Browsing by Subject "Neurodevelopment"

Astrocytes, the perisynaptic glial cells of the brain, display a complex morphology that is strongly linked to their functions at the synapse. Primary processes radiating from the astrocyte cell soma branch out to secondary and tertiary processes, which further ramify into tiny perisynaptic astrocyte processes, giving a mature astrocyte its characteristic arborized structure. Astrocyte processes dynamically ensheath the pre- and post-synapse to provide instructive cues for synapse formation, maturation, and function. Perturbations in astrocyte-synapse interactions result in synaptic deficits, leading to excitation/inhibition imbalance and aberrant neural circuitry. However, the mechanisms linking astrocyte morphology and function to neuronal contact and synaptic adhesion are poorly understood. In a candidate-based reverse genetic screen utilizing rodent cortical neurons and astrocytes, I identified two genes, HepaCAM and CTNND2, as regulators of astrocyte morphogenesis in response to neuronal adhesion.HepaCAM is an astrocyte-enriched cell adhesion molecule that participates in cell-cell and cell-ECM interactions to regulate cell migration and proliferation. shRNA-mediated silencing of hepaCAM expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. HepaCAM stabilizes the gap junction protein connexin 43 (Cx43) at cell-cell junctions. We used stimulated emission depletion (STED) microscopy to show that hepaCAM and Cx43 colocalize at astrocyte processes in the mouse cortex and performed native affinity purifications followed by liquid chromatography-coupled high-resolution mass spectrometry (AP-MS) to demonstrate that Cx43 binds to hepaCAM. Finally, utilizing the same shRNA silencing approach, we found that hepaCAM and Cx43 were epistatic to each other in the regulation of astrocyte morphogenesis. Through mosaic analysis with double markers (MADM), we found that hepaCAM knockout astrocytes lost their ability to tile and had mislocalized Cx43. Consequently, gap junction coupling is impaired in astrocytes without hepaCAM. Additionally, we found decreased colocalization of hepaCAM puncta with synapses, a marked decrease in inhibitory synapses density, and a significant decrease in amplitude of miniature inhibitory postsynaptic currents, suggesting that loss of astrocytc hepaCAM disrupts the balance between synaptic excitation and inhibition. During development, astrocytes need to form non-overlapping territories within which they dynamically ensheathe synapses within discrete regions of neuropil. Taken together, our findings suggest that hepaCAM and Cx43 are critical proteins at the intersection of these two processes to ensure the proper molecular regulation of astrocyte self-organization and territory formation for normal circuit formation and function. Next, we identified Ctnnd2 (protein: δ-catenin) as another key regulator of astrocyte morphological complexity. δ-catenin was previously thought to be a neuron-specific protein that regulates dendrite morphology. Utilizing RNA fluorescence in situ hybridization (RNA-FISH) and immunohistochemistry, we found Ctnnd2 mRNA and δ-catenin is also highly expressed by astrocytes during the critical period of astrocyte morphological maturation and synapse formation during cortical development. shRNA-mediated silencing of Ctnnd2 expression in astrocytes resulted in decreased astrocyte complexity in vitro and in vivo. δ-catenin is hypothesized to mediate transcellular interactions through the cadherin family of cell adhesion proteins. We used structural modeling and surface biotinylation assays in both HEK293T and purified astrocyte cultures to reveal that δ-catenin interacts with N-cadherin juxtamembrane domain to promote N-cadherin surface expression. An autism-linked δ-catenin point mutation impaired N-cadherin cell surface expression and reduced astrocyte complexity. In the developing mouse cortex, only lower-layer cortical neurons express N-cadherin. Remarkably, when we silenced astrocytic N-cadherin throughout the cortex, only lower-layer astrocyte morphology was disrupted. These findings show that δ-catenin controls astrocyte-neuron cadherin interactions that regulate layer-specific astrocyte morphogenesis.

In both insects and mammals, odor detection depends heavily on diverse classes of olfactory neurons that organize their axons to converge in a class-specific manner within the central brain’s olfactory bulb, or antennal lobe in flies. The olfactory sensory circuit is characterized by its unique and essential feature—a functionally organized topographic map. This map relies on the convergence of axons from dispersed olfactory sensory neurons of the same type into specific regions known as class-specific glomeruli. Exploring how the identity of neurons shapes this circuit organization is a central pursuit in neurobiology, given its significant implications for neurodegenerative diseases and neuronal dysfunction.In the olfactory system, various cell surface proteins, such as Robo/Slit and Toll receptors, govern numerous aspects of circuit organization, including axon guidance and synaptic matching. In our study, we have identified an atypical cadherin protein called Fat2 (also known as Kugelei) as a regulator of axon organization specific to neuronal classes. Fat2 is expressed in olfactory receptor neurons (ORNs) and local interneurons (LNs) within olfactory circuits, with minimal expression in projection neurons (PNs). Notably, Fat2 expression levels vary depending on neuronal class and peak during pupal development. In cases of fat2 gene mutations, we observed varying degrees of phenotypic presentations in ORN axon terminals belonging to different classes, with a notable trend toward more severe effects in classes with higher Fat2 expression. In the most extreme cases, fat2 mutations resulted in ORN degeneration. Our findings suggest that the intracellular domain of Fat2 is crucial for its role in organizing ORN axons. Specifically, during early stages of olfactory circuit development, Fat2 plays a pivotal role in coordinating axons precisely, facilitating the formation of class-specific glomerular structures. Importantly, our research indicates that the expression of fat2 by PNs and LNs does not significantly contribute to ORN organization. Finally, we have identified potential interactors of the Fat2 intracellular domain, namely APC family proteins (Adenomatous polyposis coli) and dop (Drop out), which likely coordinate cytoskeletal remodeling essential for axon retraction during protoglomerular development. In summary, our study establishes a foundational understanding of Fat2's role in organizing the olfactory circuit and underscores the critical importance of axon behavior in the maturation of glomeruli.

Background: Silver nanoparticles (AgNPs) act as antimicrobials by releasing monovalent silver (Ag+) and are increasingly used in consumer products, thus elevating exposures in human and environmental populations. Materials and Methods: We evaluated Ag+ in a standard model of neuronal cell replication and differentiation, and then determined whether there were similar effects of the ion in vivo using zebrafish. Next, we compared Ag+ and AgNP exposures in the same two models and incorporated the effects of particle coating, size and composition. Conclusions: This work is the first to show that both Ag+ and AgNPs are developmental neurotoxicants in vitro and in vivo. Moreover, although both the soluble ion and the particles impair measures of neurodevelopment, the outcomes and underlying mechanisms of each toxicant are often wholly distinct. Superimposed on the dichotomies between Ag+ and AgNP exposures are clear effects of particle coating, size and composition that will necessitate evaluation of individual AgNP types when considering potential environmental and human health effects. The results presented here provide hazard identification that can help isolate the models and endpoints necessary for developing a risk assessment framework for the growing use of AgNPs.

Following the phase out of polybrominated diphenyl ethers (PBDEs), manufacturers turned to several alternative flame retardants (FRs) to meet flammability standards. Organophosphate FRs (OPFRs), and in particular tris (1,3-dichloropropyl) phosphate (TDCPP), have been increasingly detected in textiles and foam padding used in a variety of consumer products including camping equipment, upholstered furniture, and baby products. Like PBDEs, OPFRs are additive, meaning that they are not chemically bound to the treated material and can more readily leach out into the surrounding environment. Indeed, OPFRs have been detected in numerous environmental and biological matrices, often at concentrations similar to or exceeding that of PBDEs.

Although OPFRs have been in use for several decades, relatively little is known regarding their potential for adverse human and environmental health consequences. However, based on their structural similarity to OP pesticides, they may have analogous mechanisms of toxicity. OP pesticide toxicity is classically associated with cholinesterase inhibition, resulting in cholinergic intoxication syndrome. OPFRs have been shown to be ineffective cholinesterase inhibitors, however chlorpyrifos (CPF) and other OP pesticides have been shown to elicit adverse effects on developing organisms through other mechanisms.

The main objective of this research project was to evaluate the toxicity of four structurally similar OPFRs (TDCPP; tris (2,3-dibromopropyl) phosphate, (TDBPP); tris (1-chloropropyl) phosphate (TCPP) and tris (2-chloroethyl) phosphate (TCEP)) in comparison to chlorpyrifos (CPF), a well-studied OP pesticide. A combination of in vitro and in vivo models was used to elucidate potential mechanisms as well as functional consequences of exposure in developing organisms.

In the first research aim, a series of in vitro experiments with neurotypic PC12 cells was used to evaluate the effects of four structurally similar OPFRs (TDCPP, TDBPP, TCEP, or TCPP) and CPF on neurodevelopment. The effects of TDCPP were also compared to that of BDE-47, a major component of the commercial PentaBDE mixture. In general, TDCPP elicited similar or greater effects when compared to an equimolar concentration of CPF. All OPFRs tested produced similar decrements in cell number and altered phenotypic differentiation, while BDE-47 had no effect on cell number, cell growth, or neurite growth.

For the second research aim, zebrafish (Danio rerio) were used to evaluate the effects of the same suite of chemicals on early development. TDCPP, TDBPP, and CPF elicited overt toxicity (e.g., malformations or death) within the concentration range tested (0.033-100 µM). TDBPP was the most potent with 100% mortality by 6 days post fertilization (dpf) at ≥3.3 µM. CPF and TDCPP showed equivalent toxicity with malformations observed in at 10 µM and significant mortality (≥75%) at ≥33 µM. There was no overt toxicity among TCEP- and TCPP-exposed fish. All test chemicals affected larval swimming behavior on 6 dpf at concentrations below the overt toxicity threshold. Parent chemical was detected in all in embryonic (1 dpf) and larval (5 dpf) tissues. TDCPP and TDBPP showed rapid and extensive metabolism.

Finally, for the third aim, juvenile (45-55 dpf) zebrafish were exposed to CPF (1 µg/g food) or TDCPP (Low TDCPP = 1 µg/g food; High TDCPP = 40 µg/g food) via diet for 28 days followed by a 7 day depuration period where all treatments received clean food. A dietary exposure was chosen to more closely recapitulate exposure in humans. Samples were collected at seven time points throughout the experiment: days 0, 7, 14, 21, 28, 30, 35. Whole tissues were collected for tissue accumulation and histopathology endpoints. Viscera and brain were dissected and flash frozen separately for DNA damage analyses.

Tissue measurements of CPF, TDCPP, and the metabolite bis (1,3-dichloropropyl) phosphate (BDCPP) were often below the method detection limit, however when present there was a trend towards increased accumulation with treatment and time. On Day 7 Low TDCPP caused a dramatic but transient increase in DNA damage in both viscera and brain that returned to control levels by Day 14. Similar results have been seen previously with other genotoxicants and may be due to CPF and High TDCPP inducing an adaptive response prior to the 7 day sampling point. All treatments shifted the neurohypophysis to adenohypophysis ratio (NH/AH; Day 7 only) and significantly increased thyroid follicle activation (Day 14). Finally High TDCPP affected gonad maturation, causing a significant increase in ovary follicle development (Day 14) and a transient but marked decrease in testes maturity (Day 7). Taken together these data suggest that dietary exposure to TDCPP and CPF elicits DNA damage in brain and viscera and alters endocrine function in juvenile zebrafish. Importantly, analyses were restricted to the first three time points (Days 0, 7, and 14) due to the emergence a disease among the experimental colony. Although these samples were collected prior to the disease becoming apparent, it remains a potential confounder of the current results.

N6-methyladenosine (m6A) is deposited co-transcriptionally on thousands of cellular mRNAs and plays important roles in mRNA processing and cellular function. m6A is particularly abundant within the brain and is critical for neurodevelopment. However, the mechanisms through which m6A contributes to brain development are inco¬¬mpletely understood. Here, we discover serine-/arginine-rich splicing factor 7 (SRSF7) and RNA-binding motif-containing protein 45 (RBM45) as m6A-binding proteins in transformed hippocampal neurons. We find that SRSF7 binds to exon-intron junctions in methylated pre-mRNA targets and regulates the gene expression of thousands of cellular mRNAs, including the m6A RNA methyltransferase, METTL3. We find that RBM45 binds to thousands of cellular RNAs, predominantly within intronic regions. Rbm45 depletion disrupts the constitutive splicing of a subset of target pre-mRNAs, leading to altered mRNA and protein levels through both m6A-dependent and m6A-independent mechanisms. Finally, we find that RBM45 is highly expressed during embryonic neurodevelopment, demonstrating that expression of RBM45 is necessary for neuroblastoma cell differentiation and that its depletion impacts the expression of genes involved in several neurodevelopmental signaling pathways. Altogether, our findings identify roles for SRSF7 and RBM45 in gene expression regulation, and highlight a previously unknown function for RBM45 in the control of pre-mRNA processing and neuronal differentiation, mediated in part by the recognition of methylated RNA.

Excitatory synapse formation during development involves the complex orchestration of both structural and functional alterations at the postsynaptic side, beginning with the formation of transient dendritic filopodia. Abnormalities in synapse development are linked to developmental brain disorders such as autism spectrum disorders, schizophrenia, and intellectual disability. However, the molecular mechanisms that underlie excitatory synaptogenesis remain elusive, in part because the internal machinery of developing synapses is largely unknown. Unlike mature excitatory synapses, there is currently no way to biochemically isolate the dendritic filopodia of nascent synapses. This lack of understanding is a critical barrier to our grasp of synapse development as well as the etiology of many neurodevelopmental disorders. This dissertation work focuses on the detection and analysis of proteins which localize to and are critical for spinogenesis and synaptogenesis. Using state-of-the-art in vivo proteomics, we identified a network of proteins which localize to the receiving end of the developing excitatory synapse, the dendritic filopodia. We then used the CRISPR/Cas9 system to identify candidates which drive the formation and maturation of dendritic filopodia. We finally did careful functional analysis of CARMIL3 and the Arp2/3 complex to identify their critical and diverse roles in synaptogenesis.

In our analysis, we found that CARMIL3 is expressed in the brain predominately during synaptogenesis, localizes to developing dendritic protrusions, and is important for the morphological and functional maturation of synapses, likely through its role in recruiting capping protein to maturing synapses. Loss of CARMIL3 leads to structurally and functionally immature synapses that are capping protein deficient. Further, we found that the Arp2/3 complex, a critical regulator of the actin cytoskeleton which creates branched actin networks, is required for both the functional and morphological maturation of dendritic spines. In the absence of the Arp2/3 complex, dendritic protrusions make presynaptic contact, recruit key proteins such as MAGUKs, and recruit certain receptors such as NMDA receptors, but lack AMPA receptors which are required for synapse unsilencing.

Together, this work demonstrates that the actin cytoskeleton controls the functional maturation of synapses by altering the cytoskeletal dynamics towards the creation of a branched actin network. CARMIL3 contributes to this process by providing capping protein, which biases actin nucleation towards branched actin networks. Arp2/3 creates the branched actin network. Without this network, there is not a sufficient framework to dock AMPA receptors in the post-synaptic density, and without AMPA receptors, dendritic protrusions remain functionally silent. Together, this work shows that the dynamics of the actin cytoskeleton drive synapse unsilencing.

Polybrominated diphenyl ether (PBDE) flame retardants are ubiquitous contaminants in the environment due to their heavy usage in plastics, foam, and textiles to comply with flammability standards from the 1970s through the late 2000s. Due to their toxicity and persistence in the environment, two of the three PBDE commercial mixtures (PentaBDE and OctaBDE) were banned by the Stockholm Convention on Persistent Organic Pollutants in 2009. The DecaBDE commercial mixture, which consists primarily of the fully brominated congener BDE-209, has been banned or phased out in the United States and Europe but is still in use in other parts of the world. Human exposure to PBDEs persists via environmental reservoirs of PBDEs and products produced before the bans/phase-outs. PBDEs disrupt thyroid hormone levels and neurodevelopment in fish and rodents and are associated with altered thyroid hormone levels and neurodevelopmental impairments in humans. However, the mechanism by which PBDEs alter neurodevelopment remains unclear. Knowledge of the mechanisms and molecular targets of PBDEs is necessary for a causal link to be established between PBDEs and neurodevelopmental impairments. The hypothesis of this thesis research is that PBDEs alter thyroid hormone levels in the brain by interfering with the activity of PBDE-metabolizing deiodinase enzymes in brain cells, which may result in decreased levels of thyroid hormones in the brain and impaired neurodevelopment.

In the first aim of this thesis research, the biotransformation of PBDEs was examined to determine whether hydroxylated PBDEs (OH-BDEs) are formed in the human brain. In biotransformation assays performed with human astrocytes, which are cells located at the blood brain barrier, no debrominated or OH-BDE metabolites were identified. The results indicate that the enzyme responsible for PBDE hydroxylation (CYP2B6) was not expressed in sufficient quantities to metabolize PBDEs in the astrocyte cells used in this study, but future studies should analyze the potential for PBDE hydroxylation in other brain cells.

In the second aim of this thesis research, the effects of PBDEs on the thyroid-activating enzyme Type 2 deiodinase (DIO2) were determined in human astrocyte cells. DIO2 converts thyroxine (T4) into triiodothyronine (T3), which is the primary ligand that binds to the thyroid nuclear receptors, and is a very important signaling molecule during neurodevelopment. Cultured primary astrocytes and a human glioma cell line (H4 cells) were exposed to PBDEs and OH-BDEs, and changes in DIO2 activity were measured using liquid chromatography with tandem mass spectrometry (LC/MS/MS). Exposure to BDE-99, -153, and -209, 3-OH-BDE-47, and 5'-OH-BDE-99 all resulted in significant decreases in DIO2 activity in the H4 cells by up to 80% at doses of 500-1,000 nM. Further experiments deduced that the primary mechanism responsible for this decrease in activity was attributed to decreased DIO2 mRNA expression, increased post-translational degradation of DIO2, and competitive inhibition of DIO2. The reduction in DIO2 activity by PBDE and OH-BDE exposures could potentially reduce the concentration of T3 in the brain, which may be responsible for the neurodevelopmental impairments produced by exposure to this class of compounds and needs to be further explored.

In the third aim of this thesis research, the effects of PBDEs and OH-BDEs were examined in the H4 cells and in a mixed culture containing a human neuroblastoma cell line (SK-N-AS cells). The SK-N-AS cells express the thyroid hormone-inactivating enzyme Type 3 deiodinase (DIO3), which works in concert with DIO2 to buffer the concentration of T3 in the brain. Exposure to BDE-99 decreased the concentration of T3 and the inactive thyroid hormone rT3 in the cell culture medium of co-cultured cells by 59-76%. 3-OH-BDE-47 competitively inhibited DIO3 with an IC50 of 19 uM. 5'-OH-BDE-99 increased the rT3 concentrations in cell culture medium by 400%, increased DIO3 activity in exposed cells by 50%, and increased DIO3 catalytic activity in cellular homogenates by over 500%. Further effects on the mRNA expression of several thyroid-regulated genes (DIO3, TR-a, TR-b, MCT8, and ENPP2) and oxidative respiration were also assessed in the SK-N-AS cells. DIO3 mRNA expression increased by 9 fold in cells exposed to 400 nM BDE-99, and ENPP2 mRNA expression increased by 2 fold in cells exposed to 500 nM BDE-99 and a mixture of the three congeners, but no other significant effects on mRNA expression were observed. The basal respiration rates and other parameters of oxidative respiration were also not significantly altered by exposure to PBDEs or OH-BDEs, but proton leak was increased by over 400% in cells exposed to 2 uM 5'-OH-BDE-99.

This was the first study to examine the effects of an environmental contaminant on human DIO2 and DIO3 in cultured cells. The results indicated that BDE-99 and OH-BDEs decreased the activity of DIO2 and 5'-OH-BDE-99 increased the activity of DIO3, which combined would lead to decreased levels of T3 exported from the cells into the extracellular environment. These results provide more evidence that disruption of DIO2 and DIO3 by PBDEs during development may mediate the neurodevelopment effects associated with PBDEs.

The epigenome underlies cell type and state and in post-mitotic neurons, and it regulates the ability for rapid response to activity. Since neurons exit the cell cycle early in development and are long lived, remodeling of brain function requires that neurons show transcriptional plasticity to let then change in function in response to stimuli including psychostimulants and developmental cues. This response is driven by the epigenomic regulation in a cell-type-specific manner. Many studies assessing experience driven genomic responses have been carried out in bulk tissues so cell-type-specific genomic responses to stimuli that drive neuronal plasticity remains poorly understood. To understand the epigenomic and transcriptomic mechanisms driving neuronal plasticity, here we study multi-omic genomic data from two contexts in the mouse brain: 1) psychostimulant responses in the nucleus accumbens and 2)the postnatal and postmitotic maturation of developing cerebellar granule neurons. In both systems, I implemented integrative bioinformatic approaches to predict transcription factor (TF) activity in regulating the transcriptome. I elucidated cell-type-specific amphetamine induced transcriptomic responses, identified canonical activity regulated transcription factors regulating those responses, and determined collaborators and developmental targets of the Zic family TFs, revealing novel roles of Zics regulating migration and synaptic maturation in CGN development. The studies reveal novel mechanistic insights into neuronal plasticity in different neuronal cell types by using integrative computational approaches to model chromatin topology, chromatin accessibility, gene expression, and TF binding.

We have a rudimentary understanding of the consequences of preconception exposure to cannabis. As the most commonly used illicit psychoactive drug, cannabis prevalence is rapidly increasing across the United States (US), and consumers are increasingly perceiving it as safe. Recreational cannabis use is especially common among American men, rendering the paternal preconception environment potentially vulnerable to deleterious effects. Parental cannabis use has been associated with adverse developmental outcomes in offspring, but little is known about how such phenotypes are transmitted. Gametic epigenetic changes – the collection of molecular modifications made to DNA and histone proteins that play a role in regulating gene activity – provide one potential explanation. In a pilot study, our group recently demonstrated that cannabis use in humans, and exposure to delta-9-tetrahydrocannabinol (THC, the main psychoactive component of cannabis) in rats, is associated with significantly altered levels of DNA methylation in sperm. Epidemiological studies have further associated prenatal cannabis use with an increased risk of numerous teratologies including neurodevelopmental disorders and cardiovascular defects. While these studies illuminate the risks associated with cannabis and the prenatal environment, little attention has been paid to the effects of paternal preconception exposures alone on such congenital anomalies. There remains an urgent need to investigate the effects of cannabis on the sperm epigenome as use increases across the globe. Further, it is critical to investigate these effects on genes that are uniquely positioned to contribute to early development. The hypothesis of this research was that cannabis exposure is associated with heritable, but reversible, changes in DNA methylation in sperm at genes important for early life development. Broadly, the main objectives of this thesis research were to generate meaningful data to contribute to the gaps in knowledge of how cannabis can impact sperm DNA methylation. The results of this thesis research are described beginning in chapter 2. An initial pilot study from our group used reduced representation bisulfite sequencing (RRBS) to analyze methylation changes in sperm between cannabis users and controls. One gene identified as being significantly differentially methylated was an autism candidate gene, Discs-Large Associated Protein 2 (DLGAP2). Quantitative bisulfite pyrosequencing confirmed that an intronic region of DLGAP2 was significantly hypomethylated in the sperm of cannabis exposed men compared to controls. Use of human fetal brain tissues demonstrated that there is a significant, sex-specific, inverse relationship between DNA methylation and gene expression at this locus. A paternal rat model of THC exposure was used to determine whether these effects at Dlgap2 were heritable. Bisulfite pyrosequencing identified significant changes in rat sperm DNA methylation at Dlgap2, as well as significant losses of methylation at the same locus in F1 nucleus accumbens (NAc) tissues. Having demonstrated that autism candidate gene DLGAP2 shows functional changes in DNA methylation, the next question addressed was whether different routes of THC exposure, and exposure to different drugs, could similarly impact DNA methylation at a select group of neuroactive genes. Sperm DNA from rats exposed to THC or vehicle control via oral gavage underwent RRBS. Bisulfite pyrosequencing of sperm DNA from rats exposed to injected THC or vehicle control was performed to examine methylation at regions identified by RRBS. Sperm DNA from rats exposed to nicotine or vehicle control underwent pyrosequencing at the same regions. Lastly, two publicly available datasets were investigated to determine significant overlap between a known list of autism candidate genes and a list of genes with bivalent chromatin, a unique epigenetic feature. In the sperm of rats injected with THC and those nicotine-exposed, significant differential methylation at five of seven neurodevelopmentally active genes that were initially identified as significantly altered by oral gavage was identified. It was further discovered that autism candidate genes are significantly enriched for genes containing bivalent chromatin. Enrichment of both autism candidate genes and genes possessing bivalent chromatin was identified in the human RRBS dataset of genes significantly differentially methylated in sperm of cannabis users. These studies demonstrated THC and nicotine exposure in rats can impact DNA methylation in sperm at neuroactive genes. Further, this work provides initial evidence that genes with bivalent chromatin may be particularly vulnerable to DNA methylation changes resulting from environmental exposures. The fourth chapter of this thesis research employed a novel in vitro model of human spermatogenesis to identify the impact of exposure to a cannabis smoke extract (CSE) on DNA methylation at two groups of genes important for early life development. Human embryonic stem cells (hECS) exposed to CSE or vehicle control were differentiated into a mixed population of spermatogonial stem-like cells (SSCs), primary spermatocyte-like cells, secondary spermatocyte-like cells, and round haploid spermatid-like cells over a ten-day period. Following differentiation, flow cytometry was performed to isolate SSC-like cells and haploid spermatid-like cells for DNA methylation analyses. Methylation was first analyzed at a group of imprinted genes. Significant effects of exposure were identified in SSC-like cells at Sarcoglycan Epsilon (SGCE) and in haploid spermatid-like cells at Paternally Expressed 3 (PEG3) and Growth Factor Receptor-Bound Protein 10 (GRB10). Next, methylation was assessed at a group of genes randomly chosen genes from the Simons Foundation Autism Research Initiative (SFARI) autism candidate gene list, half of which possessed bivalent chromatin at the specific CpG sites analyzed and half of which did not. Significant methylation changes were identified in SSC-like cells at genes from the SFARI list possessing bivalent chromatin, but not at genes from the SFARI list without this epigenetic modification. These results support the hypothesis that bivalent chromatin may make genes more vulnerable to environmental exposures. Chapter five of this thesis research addressed the potential heritability of the impacts of exposure to CSE. Changes in F0 sperm DNA methylation were initially identified via whole genome bisulfite sequencing (WGBS) and methylation changes were validated at select genes via bisulfite pyrosequencing. Methylation changes validated in F0 sperm at the gene 2-Phosphoxylose Phosphatase 1 (Pxylp1) were similarly present in F1 sperm, while changes validated in F0 sperm at Gamma-Aminobutyric Acid Type A Receptor Subunit Beta2 (Gabrb2) and Metastasis Suppressor 1-Like Protein (Mtss1l) were similarly present in F1 hippocampal, and NAc and hippocampal tissues, respectively. Further, for Mtss1l a significant, sex-specific relationship between DNA methylation and gene expression in offspring NAc was demonstrated. Phenotypically, rats born to CSE-exposed fathers exhibited significant cardiomegaly relative to those whose fathers were CSE-naïve. Finally, chapter six of this thesis research addressed whether or not the effects of cannabis on human sperm DNA methylation were reversible. Men were recruited to participate in the study as cannabis users and non-user controls. Semen samples were collected at baseline, and then again following an 11-week cannabis-abstinence period. WGBS was performed on all sperm samples. There were no significant differences between users and controls based on demographic information or measured semen parameters. WGBS quantified DNA methylation changes in sperm. Importantly, a reduction in the magnitude of methylation difference between users and controls after the abstinence period relative to the methylation difference present before abstinence was observed. However, select genes retained their altered methylation patterns after abstinence, suggesting not all cannabis-induced effects were ameliorated. Bioinformatic analysis of genes associated with significantly differentially methylated CpG sites revealed terms associated with nervous system development, cardiovascular system development, and embryonic development. Together, this suggests that the abstinence period is at least partially effective at resolving the methylation changes observed following cannabis use at genes important for early development. This research adds to the emergent literature that cannabis is able to impact DNA methylation in sperm at genes important for early life development. It demonstrates in rodents the ability of this exposure to induce heritable epigenetic and phenotypic effects in offspring. Further, it provides the first evidence that abstinence from cannabis use might help resolve the methylation changes that arise in sperm following this exposure. Future work should assess the ability of this exposure to impact offspring methylation and neurodevelopmental outcomes in children and should define how long abstinence from cannabis use must last to produce the most robust amelioration effects.

Maternal smoking is a deleterious and preventable risk to fetal health. Maternal tobacco smoke (TS) exposure in humans has been linked to impaired fetal growth, preterm birth, sudden infant death syndrome, and neurobehavioral disorders including cognitive dysfunction, attention-deficit hyperactivity disorder (ADHD) and autism spectrum disorder (ASD). In the United States, nearly 10% of pregnant women smoke despite ongoing public health efforts to reduce the incidence of smoking. Of additional concern is the steady rise of electronic nicotine delivery systems (ENDS) use among pregnant women over the past decade. Further, ENDS are often used in conjunction with tobacco cigarettes, compounding exposure effects. In animal models, both maternal TS and nicotine exposure lead to adverse neurodevelopmental outcomes, including increased anxiety and ADHD-like behavior, that are transmitted to subsequent generations. A likely explanation for this phenomenon lies in early developmental epigenetic programming. Epigenetic markers that are established early in development, like DNA methylation, can persist throughout somatic cell division and gametogenesis. During early development, zygotic DNA methylation is reprogrammed following a wave of global demethylation, and only re-established during the peri-implantation period. Environmental perturbations during these critical phases of reprogramming have been associated with persistent, and even transgenerationally-inherited effects, underscoring the importance of examining these associations in the context of human health and disease.

The broader goals of this dissertation were to identify alterations in DNA methylation patterning in the brain as a result of maternal TS exposure, and assess their neurodevelopmental significance. In an effort to better understand the effects of nicotine alone, especially given the increasing usage of ENDS during pregnancy, we chose to examine developmental nicotine exposure in addition to TS exposure. Finally, we evaluated the translational significance of these alterations by examining correlations in humans developmentally exposed to TS. Using a rat model for gestational nicotine exposure, DNA methylation levels were measured in the brains of neonatal and adult rats to determine the persistence of exposure effects. Specific brain regions, including the rat preoptic area (POA), hippocampus and cortex were targeted for evaluation based on their neurobehavioral significance. A rat gestational exposure model for tobacco smoke extract (TSE) was further employed to determine potential overlap with methylomic regions affected by developmental nicotine exposure. Finally, to derive translational implications, DNA methylation was examined on both epigenome-wide and targeted levels in human cord blood from newborns exposed to maternal TS.

In neonatal rats developmentally exposed to nicotine, DNA methylation was reduced in regions implicated in masculinization of the preoptic area (POA), a region of the brain that requires epigenetic reprogramming events to sexually differentiate. Subsequent behavioral analyses in adulthood revealed that these alterations may have contributed to the developmental masculinization of the POA in nicotine-exposed females. In adults males developmentally exposed to nicotine, alterations to DNA methylation were observed in the hippocampus and cortex, two brain regions that are also associated with ADHD- and ASD-like behaviors, respectively. Further, a comparison of differentially-methylated regions (DMRs) between the brains of animals exposed to developmental nicotine and TSE revealed significant overlap, indicating that nicotine is largely driving the developmental alterations to DNA methylation observed in TSE-exposed animals. Examination of DNA methylation alterations in human infant cord blood as a result of maternal TS exposure indicated significant overlap with those revealed in rats, supporting common impacts on developmental epigenetic reprogramming across species. Moreover, nearly half of these common regions were implicated in neurodevelopmental disorders, namely ASD and ADHD. Alterations to DNA methylation at human metastable epialleles, or regions for which DNA methylation is stochastically established during early development, were observed in the cord blood of infants exposed to TS in utero, supporting the ability of TS-exposure to alter vulnerable regions of the epigenome during early developmental reprogramming.

The IFN-γ/STAT1 response is an immune signaling pathway well known for its potent pro-inflammatory and anti-viral functions. However, IFN-γ/STAT1 signaling also impacts many homeostatic and pathological aspects in the central nervous system, beyond its canonical role in controlling intracellular pathogens. IFN-γ can modulate neuronal excitability, synaptic pruning, and gene expression of pathways associated with neurodevelopmental disorders, including autism spectrum disorder (ASD) and schizophrenia (SZ). Surprisingly, the IFN response was recently identified as the most highly enriched pathway in brains of individuals with ASD and SZ. Children born to mothers who are hospitalized for infection during pregnancy are at a higher risk of developing ASD, and mouse models demonstrate that elevating cytokines during embryonic neurodevelopment cause ASD-like phenotypes. While microglia are thought to be the major targets of IFNs in the brain, neurons can respond to IFNs and require physiological levels of IFN-γ for proper function. The IFN-γ/STAT1 pathway is rapidly activated then deactivated to prevent excessive inflammation; however, neurons utilize unique IFN-γ/STAT1 activation patterns, which may contribute to the non-canonical neuron-specific downstream effects. We hypothesized that pathological IFN-γ signaling in neurons leads to neuronal dysfunction and behavioral deficits through non-canonical STAT1 signaling. Using primary neuron cultures, we demonstrated that developing neurons have differential STAT1 activation downstream of physiological versus pathological IFN-γ. Physiological levels of IFN-γ caused brief and transient STAT1 activation, while high pathological levels of IFN-γ caused robust and prolonged activation of STAT1 in neurons, but not in microglia or astrocytes. To determine the effects of prolonged STAT1 activation in vivo, we developed a novel mouse model in which STAT1 signaling is prolonged in neurons. These mice displayed hyperactive behavior and neural hypoactivity, which are common comorbidities of neurodevelopmental disorders like ASD and attention deficit hyperactivity disorder (ADHD). Moreover, we demonstrated that this phenotype is neuron specific, as mice with prolonged STAT1 activation in microglia did not have behavior deficits. Our findings suggest pathological activation of the IFN-γ/STAT1 pathway contributes to neuronal dysfunction through non-canonical STAT1 activation. Overall, the IFN-γ/STAT1 pathway is critical for normal neurodevelopment and neuronal function in adulthood and provides new insight into a neuron specific neuroimmune mechanism which may contribute to the pathophysiology of neurodevelopmental disorders.