent with the development of anatomical differences in the brains of pups from KD-fed versus rodent chow–fed controls. A thorough evaluation of the effects of different lipid and protein profiles (amount and type) will be necessary if KDs are going to be safe and effective long-term therapies for both sexes and in neurological disorders besides pediatric intractable epilepsy.

Animal models of early-life stress and variation in social experience across the lifespan have contributed signifi cantly to our understanding of the environmental regulation of the developing brain. Plasticity in neurobiological pathways regulating stress responsivity, cognition, and reproductive behavior is apparent during the prenatal period and continues into adulthood, suggesting a lifelong sensitivity to environmental cues. Recent evidence suggests that dynamic epigenetic changes—molecular modifi cations that alter gene expression without altering the underlying DNA sequence— account for this plasticity. In this review, we highlight studies of laboratory rodents that illustrate the association between the experience of prenatal stress, maternal separation, maternal care, abusive caregiving in infancy, juvenile social housing, and adult social stress and variation in DNA methylation and histone modifi cation. Moreover, we discuss emerging evidence for the transgenerational impact of these experiences. These experimental paradigms have yielded insights into the potential role of epigenetic mechanisms in mediating the effects of the environment on human development and also indicate that consideration of the sensitivity of laboratory animals to environmental cues may be an important factor in predicting long-term health and welfare.

Experiences occurring in early development can exert long-term effects that lead to either heightened or attenuated risk of physical and psychiatric disease. Of particular importance for these outcomes is the quality of the relationship between a parent and an infant. Parental stress, reduced sensitivity of parents to infant cues, and childhood neglect/abuse may lead to a cascade of biological changes that compromise the func- tioning of infants, leading to effects that persist into adolescence and adulthood. Moreover, genetic and environmental factors may heighten the vulnerability of infants to these effects. For the high-risk neonate, both underlying vulnerability and adverse early-life experiences are characteristic and may ultimately lead to divergent develop- mental pathways that compromise future health and well-being. The deprivation or reduction in parental contact that is often experienced by these infants during the neonatal period may be a significant factor in determining the severity of these outcome measures. Thus, understanding the mechanisms through which parental care can alter infant development may provide insight into the potential interventions and practices that are critical in promoting healthy children, adolescents, and adults. During fetal and infant development, the brain is rapidly changing, leading to prolif- eration and refinement of neural pathways. The sensitive period created by this time of neuronal plasticity creates a window of opportunity during which experiences can exert long-term effects. Although the study of human brain development and the effects of experiences on these processes has been limited by the availability of appropriate methodological tools, decades of work using animal models has provided some valuable insights. Laboratory studies of rodents suggest that adverse experi- ences occurring during fetal and/or infant development lead to changes in brain architecture and function. These effects are particularly evident when the quality of the parent-infant interaction is affected, either through parental stress or through

Individual differences in maternal behavior in rodents are associated with altered physiology and behavior in offspring across their lifespan and across generations. Offspring of rat dams that engage in high frequencies of high-arched-back nursing and pup-licking (High-LG) show attenuated stress responses compared to those engaging in lower frequencies (Low-LG). Selective breeding also produces widespread alterations in physiology and behavior that are stable over generations. To examine processes underlying generational and developmental influences on anxiety in an animal model, we developed two lines of rats that emit either extremely high (High-USV) or low (Low-USV) rates of 45kHz ultrasonic vocalizations in isolation at postnatal day 10. Compared to the Low-USV line, High-USV rats display increased indices of anxiety- and depression-like behavior in adulthood. The current study assessed maternal behaviors as well as oxytocin and vasopressin receptor density in High-USV and Low-USV dams to determine if selective breeding had produced differences that paralleled those found in Low- and High-LG dams. We found that Low-USV dams engage in more high-arched nursing and pup-licking than High-USV dams. Differences in oxytocin and vasopressin receptor levels were not widespread throughout the brain, with line differences in the piriform cortex and nucleus accumbens. This research illustrates the potential interplay between genetically determined (USV line) and environmental (postnatal mother-infant interactions) factors in accounting for the phenotypes associated with maternal separation induced postnatal vocalizations.

Early-life adversity increases the risk for psychopathology in later life. The underlying mechanism(s) is unknown, but epigenetic variation represents a plausible candidate. Early-life exposures can disrupt epigenetic programming in the brain, with lasting consequences for gene expression and behavior. This evidence is primarily derived from animal studies, with limited study in humans due to inaccessibility of the target brain tissue. In humans, although there is evidence for DNA methylation changes in the peripheral blood of psychiatric patients, a fundamental question remains as to whether epigenetic markers in the blood can predict epigenetic changes occurring in the brain. We used in utero bisphenol A (BPA) exposure as a model environmental exposure shown to disrupt neurodevelopment and exert long-term effects on behavior in animals and humans. We show that prenatal BPA induces lasting DNA methylation changes in the transcriptionally relevant region of the Bdnf gene in the hippocampus and blood of BALB/c mice and that these changes are consistent with BDNF changes in the cord blood of humans exposed to high maternal BPA levels in utero. Our data suggest that BDNF DNA methylation in the blood may be used as a predictor of brain BDNF DNA methylation and gene expression as well as behavioral vulnerability induced by early-life environmental exposure. Because BDNF expression and DNA methylation are altered in several psychiatric disorders that are associated with early-life adversity, including depression, schizophrenia, bipolar disorder, and autism, BDNF DNA methylation in the blood may represent a novel biomarker for the early detection of psychopathology.

Bisphenol A (BPA) is an estrogenic endocrine disruptor widely used in the production of plastics. Increasing evidence indicates that in utero BPA exposure affects sexual differentiation and behavior; however, the mechanisms underlying these effects are unknown. We hypothesized that BPA may disrupt epigenetic programming of gene expression in the brain. Here, we provide evidence that maternal exposure during pregnancy to environmentally relevant doses of BPA (2, 20, and 200 µg/kg/d) in mice induces sex-specific, dose-dependent (linear and curvilinear), and brain region-specific changes in expression of genes encoding estrogen receptors (ERs; ERα and ERβ) and estrogen-related receptor-γ in juvenile offspring. Concomitantly, BPA altered mRNA levels of epigenetic regulators DNA methyltransferase (DNMT) 1 and DNMT3A in the juvenile cortex and hypothalamus, paralleling changes in estrogen-related receptors. Importantly, changes in ERα and DNMT expression in the cortex (males) and hypothalamus (females) were associated with DNA methylation changes in the ERα gene. BPA exposure induced persistent, largely sex-specific effects on social and anxiety-like behavior, leading to disruption of sexually dimorphic behaviors. Although postnatal maternal care was altered in mothers treated with BPA during pregnancy, the effects of in utero BPA were not found to be mediated by maternal care. However, our data suggest that increased maternal care may partially attenuate the effects of in utero BPA on DNA methylation. Overall, we demonstrate that low-dose prenatal BPA exposure induces lasting epigenetic disruption in the brain that possibly underlie enduring effects of BPA on brain function and behavior, especially regarding sexually dimorphic phenotypes.

Early life adversity can have a significant long-term impact with implications for the emergence of psychopathology. Disruption to mother-infant interactions is a form of early life adversity that may, in particular, have profound programming effects on the developing brain. However, despite converging evidence from human and animal studies, the precise mechanistic pathways underlying adversity-associated neurobehavioral changes has yet to be elucidated. One approach to the study of mechanism is exploration of epigenetic changes associated with early life experience. In the current study, we examined the effects of postnatal maternal separation in mice and assessed the behavioral, brain gene expression, and epigenetic effects of this manipulation in offspring. Importantly, we included two different mouse strains (B6 and Balb/c) and both male and female offspring to determine strain- and/or sex-associated differential response to maternal separation. We found both strain-specific and sex-dependent effects of maternal separation in early adolescent offspring on measures on open-field exploration, sucrose preference, and social behavior. Analyses of cortical and hippocampal mRNA levels of the glucocorticoid receptor (Nr3c1) and brain-derived neurotrophic factor (Bdnf) genes revealed decreased hippocampal Bdnf expression in maternally-separated B6 females and increased cortical Bdnf expression in maternally separated male and female Balb/c offspring. Analyses of Nr3c1and Bdnf (IV and IX) CpG methylation indicated increased hippocampal Nr3c1 methylation in maternally separated B6 males and increased hippocampal Bdnf IX methylation in male and female maternally separated Balb/c mice. Overall, though effect sizes were modest, these findings suggest a complex interaction between early life adversity, genetic background, and sex in the determination of neurobehavioral and epigenetic outcomes that may account for differential vulnerability to later life disorder.

In nonprimate mammals, the dorsal cochlear nucleus (DCN) is thought to play a role in the orientation of the head toward sounds of interest by integrating acoustic and somatosensory information. Humans and higher primates might not use this system because of reported phylogenetic changes in DCN cytoarchitecture [Moskowitz N (1969) Comparative aspects of some features of the central auditory system of primates. Ann N Y Acad Sci 167:357-369; Moore JK, Osen KK (1979) The cochlear nuclei in man. Am J Anat 154:393-418; Moore JK (1980) The primate cochlear nuclei: loss of lamination as a phylogenetic process. J Comp Neurol 193:609-629]. In this study, we re-evaluated this question from a comparative perspective and examined the rhesus monkey (cercopithecoid primate) using more sensitive probes and higher resolution imaging methods. We used electron microscopy to identify parallel fibers and their synapses, and molecular markers to determine that primates exhibit the main components of excitatory neurotransmission as other mammals. We observed that characteristics of the monkey molecular layer resembled what has been reported for nonprimates: (1) immunohistochemistry revealed many unmyelinated, thin axons and en passant glutamatergic synapses on dendritic spines; (2) immunohistochemistry for phosphodiesterase (PDE10A) showed the nuclei of granule cells distributed in the external molecular layer and the deep layers in the DCN; (3) antibodies for the inositol trisphosphate receptor (IP3r) and calbindin immunostained cartwheel cells; (4) postembedding immunogold labeling revealed synaptic expression of AMPA and delta glutamate receptor subunits on spines in parallel fiber endings; and (5) parallel fibers use vesicular glutamate transporter 1 (VGLUT1) to package glutamate into the synaptic vesicles and to mediate glutamate transport. These observations are consistent with the argument that the rhesus monkey DCN has neuronal features similar to those of other nonprimate mammals.

SummaryDevelopmental alterations of excitatory synapses are implicated in autism spectrum disorders (ASDs). Here, we report increased dendritic spine density with reduced developmental spine pruning in layer V pyramidal neurons in postmortem ASD temporal lobe. These spine deficits correlate with hyperactivated mTOR and impaired autophagy. In Tsc2+/− ASD mice where mTOR is constitutively overactive, we observed postnatal spine pruning defects, blockade of autophagy, and ASD-like social behaviors. The mTOR inhibitor rapamycin corrected ASD-like behaviors and spine pruning defects in Tsc2+/ mice, but not in Atg7CKO neuronal autophagy-deficient mice or Tsc2+/−:Atg7CKO double mutants. Neuronal autophagy furthermore enabled spine elimination with no effects on spine formation. Our findings suggest that mTOR-regulated autophagy is required for developmental spine pruning, and activation of neuronal autophagy corrects synaptic pathology and social behavior deficits in ASD models with hyperactivated mTOR.