Benjamin L. Prosser

BACKGROUND: No therapies exist to reverse right ventricular failure (RVF), and the molecular mechanisms that drive RVF remain understudied. We recently reported that the developmentally restricted noncanonical WNT receptor ROR2 (receptor tyrosine kinase-like orphan receptor 2) is upregulated in human RVF in proportion to severity of disease. Here we test the mechanistic role of ROR2 in RVF pathogenesis. METHODS: ROR2 was overexpressed or knocked down in neonatal rat ventricular myocytes and then characterized using confocal microscopy, RNA sequencing, proteomics, proteostatic functional assays, and pacing to assess contractile properties. The impact of cardiac ROR2 expression was evaluated in mice by AAV9-mediated overexpression and by AAV9-mediated delivery of short hairpin RNA to knockdown ROR2 in a pulmonary artery banded pressure overload model of RVF. ROR2 -modified mice were evaluated by echocardiography, histology, and right ventricle protein synthesis and proteasome capacity. RESULTS: In neonatal rat ventricular myocytes, we find that ROR2 profoundly dysregulates the coordination between protein translation and folding. This imbalance leads to excess protein clearance by the ubiquitin proteasome system with dramatic impacts on sarcomere and cytoskeletal structure and function. Inhibiting the ubiquitin proteasome system or restoring chaperone expression is sufficient to partially rescue ROR2-induced structural and contractile deficits in cardiomyocytes. In mice, forced cardiac ROR2 expression is sufficient to disrupt proteostasis and drive RVF, whereas conversely Ror2 knockdown partially rescues proteostasis and right ventricle structure and function in a pressure overload model of RVF. CONCLUSIONS: In sum, ROR2 is a key driver of RVF pathogenesis through proteostatic disruption and thus provides a promising target to treat RVF.

Objective: STXBP1 ‐related disorders ( STXBP1 ‐RD) are among the most common genetic neurodevelopmental disorders, marked by early onset epilepsy, global developmental delay, and motor impairments. Many missense variants remain uncharacterized, limiting accurate variant interpretation and hindering development of precision therapies. Current American College of Medical Genetics and Genomics (ACMG)/Association for Molecular Pathology (AMP) variant curation frameworks provide limited discriminatory power for STXBP1 missense variants due to limited functional data and frequently incomplete parental testing, leaving a substantial proportion of variants classified as variants of uncertain significance. Methods: We developed a fluorometric assay that leverages split fluorescence complementation to simultaneously measure STXBP1 protein stability and its binding affinity to a primary interaction partner, syntaxin‐1A (STX1A), including genetically engineered closed and open STX1A conformations, which are essential for SNARE complex function. Using this platform, we evaluated five benign variants, six pathogenic variants, and four variants that result in an atypically mild clinical presentation. Results: Benign population variants showed minimal effects on stability or binding, whereas known pathogenic variants consistently demonstrated reduced protein stability and diminished binding to STX1A, with some exhibiting STX1A conformation‐specific binding deficits. Variants associated with atypically mild phenotypes displayed intermediate, variant‐specific profiles. To quantify discriminatory performance, we developed and trained an exploratory support vector machine classifier using stability and binding metrics. With the exception of a single STXBP1 variant in an individual with a mild clinical presentation, the model correctly classified benign and pathogenic variants using leave‐one‐out cross‐validation. For variants with available clinical data, classifier scores showed a statistically significant correlation with clinical severity. Significance: Together, these findings demonstrate that a scalable, quantitative assay capturing STXBP1 stability and syntaxin‐binding behavior can differentiate benign from pathogenic variants and provide functional insights into mechanisms of pathogenicity. This platform offers a path toward improved variant interpretation, supports refinement of ACMG/AMP criteria for synaptic disorders, and may inform future therapeutic targeting strategies for STXBP1 ‐RD.

BACKGROUND: Translational control of gene expression is crucial in cardiomyocytes, particularly in response to hypertrophic stimuli. The ERK (extracellular signal-regulated kinase) pathway plays a key role in inducing cardiac hypertrophy and regulating specific protein translation. However, it remains unclear how this specificity is achieved, and the spatiotemporal regulation of protein translation is not fully understood. METHODS: We used SINAP (single-molecule imaging of nascent peptide) reporters to visualize and analyze the translation dynamics in single adult rat ventricular cardiomyocytes and tracked active translation sites at high spatiotemporal resolution. We also examined the effects of adrenergic stimulation and the role of the ERK pathway in translation localization. RESULTS: Our findings revealed that translation sites are primarily localized near Z-lines in cardiomyocytes, with some sites being highly dynamic and moving during translation. The 3′ untranslated regions did not significantly change the localization of translation. Many translation sites colocalized with microtubules, and their movement predominantly occurred along microtubular tracks. Adrenergic stimulation led to a transient shift in translation activity toward the perinuclear region, peaking at 12 hours and requiring ERK pathway activity for this localization change. This shift is part of the hypertrophic response and is required for early translation of genes such as Nppa . CONCLUSIONS: Our high-resolution single-cell study demonstrates that protein translation in cardiomyocytes is dynamic and responsive to hypertrophic stimuli in an ERK-dependent manner. The localized translation mechanism allows cardiomyocytes to rapidly adapt to changing environments by preferentially translating mRNAs in the perinuclear region. These findings provide new insights into the spatial regulation of translation in cardiomyocytes and its role in cardiac hypertrophy.

Aims: To describe the developmental phenotype of individuals with STXBP1 -related disorders ( STXBP1 -RD) in a prospective natural history study using standardized outcome measures. Methods: Data were collected from 155 individuals with a mean age of 8.2 years. The Bayley Scales of Infant and Toddler Development-Fourth Edition (Bayley-4), Hand Manipulation and Eye Hand Coordination subtests of the Peabody Developmental Motor Scales-Third Edition (PDMS-3), and Gross Motor Function Measure-66 (GMFM-66) were assessed. The Gross Motor Function Classification System (GMFCS), Mini-Manual Ability Classification System (MiniMACS), Manual Ability Classification System (MACS), and Communication Function Classification System (CFCS) were used to classify participants. Results: Assessments were well tolerated with 0-6.9% incomplete assessments. Floor or ceiling effects were not common with raw scores, age equivalents, or growth scales values but were frequently observed with scaled scores. Significant differences were found between the Bayley-4, GMFM-66, and PDMS-3 when participants were divided into groups by corresponding developmental domain classification scale. Strong associations ( p <.05) were seen between all measures across Bayley-4 domains, PDMS-3 domains, and GMFM-66 total scores. Interpretation: The Bayley-4, GMFM-66, and PDMS-3 are suitable for use in clinical practice and future clinical trials in STXBP1 -RD.

The nervous system relies on billions of neurons connected through trillions of synapses to support vital functions. Despite the importance of this synaptic network, cellular mechanisms dictating synapse formation during human neurodevelopment remain unclear. Long-distance trafficking by the microtubule motor KIF1A is crucial for synaptogenesis and downstream synapse maintenance. Mutations in KIF1A cause KIF1A-associated neurological disorder (KAND). We employed isogenic gene-edited human-induced pluripotent stem cell -derived neurons to assess the effects of disparate pathogenic mutations in KIF1A on synaptic trafficking and function. Null (p.C92*) and hypoactive (p.P305L) mutations delay neurite outgrowth, mislocalize synaptic cargoes, and decrease synapse density. Conversely, a hyperactive (p.R350G) mutation supports neurite outgrowth but causes aberrant motility of synaptic vesicle precursors and deficits in microtubule-dependent presynaptic patterning. Functional analysis of neuronal activity reveals delayed synaptic maturation in loss-of-function mutations (p.P305L, p.C92*) and precocious activity in the hyperactive p.R350G mutation. These data provide insights into how KIF1A mutations with distinct molecular-level impacts lead to significant downstream synaptic deficits in human neurons.

Heterozygous variants in SYNGAP1 and STXBP1 cause distinct neurodevelopmental disorders due to haploinsufficiency of essential synaptic proteins. As gene targeted approaches to correct these disorders often target non-conserved genomic regions, thus limiting their clinical translation, we generated humanized mouse models wherein the entire Syngap1 or Stxbp1 loci were replaced with their human counterparts. Stxbp1 humanized mice exhibited impaired viability, while Stxbp1 hybrid mice ( Stxbp1 ^Hu/+ ) were viable and suitable for evaluating target engagement of human-specific therapeutics. Syngap1 humanized mice were viable and successfully crossed with Syngap1 heterozygous mice to produce a Syngap1 humanized-haploinsufficient model ( Syngap1 ^Hu/− ). Syngap1 ^Hu/− mice displayed haploinsufficient levels of human SYNGAP1, disease-relevant behaviors, and EEG abnormalities including epileptiform activity and generalized slowing. Importantly, parallel analysis in a cohort of patients with SYNGAP1 -disorder revealed similar electrophysiological signatures. Finally, we showed that human gene-targeted antisense oligonucleotides modulate human SYNGAP1 expression in Syngap1 ^Hu/− neurons. Together, we describe new models to support pre-clinical therapeutic development for SYNGAP1 and STXBP1 disorders and identify translational biomarkers of SYNGAP1 -disorder in mice and humans to benchmark therapeutic testing.

Objective: STXBP1‐ related disorder ( STXBP1 ‐RD) and SYNGAP1 ‐related disorder ( SYNGAP1 ‐RD) are two common genetic synaptopathies that are associated with epilepsy, developmental delay, intellectual developmental disorder, and behavioral problems. Both STXBP1 ‐RD and SYNGAP1 ‐RD are potential targets for disease‐modifying therapies, but there is limited information in the literature describing the natural history of either disorder, which impedes outcome selection for future clinical trials. The objective of this study is to develop a framework to better define and outline the clinical spectrum and longitudinal trajectories of STXBP1 ‐RD and SYNGAP1 ‐RD natural history, including development, behavior, seizure histories, and electrophysiology. Methods: Here, we describe a protocol, regulatory structure, and supportive preliminary data for multicenter, prospective natural history studies of STXBP1 ‐RD (STARR) and SYNGAP1 ‐RD (ProMMiS). The protocols incorporate gold standard clinician‐administered outcome measures including the Bayley Scales of Infant and Toddler Development 4th edition, Gross Motor Function Measure‐66, fine motor domains of the Peabody Developmental Motor Scales 3rd edition, parent‐reported outcome measures, epilepsy histories, and biomarker exploration. To date, the study has enrolled 164 individuals with STXBP1 ‐RD and 159 with SYNGAP1 ‐RD, with ongoing longitudinal assessments every 6 months in a subset of approximately 200 total individuals across both disorders. Results: Our data support that existing developmental measures are feasible, informative, and show minimal floor or ceiling effects. Furthermore, we demonstrate that medical record‐based seizure history reconstruction reveals unique epilepsy trajectories while minimizing burden to families. We observe disease‐specific patterns of developmental performance and distinct longitudinal seizure dynamics, highlighting the need for data generation in a gene/disorder‐specific manner for clinical trial readiness. Significance: In summary, we present a feasible natural history protocol with prospective data for two complex neurodevelopmental disorders with natural histories that have previously been incompletely characterized, within a regulatory framework that will support the use of these data to expedite clinical trial development.

The adult heart grows by the addition of sarcomeres along the length or width of individual cardiomyocytes, yet how directional growth is spatially coordinated remains unclear. We found that microtubule dynamics could act as a toggle to direct cardiomyocyte growth. Increasing microtubule stability drove cellular widening, concomitant with redirecting messenger RNA (mRNA) export and translation along the width of the cell and reinforcement of the intercalated disc. Conversely, decreasing microtubule stability promoted cellular lengthening, disrupting the intercalated disc and biasing translation and incorporation of new sarcomeric protein toward this structure. Notably, disrupting intercalated disc adhesion was sufficient for cardiomyocyte elongation yet dispensable for cardiomyocyte widening. Thus, the heart coordinates local translation and structural remodeling to orchestrate bidirectional growth.

Cardiomyocytes depend on local translation for growth and can undergo directed growth in length or width in response to different stimuli. Protein synthesis is augmented during concentric hypertrophy, which leads to thickening of the heart muscle by increasing cardiomyocyte width. Protein synthesis is controlled at the translation initiation step, when ribosome loading onto transcripts is regulated by the sequential phosphorylation of the eukaryotic initiation factor 4E–binding protein 1 (4EBP1). Here, we identified a mode of 4EBP1 phosphorylation that was associated with concentric hypertrophy in cultured cardiomyocytes and mouse hearts. Whereas canonical phosphorylation of 4EBP1 by mTORC1 regulates global protein synthesis rates, mTORC1- and nuclear ERK–dependent phosphorylation of 4EBP1 was specifically activated during concentric but not eccentric hypertrophy. Nuclear ERK–dependent phosphorylation of 4EBP1 at Ser ⁶⁴ was necessary and sufficient to relocalize translation initiation sites closer to the nuclei. ERK activation drove redistribution of ribosomes and nascent translation toward the center of the cardiomyocyte without altering global mRNA distribution, leading to spatially enriched deposition of new sarcomeric protein in the cardiomyocyte interior. Together, these findings demonstrate that global protein synthesis can be spatially regulated by the activation of different kinases in distinct subcellular compartments and identify a mechanism that drives concentric hypertrophy.