Recurrent deletions and duplications at chromosomal region 16p11.2 are variably associated with speech delay, autism spectrum disorder, developmental delay, schizophrenia, and cognitive impairments. Social communication deficits are a primary diagnostic symptom of autism. Here we investigated ultrasonic vocalizations (USVs) in young adult male 16p11.2 deletion mice during a novel three-phase male-female social interaction test that detects vocalizations emitted by a male in the presence of an estrous female, how the male changes its calling when the female is suddenly absent, and the extent to which calls resume when the female returns. Strikingly fewer vocalizations were detected in two independent cohorts of 16p11.2 heterozygous deletion males (+/-) during the first exposure to an unfamiliar estrous female, as compared to wildtype littermates (+/+). When the female was removed, +/+ emitted calls, but at a much lower level, whereas +/- males called minimally. Sensory and motor abnormalities were detected in +/-, including higher nociceptive thresholds, a complete absence of acoustic startle responses, and hearing loss in all +/- as confirmed by lack of auditory brainstem responses to frequencies between 8 and 100 kHz. Stereotyped circling and backflipping appeared in a small percentage of individuals, as previously reported. However, these sensory and motor phenotypes could not directly explain the low vocalizations in 16p11.2 deletion mice, since (a) +/- males displayed normal abilities to emit vocalizations when the female was subsequently reintroduced, and (b) +/- vocalized less than +/+ to social odor cues delivered on an inanimate cotton swab. Our findings support the concept that mouse USVs in social settings represent a response to social cues, and that 16p11.2 deletion mice are deficient in their initial USVs responses to novel social cues.

Background

Common genetic variation and rare mutations in genes encoding calcium channel subunits have pleiotropic effects on risk for multiple neuropsychiatric disorders, including autism spectrum disorder (ASD) and schizophrenia. To gain further mechanistic insights by extending previous gene expression data, we constructed co-expression networks in Timothy syndrome (TS), a monogenic condition with high penetrance for ASD, caused by mutations in the L-type calcium channel, Cav1.2.

Methods

To identify patient-specific alterations in transcriptome organization, we conducted a genome-wide weighted co-expression network analysis (WGCNA) on neural progenitors and neurons from multiple lines of induced pluripotent stem cells (iPSC) derived from normal and TS (G406R in CACNA1C) individuals. We employed transcription factor binding site enrichment analysis to assess whether TS associated co-expression changes reflect calcium-dependent co-regulation.

Results

We identified reproducible developmental and activity-dependent gene co-expression modules conserved in patient and control cell lines. By comparing cell lines from case and control subjects, we also identified co-expression modules reflecting distinct aspects of TS, including intellectual disability and ASD-related phenotypes. Moreover, by integrating co-expression with transcription factor binding analysis, we showed the TS-associated transcriptional changes were predicted to be co-regulated by calcium-dependent transcriptional regulators, including NFAT, MEF2, CREB, and FOXO, thus providing a mechanism by which altered Ca(2+) signaling in TS patients leads to the observed molecular dysregulation.

Conclusions

We applied WGCNA to construct co-expression networks related to neural development and depolarization in iPSC-derived neural cells from TS and control individuals for the first time. These analyses illustrate how a systems biology approach based on gene networks can yield insights into the molecular mechanisms of neural development and function, and provide clues as to the functional impact of the downstream effects of Ca(2+) signaling dysregulation on transcription.

Apelin is a peptide ligand for an orphan G-protein coupled receptor (APJ receptor) and serves as a critical gradient for migration of mesodermal cells fated to contribute to the myocardial lineage. The present study was designed to establish a robust cardiac differentiation protocol, specifically, to evaluate the effect of apelin on directed differentiation of mouse and human embryonic stem cells (mESCs and hESCs) into cardiac lineage. Different concentrations of apelin (50, 100, 500 nM) were evaluated to determine its differentiation potential. The optimized dose of apelin was then combined with mesodermal differentiation factors, including BMP-4, activin-A, and bFGF, in a developmentally specific temporal sequence to examine the synergistic effects on cardiac differentiation. Cellular, molecular, and physiologic characteristics of the apelin-induced contractile embryoid bodies (EBs) were analyzed. It was found that 100 nM apelin resulted in highest percentage of contractile EB for mESCs while 500 nM had the highest effects on hESCs. Functionally, the contractile frequency of mESCs-derived EBs (mEBs) responded appropriately to increasing concentration of isoprenaline and diltiazem. Positive phenotype of cardiac specific markers was confirmed in the apelin-treated groups. The protocol, consisting of apelin and mesodermal differentiation factors, induced contractility in significantly higher percentage of hESC-derived EBs (hEBs), up-regulated cardiac-specific genes and cell surface markers, and increased the contractile force. In conclusion, we have demonstrated that the treatment of apelin enhanced cardiac differentiation of mouse and human ESCs and exhibited synergistic effects with mesodermal differentiation factors.

Monogenic neurodevelopmental disorders provide key insights into the pathogenesis of disease and help us understand how specific genes control the development of the human brain. Timothy syndrome is caused by a missense mutation in the L-type calcium channel Ca(v)1.2 that is associated with developmental delay and autism. We generated cortical neuronal precursor cells and neurons from induced pluripotent stem cells derived from individuals with Timothy syndrome. Cells from these individuals have defects in calcium (Ca(2+)) signaling and activity-dependent gene expression. They also show abnormalities in differentiation, including decreased expression of genes that are expressed in lower cortical layers and in callosal projection neurons. In addition, neurons derived from individuals with Timothy syndrome show abnormal expression of tyrosine hydroxylase and increased production of norepinephrine and dopamine. This phenotype can be reversed by treatment with roscovitine, a cyclin-dependent kinase inhibitor and atypical L-type-channel blocker. These findings provide strong evidence that Ca(v)1.2 regulates the differentiation of cortical neurons in humans and offer new insights into the causes of autism in individuals with Timothy syndrome.

Neurodevelopmental neuropsychiatric disorders, such as autism spectrum disorder and schizophrenia, have strong genetic risk components, but the underlying mechanisms have proven difficult to decipher. Rare, high-risk variants may offer an opportunity to delineate the biological mechanisms responsible more clearly for more common idiopathic diseases. Indeed, different rare variants can cause the same behavioral phenotype, demonstrating genetic heterogeneity, while the same rare variant can cause different behavioral phenotypes, demonstrating variable expressivity. These observations suggest convergent underlying biological and neurological mechanisms; identification of these mechanisms may ultimately reveal new therapeutic targets. At the 2021 Keystone eSymposium "Neuropsychiatric and Neurodevelopmental Disorders: Harnessing Rare Variants" a panel of experts in the field described significant progress in genomic discovery and human phenotyping and raised several consistent issues, including the need for detailed natural history studies of rare disorders, the challenges in cohort recruitment, and the importance of viewing phenotypes as quantitative traits that are impacted by rare variants.

22q11.2 deletion syndrome (22q11DS) is a highly penetrant and common genetic cause of neuropsychiatric disease. Here we generated induced pluripotent stem cells from 15 individuals with 22q11DS and 15 control individuals and differentiated them into three-dimensional (3D) cerebral cortical organoids. Transcriptional profiling across 100 days showed high reliability of differentiation and revealed changes in neuronal excitability-related genes. Using electrophysiology and live imaging, we identified defects in spontaneous neuronal activity and calcium signaling in both organoid- and 2D-derived cortical neurons. The calcium deficit was related to resting membrane potential changes that led to abnormal inactivation of voltage-gated calcium channels. Heterozygous loss of DGCR8 recapitulated the excitability and calcium phenotypes and its overexpression rescued these defects. Moreover, the 22q11DS calcium abnormality could also be restored by application of antipsychotics. Taken together, our study illustrates how stem cell derived models can be used to uncover and rescue cellular phenotypes associated with genetic forms of neuropsychiatric disease.