Assessment of the FRET-based Teen sensor to monitor ERK activation changes preceding morphological defects in a RASopathy zebrafish model and phenotypic rescue by MEK inhibitor.

BACKGROUND: RASopathies are genetic syndromes affecting development and having variable cancer predisposition. These disorders are clinically related and are caused by germline mutations affecting key players and regulators of the RAS-MAPK signaling pathway generally leading to an upregulated ERK activity. Gain-of-function (GOF) mutations in PTPN11, encoding SHP2, a cytosolic protein tyrosine phosphatase positively controlling RAS function, underlie approximately 50% of Noonan syndromes (NS), the most common RASopathy. A different class of these activating mutations occurs as somatic events in childhood leukemias. METHOD: Here, we evaluated the application of a FRET-based zebrafish ERK reporter, Teen, and used quantitative FRET protocols to monitor non-physiological RASopathy-associated changes in ERK activation. In a multi-level experimental workflow, we tested the suitability of the Teen reporter to detect pan-embryo ERK activity correlates of morphometric alterations driven by the NS-causing Shp2D61G allele. RESULTS: Spectral unmixing- and acceptor photobleaching (AB)-FRET analyses captured pathological ERK activity preceding the manifestation of quantifiable body axes defects, a morphological pillar used to test the strength of SHP2 GoF mutations. Last, the work shows that by multi-modal FRET analysis, we can quantitatively trace back the modulation of ERK phosphorylation obtained by low-dose MEK inhibitor treatment to early development, before the onset of morphological defects. CONCLUSION: This work proves the usefulness of FRET imaging protocols on both live and fixed Teen ERK reporter fish to readily monitor and quantify pharmacologically- and genetically-induced ERK activity modulations in early embryos, representing a useful tool in pre-clinical applications targeting RAS-MAPK signaling.

Dominantly acting KIF5B variants with pleiotropic cellular consequences cause variable clinical phenotypes.

Kinesins are motor proteins involved in microtubule (MT)-mediated intracellular transport. They contribute to key cellular processes, including intracellular trafficking, organelle dynamics and cell division. Pathogenic variants in kinesin-encoding genes underlie several human diseases characterized by an extremely variable clinical phenotype, ranging from isolated neurodevelopmental/neurodegenerative disorders to syndromic phenotypes belonging to a family of conditions collectively termed as 'ciliopathies.' Among kinesins, kinesin-1 is the most abundant MT motor for transport of cargoes towards the plus end of MTs. Three kinesin-1 heavy chain isoforms exist in mammals. Different from KIF5A and KIF5C, which are specifically expressed in neurons and established to cause neurological diseases when mutated, KIF5B is an ubiquitous protein. Three de novo missense KIF5B variants were recently described in four subjects with a syndromic skeletal disorder characterized by kyphomelic dysplasia, hypotonia and DD/ID. Here, we report three dominantly acting KIF5B variants (p.Asn255del, p.Leu498Pro and p.Leu537Pro) resulting in a clinically wide phenotypic spectrum, ranging from dilated cardiomyopathy with adult-onset ophthalmoplegia and progressive skeletal myopathy to a neurodevelopmental condition characterized by severe hypotonia with or without seizures. In vitro and in vivo analyses provide evidence that the identified disease-associated KIF5B variants disrupt lysosomal, autophagosome and mitochondrial organization, and impact cilium biogenesis. All variants, and one of the previously reported missense changes, were shown to affect multiple developmental processes in zebrafish. These findings document pleiotropic consequences of aberrant KIF5B function on development and cell homeostasis, and expand the phenotypic spectrum resulting from altered kinesin-mediated processes.

A minimally invasive fin scratching protocol for fast genotyping and early selection of zebrafish embryos.

Current genetic modification and phenotyping methods in teleost fish allow detailed investigation of vertebrate mechanisms of development, modeling of specific aspects of human diseases and efficient testing of drugs at an organ/organismal level in an unparalleled fast and large-scale mode. Fish-based experimental approaches have boosted the in vivo verification and implementation of scientific advances, offering the quality guaranteed by animal models that ultimately benefit human health, and are not yet fully replaceable by even the most sophisticated in vitro alternatives. Thanks to highly efficient and constantly advancing genetic engineering as well as non-invasive phenotyping methods, the small zebrafish is quickly becoming a popular alternative to large animals' experimentation. This approach is commonly associated to invasive procedures and increased burden. Here, we present a rapid and minimally invasive method to obtain sufficient genomic material from single zebrafish embryos by simple and precise tail fin scratching that can be robustly used for at least two rounds of genotyping already from embryos within 48 h of development. The described protocol betters currently available methods (such as fin clipping), by minimizing the relative animal distress associated with biopsy at later or adult stages. It allows early selection of embryos with desired genotypes for strategizing culturing or genotype-phenotype correlation experiments, resulting in a net reduction of "surplus" animals used for mutant line generation.

Targeting Oncogenic Src Homology 2 Domain-Containing Phosphatase 2 (SHP2) by Inhibiting Its Protein-Protein Interactions.

We developed a new class of inhibitors of protein-protein interactions of the SHP2 phosphatase, which is pivotal in cell signaling and represents a central target in the therapy of cancer and rare diseases. Currently available SHP2 inhibitors target the catalytic site or an allosteric pocket but lack specificity or are ineffective for disease-associated SHP2 mutants. Considering that pathogenic lesions cause signaling hyperactivation due to increased levels of SHP2 association with cognate proteins, we developed peptide-based molecules with nanomolar affinity for the N-terminal Src homology domain of SHP2, good selectivity, stability to degradation, and an affinity for pathogenic variants of SHP2 that is 2-20 times higher than for the wild-type protein. The best peptide reverted the effects of a pathogenic variant (D61G) in zebrafish embryos. Our results provide a novel route for SHP2-targeted therapies and a tool for investigating the role of protein-protein interactions in the function of SHP2.

SPRED2 loss-of-function causes a recessive Noonan syndrome-like phenotype.

Upregulated signal flow through RAS and the mitogen-associated protein kinase (MAPK) cascade is the unifying mechanistic theme of the RASopathies, a family of disorders affecting development and growth. Pathogenic variants in more than 20 genes have been causally linked to RASopathies, the majority having a dominant role in promoting enhanced signaling. Here, we report that SPRED2 loss of function is causally linked to a recessive phenotype evocative of Noonan syndrome. Homozygosity for three different variants-c.187C>T (p.Arg63∗), c.299T>C (p.Leu100Pro), and c.1142_1143delTT (p.Leu381Hisfs∗95)-were identified in four subjects from three families. All variants severely affected protein stability, causing accelerated degradation, and variably perturbed SPRED2 functional behavior. When overexpressed in cells, all variants were unable to negatively modulate EGF-promoted RAF1, MEK, and ERK phosphorylation, and time-course experiments in primary fibroblasts (p.Leu100Pro and p.Leu381Hisfs∗95) documented an increased and prolonged activation of the MAPK cascade in response to EGF stimulation. Morpholino-mediated knockdown of spred2a and spred2b in zebrafish induced defects in convergence and extension cell movements indicating upregulated RAS-MAPK signaling, which were rescued by expressing wild-type SPRED2 but not the SPRED2Leu381Hisfs∗95 protein. The clinical phenotype of the four affected individuals included developmental delay, intellectual disability, cardiac defects, short stature, skeletal anomalies, and a typical facial gestalt as major features, without the occurrence of the distinctive skin signs characterizing Legius syndrome. These features, in part, characterize the phenotype of Spred2-/- mice. Our findings identify the second recessive form of Noonan syndrome and document pleiotropic consequences of SPRED2 loss of function in development.

In vivo Functional Genomics for Undiagnosed Patients: The Impact of Small GTPases Signaling Dysregulation at Pan-Embryo Developmental Scale.

While individually rare, disorders affecting development collectively represent a substantial clinical, psychological, and socioeconomic burden to patients, families, and society. Insights into the molecular mechanisms underlying these disorders are required to speed up diagnosis, improve counseling, and optimize management toward targeted therapies. Genome sequencing is now unveiling previously unexplored genetic variations in undiagnosed patients, which require functional validation and mechanistic understanding, particularly when dealing with novel nosologic entities. Functional perturbations of key regulators acting on signals' intersections of evolutionarily conserved pathways in these pathological conditions hinder the fine balance between various developmental inputs governing morphogenesis and homeostasis. However, the distinct mechanisms by which these hubs orchestrate pathways to ensure the developmental coordinates are poorly understood. Integrative functional genomics implementing quantitative in vivo models of embryogenesis with subcellular precision in whole organisms contribute to answering these questions. Here, we review the current knowledge on genes and mechanisms critically involved in developmental syndromes and pediatric cancers, revealed by genomic sequencing and in vivo models such as insects, worms and fish. We focus on the monomeric GTPases of the RAS superfamily and their influence on crucial developmental signals and processes. We next discuss the effectiveness of exponentially growing functional assays employing tractable models to identify regulatory crossroads. Unprecedented sophistications are now possible in zebrafish, i.e., genome editing with single-nucleotide precision, nanoimaging, highly resolved recording of multiple small molecules activity, and simultaneous monitoring of brain circuits and complex behavioral response. These assets permit accurate real-time reporting of dynamic small GTPases-controlled processes in entire organisms, owning the potential to tackle rare disease mechanisms.