Effects of Heterozygous Deletion of Autism-related gene Cullin-3 in mice

Autism Spectrum Disorder (ASD) is a developmental disorder in which children display repetitive behavior, restricted range of interests, and atypical social interaction and communication. CUL3, coding for a Cullin family scaffold protein mediating assembly of ubiquitin ligase complexes through BTB domain substrate-recruiting adaptors, has been identified as a high-risk gene for autism. Although complete knockout of Cul3 results in embryonic lethality, Cul3 heterozygous mice have reduced CUL3 protein, demonstrate comparable body weight, and display minimal behavioral differences including decreased spatial object recognition memory. In measures of reciprocal social interaction, Cul3 heterozygous mice behaved similarly to their wild-type littermates. In area CA1 of hippocampus, reduction of Cul3 significantly increased mEPSC frequency but not amplitude nor baseline evoked synaptic transmission or paired-pulse ratio. Sholl and spine analysis data suggest there is a small yet significant difference in CA1 pyramidal neuron dendritic branching and stubby spine density. Unbiased proteomic analysis of Cul3 heterozygous brain tissue revealed dysregulation of various cytoskeletal organization proteins, among others. Overall, our results suggest that Cul3 heterozygous deletion impairs spatial object recognition memory, alters cytoskeletal organization proteins, but does not cause major hippocampal neuronal morphology, functional, or behavioral abnormalities in adult global Cul3 heterozygous mice

Pathophysiological mechanisms in neurodevelopmental disorders caused by rac GTPases dysregulation: What’s behind neuro-RACopathies

Rho family guanosine triphosphatases (GTPases) regulate cellular signaling and cytoskele-tal dynamics, playing a pivotal role in cell adhesion, migration, and cell cycle progression. The Rac subfamily of Rho GTPases consists of three highly homologous proteins, Rac 1–3. The proper function of Rac1 and Rac3, and their correct interaction with guanine nucleotide-exchange factors (GEFs) and GTPase-activating proteins (GAPs) are crucial for neural development. Pathogenic variants affecting these delicate biological processes are implicated in different medical conditions in humans, primarily neurodevelopmental disorders (NDDs). In addition to a direct deleterious effect produced by genetic variants in the RAC genes, a dysregulated GTPase activity resulting from an abnormal function of GEFs and GAPs has been involved in the pathogenesis of distinctive emerging conditions. In this study, we reviewed the current pertinent literature on Rac-related disorders with a primary neurological involvement, providing an overview of the current knowledge on the pathophysiological mechanisms involved in the neuro-RACopathies

mTOR-dependent spine dynamics in autism

Autism Spectrum Conditions (ASC) are a group of neurodevelopmental disorders characterized by deficits in social communication and interaction as well as repetitive behaviors and restricted range of interests. ASC are complex genetic disorders with moderate to high heritability, and associated with atypical patterns of neural connectivity. Many of the genes implicated in ASC are involved in dendritic spine pruning and spine development, both of which can be mediated by the mammalian target of rapamycin (mTOR) signaling pathway. Consistent with this idea, human postmortem studies have shown increased spine density in ASC compared to controls suggesting that the balance between autophagy and spinogenesis is altered in ASC. However, murine models of ASC have shown inconsistent results for spine morphology, which may underlie functional connectivity. This review seeks to establish the relevance of changes in dendritic spines in ASC using data gathered from rodent models. Using a literature survey, we identify 20 genes that are linked to dendritic spine pruning or development in rodents that are also strongly implicated in ASC in humans. Furthermore, we show that all 20 genes are linked to the mTOR pathway and propose that the mTOR pathway regulating spine dynamics is a potential mechanism underlying the ASC signaling pathway in ASC. We show here that the direction of change in spine density was mostly correlated to the upstream positive or negative regulation of the mTOR pathway and most rodent models of mutant mTOR regulators show increases in immature spines, based on morphological analyses. We further explore the idea that these mutations in these genes result in aberrant social behavior in rodent models that is due to these altered spine dynamics. This review should therefore pave the way for further research on the specific genes outlined, their effect on spine morphology or density with an emphasis on understanding the functional role of these changes in ASC

The Role Of The Nmda Receptor In Shaping Cortical Activity During Development

Currently, it is estimated that neuropsychiatric disorders will affect 20-25% of humans in their lifetime. These disorders are a major cause of mortality, suffering, and economic cost to society. Within this broad class, neurodevelopmental disorders (NDDs), including intellectual disability, autism spectrum disorder, and schizophrenia, are estimated to affect 2-5% percent of the world population. Devastatingly, we lack fundamental treatments for NDDs, which have proved some of the most imposing disorders to understand scientifically. The challenge is twofold: first, NDDs affect the most complex aspects of human cognition; second, pathogenesis begins early in neural circuit development, but we lack predictive biomarkers before overt behavioral deficits are apparent. Although we have identified many genes associated with these disorders, how underlying genetic disruptions lead to pathological neural network development and function remains unclear. The overarching framework of this dissertation is that all NPDs are disorders of distributed neural networks, and pathophysiology must be understood at this level to effectively intervene clinically. The cerebral cortex is necessary for complex human capacities, and cortical dysfunction is hypothesized to be central to the pathophysiology of NDDs. NMDA glutamate receptors (NMDARs) are important for the development of local circuit features in the cortex, for normal neurocognitive function, and are strongly implicated in NDDs. However, the role of NMDARs in the development of the large-scale cortical network dynamics that underly higher cognition has not been well examined. Understanding the role of NMDARs at this network level is critical because large-scale “functional connectivity” patterns are thought to be hallmarks of normal cortical function, are hypothesized to be disrupted in NDDs, and may be detectable in humans using non-invasive neuroimaging or electrophysiology. In the studies presented in this dissertation, I (in collaboration and with the support of my colleagues) tested the role of the NMDAR in shaping large-scale cortical network organization using in vivo widefield imaging of whole cortex spontaneous activity in developing mice. I found that NMDAR function in the lineage that includes cortical excitatory neurons and glia, specifically, was critical for the elaboration of normal cortical activity patterns and dynamic network organization. In the first set of experiments, NMDARs were deleted in glutamatergic excitatory neurons (Emx1-cre+/WT/Grin1f/f ; referred to as EX-NMDAR KO mice) or GABAergic inhibitory neurons (Nkx2.1+/WT/Grin1f/f; referred to as IN-NMDAR KO mice). The developing cortex normally exhibits a diverse range of spatio-temporal patterns, reflecting the emergence of functionally associated sub-networks. In EX-NMDAR KO mice, normal patterns of spontaneous activity were severely disrupted and reduced to a nearly one-dimensional dynamic space dominated by large, cortex-wide events. Interestingly, in IN-NMDAR KO mice, the structure and complexity of spontaneous activity was largely normal. In the next set of experiments, I tested the role of extrinsic thalamic neurotransmission on cortical activity during development. Deleting the vesicular glutamate transporter from thalamic neurons while leaving cortical NMDARs intact (Sert-Cre+/−,vglut1−/−,vglut2fl/fl; referred to as TH-VG KO mice) led to a shift in cortical activity patterns towards large domains of activity, reminiscent of patterns observed in EX-NMDAR KO mice. This manipulation also reduced the dimensionality of cortical activity, though not as severally as in EX-NMDAR KO mice. In a final set of experiments, I tested cortical activity in three established mouse models of mono-genetic causes of NDDs in humans: the FMR1-KO mouse based on Fragile X Syndrome, the CNTNAP2-KO mouse, and the TS2-neo mouse based on Timothy Syndrome. In all three of these mouse models, I found that large-scale cortical activity patterns were largely normal, but there was a statistically significant shift towards reduced cortex-wide synchrony and increased dimensionality of spontaneous activity, which may be consistent with the disconnectivity hypothesis of autism. In a final set of experiments, we tested our hypothesis, based on past literature and our results in EX-NMDAR KO and TH-VG KO mice, that the disruptions in cortical activity was predominantly due to the developmental loss of activity-dependent wiring of circuits. To test the developmental versus acute role of NMDAR function in shaping cortical activity, I blocked NMDAR pharmacologically in wild-type mice. I found that acute NMDAR blockade shifted cortical activity to a restricted dynamic space similar to that observed in EX-NMDAR KO mice and more extreme than that observed in TH-VG KO mice. These results strongly reinforce the critical role of NMDAR in shaping cortical activity during development, and suggest that a substantial component of that may be through NMDAR’s role in synaptic transmission and moment to moment cortex-wide circuit function. Overall, these results provide critical insight into the role of NMDARs and the glutamatergic system in cortical network functional organization during development. Specifically, they highlight the essential role of NMDARs in excitatory neurons on the functional connectivity and dynamic repertoire of the cortical network during development. These results make novel contribution to our understanding of how NMDARs may contribute to the pathophysiology of NDDs. Specifically, they contribute powerful new insight into to a critical mechanistic question about the cell-specific role of NMDARs in the pathophysiology of schizophrenia and the mechanisms of NMDAR antagonists, which have transformed psychiatry recently due to their rapid-acting anti-depressant and anti-suicidal properties. Furthermore, they identify a patterns of large-scale network dysfunction that might be detectable in humans using noninvasive functional imaging or electrophysiology

Can biological quantum networks solve NP-hard problems?

There is a widespread view that the human brain is so complex that it cannot be efficiently simulated by universal Turing machines. During the last decades the question has therefore been raised whether we need to consider quantum effects to explain the imagined cognitive power of a conscious mind. This paper presents a personal view of several fields of philosophy and computational neurobiology in an attempt to suggest a realistic picture of how the brain might work as a basis for perception, consciousness and cognition. The purpose is to be able to identify and evaluate instances where quantum effects might play a significant role in cognitive processes. Not surprisingly, the conclusion is that quantum-enhanced cognition and intelligence are very unlikely to be found in biological brains. Quantum effects may certainly influence the functionality of various components and signalling pathways at the molecular level in the brain network, like ion ports, synapses, sensors, and enzymes. This might evidently influence the functionality of some nodes and perhaps even the overall intelligence of the brain network, but hardly give it any dramatically enhanced functionality. So, the conclusion is that biological quantum networks can only approximately solve small instances of NP-hard problems. On the other hand, artificial intelligence and machine learning implemented in complex dynamical systems based on genuine quantum networks can certainly be expected to show enhanced performance and quantum advantage compared with classical networks. Nevertheless, even quantum networks can only be expected to efficiently solve NP-hard problems approximately. In the end it is a question of precision - Nature is approximate.Comment: 38 page

Genetic Controls Balancing Excitatory and Inhibitory Synaptogenesis in Neurodevelopmental Disorder Models

Proper brain function requires stringent balance of excitatory and inhibitory synapse formation during neural circuit assembly. Mutation of genes that normally sculpt and maintain this balance results in severe dysfunction, causing neurodevelopmental disorders including autism, epilepsy and Rett syndrome. Such mutations may result in defective architectural structuring of synaptic connections, molecular assembly of synapses and/or functional synaptogenesis. The affected genes often encode synaptic components directly, but also include regulators that secondarily mediate the synthesis or assembly of synaptic proteins. The prime example is Fragile X syndrome (FXS), the leading heritable cause of both intellectual disability and autism spectrum disorders. FXS results from loss of mRNA-binding FMRP, which regulates synaptic transcript trafficking, stability and translation in activity-dependent synaptogenesis and plasticity mechanisms. Genetic models of FXS exhibit striking excitatory and inhibitory synapse imbalance, associated with impaired cognitive and social interaction behaviors. Downstream of translation control, a number of specific synaptic proteins regulate excitatory versus inhibitory synaptogenesis, independently or combinatorially, and loss of these proteins is also linked to disrupted neurodevelopment. The current effort is to define the cascade of events linking transcription, translation and the role of specific synaptic proteins in the maintenance of excitatory versus inhibitory synapses during neural circuit formation. This focus includes mechanisms that fine-tune excitation and inhibition during the refinement of functional synaptic circuits, and later modulate this balance throughout life. The use of powerful new genetic models has begun to shed light on the mechanistic bases of excitation/inhibition imbalance for a range of neurodevelopmental disease states

SH3 AND MULTIPLE ANKYRIN REPEAT DOMAIN 3 (SHANK3) AFFECTS THE EXPRESSION OF HYPERPOLARIZATION-ACTIVATED CYCLIC NUCLEOTIDE-GATED (HCN) CHANNELS IN MOUSE MODELS OF AUTISM

SH3 and multiple ankyrin repeat domains 3 (SHANK3) is a multidomain scaffold protein that is highly augmented in the postsynaptic density (PSD) of excitatory glutamatergic synapses within the central and peripheral nervous systems. SHANK3 links neurotransmitter receptors, ion channels, and other critical membrane proteins to intracellular cytoskeleton and signal transduction pathways. Mutations in SHANK3 are linked with a number neuropsychiatric disorders including autism spectrum disorders (ASDs). Intellectual disability, impaired memory and learning, and epilepsy are some of the deficits commonly associated with ASDs that result from mutations in SHANK3. Interestingly, these symptoms show some clinical overlap with presentations of human neurological disorders involving hyperpolarization-activated cyclin nucleotide-gated (HCN) channels. In fact, it has recently been demonstrated in human neurons that SHANK3 haploinsufficiency causes Ih-channel dysfunction, and that SHANK3 has a physical interaction with HCN channels via its ANKYRIN repeat domain. These insights suggest that SHANK3 may play important roles in HCN channel expression and function, and put forward the idea that HCN channelopathies may actually encourage some of the symptoms observed in patients with SHANK-deficiency related ASDs. In this study, we provide preliminary data that suggests the ANK domain of SHANK3 interacts with COOH portion of HCN1. We also exploited the differences between two mouse models of autism to show that a subset of SHANK3 isoforms may be involved in the proper expression and function of HCN channels. We found that HCN2 expression is significantly decreased in a mouse model lacking all major isoforms of SHANK3 (exons 13-16 deleted; Δ13-16), while HCN2 expression is unaltered in a mouse model only lacking SHANK3a and SHANK3b (exons 4-9 deleted; Δ4-9). Surprisingly, we also found that HCN4 expression is altered in SHANK3Δ13-16, but not SHANK3Δ4-9. Taken together, our results show HCN channelopathy as a major downstream carrier of SHANK3 deficiency

Investigation of the human X-linked autism protein KIDLIA in neuronal development and brain function

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.2018-11-08T00:00:00

MicroRNA Dysregulation in Neuropsychiatric Disorders and Cognitive Dysfunction

MicroRNAs (miRNAs) are evolutionarily-conserved small non-coding RNAs that are important posttranscriptional regulators of gene expression. Genetic Variants may cause microRNA dysregulation and the concomitant aberrant target expression. The dysregulation of one or a few targets may in turn lead to functional consequences ranging from phenotypic variations to disease conditions. In this thesis, I present our studies of mouse models of two human genetic variants - a rare copy number variant (CNV), 22q11.2 microdeletions, and a common single nucleotide polymorphism (SNP), BDNF Val66Met. 22q11.2 microdeletions result in specific cognitive deficits and high risk to develop schizophrenia. Analysis of Df(16)A+/- mice, which model this microdeletion, revealed abnormalities in the formation of neuronal dendrites and spines as well as microRNA dysregulation in brain. We show a drastic reduction of miR- 185, which resides within the 22q11.2 locus, to levels more than expected by a hemizygous deletion and demonstrate that this reduction impairs dendritic and spine development. miR-185 targets and represses, through an evolutionary conserved target site, a previously unknown inhibitor of these processes that resides in the Golgi apparatus. Sustained derepression of this inhibitor after birth represents the most robust transcriptional disturbance in the brains of Df(16)A+/- mice and could affect the formation and maintenance of neural circuits. Reduction of miR-185 also has milder effects on the expression of a group of Golgi-related genes. One the other hand, BNDF Val66Met results in impaired activity-dependent secretion of BDNF from neuronal terminals and affects episodic memory and affective behaviors. We found a modest reduction of miR-146b which causes derepression of mRNA and/or protein levels of a few targets. Our findings add to the growing evidence of the pivotal involvement of miRNAs in the development of neuropsychiatric disorders and cognitive dysfunction. In addition, the identification of key players in miRNA dysregulation has implications for both basic and translational research in psychiatric disorders and cognitive dysfunction