Previous studies of autism spectrum disorder (ASD) have shown abnormalities in brain development, synaptic plasticity, and social and learning behavior. Our previous study has identified the X-linked gene KIAA2022, and its protein product KIDLIA, as the etiological factor in a particular group of patients with intellectual disability and ASD phenotypes. I found that KIDLIA is neuron specific and localized exclusively in the nucleus, indicating a possible role for KIDLIA in neuronal gene regulation. Using rat hippocampal neurons, I found that shRNA-mediated knockdown of KIDLIA resulted in a marked impairment of neurite outgrowth via the disruption of the N-cadherin/δ-catenin/RhoA signaling pathway. Additionally, I showed that loss of KIDLIA expression decreases synapse formation and synaptic transmission. To investigate the role of KIDLIA in vivo, I generated and characterized KIDLIA knockout (KO) mice. KIDLIA KO mice demonstrated significant impairments in social interactions, increased repetitive behaviors and deficits in learning and memory, consistent with symptoms observed in human ASD patients and validate this mouse line as a valuable new model for ASD. The KIDLIA KO mice showed reduced synapse formation and abnormal expression of synaptic components such as the GluA1 subunit of AMPA receptors. To understand the potential role of KIDLIA in gene regulation, I used RNAseq to identify major candidates involved in synapse formation and function and discovered the synapse-enriched Ca2+-mediated protein, neurogranin, as the most down-regulated synaptic transcript. I showed that over-expression of neurogranin can rescue KIDLIA-dependent structural and functional synaptic deficits. This study provides valuable insight into the cellular and molecular mechanisms underlying KIDLIA-dependent autism and intellectual disability phenotypes. / 2018-11-08T00:00:00Z

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / A esquizofrenia à um transtorno psiquiÃtrico grave e, talvez, por isso, um dos mais pesquisados. Apesar dos importantes avanÃos realizados nos estudos sobre a fisiopatologia da doenÃa, durante o sÃculo passado, poucos benefÃcios foram significativos no tratamento pacientes. A elaboraÃÃo de novos modelos animais de esquizofrenia à uma ferramenta importante para a compreensÃo da fisiopatologia da doenÃa e para o desenvolvimento de novas terapias, uma vez que atà mesmo os antipsicÃticos atÃpicos nÃo mostraram os resultados esperados em relaÃÃo à melhora dos sintomas negativos e cognitivos. O objetivo desta pesquisa foi investigar os efeitos da cetamina no sistema nervoso central como um possÃvel agente em um modelo neurodesenvolvimental da esquizofrenia. Procurou-se determinar as alteraÃÃes de comportamento e estresse oxidativo pela administraÃÃo de cetamina neonatal, bem como a reversÃo e prevenÃÃo destes efeitos pelo tratamento com clozapina, um antipsicÃtico atÃpico. TambÃm realizou-se a avaliaÃÃo do sexo no desenvolvimento da esquizofrenia, dividindo as fÃmeas pelo ciclo estral, cujas fases apresentam concentraÃÃes altas e baixas de estrogÃnio, proestro e diestro, respectivamente, visto que estrogÃnio apresenta um efeito neuroprotetor. O delineamento experimental seguiu os critÃrios para determinar um modelo animal: a validade de face (onde se procura imitar o comportamento caracterÃstico da doenÃa no animal), validade do constructo (a fisiopatologia da doenÃa) e validade preditiva (se os medicamentos estabelecidos para a doenÃa sÃo capazes de inverter e prevenir os efeitos que a droga pesquisada induz). Os animais utilizados no protocolo experimental foram ratos Wistar, divididos em seis grupos e tratados com soluÃÃo salina ou cetamina intraperitoneal (i.p.) durante cinco dias (7Â- 11 dias apÃs o nascimento). Grupo 1 (machos tratados com soluÃÃo salina); Grupo 2 (fÃmeas tratadas com soluÃÃo salina) ; Grupo 3 (machos tratados com cetamina 2,5 mg/kg), Grupo 4 (fÃmeas tratadas com cetamina 2,5 mg/kg), grupo 5 (machos tratados com cetamina 5 mg/kg); Grupo 6 (fÃmeas tratadas com cetamina 5 mg/kg). Os testes de inibiÃÃo prÃ-pulso e y-maze (testes comportamentais) foram realizados na adolescÃncia (35 dias apÃs o nascimento), e repetidos na fase adulta (65 dias apÃs o nascimento). Na fase adulta, as fÃmeas foram divididas de acordo com o ciclo menstrual (diestro e de proestro) para observar a interferÃncia do estrogÃnio. No 66 dia apÃs o nascimento, os animais tratados com cetamina 5 mg/kg foram submetidos ao tratamento de reversÃo pela administraÃÃo de clozapina 10 mg/kg (i.p.), uma vez ao dia, durante 10 dias. ApÃs este perÃodo, os testes comportamentais e neuroquÃmicos (dosagem de BDNF e GSH) foram realizados. Os animais tratados com cetamina 2,5 mg/kg nÃo apresentaram qualquer diferenÃa significativa nos testes comportamentais realizados na fase adulta, quando comparados com os animais tratados com soluÃÃo salina, e, portanto, a dose escolhida para medir os outros parÃmetros foi de 5 mg/kg. O tratamento com cetamina 5 mg/kg reduziu significativamente os parÃmetros observados nos testes comportamentais, em machos e fÃmeas no diestro, na fase adulta. Os machos e as fÃmeas no diestro tambÃm apresentaram uma reduÃÃo significativa nos nÃveis de GSH. Os machos adultos tratados com cetamina 5 mg/kg apresentaram um aumento significativo de BDNF, enquanto que as fÃmeas no diestro os apresentaram uma reduÃÃo neste parÃmetro. As fÃmeas no proestro alcanÃaram um melhor desempenho em todos os testes. No protocolo de prevenÃÃo, o tratamento com clozapina 10mg/kg reverteu os efeitos da cetamina para os ensaios de inibiÃÃo prÃ-pulso e nos testes neuroquÃmicos, em machos e fÃmeas no diestro, tratados com cetamina 5 mg/kg. Os resultados observados nos testes comportamentais e neuroquÃmicas demonstraram que a cetamina induziu os parÃmetros da esquizofrenia, apresentando-se como uma nova ferramenta para a pesquisa de modelos animais esquizofrenia neonatal. O modelo desenvolvido conseguiu, pela primeira vez, mimetizar as diferenÃas entre as fases do ciclo estral dos animais, simulando o que acontece em humanos. / Schizophrenia is a severe psychiatric disorder, and maybe, because of this, one of the most researched. Despite the important advances achieved in the studies about the physiopathology of the disease during the last century, few benefits were significant in the treatment of these patients. The construction of new animal models of schizophrenia is an important tool for the comprehension of the physiopathology of the disease and the development of new therapies, since even the atypical antipsychotics did not show the expected results regarding the improvement of negative and cognitive symptoms. The objective of this research was to investigate the effects of ketamine in the central nervous system as a possible agent in a neurodevelopmental model of schizophrenia. We sought to determinate the behavioral changes and oxidative stress by the neonatal administration of ketamine, as well as the reversion and prevention of these effects by the treatment with clozapine, an atypical antipsychotic. We also conducted an analysis of the role of sex at the development of schizophrenia, dividing the females by the estrous cycle, that includes phases presenting high and low concentrations of estrogen, proestrus and diestrus, respectively, since estrogen presents a neuroprotective effect. The experimental design followed the criteria to determine an animal model: the face validity (where one seeks to mimic the characteristic behavior of the disease in the animal), construct validity (the physiopathology of the disease) and predictive validity (if the established medicines for the disease are able to reverse and prevent the effects that the researched drug induces). The animals used at the experimental protocol were Wistar rats, divided in six groups and treated with saline or ketamine intraperitoneal (i.p.) for five days (7th-11th day after birth). Group 1: (males treated with saline) Group 2 (females treated with saline); Group 3 (males treated with ketamine 2,5mg/kg); Group 4 (females treated with ketamine 2,5mg/kg); Group 5 (males treated with ketamine 5 mg/kg); Group 6 (females treated with ketamine 5 mg/kg).The tests of prepulse inhibition and y-maze (behavioral tests) were conducted in adolescence (35 days after birth), and repeated at the adult phase (65 days after birth). At the adult phase, the females were divided according with the estrous cycle (diestrus and proestrus) to observe the influence of estrogen. At the 66th day after birth, the animals trated with ketamine 5 mg/kg were submitted to a reversion treatment by clozapine 10 mg/kg i.p. once a day during 10 days. After this period, the behavioral and neurochemical (dosage of BDNF and GSH) tests were performed. The animals treated with ketamine 2,5 mg/kg did not show any significant difference in the behavioral tests conducted in the adult phase when compared with the animals treated with saline and, therefore, the dose selected to measure the other parameters was 5,0 mg/kg. The treatment with ketamine 5 mg/kg significantly reduced the parameters observed at the behavioral tests, both in adult males and females at the diestrus. The males and females at the diestrus also presented a significant reduction in the GSH levels. The adult males treated with ketamine 5 mg/kg presented a significant increase in BDNF, whereas the females at the diestrus presented a reduction in this parameter. The females at the proestrus achieved a better performance in all tests. In the prevention protocol, the treatment with clozapine 10mg/kg reverted the effects of ketamine in the tests of prepulse inhibition and in the neurochemical testes in males and females in the diestrus that were treated with ketamine 5 mg/kg.The results observed in the behavioral and neurochemical tests showed that ketamine induced the parameters of schizophrenia, presenting itself as a new tool available to the research of animal models of neonatal schizophrenia. The developed model succeeded for the first time in mimicking the differences between the phases of the estrous cycle of the animals, in a close approximation of what happens in humans

Functional magnetic resonance imaging studies have long demonstrated a high degree of correlated activity between the left and right hemispheres of the brain. Interregional correlations between the time series of each brain voxel or region and its homotopic pair have recently been identified by methods such as homotopic resting-state functional connectivity (H-RSFC). However, little is known about whether interhemispheric regions in patients with Attention-deficit/hyperactivity disorder (ADHD) are functionally abnormal. The aim of this thesis is to examine the association between H-RSFC and medication status, age, sex, and volumetric asymmetry index (AI). In our approach, region-based activity was obtained using three different methods. To test for associations, two linear mixed-effects models were used. Across results, H-RSFC variation was found in subcortical regions and portions of cortical regions. In addition, changes in functional connectivity were found to be linked with structural asymmetry in two cortical regions. More importantly, shifting in homotopic functional activation was found as a result of medication intake in youths with ADHD. These findings demonstrate the utility of homotopic resting-state functional connectivity for measuring differences among pharmacotherapy intake, gender, neurodevelopment, and structural asymmetry.

<p>  </p> <p>Recent advancements in genetics have revealed that <em>SCN2A</em> is one of the leading genes associated with neurodevelopmental disorders including autism spectrum disorder and epilepsy. In particular, loss-of-function and truncation variants account for a majority of cases. As there are no current treatments specific for <em>SCN2A</em>, the neuropharmacogenomics field has strived to further elucidate the role of <em>SCN2A</em> in neurodevelopment to identify intervention targets. Rodent models offer <em>in vivo</em>, pre-clinical insight into the effects of genetic variation on behavior, biochemistry, and electrophysiology as well as the mechanisms on molecular, cellular, and circuitry levels. Due to <em>SCN2A</em>’s critical involvement in the initiation and propagation of action potential neuronal firing early in neurological development, full null homozygous knockout of <em>Scn2a</em> in mice is perinatal lethal. Furthermore, canonical heterozygous knockout of <em>Scn2a </em>in mice does not render phenotypes that recapitulate <em>SCN2A</em> deficiency in humans. Therefore my dissertation aims at developing a mouse model that better parallels the human condition, then using that pre-clinical platform to explore precision medicine.</p> <p>  </p> <p>Using the unconventional strategy of gene trapping, we generated mice with a severe reduction in <em>Scn2a</em> expression, resulting in significant behavioral and electrophysiological differences from neurotypical wild-type mice with full <em>Scn2a</em> expression, but enough residual expression that the <em>Scn2a</em>-deficient mice survived into adulthood. The severely decreased sociability accompanied by increased high and low order repetitive behaviors observed with the <em>Scn2a</em>-deficient mice suggest autism-like phenotypes. In addition, <em>Scn2a</em>-deficient mice also displayed other co-morbidities of neurodevelopmental disorders including atypical innate behavior, increased anxiety, increased sensitivity to stimuli, motor discoordination, and impaired learning and memory. On the electrophysiological level, these mice displayed enhanced intrinsic excitabilities of principal neurons in the prefrontal cortex and striatum, brain regions known to be involved in seizures and social behavior. This increased excitability was autonomous and reversible by the genetic restoration of <em>Scn2a</em> expression in adult mice. Further, RNA-sequencing revealed a downregulation of multiple potassium channels as well as differential expression of glutamate excitatory and GABA inhibitory signaling, which led to the pursuit of targeting these pathways. Indeed, the use of potassium channel openers alleviated the hyperexcitability of <em>Scn2a</em>-deficient neurons, thus supporting the pursuit of these targets.  </p> <p>Since characterization of the <em>Scn2a</em>-deficient mouse model revealed disruption in excitatory and inhibitory pathways, excitatory/inhibitory balance was examined further as a precision medicine target. Increasing <em>Scn2a</em> expression throughout the whole brain by excising the gene trap, as well as specific targeting of the striatum and the neurons that project to it using a retrograde viral vector, rescued social deficits. However the striatum-specific injection did not lead to a social rescue. This shifted the focus to the neurons that project to the striatum such as the medial prefrontal cortex. Using chemogenetics to reduce excitatory signaling in the prelimbic region of the medial prefrontal cortex, we were able to increase the social behavior in <em>Scn2a</em>-deficient mice. Synthesizing the results from the retrograde striatum and prelimbic-specific rescue, the next hypothesis tested was a circuity-level manipulation of the medial prefrontal cortex projections to the striatum. Retrograde control (striatum) of chemogenetics (medial prefrontal cortex) decreased the excitatory signaling in the medial prefrontal cortex neurons that project to the striatum, which also led to improved sociability. On the other side of the excitatory/inhibitory balance, increasing inhibitory signaling through acute exposure to small-molecule GABA receptor positive allosteric modulators, clonazepam and AZD7325, rescued sociability.</p> <p>This dissertation opens up new avenues of research by supporting the use of a pre-clinical mouse model of <em>Scn2a</em> deficiency to advance the study of underlying mechanisms behind <em>SCN2A</em>-related neurodevelopmental disorders. Although the results of this dissertation need additional validation such as cellular support, the data and results in this dissertation can serve as a guide to further explore excitatory/inhibitory balance as a neuropharmacogenomics precision medicine target to treat <em>SCN2A</em>-related neurodevelopmental disorders. </p> <p><br></p> <p><br></p>

Large-scale transcriptomic studies are among of the most comprehensive accounts we have of the biological processes underlying human brain development and ageing. However, many analyses and descriptive models applied to gene expression data implicitly assume that developmental change is continuous and uninterrupted. Perhaps this bias is often overlooked because the emphasis is on what is changing during development rather than how development itself is changing. Indeed, despite the richness of transcriptomic data and its capacity to recapitulate higher-order functions, few have used it to understand the dynamics of brain development. Gene expression is determined by complex, high-dimensional interactions of the gene regulatory network. Dynamic systems theory states that the interactions of components in any complex systems will converge on certain stable patterns, also known as attractor states. To approximate these stable states, the current study leveraged robust and sparse k-means clustering to identify tissue samples with similar patterns of gene expression across the transcriptome. Sample ages were then used to visualize when in developmental time these stable patterns are present. The resulting model describes the developmental dynamics of the brain transcriptome as a series of non-linear, overlapping states that progress across the lifespan. / Thesis / Master of Science (MSc)

Neurodevelopmental disorders encompass a spectrum of different conditions with both genetic and environmental etiologies. Although rapid progress has been made in deciphering the genetic landscape of these disorders, in most cases, it remains unclear how mutations undermine neurodevelopmental mechanisms. However, increasing identification of risk genes suggests chromatin remodelling is frequently impacted. For instance, de novo variants encoding the chromatin remodeller CHD4 causes Sifrim-Hitz-Weiss syndrome, which manifests as an overgrowth-intellectual disability syndrome. To further understand Chd4’s role during cortical development, we excised the ATPase domain of Chd4 in the germline or specifically in the developing telencephalon, creating three mouse models. Germline heterozygotes presented a slight decrease in brain weight, cortex area and Ctip2+ cells, with females displaying more overt impairments in learning and memory. Telencephalon-specific conditional heterozygotes exhibited slight changes in white matter, increased repetitive movements and altered social behaviours. Telencephalon-specific conditional knockouts presented with decreased brain size, brain weight, and cortex thickness due to decreased upper layer neurons, and anxiety phenotypes. These data reveal an unexpected complexity in the impacts of Chd4 mutations on neurodevelopmental processes and behaviour.

BACKGROUND: While manganese (Mn) is essential for growth and development, evidence for Mn as a developmental neurotoxicant is mounting. However, inconsistencies in exposure metrics, susceptibility factors and neurobehavioral outcomes muddle the understanding of Mn effects on the developing nervous system. OBJECTIVE: To estimate Mn-neurobehavioral associations in varied neurobehavioral tasks and evaluate susceptibility factors (sex differences, co-exposures, exposure timing). METHODS: Research aims were conducted using the Public Health Impact of Mixed element Exposure (PHIME) study, including 720 Italian adolescents living near ferro-manganese industry. Blood, hair, nails, urine and saliva were collected in adolescence and metals [Mn, lead (Pb), copper (Cu), chromium (Cr)] were measured using ICP-MS. Mn was measured in deciduous teeth in a subset of participants (n=195) using LA ICP-MS to represent prenatal, postnatal and childhood exposure periods. Trained neuropsychologists administered a neurobehavioral battery to adolescents. In the first aim, we estimated associations of early-life Mn levels measured in deciduous teeth with visuospatial learning and memory, assessed using the Virtual Radial Arm Maze (VRAM), a novel animal-human translational neurobehavioral task. The second aim estimated associations of prenatal, early postnatal (0-1 year) and childhood (1~6 years) Mn measured in deciduous teeth with IQ scores and subtests measured by the Wechsler Intelligence Scale for Children (WISC-III). The third aim evaluated the association of a mixture of metals (Mn, Pb, Cu, Cr) measured in multiple biomarkers (hair, blood, urine, nails, saliva) and IQ. Data analysis included generalized additive models, linear regression, zero-inflated Poisson regression (for VRAM count outcomes), and Bayesian kernel machine regression (BKMR). RESULTS: In the first aim we observed U-shaped associations between prenatal Mn and VRAM outcomes among girls only, suggesting that low and high prenatal Mn levels may be harmful to visuospatial learning and memory. In the second aim, several associations were found with specific subtests that assess visuospatial ability, working memory, problem solving and attention, wherein estimates from the prenatal period suggested beneficial Mn associations, unlike the early postnatal and childhood periods. For the third aim we found inverse associations of adolescent Mn, measured either in hair or saliva, with verbal IQ (VIQ) scores, and an inverted U-shaped association for hair Cu. Strongest associations for the overall metals mixture were estimated with VIQ, where the joint increase in metals concentrations was associated with lower VIQ scores. / 2022-09-25T00:00:00Z

Methylmercury (MeHg) is a global pollutant that can elicit a range of adverse health effects in both humans and wildlife populations. Humans are often exposed to MeHg through the consumption of contaminated seafood. Developing fetuses are especially susceptible to the effects of MeHg as it can cross the blood-brain barrier and the placenta. At high doses in utero MeHg causes developmental disorders and congenital disabilities, but long-term low-dose effects are still not fully known. Using a culture model of cerebral cortex development, our lab has shown that low-dose MeHg promotes premature neuronal differentiation. Autism spectrum disorder (ASD) has been associated with prenatal MeHg exposure and is correlated with neuronal overproduction, but a cause-effect relationship has not been shown. In this thesis, I aim to test the hypothesis that prenatal exposure to low-dose MeHg can cause ASD-like symptoms in the offspring following premature neuronal differentiation. My results showed that adult mice prenatally exposed to MeHg exhibited key ASD characteristics including impaired communication, reduced sociability, and increased restrictive repetitive behaviours. Furthermore, I explored the underlying cellular and molecular mechanism that promotes premature neuronal differentiation caused by prenatal MeHg exposure. To reverse the MeHg-induced premature neuronal differentiation, I utilized metformin, an FDA-approved diabetes drug. Overall, these findings provide insights into the toxicology of MeHg and its relationship with ASD etiology, including the underlying mechanism, and a potential therapeutic strategy.

No description available.

<p dir="ltr">Down syndrome (DS) arises from the triplication of human chromosome 21 (Hsa21), leading to a spectrum of phenotypes characterized by neurodevelopmental and cognitive abnormalities. The Ts65Dn mouse model emulates DS by harboring three copies of genes found on Hsa21 resulting in trisomy 21 (Ts21)- like traits, including disruptions in neuronal pathways, delays in sensorimotor and behavior milestones, and deficits in learning and memory tasks. There is no cure for DS and available therapies primarily address symptoms stemming from Ts21-associated phenotypes. <i>DYRK1A</i>, a gene triplicated in Ts21, has a pivotal role in pathways of neurodevelopment and has been a focus of inhibition treatment research aimed at preempting abnormal brain phenotypes. This study aimed to find a point of substantial <i>Dyrk1a </i>expression dysregulation during a period of critical neonatal neurodevelopment and employ targeted pharmacological and genetic knockdown methods to alleviate the presence or severity of characteristically abnormal brain and behavior phenotypes. The hypothesis of this study was that administering a targeted intervention prior to a point of known overexpression in trisomic pups would ameliorate molecular, sensorimotor, and neurobehavioral deficits, redirecting growth trajectories of Ts65Dn neonatal pups towards more neurotypical outcomes. To test this hypothesis, the spatiotemporal pattern of DYRK1A expression was quantified during the first three weeks of neonatal development across the hippocampus, cerebral cortex, and cerebellum of the Ts65Dn mouse model and found to fluctuate according to the genotype, age, sex, and brain region of the subject. <i>Dyrk1a </i>protein and mRNA expression levels were delineated in trisomic animals by age, exploring the correlation between expression and age, sex, genotype, and brain region. Next a constitutive <i>Dyrk1a </i>knockdown model was integrated with the Ts65Dn model to investigate the impact of gene copy number reduction on protein and mRNA expression levels during phases of known DYRK1A dysregulation. On postnatal day 6, protein expression was rescued in all three brain regions of male animals but was rescued only in the cerebellum of females. There were no significant differences in mRNA transcript levels in either sex at this age. Finally, genetic elements were introduced into the Ts65Dn model to facilitate a spatiotemporally controlled functional reduction of <i>Dyrk1a</i> and discern how the timing of gene copy number reduction affects molecular and neurobehavioral development in a trisomic system. Results from these studies suggest that only functionally reducing <i>Dyrk1a </i>gene copy number on the day of birth is not sufficient to rescue the majority of deficits and delays present in the Ts65Dn mouse model of DS. These findings significantly enhance the understanding of trisomic <i>Dyrk1a </i>expression dynamics during neonatal development and shed light on tailored therapeutic approaches to modulate intrinsic DS characteristics based on age, sex, and phenotypic considerations.</p>