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Title:
Human models for the investigation of proteins associated with birth defects affecting face and long bone skeleton and cognitive development
Supervisors:
Patrizia Ferretti, Oliver Gardner and Simon Eaton
Project Description:
Background:
Mutations and/or genetic variations in some phosphodiesterases (PDEs), including members of the PDE4 family, have been implicated in intellectual disability in neurodevelopmental disorders (e.g. autism, Fragile X Syndrome), neurodegenerative diseases, and in bone formation disorders. Autosomal dominant mutations in PDE4D (cAMP-specific-phosphodiesterase-4D) and PRKAR1A (type-1A-regulatory subunit of protein-kinase-A, PKA), both components of the cAMP/PKA signalling pathway, underly two types of the rare disease, acrodysostosis (ACRDYS), ACRDYS2 and ACRDYS1, respectively. They share many distinctive features, including facial hypoplasia, brachydactyly, and obesity, but present important differences including hormonal stimulation resistance of growth plate chondrocytes and reduced height (predominantly ACRDYS1), and mental retardation (predominantly ACRDYS2). The mechanisms underlying these differences are not well understood and there is no targeted care for these patients. Spatio-temporal and quantitative differences in PRKAR1A and PDE4D expression and activity in different cell types during human development may contribute to differences in disease presentation, but have yet to be investigated. Crucially, no study has been carried out in human chondrocytes and neural cells directly affected by PRKAR1A and PDE4D mutations resulting in ACRDYS. Understanding the mechanisms underlying the two ACRDYS types will guide development of novel tissue/patient-specific therapeutic approaches and can be also relevant to other diseases affecting cAMP/PKA signalling.
Aims/Objectives
• The overall aim is to use human embryonic/foetal brain and skeletal tissues and human cell models of ACRDYS to understand how mutations in genes along the same signalling pathway, the cAMP/PKA pathway, lead to converging and diverging phenotypes in ACRDYS1 and ACRDYS2.
Hypotheses
• Cell type-specific differences in expression levels of PRKAR1A and PDE4D, and of other members of the PDE4 family, underlie phenotypic differences between ACRDYS1 and ACRDYS2.
• ACRDYS2 changes in PDE4D stability and its intracellular localization, and consequent dysregulation of other PDE4 family members, contribute to disrupting cAMP signalling possibly making it unavailable to inactivation by PKA.
Methods
The student will use cells from healthy individuals and ACRDYS1 and ACRDYS2 patients recently reprogrammed (iPSC); isogenic lines carrying patients' mutations are being generated. We have already extensive experience both in skeletal and neural stem cell differentiation. Refinement of the growth plate model from iPSCs will be built on our current chondrogenic spheroid culture model.
Immunostaining, western blotting and RT-qPCR will be used to assess developmental changes in developing human embryos/foetuses (from the HDBR) as well as changes in cell localization/gene/protein expression in iPSC-derived healthy and ACRYDS cell models.
Interactions between PDE4D and PKA in different cell lines and under different experimental conditions will be tested by immunoprecipitation.
Biochemical evaluations of enzymatic activities will be carried out either using commercially available kits or assay established in Prof. Eaton’s laboratory.
A range of small molecules, including PDE4D-specific PROTACs, will be assessed for their ability to regulate relevant components of the cAMP signalling pathway, and normalize growth/differentiation at the growth plate and the altered expression of PDE4 isoforms.
Timeline
Year-1: Learn key techniques; assess PDE4D and PRKAR1A during human development/cell differentiations; establish growth plate model (“assembloid approach”).
Year-2: investigate hypothesised disease mechanism in growth plate/neural models
Year-3: assess possible pharmacological interventions; write papers/thesis.
Refs (DOI)
10.1093/braincomms/fcae225
10.1038/nrendo.2016.52
10.1038/s41431-018-0135-1
10.1038/s41573-019-0033-4
10.1016/j.cellsig.2014.07.025
Contact Information:
Patrizia Ferretti and/or Oliver Gardner