Neuronal deletion of Gtf2i results in developmental microglial alterations in a mouse model related to Williams syndrome.

Williams syndrome (WS) is a genetic neurodevelopmental disorder caused by a heterozygous microdeletion, characterized by hypersociability and unique neurocognitive abnormalities. Of the deleted genes, GTF2I has been linked to hypersociability in WS. We have recently shown that Gtf2i deletion from forebrain excitatory neurons, referred to as Gtf2i conditional knockout (cKO) mice leads to multi-faceted myelination deficits associated with the social behaviors affected in WS. These deficits were potentially mediated also by microglia, as they present a close relationship with oligodendrocytes. To study the impact of altered myelination, we characterized these mice in terms of microglia over the course of development. In postnatal day 30 (P30) Gtf2i cKO mice, cortical microglia displayed a more ramified state, as compared with wild type (controls). However, postnatal day 4 (P4) microglia exhibited high proliferation rates and an elevated activation state, demonstrating altered properties related to activation and inflammation in Gtf2i cKO mice compared with control. Intriguingly, P4 Gtf2i cKO-derived microglial cells exhibited significantly elevated myelin phagocytosis in vitro compared to control mice. Lastly, systemic injection of clemastine to P4 Gtf2i cKO and control mice until P30, led to a significant interaction between genotypes and treatments on the expression levels of the phagocytic marker CD68, and a significant reduction of the macrophage/microglial marker Iba1 transcript levels in the cortex of the Gtf2i cKO treated mice. Our data thus implicate microglia as important players in WS, and that early postnatal manipulation of microglia might be beneficial in treating inflammatory and myelin-related pathologies.

Robust reconstruction of single-cell RNA-seq data with iterative gene weight updates.

MOTIVATION: Single-cell RNA-sequencing technologies have greatly enhanced our understanding of heterogeneous cell populations and underlying regulatory processes. However, structural (spatial or temporal) relations between cells are lost during cell dissociation. These relations are crucial for identifying associated biological processes. Many existing tissue-reconstruction algorithms use prior information about subsets of genes that are informative with respect to the structure or process to be reconstructed. When such information is not available, and in the general case when the input genes code for multiple processes, including being susceptible to noise, biological reconstruction is often computationally challenging. RESULTS: We propose an algorithm that iteratively identifies manifold-informative genes using existing reconstruction algorithms for single-cell RNA-seq data as subroutine. We show that our algorithm improves the quality of tissue reconstruction for diverse synthetic and real scRNA-seq data, including data from the mammalian intestinal epithelium and liver lobules. AVAILABILITY AND IMPLEMENTATION: The code and data for benchmarking are available at github.com/syq2012/iterative_weight_update_for_reconstruction.

In individuals with Williams syndrome, dysregulation of methylation in non-coding regions of neuronal and oligodendrocyte DNA is associated with pathology and cortical development.

Williams syndrome (WS) is a neurodevelopmental disorder caused by a heterozygous micro-deletion in the WS critical region (WSCR) and is characterized by hyper-sociability and neurocognitive abnormalities. Nonetheless, whether and to what extent WSCR deletion leads to epigenetic modifications in the brain and induces pathological outcomes remains largely unknown. By examining DNA methylation in frontal cortex, we revealed genome-wide disruption in the methylome of individuals with WS, as compared to typically developed (TD) controls. Surprisingly, differentially methylated sites were predominantly annotated as introns and intergenic loci and were found to be highly enriched around binding sites for transcription factors that regulate neuronal development, plasticity and cognition. Moreover, by utilizing enhancer-promoter interactome data, we confirmed that most of these loci function as active enhancers in the human brain or as target genes of transcriptional networks associated with myelination, oligodendrocyte (OL) differentiation, cognition and social behavior. Cell type-specific methylation analysis revealed aberrant patterns in the methylation of active enhancers in neurons and OLs, and important neuron-glia interactions that might be impaired in individuals with WS. Finally, comparison of methylation profiles from blood samples of individuals with WS and healthy controls, along with other data collected in this study, identified putative targets of endophenotypes associated with WS, which can be used to define brain-risk loci for WS outside the WSCR locus, as well as for other associated pathologies. In conclusion, our study illuminates the brain methylome landscape of individuals with WS and sheds light on how these aberrations might be involved in social behavior and physiological abnormalities. By extension, these results may lead to better diagnostics and more refined therapeutic targets for WS.

SH3- and actin-binding domains connect ADNP and SHANK3, revealing a fundamental shared mechanism underlying autism.

De novo heterozygous mutations in activity-dependent neuroprotective protein (ADNP) cause autistic ADNP syndrome. ADNP mutations impair microtubule (MT) function, essential for synaptic activity. The ADNP MT-associating fragment NAPVSIPQ (called NAP) contains an MT end-binding protein interacting domain, SxIP (mimicking the active-peptide, SKIP). We hypothesized that not all ADNP mutations are similarly deleterious and that the NAPV portion of NAPVSIPQ is biologically active. Using the eukaryotic linear motif (ELM) resource, we identified a Src homology 3 (SH3) domain-ligand association site in NAP responsible for controlling signaling pathways regulating the cytoskeleton, namely NAPVSIP. Altogether, we mapped multiple SH3-binding sites in ADNP. Comparisons of the effects of ADNP mutations p.Glu830synfs*83, p.Lys408Valfs*31, p.Ser404* on MT dynamics and Tau interactions (live-cell fluorescence-microscopy) suggested spared toxic function in p.Lys408Valfs*31, with a regained SH3-binding motif due to the frameshift insertion. Site-directed-mutagenesis, abolishing the p.Lys408Valfs*31 SH3-binding motif, produced MT toxicity. NAP normalized MT activities in the face of all ADNP mutations, although, SKIP, missing the SH3-binding motif, showed reduced efficacy in terms of MT-Tau interactions, as compared with NAP. Lastly, SH3 and multiple ankyrin repeat domains protein 3 (SHANK3), a major autism gene product, interact with the cytoskeleton through an actin-binding motif to modify behavior. Similarly, ELM analysis identified an actin-binding site on ADNP, suggesting direct SH3 and indirect SHANK3/ADNP associations. Actin co-immunoprecipitations from mouse brain extracts showed NAP-mediated normalization of Shank3-Adnp-actin interactions. Furthermore, NAP treatment ameliorated aberrant behavior in mice homozygous for the Shank3 ASD-linked InsG3680 mutation, revealing a fundamental shared mechanism between ADNP and SHANK3.

Hyperbaric Oxygen Therapy Alleviates Social Behavior Dysfunction and Neuroinflammation in a Mouse Model for Autism Spectrum Disorders.

Autism spectrum disorder (ASD) is a multifactorial neurodevelopmental disorder (NDD) characterized by impaired social communication and repetitive behavior, among other symptoms. ASD is highly heritable, with SHANK3 being one of the high-risk genes for ASD. In recent years, knowledge has been growing regarding the neuroplasticity effect induced by hyperbaric oxygen therapy (HBOT) and its potential use for ASD. Here, we characterized the effect of HBOT on a mouse model for ASD with the human genetic condition of InsG3680 mutation in the Shank3 gene. As compared to placebo, HBOT improved social behavior and reduced neuroinflammation in the cortex of the InsG3680(+/+) mice. Specifically, HBOT induced upregulation of Insulin-like growth factor 1 (Igf1) expression levels and reduced the number of Iba1-positive cells in the mouse model for ASD compared to placebo control. Together, our research suggests that HBOT has the potential to improve the clinical outcome of ASD by ameliorating some of the core pathophysiological processes responsible for the development of the disorder.

Noisy tensor completion via the sum-of-squares hierarchy.

In the noisy tensor completion problem we observe m entries (whose location is chosen uniformly at random) from an unknown n 1 × n 2 × n 3 tensor T. We assume that T is entry-wise close to being rank r. Our goal is to fill in its missing entries using as few observations as possible. Let n = max ( n 1 , n 2 , n 3 ) . We show that if m ≳ n 3 / 2 r then there is a polynomial time algorithm based on the sixth level of the sum-of-squares hierarchy for completing it. Our estimate agrees with almost all of T's entries almost exactly and works even when our observations are corrupted by noise. This is also the first algorithm for tensor completion that works in the overcomplete case when r > n , and in fact it works all the way up to r = n 3 / 2 - ϵ . Our proofs are short and simple and are based on establishing a new connection between noisy tensor completion (through the language of Rademacher complexity) and the task of refuting random constraint satisfaction problems. This connection seems to have gone unnoticed even in the context of matrix completion. Furthermore, we use this connection to show matching lower bounds. Our main technical result is in characterizing the Rademacher complexity of the sequence of norms that arise in the sum-of-squares relaxations to the tensor nuclear norm. These results point to an interesting new direction: Can we explore computational vs. sample complexity tradeoffs through the sum-of-squares hierarchy?

White matter alterations in Williams syndrome related to behavioral and motor impairments.

Myelin is the electrical insulator surrounding the neuronal axon that makes up the white matter (WM) of the brain. It helps increase axonal conduction velocity (CV) by inducing saltatory conduction. Damage to the myelin sheath and WM is associated with many neurological and psychiatric disorders. Decreasing myelin deficits, and thus improving axonal conduction, has the potential to serve as a therapeutic mechanism for reducing the severity of some of these disorders. Myelin deficits have been previously linked to abnormalities in social behavior, suggesting an interplay between brain connectivity and sociability. This review focuses on Williams syndrome (WS), a genetic disorder characterized by neurocognitive characteristics and motor abnormalities, mainly known for its hypersociability characteristic. We discuss fundamental aspects of WM in WS and how its alterations can affect motor abilities and social behavior. Overall, findings regarding changes in myelin genes and alterations in WM structure in WS suggest new targets for drug therapy aimed at improving conduction properties and altering brain-activity synchronization in this disorder.

Shank3 mutation in a mouse model of autism leads to changes in the S-nitroso-proteome and affects key proteins involved in vesicle release and synaptic function.

Mutation in the SHANK3 human gene leads to different neuropsychiatric diseases including Autism Spectrum Disorder (ASD), intellectual disabilities and Phelan-McDermid syndrome. Shank3 disruption in mice leads to dysfunction of synaptic transmission, behavior, and development. Protein S-nitrosylation, the nitric oxide (NO•)-mediated posttranslational modification (PTM) of cysteine thiols (SNO), modulates the activity of proteins that regulate key signaling pathways. We tested the hypothesis that Shank3 mutation would generate downstream effects on PTM of critical proteins that lead to modification of synaptic functions. SNO-proteins in two ASD-related brain regions, cortex and striatum of young and adult InsG3680(+/+) mice (a human mutation-based Shank3 mouse model), were identified by an innovative mass spectrometric method, SNOTRAP. We found changes of the SNO-proteome in the mutant compared to WT in both ages. Pathway analysis showed enrichment of processes affected in ASD. SNO-Calcineurin in mutant led to a significant increase of phosphorylated Synapsin1 and CREB, which affect synaptic vesicle mobilization and gene transcription, respectively. A significant increase of 3-nitrotyrosine was found in the cortical regions of the adult mutant, signaling both oxidative and nitrosative stress. Neuronal NO• Synthase (nNOS) was examined for levels and localization in neurons and no significant difference was found in WT vs. mutant. S-nitrosoglutathione concentrations were higher in mutant mice compared to WT. This is the first study on NO•-related molecular changes and SNO-signaling in the brain of an ASD mouse model that allows the characterization and identification of key proteins, cellular pathways, and neurobiological mechanisms that might be affected in ASD.

A zero-knowledge protocol for nuclear warhead verification.

The verification of nuclear warheads for arms control involves a paradox: international inspectors will have to gain high confidence in the authenticity of submitted items while learning nothing about them. Proposed inspection systems featuring 'information barriers', designed to hide measurements stored in electronic systems, are at risk of tampering and snooping. Here we show the viability of a fundamentally new approach to nuclear warhead verification that incorporates a zero-knowledge protocol, which is designed in such a way that sensitive information is never measured and so does not need to be hidden. We interrogate submitted items with energetic neutrons, making, in effect, differential measurements of both neutron transmission and emission. Calculations for scenarios in which material is diverted from a test object show that a high degree of discrimination can be achieved while revealing zero information. Our ideas for a physical zero-knowledge system could have applications beyond the context of nuclear disarmament. The proposed technique suggests a way to perform comparisons or computations on personal or confidential data without measuring the data in the first place.

Microglia roles in synaptic plasticity and myelination in homeostatic conditions and neurodevelopmental disorders.

Microglia are the immune cells of the brain, involved in synapse formation, circuit sculpting, myelination, plasticity, and cognition. Being active players during early development as well as in adulthood, microglia affect other cells directly by their long processes and unique receptors and indirectly by secreting growth factors and cytokines. In this review, we discuss the roles of microglia in neurodevelopmental disorders, synaptic plasticity, myelination, and homeostatic conditions throughout human and mouse development. Within these processes, we specifically focus on the contribution of altered microglial interactions with neurons and oligodendrocytes, altered cytokine and growth factor activities, and alterations in the complement system. We conclude by highlighting future perspectives and providing an overview of future research on microglia.

Dichotomous parvalbumin interneuron populations in dorsolateral and dorsomedial striatum.

KEY POINTS: There are two electrophysiological dichotomous populations of parvalbumin (PV) interneurons located in the dorsal striatum. Striatal PV interneurons in medial and lateral regions differ significantly in their intrinsic excitability. Parvalbumin interneurons in the dorsomedial striatum, but not in the dorsolateral striatum, receive afferent glutamatergic input from cingulate cortex. ABSTRACT: Dorsomedial striatum circuitry is involved in goal-directed actions or movements that become habits upon repetition, as encoded by the dorsolateral striatum. An inability to shift from habits can compromise action-control and prevent behavioural adaptation. Although these regions appear to be clearly behaviourally distinct, little is known about their distinct physiology. Parvalbumin (PV) interneurons are a major source of striatal inhibition and are usually considered as a homogeneous population in the entire dorsal striatum. In the present study, we recorded PV interneurons in dorsal striatum slices from wild-type male mice and suggest the existence of two electrophysiological dichotomous populations. We found that PV interneurons located at the dorsomedial striatum region have increased intrinsic excitability compared to PV interneurons in dorsolateral region. We also found that PV interneurons in the dorsomedial region, but not in the dorsolateral striatum region, receive short-latency excitatory inputs from cingulate cortex. Therefore, the results of the present study demonstrate the importance of considering region specific parvalbumin interneuron populations when studying dorsal striatal function.

Tackling myelin deficits in neurodevelopmental disorders using drug delivery systems.

Interest in myelin and its roles in almost all brain functions has been greatly increasing in recent years, leading to countless new studies on myelination, as a dominant process in the development of cognitive functions. Here, we explore the unique role myelin plays in the central nervous system and specifically discuss the results of altered myelination in neurodevelopmental disorders. We present parallel developmental trajectories involving myelination that correlate with the onset of cognitive impairment in neurodevelopmental disorders and discuss the key challenges in the treatment of these chronic disorders. Recent developments in drug repurposing and nano/micro particle-based therapies are reviewed as a possible pathway to circumvent some of the main hurdles associated with early intervention, including patient's adherence and compliance, side effects, relapse, and faster route to possible treatment of these disorders. The strategy of drug encapsulation overcomes drug solubility and metabolism, with the possibility of drug targeting to a specific compartment, reducing side effects upon systemic administration.

Toll-like receptor 3 inhibits memory retention and constrains adult hippocampal neurogenesis.

Toll-like receptors (TLRs) are innate immune receptors that have recently emerged as regulators of neuronal survival and developmental neuroplasticity. Adult TLR3-deficient mice exhibited enhanced hippocampus-dependent working memory in the Morris water maze, novel object recognition, and contextual fear-conditioning tasks. In contrast, TLR3-deficient mice demonstrated impaired amygdala-related behavior and anxiety in the cued fear-conditioning, open field, and elevated plus maze tasks. Further, TLR3-deficient mice exhibited increased hippocampal CA1 and dentate gyrus volumes, increased hippocampal neurogenesis, and elevated levels of the AMPA receptor subunit GluR1 in the CA1 region of the hippocampus. In addition, levels of activated forms of the kinase ERK and the transcription factor CREB were elevated in the hippocampus of TLR3-deficient mice, suggesting that constitutive TLR3 signaling negatively regulates pathways known to play important roles in hippocampal plasticity. Direct activation of TLR3 by intracerebroventricular infusion of a TLR3 ligand impaired working memory, but not reference memory. Our findings reveal previously undescribed roles for TLR3 as a suppressor of hippocampal cellular plasticity and memory retention.

Deletion of Gtf2i via Systemic Administration of AAV-PHP.eB Virus Increases Social Behavior in a Mouse Model of a Neurodevelopmental Disorder.

Williams syndrome (WS) is a neurodevelopmental disorder characterized by distinctive cognitive and personality profiles which also impacts various physiological systems. The syndrome arises from the deletion of about 25 genes located on chromosome 7q11.23, including Gtf2i. Prior research indicated a strong association between pre-natal Gtf2i deletion, and the hyper-social phenotypes observed in WS, as well as myelination deficits. As most studies addressed pre-natal Gtf2i deletion in mouse models, post-natal neuronal roles of Gtf2i were unknown. To investigate the impact of post-natal deletion of neuronal Gtf2i on hyper-sociability, we intravenously injected an AAV-PHP.eB virus expressing Cre-recombinase under the control of αCaMKII, a promoter in a mouse model with floxed Gtf2i. This targeted deletion was performed in young mice, allowing for precise and efficient brain-wide infection leading to the exclusive removal of Gtf2i from excitatory neurons. As a result of such gene deletion, the mice displayed hyper-sociability, increased anxiety, impaired cognition, and hyper-mobility, relative to controls. These findings highlight the potential of systemic viral manipulation as a gene-editing technique to modulate behavior-regulating genes during the post-natal stage, thus presenting novel therapeutic approaches for addressing neurodevelopmental dysfunction.

Remote inspection of adversary-controlled environments.

Remotely monitoring the location and enduring presence of valuable items in adversary-controlled environments presents significant challenges. In this article, we demonstrate a monitoring approach that leverages the gigahertz radio-wave scattering and absorption of a room and its contents, including a set of mirrors with random orientations placed inside, to remotely verify the absence of any disturbance over time. Our technique extends to large physical systems the application of physical unclonable functions for integrity protection. Its main applications are scenarios where parties are mutually distrustful and have privacy and security constraints. Examples range from the verification of nuclear arms-control treaties to the securing of currency, artwork, or data centers.

Impaired myelin ultrastructure is reversed by citalopram treatment in a mouse model for major depressive disorder.

Major depressive disorder (MDD) is the most common and widespread mental disorder. Selective serotonin reuptake inhibitors (SSRIs) are the first-line treatment for MDD. The relation between the inhibition of serotonin reuptake in the central nervous system and remission from MDD remains controversial, as reuptake inhibition occurs rapidly, but remission from MDD takes weeks to months. Myelination-related deficits and white matter abnormalities were shown to be involved in psychiatric disorders such as MDD. This may explain the delay in remission following SSRI administration. The raphe nuclei (RN), located in the brain stem, consist of clusters of serotonergic (5-HT) neurons that project to almost all regions of the brain. Thus, the RN are an intriguing area for research of the potential effect of SSRI on myelination, and their involvement in MDD. MicroRNAs (miRNAs) regulate many biological features that might be altered by antidepressants. Two cohorts of chronic unpredictable stress (CUS) mouse model for depression underwent behavioral tests for evaluating stress, anxiety, and depression levels. Following application of the CUS protocol and treatment with the SSRI, citalopram, 48 mice of the second cohort were tested via magnetic resonance imaging and diffusion tensor imaging for differences in brain white matter tracts. RN and superior colliculus were excised from both cohorts and measured for changes in miRNAs, mRNA, and protein levels of candidate genes. Using MRI-DTI scans we found lower fractional anisotropy and axial diffusivity in brains of stressed mice. Moreover, both miR-30b-5p and miR-101a-3p were found to be downregulated in the RN following CUS, and upregulated following CUS and citalopram treatment. The direct binding of these miRNAs to Qki, and the subsequent effects on mRNA and protein levels of myelin basic protein (Mbp), indicated involvement of these miRNAs in myelination ultrastructure processes in the RN, in response to CUS followed by SSRI treatment. We suggest that SSRIs are implicated in repairing myelin deficits resulting from chronic stress that leads to depression.

Neuronal Gtf2i deletion alters mitochondrial and autophagic properties.

Gtf2i encodes the general transcription factor II-I (TFII-I), with peak expression during pre-natal and early post-natal brain development stages. Because these stages are critical for proper brain development, we studied at the single-cell level the consequences of Gtf2i's deletion from excitatory neurons, specifically on mitochondria. Here we show that Gtf2i's deletion resulted in abnormal morphology, disrupted mRNA related to mitochondrial fission and fusion, and altered autophagy/mitophagy protein expression. These changes align with elevated reactive oxygen species levels, illuminating Gtf2i's importance in neurons mitochondrial function. Similar mitochondrial issues were demonstrated by Gtf2i heterozygous model, mirroring the human condition in Williams syndrome (WS), and by hemizygous neuronal Gtf2i deletion model, indicating Gtf2i's dosage-sensitive role in mitochondrial regulation. Clinically relevant, we observed altered transcript levels related to mitochondria, hypoxia, and autophagy in frontal cortex tissue from WS individuals. Our study reveals mitochondrial and autophagy-related deficits shedding light on WS and other Gtf2i-related disorders.

Altered White Matter and microRNA Expression in a Murine Model Related to Williams Syndrome Suggests That miR-34b/c Affects Brain Development via Ptpru and Dcx Modulation.

Williams syndrome (WS) is a multisystem neurodevelopmental disorder caused by a de novo hemizygous deletion of ~26 genes from chromosome 7q11.23, among them the general transcription factor II-I (GTF2I). By studying a novel murine model for the hypersociability phenotype associated with WS, we previously revealed surprising aberrations in myelination and cell differentiation properties in the cortices of mutant mice compared to controls. These mutant mice had selective deletion of Gtf2i in the excitatory neurons of the forebrain. Here, we applied diffusion magnetic resonance imaging and fiber tracking, which showed a reduction in the number of streamlines in limbic outputs such as the fimbria/fornix fibers and the stria terminalis, as well as the corpus callosum of these mutant mice compared to controls. Furthermore, we utilized next-generation sequencing (NGS) analysis of cortical small RNAs' expression (RNA-Seq) levels to identify altered expression of microRNAs (miRNAs), including two from the miR-34 cluster, known to be involved in prominent processes in the developing nervous system. Luciferase reporter assay confirmed the direct binding of miR-34c-5p to the 3'UTR of PTPRU-a gene involved in neural development that was elevated in the cortices of mutant mice relative to controls. Moreover, we found an age-dependent variation in the expression levels of doublecortin (Dcx)-a verified miR-34 target. Thus, we demonstrate the substantial effect a single gene deletion can exert on miRNA regulation and brain structure, and advance our understanding and, hopefully, treatment of WS.

Friends and foes in synaptic transmission: the role of tomosyn in vesicle priming.

Priming is the process by which vesicles become available for fusion at nerve terminals and is modulated by numerous proteins and second messengers. One of the prominent members of this diverse family is tomosyn. Tomosyn has been identified as a syntaxin-binding protein; it inhibits vesicle priming, but its mode of action is not fully understood. The inhibitory activity of tomosyn depends on its N-terminal WD40-repeat domain and is regulated by the binding of its SNARE motif to syntaxin. Here, we describe new physiological information on the function of tomosyn and address possible interpretations of these results in the framework of the recently described crystal structure of the yeast tomosyn homolog Sro7. We also present possible molecular scenarios for vesicle priming and the involvement of tomosyn in these processes.

Postnatal therapeutic approaches in genetic neurodevelopmental disorders.

Genetic neurodevelopmental disorders are characterized by abnormal neurophysiological and behavioral phenotypes, affecting individuals worldwide. While the subject has been heavily researched, current treatment options relate mostly to alleviating symptoms, rather than targeting the altered genome itself. In this review, we address the neurogenetic basis of neurodevelopmental disorders, genetic tools that are enabling precision research of these disorders in animal models, and postnatal gene-therapy approaches for neurodevelopmental disorders derived from preclinical studies in the laboratory.