SYNGAP1 deficiency disrupts synaptic neoteny in xenotransplanted human cortical neurons in vivo

We thank members of the P.V. lab and CBD for helpful discussions and precious help. We thank Alexandre Gehanno, Daan Remans, and Arissa Jokhio for precious help. This work was funded by grants from the European Research Council (NEUROTEMPO), the C1 KU Leuven Internal Funds Program, the EOS Program, the Foundation GENERET, ERANET EPINEURO-DEVO, NSC-Reconstruct, the Belgian FWO, and the Belgian Queen Elizabeth Foundation (P.V.). The authors gratefully acknowledge the VIB Bio Imaging Core for their support and assistance in this work. R.I. was supported by a postdoctoral fellowship of the FRS/FNRS

Integrating the Neurodevelopmental and Dopamine Hypotheses of Schizophrenia and the Role of Cortical Excitation-Inhibition Balance

The neurodevelopmental and dopamine hypotheses are leading theories of the pathoetiology of schizophrenia, but they were developed in isolation. However, since they were originally proposed, there have been considerable advances in our understanding of the normal neurodevelopmental refinement of synapses and cortical excitation-inhibition (E/I) balance, as well as preclinical findings on the interrelationship between cortical and subcortical systems and new in vivo imaging and induced pluripotent stem cell evidence for lower synaptic density markers in patients with schizophrenia. Genetic advances show that schizophrenia is associated with variants linked to genes affecting GABA (gamma-aminobutyric acid) and glutamatergic signaling as well as neurodevelopmental processes. Moreover, in vivo studies on the effects of stress, particularly during later development, show that it leads to synaptic elimination. We review these lines of evidence as well as in vivo evidence for altered cortical E/I balance and dopaminergic dysfunction in schizophrenia. We discuss mechanisms through which frontal cortex circuitry may regulate striatal dopamine and consider how frontal E/I imbalance may cause dopaminergic dysregulation to result in psychotic symptoms. This integrated neurodevelopmental and dopamine hypothesis suggests that overpruning of synapses, potentially including glutamatergic inputs onto frontal cortical interneurons, disrupts the E/I balance and thus underlies cognitive and negative symptoms. It could also lead to disinhibition of excitatory projections from the frontal cortex and possibly other regions that regulate mesostriatal dopamine neurons, resulting in dopamine dysregulation and psychotic symptoms. Together, this explains a number of aspects of the epidemiology and clinical presentation of schizophrenia and identifies new targets for treatment and prevention.</p

Investigation of sex-specific differences in visual cortical function in a mouse model of syngap1-related intellectual disability

SYNGAP1-related intellectual disability is a neurodevelopmental disorder caused by mutations in one copy of the SYNGAP1 gene. It accounts for around 1% of all intellectual disabilities, with a high penetrance of co-morbidities such as epilepsy and autism spectrum disorders. Care giver accounts of the disease symptoms commonly cite atypical sensory perception as one of the symptoms of SYNGAP1-related intellectual disability. Abnormal perception affects the ability of individuals to participate in a shared view of the world and likely underpins some of the difficulties that individuals with intellectual disability and autism spectrum disorders face in social interactions. Increasingly, evidence is supporting sexual differentiation in the severity of symptoms, with girls and women more likely to experience severe sensory processing atypicality and intractable epilepsy in cases where intellectual disability and autism spectrum disorder are co-morbid. Here we attempt to investigate potential sex differences in visual processing using a mouse model of Syngap1 haploinsufficiency. Using 2-photon microscopy and a virally expressed calcium indicator (GCaMP6f) we recorded in-vivo activity of layer 2/3 neurons in the primary visual cortex of female mice whilst they were presented with drifting grating visual stimuli. We quantified the discriminability, selectivity, reliability, depth of modulation, of neuronal population activity in the primary visual cortex of Syngap1 haploinsufficient mice and control littermates. Results obtained from female mice were compared with data previously acquired from male mice using the same methodology. Compared to control littermates, Syngap1+/- mice less reliably encode visual information in primary visual networks of layer 2/3, as measured by the decoding accuracy of visual stimuli, which is reduced in Syngap1+/- mice due to an increase in the variability of neuronal activity. However, no conclusive difference in visual response properties was found between female and male Syngap1 heterozygous mice, indicating that Syngap1 haploinsufficiency produces equivalent deficits in cortical visual processing for both male and female mice

Dysfunction of homeostatic control of dopamine by astrocytes in the developing prefrontal cortex leads to cognitive impairments

Astrocytes orchestrate neural development by powerfully coordinating synapse formation and function and, as such, may be critically involved in the pathogenesis of neurodevelopmental abnormalities and cognitive deficits commonly observed in psychiatric disorders. Here, we report the identification of a subset of cortical astrocytes that are competent for regulating dopamine (DA) homeostasis during postnatal development of the prefrontal cortex (PFC), allowing for optimal DA-mediated maturation of excitatory circuits. Such control of DA homeostasis occurs through the coordinated activity of astroglial vesicular monoamine transporter 2 (VMAT2) together with organic cation transporter 3 and monoamine oxidase type B, two key proteins for DA uptake and metabolism. Conditional deletion of VMAT2 in astrocytes postnatally produces loss of PFC DA homeostasis, leading to defective synaptic transmission and plasticity as well as impaired executive functions. Our findings show a novel role for PFC astrocytes in the DA modulation of cognitive performances with relevance to psychiatric disorders

Molecular mechanisms of human presynapse assembly - role of liprin-alpha proteins in synapse organization and neurological disease

Synapses are the essential transmission units of neuronal circuits and represent the cornerstone of our unique cognitive abilities. During synapse formation, contact between two neurons induce the assembly of pre- and postsynaptic compartments. On the presynaptic side, this involves the recruitment of intracellular proteins to form an active zone protein network that precisely orchestrates clustering of synaptic vesicles and their tightly controlled release. Cell-adhesion receptors present at the synapse, for example members of the neurexin (NRXN) and leukocyte common antigen-related protein tyrosine phosphatase receptor (LAR-PTPR) families, are thought to contribute to synapse assembly, but how extracellular interactions translate into downstream intracellular events remains unknown. Defects in synapse formation and function are involved in multiple neurodevelopmental and psychiatric disorders. Understanding how synaptic connections assemble is thus a central issue in neuroscience but, despite being extensively studied, the underlying molecular mechanisms have remained elusive. This thesis aimed at defining the contribution of the liprin-alpha family of scaffolding proteins in presynapse formation and neurological disease, by use of human stem cell-derived neuronal models. In paper I, we generated gene-edited human quadruple knockout neuronal models lacking all liprin-alpha isoforms and found that emergent synaptic connections are still formed but subsequent recruitment steps of synaptic components, are completely ablated, resulting in disruption of synaptic transmission. We thus identify liprin-alpha proteins as central master organizers of human presynapse assembly, bridging cell-adhesion receptors to downstream recruitment steps. In paper II, we set up a streamlined targeting system for the rapid generation of gene-edited human pluripotent stem cell lines integrating adeno-associated virus and CRISPR-Cas9 technologies. In paper III, we developed a glia-free protocol for the differentiation of human pluripotent stem cells into mature cortical glutamatergic neurons with preserved synchronous network synaptic activity. Finally, in paper IV we sought to characterize the role of liprin-alpha proteins during early neurodevelopment in vitro, uncovering a putative impact of liprin-alpha functions on the adherens junction of neuroepithelial progenitor cells. In conclusion, this thesis provides the first example of hierarchical protein recruitment in mammalian synapse assembly. Our findings may have implications for both the fundamental understanding of how synapses molecularly assemble and the processes underlying synaptic dysfunction in human disease

Autism Spectrum Disorders

Estimated prevalence rates of autism spectrum disorders (ASDs) have increased at an alarming rate over the past decade; current estimates stand as high as 1 in 110 persons in the population with a higher ratio of affected males to females. In addition to their emotional impact on the affected persons and their family members (in fact, the latter are often unrecognized unaffected “patients†themselves), the economic and social impacts of ASDs on society are staggering. Persons with ASDs will need interdisciplinary approaches to complex treatment and life planning, including, but not limited to, special education, speech and language therapy, vocational skills training and rehabilitation, social skills training and cognitive remediation, in addition to pharmacotherapy. The current book highlights some of the recent research on nosology, etiology, and pathophysiology. Additionally, the book touches on the implications of new research for treatment and genetic counseling. Importantly, because the field is advancing rapidly, no book can be considered the final word or finished product; thus, the availability of open access rapid publication is a mechanism that will help to assure that readers remain current and up-to-date

PYRAMIDAL CELLS: ROLE IN PRIMATE PREFRONTAL CORTEX CIRCUITRY DURING POSTNATAL DEVELOPMENT AND SCHIZOPHRENIA

Cognitive deficits constitute a core feature of schizophrenia, are persistent across the course of the illness and are the best predictor of long-term functional outcome. Dysfunction in certain cognitive processes, such as working memory, are common in subjects with schizophrenia and have been attributed to aberrant function of the dorsolateral prefrontal cortex (DLPFC). This dysfunction appears to reflect, at least in part, alterations in excitatory neurotransmission. Cortical pyramidal neurons, the principal source of cortical glutamate neurotransmission, exhibit highly robust molecular and morphological alterations in schizophrenia. These alterations appear to be most pronounced in DLPFC deep layer 3, the same microcircuit necessary for the generation of neural oscillations in the γ-frequency range that sustain working memory function. Understanding how dysfunction in DLPFC cortical circuits in deep layer 3 might give rise to the pathophysiology of altered γ-frequency oscillations and working memory deficits in schizophrenia require an interrogation of the mechanisms by which these neuropathological alterations may arise, but also the normal developmental trajectories of these vulnerable microcircuits. In this dissertation, we provide evidence for pyramidal cell type-specific molecular disturbances and synapse-specific structural impairments in DLPFC deep layer 3, and cell type-specific and layer-specific nature of postnatal developmental refinements in pyramidal cells in the DLPFC, within the circuitry that subserves γ-frequency oscillations and working memory. Accordingly, we have identified alterations in the expression of numerous molecular regulators of the actin cytoskeleton in a layer-specific and cell type-specific manner in DLPFC deep layer 3 in individuals with schizophrenia that might be a critical “upstream” cause in the pathogenesis of the illness. Additionally, using novel triple-label fluorescence immunohistochemistry and spinning-disk confocal microscopy, we characterize specific synaptic connections onto DLPFC deep layer 3 pyramidal cells in schizophrenia. Finally, we demonstrate that the developmental trajectories of primate DLPFC deep layer 3 pyramidal neurons are protracted, and layer-specific and posit that the molecular maturation of GABA synapses on pyramidal cells may account, at least in part, for the maturation of synchronized pyramidal cell firing which is crucial for γ-frequency oscillations

Functional genomics studies of human brain development and implications for autism spectrum disorder

Human neurodevelopment requires the coordinated expression of thousands of genes, exquisitely regulated in both spatial and temporal dimensions, to achieve the proper specialization and inter-connectivity of brain regions. Consequently, the dysregulation of complex gene networks in the developing brain is believed to underlie many neurodevelopmental disorders, such as autism spectrum disorders (ASD). Autism has a significant genetic etiology, but there are hundreds of genes implicated, and their functions are heterogeneous and complex. Therefore, an understanding of shared molecular and cellular pathways underlying the development ASD has remained elusive, hampering attempts to develop common diagnostic biomarkers or treatments for this disorder. I hypothesized that analyzing functional genomics relationships among ASD candidate genes during normal human brain development would provide insight into common cellular and molecular pathways that are affected in autistic individuals, and may help elucidate how hundreds of diverse genes can all be linked to a single clinical phenotype. This thesis describes a coordinated set of bioinformatics experiments that first (i) assessed for gene expression and co-expression properties among ASD candidates and other non-coding RNAs during normal human brain development to discover potential shared mechanisms; and then (ii) directly assessed for changes in these pathways in autistic post-mortem brain tissue. The results demonstrated that when examined in the context of normal human brain gene expression during early development, autism candidate genes appear to be strongly related to the neurodevelopmental pathways of synaptogenesis, mitochondrial function, glial cytokine signaling, and transcription/translation regulation. Furthermore, the known sex bias in ASD prevalence appeared to relate to differences in gene expression between the developing brains of males and females. Follow up studies in autistic brain tissue confirmed that changes in mitochondrial gene expression networks, glial pathways, and gene expression regulatory mechanisms are all altered in the brains of autistic individuals. Together, these results show that the heterogeneous set of autism candidate genes are related to each other through shared transcriptional networks that funnel into common molecular mechanisms, and that these mechanisms are aberrant in autistic brains

SYNGAP1 deficiency disrupts neoteny in human cortical neurons in vivo

AbstractIntellectual deficiency (ID) and autism spectrum disorder (ASD) originate from disrupted development of human-specific cognitive functions. Human brain ontogeny is characterized by a considerably prolonged, neotenic, cortical neuron development. Neuronal neoteny could be disrupted in ID/ASD, but this was never tested because of the difficulties to study developing human cortical circuits. Here we use xenotransplantation of human cortical neurons into the mouse cortex to study the in vivo neuronal consequences of SYNGAP1 haploinsufficiency, a frequent cause of ID/ASD. We find that SYNGAP1 deficient neurons display strong acceleration of morphological and functional synaptic development. At the circuit level, SYNGAP1 haploinsufficient neurons display disrupted neoteny, with faster integration into cortical circuits and acquisition of sensory responsiveness months ahead of time. These data link neuronal neoteny to ID/ASD, with important implications for diagnosis and treatments.</jats:p