High-impact rare genetic variants in severe schizophrenia.

Extreme phenotype sequencing has led to the identification of high-impact rare genetic variants for many complex disorders but has not been applied to studies of severe schizophrenia. We sequenced 112 individuals with severe, extremely treatment-resistant schizophrenia, 218 individuals with typical schizophrenia, and 4,929 controls. We compared the burden of rare, damaging missense and loss-of-function variants between severe, extremely treatment-resistant schizophrenia, typical schizophrenia, and controls across mutation intolerant genes. Individuals with severe, extremely treatment-resistant schizophrenia had a high burden of rare loss-of-function (odds ratio, 1.91; 95% CI, 1.39 to 2.63; P = 7.8 × 10-5) and damaging missense variants in intolerant genes (odds ratio, 2.90; 95% CI, 2.02 to 4.15; P = 3.2 × 10-9). A total of 48.2% of individuals with severe, extremely treatment-resistant schizophrenia carried at least one rare, damaging missense or loss-of-function variant in intolerant genes compared to 29.8% of typical schizophrenia individuals (odds ratio, 2.18; 95% CI, 1.33 to 3.60; P = 1.6 × 10-3) and 25.4% of controls (odds ratio, 2.74; 95% CI, 1.85 to 4.06; P = 2.9 × 10-7). Restricting to genes previously associated with schizophrenia risk strengthened the enrichment with 8.9% of individuals with severe, extremely treatment-resistant schizophrenia carrying a damaging missense or loss-of-function variant compared to 2.3% of typical schizophrenia (odds ratio, 5.48; 95% CI, 1.52 to 19.74; P = 0.02) and 1.6% of controls (odds ratio, 5.82; 95% CI, 3.00 to 11.28; P = 2.6 × 10-8). These results demonstrate the power of extreme phenotype case selection in psychiatric genetics and an approach to augment schizophrenia gene discovery efforts.

A population-based study of KCNH7 p.Arg394His and bipolar spectrum disorder.

We conducted blinded psychiatric assessments of 26 Amish subjects (52 ± 11 years) from four families with prevalent bipolar spectrum disorder, identified 10 potentially pathogenic alleles by exome sequencing, tested association of these alleles with clinical diagnoses in the larger Amish Study of Major Affective Disorder (ASMAD) cohort, and studied mutant potassium channels in neurons. Fourteen of 26 Amish had bipolar spectrum disorder. The only candidate allele shared among them was rs78247304, a non-synonymous variant of KCNH7 (c.1181G>A, p.Arg394His). KCNH7 c.1181G>A and nine other potentially pathogenic variants were subsequently tested within the ASMAD cohort, which consisted of 340 subjects grouped into controls subjects and affected subjects from overlapping clinical categories (bipolar 1 disorder, bipolar spectrum disorder and any major affective disorder). KCNH7 c.1181G>A had the highest enrichment among individuals with bipolar spectrum disorder (χ(2) = 7.3) and the strongest family-based association with bipolar 1 (P = 0.021), bipolar spectrum (P = 0.031) and any major affective disorder (P = 0.016). In vitro, the p.Arg394His substitution allowed normal expression, trafficking, assembly and localization of HERG3/Kv11.3 channels, but altered the steady-state voltage dependence and kinetics of activation in neuronal cells. Although our genome-wide statistical results do not alone prove association, cumulative evidence from multiple independent sources (parallel genome-wide study cohorts, pharmacological studies of HERG-type potassium channels, electrophysiological data) implicates neuronal HERG3/Kv11.3 potassium channels in the pathophysiology of bipolar spectrum disorder. Such a finding, if corroborated by future studies, has implications for mental health services among the Amish, as well as development of drugs that specifically target HERG3/Kv11.3.

The pattern of cortical dysfunction in a mouse model of a schizophrenia-related microdeletion.

We used a mouse model of the schizophrenia-predisposing 22q11.2 microdeletion to evaluate how this genetic lesion affects cortical neural circuits at the synaptic, cellular, and molecular levels. Guided by cognitive deficits, we demonstrated that mutant mice display robust deficits in high-frequency synaptic transmission and short-term plasticity (synaptic depression and potentiation), as well as alterations in long-term plasticity and dendritic spine stability. Apart from previously reported reduction in dendritic complexity of layer 5 pyramidal neurons, altered synaptic plasticity occurs in the context of relatively circumscribed and often subtle cytoarchitectural changes in neuronal density and inhibitory neuron numbers. We confirmed the pronounced DiGeorge critical region 8 (Dgcr8)-dependent deficits in primary micro-RNA processing and identified additional changes in gene expression and RNA splicing that may underlie the effects of this mutation. Reduction in Dgcr8 levels appears to be a major driver of altered short-term synaptic plasticity in prefrontal cortex and working memory but not of long-term plasticity and cytoarchitecture. Our findings inform the cortical synaptic and neuronal mechanisms of working memory impairment in the context of psychiatric disorders. They also provide insight into the link between micro-RNA dysregulation and genetic liability to schizophrenia and cognitive dysfunction.

Inhibition of Abl Kinase by Imatinib Can Rescue the Compromised Barrier Function of 22q11.2DS Patient-iPSC-Derived Blood-Brain Barriers.

We have previously established that the integrity of the induced blood-brain barrier (iBBB) formed by brain microvascular endothelial cells derived from the iPSC of 22q11.2 DS (22q11.2 Deletion Syndrome, also called DiGeorge Syndrome) patients is compromised. We tested the possibility that the haploinsufficiency of CRKL, a gene within the 22q11.2 DS deletion region, contributes to the deficit. The CRKL is a major substrate of the Abl tyrosine kinase, and the Abl/CRKL signaling pathway is critical for endothelial barrier functions. Imatinib, an FDA-approved drug, inhibits Abl kinase and has been used to treat various disorders involving vascular leakages. To test if imatinib can restore the compromised iBBB, we treated the patient's iBBB with imatinib. After treatment, both trans-endothelial electrical resistance and solute permeability returned to comparable levels of the control iBBB. Correspondingly, changes in tight junctions and endothelial glycocalyx of the iBBB were also restored. Western blotting showed that imatinib increased the level of active forms of the CRKL protein. A transcriptome study revealed that imatinib up-regulated genes in the signaling pathways responsible for the protein modification process and down-regulated those for cell cycling. The KEGG pathway analysis further suggested that imatinib improved the gene expression of the CRKL signaling pathway and tight junctions, which agrees with our expectations and the observations at protein levels. Our results indicate that the 22q11.2DS iBBB is at least partially caused by the haploinsufficiency of CRKL, which can be rescued by imatinib via its effects on the Abl/CRKL signaling pathway. Our findings uncover a novel disease mechanism associated with 22q11.2DS.

Aberrant pace of cortical neuron development in brain organoids from patients with 22q11.2 deletion syndrome and schizophrenia.

Adults and children afflicted with the 22q11.2 deletion syndrome (22q11.2DS) exhibit cognitive, social, and emotional impairments, and are at significantly heightened risk for schizophrenia (SCZ). The impact of this deletion on early human brain development, however, has remained unclear. Here we harness organoid models of the developing human cerebral cortex, cultivated from subjects with 22q11.2DS and SCZ, as well as unaffected control samples, to identify cell-type-specific developmental abnormalities arising from this genomic lesion. Leveraging single-cell RNA-sequencing in conjunction with experimental validation, we find that the loss of genes within the 22q11.2 locus leads to a delayed development of cortical neurons. This compromised development was reflected in an elevated proportion of actively proliferating neural progenitor cells, coupled with a decreased fraction of more mature neurons. Furthermore, we identify perturbed molecular imprints linked to neuronal maturation, observe the presence of sparser neurites, and note a blunted amplitude in glutamate-induced Ca2+ transients. The aberrant transcription program underlying impaired development contains molecular signatures significantly enriched in neuropsychiatric genetic liability. MicroRNA profiling and target gene investigation suggest that microRNA dysregulation may drive perturbations of genes governing the pace at which maturation unfolds. Using protein-protein interaction network analysis we define complementary effects stemming from additional genes residing within the deleted locus. Our study uncovers reproducible neurodevelopmental and molecular alterations due to 22q11.2 deletions. These findings have the potential to facilitate disease modeling and promote the pursuit of therapeutic interventions.

Investigations of caspr2, an autoantigen of encephalitis and neuromyotonia.

OBJECTIVE: To report clinical and immunological investigations of contactin-associated protein-like 2 (Caspr2), an autoantigen of encephalitis and peripheral nerve hyperexcitability (PNH) previously attributed to voltage-gated potassium channels (VGKC). METHODS: Clinical analysis was performed on patients with encephalitis, PNH, or both. Immunoprecipitation and mass spectrometry were used to identify the antigen and to develop an assay with Caspr2-expressing cells. Immunoabsorption with Caspr2 and comparative immunostaining of brain and peripheral nerve of wild-type and Caspr2-null mice were used to assess antibody specificity. RESULTS: Using Caspr2-expressing cells, antibodies were identified in 8 patients but not in 140 patients with several types of autoimmune or viral encephalitis, PNH, or mutations of the Caspr2-encoding gene. Patients' antibodies reacted with brain and peripheral nerve in a pattern that colocalized with Caspr2. This reactivity was abrogated after immunoabsorption with Caspr2 and was absent in tissues from Caspr2-null mice. Of the 8 patients with Caspr2 antibodies, 7 had encephalopathy or seizures, 5 neuropathy or PNH, and 1 isolated PNH. Three patients also had myasthenia gravis, bulbar weakness, or symptoms that initially suggested motor neuron disease. None of the patients had active cancer; 7 responded to immunotherapy and were healthy or only mildly disabled at last follow-up (median, 8 months; range, 6-84 months). INTERPRETATION: Caspr2 is an autoantigen of encephalitis and PNH previously attributed to VGKC antibodies. The occurrence of other autoantibodies may result in a complex syndrome that at presentation could be mistaken for a motor neuron disorder. Recognition of this disorder is important, because it responds to immunotherapy.

An in vitro model of neuronal ensembles.

Advances in 3D neuronal cultures, such as brain spheroids and organoids, are allowing unprecedented in vitro access to some of the molecular, cellular and developmental mechanisms underlying brain diseases. However, their efficacy in recapitulating brain network properties that encode brain function remains limited, thereby precluding development of effective in vitro models of complex brain disorders like schizophrenia. Here, we develop and characterize a Modular Neuronal Network (MoNNet) approach that recapitulates specific features of neuronal ensemble dynamics, segregated local-global network activities and a hierarchical modular organization. We utilized MoNNets for quantitative in vitro modelling of schizophrenia-related network dysfunctions caused by highly penetrant mutations in SETD1A and 22q11.2 risk loci. Furthermore, we demonstrate its utility for drug discovery by performing pharmacological rescue of alterations in neuronal ensembles stability and global network synchrony. MoNNets allow in vitro modelling of brain diseases for investigating the underlying neuronal network mechanisms and systematic drug discovery.

Investigation of Neurodevelopmental Deficits of 22 q11.2 Deletion Syndrome with a Patient-iPSC-Derived Blood-Brain Barrier Model.

The blood-brain barrier (BBB) is important in the normal functioning of the central nervous system. An altered BBB has been described in various neuropsychiatric disorders, including schizophrenia. However, the cellular and molecular mechanisms of such alterations remain unclear. Here, we investigate if BBB integrity is compromised in 22q11.2 deletion syndrome (also called DiGeorge syndrome), which is one of the validated genetic risk factors for schizophrenia. We utilized a set of human brain microvascular endothelial cells (HBMECs) derived from the induced pluripotent stem cell (iPSC) lines of patients with 22q11.2-deletion-syndrome-associated schizophrenia. We found that the solute permeability of the BBB formed from patient HBMECs increases by ~1.3-1.4-fold, while the trans-endothelial electrical resistance decreases to ~62% of the control values. Correspondingly, tight junction proteins and the endothelial glycocalyx that determine the integrity of the BBB are significantly disrupted. A transcriptome study also suggests that the transcriptional network related to the cell-cell junctions in the compromised BBB is substantially altered. An enrichment analysis further suggests that the genes within the altered gene expression network also contribute to neurodevelopmental disorders. Our findings suggest that neurovascular coupling can be targeted in developing novel therapeutical strategies for the treatment of 22q11.2 deletion syndrome.

Cortical overgrowth in a preclinical forebrain organoid model of CNTNAP2-associated autism spectrum disorder.

We utilized forebrain organoids generated from induced pluripotent stem cells of patients with a syndromic form of Autism Spectrum Disorder (ASD) with a homozygous protein-truncating mutation in CNTNAP2, to study its effects on embryonic cortical development. Patients with this mutation present with clinical characteristics of brain overgrowth. Patient-derived forebrain organoids displayed an increase in volume and total cell number that is driven by increased neural progenitor proliferation. Single-cell RNA sequencing revealed PFC-excitatory neurons to be the key cell types expressing CNTNAP2. Gene ontology analysis of differentially expressed genes (DEgenes) corroborates aberrant cellular proliferation. Moreover, the DEgenes are enriched for ASD-associated genes. The cell-type-specific signature genes of the CNTNAP2-expressing neurons are associated with clinical phenotypes previously described in patients. The organoid overgrowth phenotypes were largely rescued after correction of the mutation using CRISPR-Cas9. This CNTNAP2-organoid model provides opportunity for further mechanistic inquiry and development of new therapeutic strategies for ASD.

Autoimmune Encephalitis in Postpartum Psychosis.

OBJECTIVE: Significant immunological alterations have been observed in women with first-onset affective psychosis during the postpartum period. Recent studies have highlighted the possibility that a subset of patients with first-onset severe psychiatric episodes might suffer from undiagnosed autoimmune encephalitis. Therefore, the authors performed a three-step immunohistochemistry-based screening for CNS autoantibodies in a large cohort of patients with postpartum psychosis and matched postpartum comparison subjects. METHOD: Ninety-six consecutive patients with postpartum psychosis and 64 healthy postpartum women were included. Screening for antibodies in patient serum was performed using immunohistochemistry. Samples showing any staining were further examined by immunocytochemistry using live hippocampal neurons and cell-based assays to test for anti-N-methyl-d-aspartate (NMDA) receptor antibodies. Cell-based assays for all other known CNS antigens were performed in those samples with immunocytochemistry labeling but negative for NMDA receptor antibodies. RESULTS: Four patients (4%) with neuropil labeling suggestive for extracellular antigen reactivity were identified. Serum samples from all four patients showed clear extracellular labeling of live hippocampal neurons. Two women had the specific staining pattern characteristic for anti-NMDA receptor antibody positivity, which was confirmed by cell-based assays. Neither patient with anti-NMDA receptor antibody positivity had evidence of an ovarian teratoma. The other two patients tested negative by cell-based assays for all known CNS antigens. None of the matched postpartum comparison subjects had confirmed neuronal surface antibodies. The two patients with anti-NMDA receptor antibodies both showed extrapyramidal symptoms following initiation of treatment with low-dose haloperidol. CONCLUSIONS: In patients with acute psychosis during the postpartum period, systematic screening for anti-NMDA receptor autoantibodies should be considered. The acute onset of severe atypical psychiatric symptoms in young female patients should raise the index of suspicion for anti-NMDA receptor encephalitis, particularly in the setting of neurological symptoms, including extrapyramidal side effects of antipsychotic treatment.

Anti-NMDA receptor encephalitis: a cause of acute psychosis and catatonia.

Anti-N-methyl-D-aspartate receptor (anti-NMDAR) encephalitis is a newly described form of encephalitis associated with prominent psychiatric symptoms at onset. Recognition of the symptom complex is the key to diagnosis. Most patients with anti-NMDAR encephalitis develop a multistage illness that progresses from initial psychiatric symptoms to memory disturbance, seizures, dyskinesia, and catatonia. Psychiatric manifestations include anxiety, mania, social withdrawal, and psychosis (i.e., delusions, hallucinations, disorganized behavior). The disorder is more common in females (80%), in approximately half of whom it is associated with an underlying ovarian teratoma. Treatment involves immunosuppression, with steroids and intravenous immunoglobulin considered first line. The disorder is particularly relevant to psychiatrists, because most patients are initially seen by psychiatric services. Psychiatrists should consider anti-NMDAR encephalitis in patients presenting with psychosis as well as dyskinesia, seizures, and/or catatonia, especially if there is no history of a psychiatric disorder. We present the case of a 37-year-old woman who demonstrated many of the key clinical features of this potentially treatable disorder.

The 22q11.2 microdeletion: fifteen years of insights into the genetic and neural complexity of psychiatric disorders.

Over the last fifteen years it has become established that 22q11.2 deletion syndrome (22q11DS) is a true genetic risk factor for schizophrenia. Carriers of deletions in chromosome 22q11.2 develop schizophrenia at rate of 25-30% and such deletions account for as many as 1-2% of cases of sporadic schizophrenia in the general population. Access to a relatively homogeneous population of individuals that suffer from schizophrenia as the result of a shared etiological factor and the potential to generate etiologically valid mouse models provides an immense opportunity to better understand the pathobiology of this disease. In this review we survey the clinical literature associated with the 22q11.2 microdeletions with a focus on neuroanatomical changes. Then, we highlight results from work modeling this structural mutation in animals. The key biological pathways disrupted by the mutation are discussed and how these changes impact the structure and function of neural circuits is described.

Development of animal models for schizophrenia.

Schizophrenia is a devastating psychiatric disorder that affects around 1% of the population worldwide. The disease is characterized by 'positive symptoms', 'negative symptoms' and cognitive deficits. Over the last 60 years, a large number of family, twin and adoption studies have clearly demonstrated a strong genetic component for schizophrenia, but the mode of inheritance of the disease is complex and, in all likelihood, involves contribution from multiple genes in conjunction with environmental and stochastic factors. Recently, several genome-wide scans have demonstrated that rare alleles contribute significantly to schizophrenia risk. Assessments of rare variants have identified specific and probably causative, disease-associated structural mutations or copy number variants (CNVs, which result from genomic gains or losses). The fact that the effects of such lesions are transparent allows the generation of etiologically valid animal models and the opportunity to explore the molecular, cellular and circuit-level abnormalities underlying the expression of psychopathology. To date, the most common genomic structural rearrangements that are unequivocally associated with the development of schizophrenia, are de novo microdeletions of the 22q11.2 locus. Fortunately, the human 22q11.2 locus is conserved within the syntenic region of mouse chromosome 16, which harbors nearly all orthologues of the human genes. This has made it possible to engineer genetically faithful, and thus etiologically valid, animal models of this schizophrenia susceptibility locus.

Signaling pathways in schizophrenia: emerging targets and therapeutic strategies.

Dopamine D(2) receptor antagonism is a unifying property of all antipsychotic drugs in use for schizophrenia. While often effective at ameliorating psychosis, these drugs are largely ineffective at treating negative and cognitive symptoms. Increasing attention is being focused on the complex genetics of the illness and the signaling pathways implicated in its pathophysiology. We review targeted approaches for pharmacotherapy involving the glutamatergic, GABAergic and cholinergic pathways. We also describe several of the major genetic findings that identify signaling pathways representing potential targets for novel pharmacological intervention. These include genes in the 22q11 locus, DISC1, Neuregulin 1/ErbB4, and components of the Akt/GSK-3 pathway.