2019 — 2020 |
De Rubeis, Silvia |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Investigating Ddx3x as a Sex-Specific Translational Regulator Associated With Intellectual Disability @ Icahn School of Medicine At Mount Sinai
PROJECT SUMMARY There are fundamental gaps in our understanding of the genetics and neurobiology of DDX3X, a high-risk gene for X-linked intellectual disability (ID) accounting for up to 2% of unexplained diagnoses in females. DDX3X mutations display a puzzling sexual dichotomy: most mutations are de novo and found only in females; the few mutations in males are inherited from healthy mothers. DDX3X regulates mRNA translation, but the mechanisms of action in neurons, the target genes, and the impact of clinical mutations have not been studied. Also, the influence of sex remains unknown. There is a critical need to fill these gaps because, until we do so, implementing strategies that use DDX3X as a therapeutic target remains out of reach. The long-term goal is to map the synaptic mechanisms underlying ID and identify new therapeutic targets. The overall objective is to capture the sex-specific synaptic changes resulting from mutations in DDX3X. The central hypothesis is that DDX3X regulates synaptic translation and synaptogenesis in a sex-specific manner: mutations impact these processes differently in males and females, leading to sex biases in prevalence and severity of the condition. The rationale is that, once we know how DDX3X regulates synaptic translation and how sex influences it, mechanism-based precision therapeutics can be developed. The central hypothesis will be tested by pursuing two Specific Aims: 1) Analyze the role of DDX3X in synaptic translation and synaptogenesis; and, 2) Determine the effects of sex-specific DDX3X mutations. Under Aim 1, biochemical and structural methods will be applied to study DDX3X-mediated translation in mouse synapses. Viral-based Translating Ribosome Affinity Purification (vTRAP-seq) will be used to map the mRNAs regulated by DDX3X in male and female mouse synapses. Dendritic spines analyses in male and female single-embryo neurons from a novel DDX3X mouse model will be used to assess the role in synaptogenesis. Under Aim 2, two male- and three female-pathogenic mutations will be structurally and biochemically modeled. The mutations will be introduced in cultured neurons and the spine phenotype compared across mutations and sexes to understand why female-pathogenic mutations are more severe. All these methods are part of the applicant's expertise. This proposal is innovative because it addresses the biology of a novel ID high-risk gene in light of a central and yet overlooked aspect: sex. It is innovative because it models patient-specific mutations in neurons. It is also innovative because it integrates genetics, structural biology, biochemistry, molecular biology and neuroscience, and uses a novel mouse model and novel DDX3X small-molecule inhibitors. This application is significant because it will critically advance our understanding of DDX3X syndrome and ID more broadly. The results will expose fundamental aspects of synaptogenesis, thus advancing the knowledge of brain development in health and disease. The results are expected to have a positive impact because they will pinpoint novel molecular targets for precision medicine, while informing clinical genetics care, with both short-term and long-term benefits for families.
|
1 |
2021 |
De Rubeis, Silvia |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Cellular and Molecular Determits of Ddx3x Syndrome @ Icahn School of Medicine At Mount Sinai
PROJECT SUMMARY There are fundamental gaps in our understanding of DDX3X syndrome, a genetic condition accounting for up to 2% of intellectual disability (ID) in females and caused by mutations in the X-linked gene DDX3X. Most affected individuals are females with DDX3X haploinsufficiency. A recent study using cell models and in utero manipulations in mouse has shown that Ddx3x regulates cortical neurogenesis and mRNA translation in neuronal progenitors. Yet, the lack of a mouse model with construct validity for DDX3X syndrome has impeded to faithfully capture the molecular and cellular determinants of the cognitive, social and motor deficits observed in individuals with DDX3X syndrome. There is a critical need to fill these gaps because, until we do so, understanding DDX3X syndrome and developing effective therapeutics remain out of reach. To address this unmet need, a mouse modeling DDX3X haploinsufficiency (Ddx3x+/-) was generated in our laboratory. The long-term goal is to unravel the pathophysiology of ID and identify targets for therapeutics. The overall objective is to capture the molecular and cellular mechanisms underlying DDX3X syndrome. The central hypothesis is that Ddx3x dictates the formation of cortical projection neurons subserving cognitive, social, and motor functions, and does so by regulating mRNA translation in the developing cortex. The rationale is that, once we identify the mechanisms of DDX3X syndrome and reliable phenotypes in the mouse model, therapeutics can be developed and tested. The hypothesis will be tested by pursuing three Specific Aims: 1) Assess the neurodevelopmental defects in a mouse model of DDX3X syndrome; 2) Capture the molecular defects in a mouse model of DDX3X syndrome; and, 3) Correlate cellular function to behavioral outcomes in Ddx3x mutant mice. Under Aim 1, the developmental and adult cognitive, social, and motor behavior of Ddx3x+/- mice will be measured with a standardized behavioral battery. Cortical projection neurons will be studied in Ddx3x+/- mice by combining cellular and in vivo (e.g., in utero electroporation) approaches. Under Aim 2, Ddx3x targets in projection neurons will be mapped using the translating ribosome affinity purification (TRAP) method for discovery and its viral-based development (vTRAP) for independent validation. Under Aim 3, conditional Ddx3x mice with the gene ablated in the forebrain or in specific cortical layers will be tested for development and adult behavior. The proposal is innovative because it addresses the neurobiology of a largely unknown ID gene and characterizes the first mouse model of DDX3X syndrome. It is also innovative because it bridges genetics, biochemistry, molecular and cellular neuroscience, developmental biology, and behavioral neuroscience. The application is significant because it will advance our understanding of ID pathophysiology, while shedding new light on corticogenesis, a fundamental process for brain functioning. These results are expected to have a positive impact because they will pave the way for novel therapeutic interventions for ID.
|
1 |