RESUMO
Recent studies have consistently demonstrated that the regulation of chromatin and gene transcription plays a pivotal role in the pathogenesis of neurodevelopmental disorders. Among many genes involved in these pathways, KMT2C, encoding one of the six known histone H3 lysine 4 (H3K4) methyltransferases in humans and rodents, was identified as a gene whose heterozygous loss-of-function variants are causally associated with autism spectrum disorder (ASD) and the Kleefstra syndrome phenotypic spectrum. However, little is known about how KMT2C haploinsufficiency causes neurodevelopmental deficits and how these conditions can be treated. To address this, we developed and analyzed genetically engineered mice with a heterozygous frameshift mutation of Kmt2c (Kmt2c+/fs mice) as a disease model with high etiological validity. In a series of behavioral analyses, the mutant mice exhibit autistic-like behaviors such as impairments in sociality, flexibility, and working memory, demonstrating their face validity as an ASD model. To investigate the molecular basis of the observed abnormalities, we performed a transcriptomic analysis of their bulk adult brains and found that ASD risk genes were specifically enriched in the upregulated differentially expressed genes (DEGs), whereas KMT2C peaks detected by ChIP-seq were significantly co-localized with the downregulated genes, suggesting an important role of putative indirect effects of Kmt2c haploinsufficiency. We further performed single-cell RNA sequencing of newborn mouse brains to obtain cell type-resolved insights at an earlier stage. By integrating findings from ASD exome sequencing, genome-wide association, and postmortem brain studies to characterize DEGs in each cell cluster, we found strong ASD-associated transcriptomic changes in radial glia and immature neurons with no obvious bias toward upregulated or downregulated DEGs. On the other hand, there was no significant gross change in the cellular composition. Lastly, we explored potential therapeutic agents and demonstrate that vafidemstat, a lysine-specific histone demethylase 1 (LSD1) inhibitor that was effective in other models of neuropsychiatric/neurodevelopmental disorders, ameliorates impairments in sociality but not working memory in adult Kmt2c+/fs mice. Intriguingly, the administration of vafidemstat was shown to alter the vast majority of DEGs in the direction to normalize the transcriptomic abnormalities in the mutant mice (94.3 and 82.5% of the significant upregulated and downregulated DEGs, respectively, P < 2.2 × 10-16, binomial test), which could be the molecular mechanism underlying the behavioral rescuing. In summary, our study expands the repertoire of ASD models with high etiological and face validity, elucidates the cell-type resolved molecular alterations due to Kmt2c haploinsufficiency, and demonstrates the efficacy of an LSD1 inhibitor that might be generalizable to multiple categories of psychiatric disorders along with a better understanding of its presumed mechanisms of action.
RESUMO
Genetic mutations contribute to the etiology of autism spectrum disorder (ASD), a common, heterogeneous neurodevelopmental disorder characterized by impairments in social interaction, communication, and repetitive and restricted patterns of behavior. Since neuroligin3 (NLGN3), a cell adhesion molecule at the neuronal synapse, was first identified as a risk gene for ASD, several additional variants in NLGN3 and NLGN4 were found in ASD patients. Moreover, synaptopathies are now known to cause several neuropsychiatric disorders including ASD. In humans, NLGNs consist of five family members, and neuroligin1 (NLGN1) is a major component forming a complex on excitatory glutamatergic synapses. However, the significance of NLGN1 in neuropsychiatric disorders remains unknown. Here, we systematically examine five missense variants of NLGN1 that were detected in ASD patients, and show molecular and cellular alterations caused by these variants. We show that a novel NLGN1 Pro89Leu (P89L) missense variant found in two ASD siblings leads to changes in cellular localization, protein degradation, and to the impairment of spine formation. Furthermore, we generated the knock-in P89L mice, and we show that the P89L heterozygote mice display abnormal social behavior, a core feature of ASD. These results, for the first time, implicate rare variants in NLGN1 as functionally significant and support that the NLGN synaptic pathway is of importance in the etiology of neuropsychiatric disorders.
Assuntos
Transtorno do Espectro Autista/genética , Moléculas de Adesão Celular Neuronais/genética , Predisposição Genética para Doença , Comportamento Social , Coluna Vertebral/crescimento & desenvolvimento , Animais , Transtorno do Espectro Autista/fisiopatologia , Comportamento Animal/fisiologia , Modelos Animais de Doenças , Feminino , Humanos , Masculino , Camundongos , Camundongos Transgênicos , Mutação de Sentido Incorreto/genética , Neurônios/patologia , Linhagem , Proteólise , Coluna Vertebral/fisiopatologia , Sinapses/genética , Sinapses/patologiaRESUMO
[This corrects the article DOI: 10.1371/journal.pgen.1006940.].
RESUMO
PURPOSE OF REVIEW: Recent advances in genetic technologies allowed researchers to identify large numbers of candidate risk genes associated with autism spectrum disorder (ASD). Both strongly penetrant rare variants and the accumulation of common variants with much weaker penetrance contribute to the cause of ASD. To identify the highly confident candidate genes, software and resources have been applied, and functional evaluation of the variants has provided further insights for ASD pathophysiology. These studies ultimately identify the molecular and circuit alteration underlying the behavioral abnormalities in ASD. In this review, we introduce the recent genetic and genomic findings and functional approaches for ASD variants providing a deeper understanding of the etiology of ASD. RECENT FINDINGS: Integrated meta-analysis that recruited a larger number of ASD cases has helped to prioritize ASD candidate genes or genetic loci into highly confidence candidate genes for further investigation. Not only coding but also noncoding variants have been recently implicated to confer the risk of ASD. Functional approaches of genes or variants revealed the disruption of specific molecular pathways. Further studies combining ASD genetics and genomics with recent techniques in engineered mouse models show molecular and circuit mechanisms underlying the behavioral deficits in ASD. SUMMARY: Advances in ASD genetics and the following functional studies provide significant insights into ASD pathophysiology at molecular and circuit levels.
Assuntos
Transtorno do Espectro Autista/genética , Predisposição Genética para Doença , Animais , Variações do Número de Cópias de DNA , Modelos Animais de Doenças , Humanos , CamundongosRESUMO
The mammalian cortex is a highly evolved brain region, but we still lack a comprehensive understanding of the molecular mechanisms underlying primate-specific neural circuits formation. In this study, we employed spatial transcriptomics to assess gene expression dynamics in the marmoset cortex during development, focusing on key regions and time points. Spatial transcriptomics identified genes that are sexually, spatially, and temporally differentially expressed in the developing marmoset cortex. Our detailed analysis of the visual cortex unveiled dynamic changes in gene expression across layers with distinct projections and functions. Notably, we discovered numerous axon guidance molecules with spatiotemporal expression patterns unique to the developing marmoset prefrontal cortex (PFC), which control PFC neuronal circuits. Among these molecules, PRSS12 (Protease, Serine, 12 (neurotrypsin, motopsin), when ectopically expressed in the mouse prelimbic cortex, caused similar changes in connectivity as observed in the marmoset A32 area. Furthermore, PRSS12 showed similar expression patterns in both marmoset and human PFC during development, suggesting parallels between marmoset and human brain development. The differential expression of axon guidance molecules in the developing PFC, varying by region, likely contributes to the formation of unique circuits observed in primates.
RESUMO
OBJECTIVES: Auditory-perceptual evaluation frameworks, such as the grade-roughness-breathiness-asthenia-strain (GRBAS) scale, are the gold standard for the quantitative evaluation of pathological voice quality. However, the evaluation is subjective; thus, the ratings lack reproducibility due to inter- and intra-rater variation. Prior researchers have proposed deep-learning-based automatic GRBAS score estimation to address this problem. However, these methods require large amounts of labeled voice data. Therefore, this study investigates the potential of automatic GRBAS estimation using deep learning with smaller amounts of data. METHODS: A dataset consisting of 300 pathological sustained /a/ vowel samples was created and rated by eight experts (200 for training, 50 for validation, and 50 for testing). A neural network model that predicts the probability distribution of GRBAS scores from an onset-to-offset waveform was proposed. Random speed perturbation, random crop, and frequency masking were investigated as data augmentation techniques, and power, instantaneous frequency, and group delay were investigated as time-frequency representations. RESULTS: Five-fold cross-validation was conducted, and the automatic scoring performance was evaluated using the quadratic weighted Cohen's kappa. The results showed that the kappa values of the automatic scoring performance were comparable to those of the inter-rater reliability of experts for all GRBAS items and the intra-rater reliability of experts for items G, B, A, and S. Random speed perturbation was the most effective data augmentation technique overall. When data augmentation was applied, power was the most effective for items G, R, A, and S; for Item B, combining group delay and power yielded additional performance gains. CONCLUSION: The automatic GRBAS scoring achieved by the proposed model using scant labeled data was comparable to that of experts. This suggests that the challenges resulting from insufficient data can be alleviated. The findings of this study can also contribute to performance improvements in other tasks such as automatic voice disorder detection.
RESUMO
Various molecular biology techniques implementing genome editing have made it possible to generate mouse mutants for nearly all known genes; as a result, the International Mouse Phenotyping Consortium (IMPC) database listing the phenotypes of genetically modified mice has been established. Among mouse phenotypes, lethality is crucial to evaluate the importance of genes in mouse survival. Although many genes are reported to show "preweaning lethality, incomplete penetrance" in the IMPC database, the survival rates of homozygous knockout mice are highly variable. Here, we propose the lethal allele index (LAI), the ratio of the observed number of mice with homozygous knockout (KO) to the theoretically predicted number of homozygous KO mice, as a simple quantitative indicator of preweaning lethality. Among the mice mutants registered as incompletely lethal in IMPC, the LAI calculated from the genotypes of F1 mice tended to be lower in disease-related genes, and correlated with the frequency of loss-of-function (LOF) alleles in humans. In genome-edited mice using CRISPR/Cas9, the number of mice with homozygous frameshift alleles seemed to be associated with lethality. We edited the Ehd1 gene in cell lines as well as mice using CRISPR/Cas9, and found that the genotype distribution was significantly different. The LAI calculated from these data was similar to the value calculated from the IMPC data. These findings support the potential usefulness of the LAI as an index of preweaning lethality in genome-edited mice.
Assuntos
Sistemas CRISPR-Cas/genética , Proteínas de Transporte Vesicular/genética , Desmame , Animais , Edição de Genes/métodos , Genes Letais/genética , Genoma/genética , Genótipo , Camundongos , Camundongos Knockout , Mutação , FenótipoRESUMO
Knockin (KI) mouse carrying a point mutation has been an invaluable tool for disease modeling and analysis. Genome editing technologies using the CRISPR/Cas system has emerged as an alternative way to create KI mice. However, if the mice carry nucleotide insertions and/or deletions (InDels) in other genes, which could have unintentionally occurred during the establishment of the KI mouse line and potentially have larger impact than a point mutation, it would confound phenotyping of the KI mice. In this study, we performed whole exome sequencing of multiple lines of F1 heterozygous Ntrk1 KI mice generated using the CRISPR/Cas system in comparison to that of a wild-type mouse used as a control. We found three InDels in four KI mice but not in a control mouse. In vitro digestion assay suggested that each InDel occurred as a de novo mutation, was carried-over from the parental mice, or was incorporated through the Cas9 nuclease mediated off-target cleavage. These results suggest that frequency of InDels found in KI mice generated by the CRISPR/Cas technology is not high, but cannot be neglected and careful assessment of these mutations is warranted.
Assuntos
Sistemas CRISPR-Cas , Endonucleases/genética , Exoma , Mutação INDEL , RNA Guia de Cinetoplastídeos/genética , Receptor trkA/genética , Animais , Sequência de Bases , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Endonucleases/metabolismo , Feminino , Efeito Fundador , Edição de Genes , Expressão Gênica , Técnicas de Introdução de Genes , Masculino , Camundongos , Camundongos Transgênicos , RNA Guia de Cinetoplastídeos/metabolismo , Receptor trkA/metabolismo , Erro Científico Experimental/estatística & dados numéricos , Sequenciamento do ExomaRESUMO
The endothelin signaling pathway plays an important role in the migration, proliferation, and differentiation of neural crest cells. Mutations in the gene encoding the endothelin receptor type B (EDNRB) cause three symptoms: aganglionosis, pigmented disorder and hearing loss. In addition, the Ednrb null mice show abnormal splenic microarchitecture with lymphopenia. In this study, we examined whether similar phenotypes are reproduced in three Ednrb-null rat strains that we established previously. AGH-Ednrb(sl)/(sl) strain showed a low white blood cell count, significant size reduction and abnormal microarchitecture of spleen. Thymus displayed a marked reduction in the size, but maintained a normal CD4/CD8 ratio. In contrast, splenic cellularity was reduced to < 15%, and splenic B and T cell numbers were reduced, showing a splenic lymphopenia. Interestingly, Ednrb-null rats in the LE and F344 genetic background did not show these abnormalities. These data show that proper T and B cell development is dependent on the endothelin signaling pathway, however, modifier gene(s) might be differentially expressed in these strain to modulate or compensate for the effect of the Ednrb deficiency.