The ASD Living Biology: from cell proliferation to clinical phenotype.

Autism spectrum disorder (ASD) has captured the attention of scientists, clinicians and the lay public because of its uncertain origins and striking and unexplained clinical heterogeneity. Here we review genetic, genomic, cellular, postmortem, animal model, and cell model evidence that shows ASD begins in the womb. This evidence leads to a new theory that ASD is a multistage, progressive disorder of brain development, spanning nearly all of prenatal life. ASD can begin as early as the 1st and 2nd trimester with disruption of cell proliferation and differentiation. It continues with disruption of neural migration, laminar disorganization, altered neuron maturation and neurite outgrowth, disruption of synaptogenesis and reduced neural network functioning. Among the most commonly reported high-confidence ASD (hcASD) genes, 94% express during prenatal life and affect these fetal processes in neocortex, amygdala, hippocampus, striatum and cerebellum. A majority of hcASD genes are pleiotropic, and affect proliferation/differentiation and/or synapse development. Proliferation and subsequent fetal stages can also be disrupted by maternal immune activation in the 1st trimester. Commonly implicated pathways, PI3K/AKT and RAS/ERK, are also pleiotropic and affect multiple fetal processes from proliferation through synapse and neural functional development. In different ASD individuals, variation in how and when these pleiotropic pathways are dysregulated, will lead to different, even opposing effects, producing prenatal as well as later neural and clinical heterogeneity. Thus, the pathogenesis of ASD is not set at one point in time and does not reside in one process, but rather is a cascade of prenatal pathogenic processes in the vast majority of ASD toddlers. Despite this new knowledge and theory that ASD biology begins in the womb, current research methods have not provided individualized information: What are the fetal processes and early-age molecular and cellular differences that underlie ASD in each individual child? Without such individualized knowledge, rapid advances in biological-based diagnostic, prognostic, and precision medicine treatments cannot occur. Missing, therefore, is what we call ASD Living Biology. This is a conceptual and paradigm shift towards a focus on the abnormal prenatal processes underlying ASD within each living individual. The concept emphasizes the specific need for foundational knowledge of a living child's development from abnormal prenatal beginnings to early clinical stages. The ASD Living Biology paradigm seeks this knowledge by linking genetic and in vitro prenatal molecular, cellular and neural measurements with in vivo post-natal molecular, neural and clinical presentation and progression in each ASD child. We review the first such study, which confirms the multistage fetal nature of ASD and provides the first in vitro fetal-stage explanation for in vivo early brain overgrowth. Within-child ASD Living Biology is a novel research concept we coin here that advocates the integration of in vitro prenatal and in vivo early post-natal information to generate individualized and group-level explanations, clinically useful prognoses, and precision medicine approaches that are truly beneficial for the individual infant and toddler with ASD

Ethanol exposure impairs glutamatergic synaptic transmission and plasticity in the ca1 hippocampal region during the third trimester-equivalent of human pregnancy : implications for fetal alcohol spectrum disorder

Fetal alcohol spectrum disorder results from developmental exposure to ethanol. Although many organ systems are targeted by this teratogen, the central nervous system is exquisitely sensitive. Children with this disease often present with behavioral disorders as well as impairments in learning and memory. Ethanol exposure affects developmental processes throughout pregnancy; however, the third trimester-equivalent has demonstrated heightened sensitivity. Although the mechanism of action(s) of ethanol remains unknown, studies suggest that impairments in glutamatergic signaling and synaptic plasticity during the third trimester-equivalent period of development lead to alterations in synaptic formation and refinement. However, little is known about the developmental effects of ethanol in the CA1 hippocampal region, a brain region often studied in the context of learning and memory. Studies presented in this dissertation address the acute and chronic effects of ethanol during the third trimester-equivalent period. First, characterization of the acute effects of ethanol demonstrated postsynaptic inhibition of both NMDA receptor (NMDAR) and AMPA receptor (AMPAR) mediated synaptic responses, as well as the inhibition of long-term potentiation (LTP) induction. Then, an in vivo repeated ethanol exposure paradigm was used to mimic maternal drinking during this period. In contrast to the effects of acute ethanol exposure, repeated exposure did not affect AMPAR- or NMDAR-mediated synaptic strength or glutamate release; however, it impaired LTP expression and/or maintenance. Lastly, repeated in vivo third trimester-equivalent ethanol exposure did not affect expression of Ca2+-permeable AMPARs, or induce tolerance to the acute inhibitory effects of ethanol. Studies in this dissertation add to a growing body of evidence indicating that significant differences exist between the effects of ethanol on the developing versus the mature brain. Furthermore, studies presented here support the notion that ethanol-mediated impairments in synaptic plasticity mechanisms during the third trimester-equivalent developmental period could lead to inappropriate wiring of neuronal circuitry, and set the stage for the long term deficits observed in children with fetal alcohol spectrum disorder

In Utero Domoic Acid Toxicity: A Fetal Basis to Adult Disease in the California Sea Lion (Zalophus californianus)

California sea lions have been a repeated subject of investigation for early life toxicity, which has been documented to occur with increasing frequency from late February through mid-May in association with organochlorine (PCB and DDT) poisoning and infectious disease in the 1970’s and domoic acid poisoning in the last decade. The mass early life mortality events result from the concentrated breeding grounds and synchronization of reproduction over a 28 day post partum estrus cycle and 11 month in utero phase. This physiological synchronization is triggered by a decreasing photoperiod of 11.48 h/day that occurs approximately 90 days after conception at the major California breeding grounds. The photoperiod trigger activates implantation of embryos to proceed with development for the next 242 days until birth. Embryonic diapause is a selectable trait thought to optimize timing for food utilization and male migratory patterns; yet from the toxicological perspective presented here also serves to synchronize developmental toxicity of pulsed environmental events such as domoic acid poisoning. Research studies in laboratory animals have defined age-dependent neurotoxic effects during development and windows of susceptibility to domoic acid exposure. This review will evaluate experimental domoic acid neurotoxicity in developing rodents and, aided by comparative allometric projections, will analyze potential prenatal toxicity and exposure susceptibility in the California sea lion. This analysis should provide a useful tool to forecast fetal toxicity and understand the impact of fetal toxicity on adult disease of the California sea lion

The Effects of Fetal Alcohol Exposure on Gene Expression and Associated Behavioral Markers in Sprague Dawley Rats

Alcohol is a dangerous recreational substance because of its availability and the economic, social, and biological problems that it can cause. Fetal alcohol exposure, although preventable, is particularly harmful to a developing embryo. This exposure often causes fetal alcohol spectrum disorder (FASD), the symptoms of which include learning disabilities and psychological hardships with comorbidities such as anxiety, depression, and attention deficit disorder. One of the main teratogenic effects of alcohol is the effect on epigenetic modifications and altered gene expression during development. These modifications involve changes in the methylation and condensation of DNA that then alters the expression of specific genes into proteins. The first trimester is a particularly important timeframe of focus since this is a critical time in embryonic development and also a time when women may consume excessive amounts of alcohol before they know they are pregnant. In my Honors Thesis project I aimed to examine the effects of prenatal ethanol exposure on both behavior and gene expression in a rat model system. I used quantitative reverse transcriptase PCR to study the expression of two DNA methyl transferase genes critical for epigenetic modification and another gene associated with neuronal function and psychiatric disorders. Expression levels of these genes were altered in response to prenatal alcohol exposure. Behavior studies involving the Morris Water Maze and forced swim test to study to spatial learning and depression, respectively, suggested that alcohol does affect behavior, although these differences were not statistically significant. This is the first study to analyze the gene expression and behavioral effects that a realistic level of alcohol consumption has on a developing embryo during the first trimester

Murine Models for the Study of Fetal Alcohol Spectrum Disorders: An Overview.

Prenatal alcohol exposure is associated to different physical, behavioral, cognitive, and neurological impairments collectively known as fetal alcohol spectrum disorder. The underlying mechanisms of ethanol toxicity are not completely understood. Experimental studies during human pregnancy to identify new diagnostic biomarkers are difficult to carry out beyond genetic or epigenetic analyses in biological matrices. Therefore, animal models are a useful tool to study the teratogenic effects of alcohol on the central nervous system and analyze the benefits of promising therapies. Animal models of alcohol spectrum disorder allow the analysis of key variables such as amount, timing and frequency of ethanol consumption to describe the harmful effects of prenatal alcohol exposure. In this review, we aim to synthetize neurodevelopmental disabilities in rodent fetal alcohol spectrum disorder phenotypes, considering facial dysmorphology and fetal growth restriction. We examine the different neurodevelopmental stages based on the most consistently implicated epigenetic mechanisms, cell types and molecular pathways, and assess the advantages and disadvantages of murine models in the study of fetal alcohol spectrum disorder, the different routes of alcohol administration, and alcohol consumption patterns applied to rodents. Finally, we analyze a wide range of phenotypic features to identify fetal alcohol spectrum disorder phenotypes in murine models, exploring facial dysmorphology, neurodevelopmental deficits, and growth restriction, as well as the methodologies used to evaluate behavioral and anatomical alterations produced by prenatal alcohol exposure in rodents

Models for the Study of Fetal Alcohol Spectrum Disorders: An Overview

Prenatal alcohol exposure is associated to different physical, behavioral, cognitive, and neurological impairments collectively known as fetal alcohol spectrum disorder. The underlying mechanisms of ethanol toxicity are not completely understood. Experimental studies during human pregnancy to identify new diagnostic biomarkers are difficult to carry out beyond genetic or epigenetic analyses in biological matrices. Therefore, animal models are a useful tool to study the teratogenic effects of alcohol on the central nervous system and analyze the benefits of promising therapies. Animal models of alcohol spectrum disorder allow the analysis of key variables such as amount, timing and frequency of ethanol consumption to describe the harmful effects of prenatal alcohol exposure. In this review, we aim to synthetize neurodevelopmental disabilities in rodent fetal alcohol spectrum disorder phenotypes, considering facial dysmorphology and fetal growth restriction. We examine the different neurodevelopmental stages based on the most consistently implicated epigenetic mechanisms, cell types and molecular pathways, and assess the advantages and disadvantages of murine models in the study of fetal alcohol spectrum disorder, the different routes of alcohol administration, and alcohol consumption patterns applied to rodents. Finally, we analyze a wide range of phenotypic features to identify fetal alcohol spectrum disorder phenotypes in murine models, exploring facial dysmorphology, neurodevelopmental deficits, and growth restriction, as well as the methodologies used to evaluate behavioral and anatomical alterations produced by prenatal alcohol exposure in rodents

Murine models for the study of fetal alcohol spectrum disorders: An overview

Prenatal alcohol exposure is associated to different physical, behavioral, cognitive, and neurological impairments collectively known as fetal alcohol spectrum disorder. The underlying mechanisms of ethanol toxicity are not completely understood. Experimental studies during human pregnancy to identify new diagnostic biomarkers are difficult to carry out beyond genetic or epigenetic analyses in biological matrices. Therefore, animal models are a useful tool to study the teratogenic effects of alcohol on the central nervous system and analyze the benefits of promising therapies. Animal models of alcohol spectrum disorder allow the analysis of key variables such as amount, timing and frequency of ethanol consumption to describe the harmful effects of prenatal alcohol exposure. In this review, we aim to synthetize neurodevelopmental disabilities in rodent fetal alcohol spectrum disorder phenotypes, considering facial dysmorphology and fetal growth restriction. We examine the different neurodevelopmental stages based on the most consistently implicated epigenetic mechanisms, cell types and molecular pathways, and assess the advantages and disadvantages of murine models in the study of fetal alcohol spectrum disorder, the different routes of alcohol administration, and alcohol consumption patterns applied to rodents. Finally, we analyze a wide range of phenotypic features to identify fetal alcohol spectrum disorder phenotypes in murine models, exploring facial dysmorphology, neurodevelopmental deficits, and growth restriction, as well as the methodologies used to evaluate behavioral and anatomical alterations produced by prenatal alcohol exposure in rodents.This work was supported by Red de Salud Materno-Infantil y del Desarrollo (SAMID) (RD12/0026/0003 and RD16/0022/0002) from Instituto de Salud Carlos III and the PI15/01179 grant from Instituto de Salud Carlos II

Ethanol exposure during synaptogenesis in a mouse model of fetal alcohol spectrum disorders: acute and long-term effects on gene expression and behaviour

Alcohol is a neuroactive molecule that is able to exert variable and often detrimental effects on the developing brain, resulting in a broad range of physiological, behavioural, and cognitive phenotypes that characterize ‘fetal alcohol spectrum disorders’ (FASD). Factors affecting the manifestation of these phenotypes include alcohol dosage, timing of exposure, and pattern of maternal alcohol consumption; however, the biological processes that are vulnerable to ethanol at any given neurodevelopmental stage are unclear, as is how their disruption results in the emergence of specific pathological phenotypes later in life. The research included in this thesis utilizes a C57BL/6J (B6) mouse model to examine the changes to gene expression and behaviour following a binge-like exposure to ethanol during synaptogenesis, a period of neurodevelopment characterized by the rapid formation and pruning of synaptic connectivity that correlates to brain development during the human third trimester. B6 pups were treated with a high dose (5 g/kg over 2 hours) of ethanol at postnatal day 4 (P4), P7, or on both days (P4+7). Mice were evaluated using a battery of behavioural tests designed to assess FASD-relevant phenotypes, and showed delayed achievement of neuromuscular coordination, hyperactivity, increased anxiety-related traits, and impaired spatial learning and memory. Gene expression analysis identified 315 transcripts that were altered acutely (4 hours) following ethanol exposure. Up-regulated transcripts were associated with cellular stress response, including both pro- and anti-apoptotic molecules, as well as maintenance of cell structural integrity. Down-regulated transcripts were associated with energetically costly processes such as ribosome biogenesis and cell cycle progression. Genes critical to synapse formation were also affected, as well as genes important for the appropriate development of the hypothalamic-pituitary-adrenal axis. Additionally, gene expression changes within the adult brain of mice treated with ethanol at P4+7 were examined to evaluate the long-term effects of neurodevelopmental alcohol exposure. Array analysis identified 376 altered mRNA transcripts with roles in synaptic function, plasticity, and stability, as well as epigenetic processes such as folate metabolism and chromatin remodeling. MicroRNA analyses identified changes in the levels of 33 microRNA species, suggesting that that long-term changes to gene expression following may be maintained (at least in part) via epigenetic mechanisms. Taken together, these analyses illustrate the sensitivity of synaptogenesis to ethanol exposure, leading to a ‘molecular footprint’ of gene expression changes that persists into adulthood and may contribute to the emergence of long-term behavioural and cognitive phenotypes associated with FASD