Disease Models & Mechanisms

Bryant-Li-Bhoj Syndrome (BLBS; OMIM: 619720, 619721) is a Mendelian neurogenetic condition, first described in 2020, with a mixed neurodevelopmental/neurodegenerative phenotype and variable systemic features. To date, 100 affected individuals with 74 unique causative variants have been published. Clinical data and prior functional work in multiple model systems have emphasized the utility of interrogating the pathogenesis of multiple causal variants to identify a convergent, therapeutically targetable mechanism. Additionally, the ability to evaluate the efficacy of future therapeutics relies on the availability of a robustly validated preclinical model. Here, we characterize the developmental and neurobehavioral phenotypes of a novel BLBS mouse model harboring one of the most recurrent causative variants (h3-3a p.T45I). H3.3T45I mice recapitulate the BLBS natural history: perinatal growth restriction, delayed developmental milestones, and progressive motor and gait impairments. Adult mice additionally display craniofacial differences, impaired nest building, hyperactivity in a social context, and male-specific elevated aggression. The non-invasive, clinically translatable endpoints established here provide a validated preclinical platform for evaluating therapeutics for a community whose current standard of care is symptom management. Summary StatementA new mouse model mirrors the developmental delays, motor decline, and behavioral changes seen in individuals with this rare, progressive genetic brain disorder, providing a foundation for testing future therapies.

Neuroblastoma amplified sequence gene (NBAS) variants are associated with short stature, optic atrophy, and Pelger-Huet anomaly (SOPH) syndrome. We previously identified compound heterozygous variants in NBAS to cause atypical Osteogenesis Imperfecta (OI), with these patients presenting with short stature, developmental delay and recurrent long-bone fractures. However, skeletal disease progression due to these variants and the disease mechanisms underlying NBAS-associated OI remain poorly understood. Here, we provide a clinical update on previously identified patients and investigate the role of NBAS during skeletal development using zebrafish knockout and patient-specific missense variant zebrafish models. Homozygous knockout larvae exhibited delayed operculum development, reduced bone ossification, and defects in Meckels cartilage morphology and its underlying cellular structure. Homozygous missense larvae displayed milder cartilage defects without any major defects to early skeletal structures. Seemingly opposing phenotypes were observed in compound heterozygous zebrafish carrying the knockout and missense alleles in trans, with no obvious phenotypes seen in the Meckels cartilage and accelerated operculum development observed. Together, our results demonstrate that different nbas variants differentially affect skeletal development, suggesting complex spectrums of phenotypic and pathogenic mechanisms in NBAS-associated atypical OI.

Pathogenic variants in COL3A1 cause Vascular Ehlers-Danlos syndrome (vEDS), a rare connective tissue disorder characterised by vascular fragility, increasing the risk of arterial ruptures/dissection. Advances in genomic sequencing have led to an increasing number of COL3A1 variants where the clinical significance is unclear, with these being termed variants of uncertain significance (VUS). VUS creates challenges for diagnosis and clinical management. Thus major efforts have been made to reclassify these to either pathogenic or benign variants in disease causality. Functional data from model systems can provide significant evidence to clinicians on the pathogenicity of a variant. To address the increasing numbers of VUS in COL3A1, we developed a fast pipeline using F0 crispant zebrafish to provide functional evidence for variant classification despite there being no direct orthologue of COL3A1 in zebrafish. Loss of col5a1 resulted in cardiac defects, dysmorphic blood vessel structures and delayed angiogenic sprouting. Trunk haemorrhage prevalence under physical stress increased in col5a1 knockout zebrafish, recapitulating vEDS patients. Remarkably, co-injection of F0 col5a1 knockout crispants with human wildtype COL3A1 mRNA partially rescued cardiac and vascular phenotypes, indicating a level of functional conservation between zebrafish type V and human type III collagen. These findings establish a tractable in vivo platform for functional assessment of COL3A1 VUS. Phenotypic rescue with wildtype COL3A1 provides a benchmark against which the pathogenicity of variants can be evaluated, generating functional evidence for VUS reclassification. Our model provides both a valuable tool for investigating vEDS disease mechanisms and a clinically relevant platform to improve diagnoses for patients with suspected vEDS.

Mutations in the VPS45 gene are associated with a rare form of severe congenital neutropenia (SCN5), a life-threatening inherited error of immunity. We developed and characterized a novel mouse model of SCN5 by CRISPR/Cas9-mediated knock-in of pathogenic VPS45 E238K and T224N mutations. Both Vps45 mutations led to decreased protein expression in bone marrow cells. In vivo phenotyping demonstrated a non-Mendelian genetic distribution with reduced numbers of knock-in homozygotes Vps45E238K. Vps45E238K knock-in homozygous mice showed reduced body weight, reduced body condition with age, and increased mortality. As in human SCN5, Vps45E238K knock-in homozygotes demonstrated neutropenia and lymphopenia. Functionally, Vps45E238K knock-in homozygote neutrophils exhibited increased lipopolysaccharide-induced apoptosis and decreased peroxide production, phagocytic capacity and in vivo cell migration, phenocopying the functional defects reported in patients. Vps45T224N knock-in homozygous mice showed a milder phenotype or no abnormalities. In conclusion, this mouse model phenocopies, in part, human SCN5. It provides a novel platform for future studies of the pathophysiology of defects in neutrophil number and function in human SCN5, potential therapies for the disease, and the biochemistry and cell biology of VPS45. Summary statementWe report a mouse model of severe congenital neutropenia due to VPS45 missense mutations. It represents the first animal model of human neutropenia due to a defect in intracellular trafficking.

MLL-rearranged (MLLr) leukemia is an aggressive form of acute leukemia driven by chromosomal translocations fusing MLL with one of more than 100 partner genes, most commonly AF4. We previously showed that expression of the human MLL-AF4 fusion protein in the larval hematopoietic system of Drosophila melanogaster promotes hyperproliferation. Here, we report that the same oncogene elicits a strikingly opposite response in the larval fat body, inducing cell shrinkage, autophagy, and caspase-dependent cell death in a manner dependent on the intact fusion protein. Autophagy induction preceded caspase activation, yet the two programs operated in parallel rather than in series. Mechanistically, MLL-AF4-expressing fat body cells displayed elevated AMPK phosphorylation and reduced mTORC1 activity, and depletion of AMPK abolished caspase activation. Despite producing opposite phenotypes, both tissue-specific responses depended on conserved complex partners, suggesting that MLL-AF4 co-opts a shared mechanism to produce starkly different outcomes depending on cellular context. Uncovering how MLL-AF4 induces apoptosis-like phenotypes in the fat body could potentially be used to rewire leukemic signaling and remove malignant cells.

Friedreich ataxia (FRDA) is a neurodegenerative and cardiac disease caused by GAA repeat expansions within the first intron of the FXN gene, leading to reduced frataxin expression. Frataxin is required for iron sulfur cluster (ISC) biosynthesis, and its deficiency results in multiple cellular dysfunctions, including mitochondrial iron overload. Although altered iron homeostasis has been reported in several frataxin-deficient models and in FRDA patients, its contribution to disease progression remains debated. Here, we used a GAA expansion-based Drosophila model of FRDA, termed fh-GAAs, to investigate the impact of reducing intestinal iron absorption on disease progression. We first found that iron accumulation was tissue-specific and predominantly affected the central nervous system. Furthermore, glial cells were affected more severely than neurons, suggesting an increased vulnerability of glia to frataxin deficiency. Reducing intestinal iron uptake, either through treatment with bathophenanthroline disulfonic acid (BPS), an extracellular iron chelator, or by gut-specific silencing of the iron transporter Malvolio, nearly doubled fly survival. BPS treatment also improved sensitivity to dietary iron, enhanced locomotor performance, fully restored normal brain size, and prevented glial alterations. Altogether, our findings identify glial cells as early and preferential targets of frataxin deficiency in an iron-dependent manner and support the in vivo relevance of intestinal iron uptake as a potential modulator of disease severity in FRDA.

Epilepsy affects approximately 30% of individuals with autism spectrum disorder (ASD). Consistent with these observations, while PRICKLE mutations are primarily linked with epilepsy, there is an enrichment of pathogenic DNA sequence variants in PRICKLE genes carried by individuals with ASD. Nonetheless, a connection between PRICKLE function and ASD warrants further investigation. Here, we show that a seizure-prone Drosophila prickle mutant (prickle-spiny-legs, or pksple) exhibits learning and memory deficits, increased pain sensitivity, both communication and social interaction difficulties, and restrictive repetitive grooming behaviors, all of which are strongly correlated with ASD, while a non-seizure prone prickle mutant (prickle-prickle, or pkpk) does not, thereby providing a direct genetic connection between epilepsy and ASD through prickle. Comparing headed versus headless pksple mutants, we also show that the excessive grooming requires higher level cognitive processing from the brain. Finally, both pksple and pkpk mutants exhibit circadian rhythm defects, another feature correlated with ASD, as well as distinct yet overlapping neurological anomalies in processes that include innate immune response, oxidative stress response, neuronal cell death, neurodegeneration, motor dysfunction and reduced lifespan, likely reflecting the unique isoform expression patterns observed in the developing CNS. Collectively, this study highlights the broadscale effects of PRICKLE mutations that extend beyond the primary clinical features of epilepsy to include several of the core features of ASD.

Heterozygous pathogenic variants in CSNK2A1 or CSNK2B encoding the Casein Kinase 2 (CK2) protein complex, lead to pediatric neurodevelopmental disorders, Okur-Chung Neurodevelopmental Syndrome (OCNDS) and Poirier-Bienvenu Neurodevelopmental Syndrome (POBINDS). OCNDS and POBINDS are characterized by a range of symptoms, including developmental delay, intellectual disability, facial dysmorphism, and seizures. Despite over 250 reported cases of OCNDS and POBINDS, we do not fully understand how specific alterations in CK2 relate to the heterogeneity observed in patients. To investigate this, we used the fruit fly, Drosophila melanogaster, as a model system. To assess variant impact, we co-expressed human CSNK2A1 and CSNK2B reference or disease-causing variants in flies. In parallel, we determined the role of Drosophila CkII in the developing and mature nervous system, specifically in neurons and glia. We found that 12/13 variants tested act as full or partial loss-of-function with one CSNK2A1 variant showing gain-of-function. Phospho-proteomic studies in neurons revealed separate signatures for loss- and gain-of-function variants. We found that neuronal and glial CkII is critical for organismal development. Reduction of neuronal CkII in the adult nervous system causes motor and seizure-like phenotypes. Finally, given the known role of CK2 in potentiating Wnt/{beta}-catenin signalling, we show that Wnt agonists partially rescue phenotypes associated with adult-specific neuronal reduction of CkII. This work generates Drosophila models of CSNK2A1 and CSNK2B expression to functionally assess variant impact, as well as an adult-specific neuronal loss-of-function model for drug screening and mechanistic studies.

Overexpression of -synuclein (-syn), an inherently disordered protein, triggers chronic activation of the mitochondrial unfolded protein response (UPRmt) pathway in Caenorhabditis elegans with enhanced dopaminergic (DAergic) neurodegeneration. Introduction of a loss-of-function(lf) mutation in atfs-1, the main transcriptional regulator of the UPRmt, into -syn nematodes results in significant neuroprotection from -syn-induced DA neuron loss, indicating that compensatory mechanisms provide neuroprotection. We performed a F3 forward genetic screen in C. elegans atfs-1(lf) mutants to identify molecular components associated with the modulation of neurodegeneration via UPRmt signaling in -syn-expressing DA neurons. Homozygous mutant animals were examined for enhanced neurodegeneration; multiple independent alleles were uncovered. Among these, we identified new nonsense alleles encoding the histone lysine demethylases (H3K27me3), jmjd-1.2 and jmjd-3.1. Another line carried a nonsense allele of twk-14. This gene encodes a conserved protein termed KCNK12 in mammals that facilitates passive background K+ leak currents to set and stabilize resting membrane potential. To further examine the association of these gene products in DA neurodegeneration, mutants and/or RNA interference were employed. DA neurodegeneration was observed in the -syn + atfs-1(lf) background when jmjd-1.2, jmjd-3.1, or twk-14 were individually depleted. These results provide evidence that jmjd-1.2 and jmjd-3.1, which encode previously characterized H3K27me3 demethylases, and the uncharacterized twk-14 gene product, orthologous to human KCNK12, naturally confer protection from -syn neurotoxicity.

Charcot-Marie-Tooth disease type 2D (CMT2D) results from gain-of-function mutations in GARS1, which encodes glycyl-tRNA synthetase (GlyRS), the enzyme responsible for charging transfer RNA (tRNA) with glycine. There are several CMT2D mouse models, but Gars{Delta}ETAQ/+ is the only one that bears a patient-sourced mutation. Created using CRISPR/Cas9 to model a 12-nucleotide de novo GARS1 deletion identified in an unusually severe CMT2D patient, Gars{Delta}ETAQ/+ mice have previously been shown to display several neuromuscular phenotypes; motor axon loss, denervated neuromuscular junctions (NMJs) and reduced muscle function. Here, we extend these analyses to provide a more comprehensive understanding of both motor and sensory nerve deficits across hind- and fore-limbs. At 3 months, Gars{Delta}ETAQ/+ mice possess sex-independent alterations in the levels of neuropathy biomarkers - including decreased NfL and increased periaxin - alongside reduced muscle endurance and strength, and impairments in the sensory modalities of mechanosensation, proprioception and nociception. Underpinning these dysfunctions, we identified site-specific defects comprising altered sensory neuron populations, muscle spindle loss, reduced motor neuron size, disrupted NMJ innervation and maturation, and reduced axonal transport of signalling endosomes in vivo. Together, these experiments show that Gars{Delta}ETAQ/+ mice display robust and selective peripheral nerve pathology that manifests in a general distal-to-proximal fashion, priming this CMT2D allele for testing treatments and evaluating mechanisms underlying peripheral nerve vulnerability. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=200 SRC="FIGDIR/small/736541v1_ufig1.gif" ALT="Figure 1"> View larger version (70K): org.highwire.dtl.DTLVardef@2b25d2org.highwire.dtl.DTLVardef@c00d77org.highwire.dtl.DTLVardef@b41173org.highwire.dtl.DTLVardef@1e5cdcc_HPS_FORMAT_FIGEXP M_FIG C_FIG

Gene variants, secondary mutations, and stochastic individual variability complicate cancer diagnosis, prognosis, and treatments. Here, we systematically assess the functional impact of PTEN cancer-related missense mutations in mammalian cell lines, yeast, and Caenorhabditis elegans. While cell-based assays revealed alterations in lipid phosphatase activity, CRISPR-based engineering of orthologous mutations in C. elegans enabled classification of variants based on organismal phenotypes and transcriptional profiles, providing a rapid framework to predict oncogenic potential. We further show that secondary mutations, such as gain-of-function of cdc-25.1/CDC25A, can enhance the phenotypic impact of specific daf-18/PTEN variants, revealing context-dependent oncogenicity. Finally, single-worm transcriptomic analyses uncovered substantial interindividual variability among isogenic animals with identical cdc-25.1 and daf-18 mutations, linking transcriptional states to divergent phenotypic outcomes. Together, our results establish C. elegans as a powerful in vivo platform to integrate genetic, functional, and transcriptional information for the interpretation of cancer-associated variants.

Bradycardia occurs when the heart rate is lower than normal resulting in reduced cerebral blood flow and contributing to neurodegeneration in adults but how it affects embryonic cerebrovascular development is not well studied. We induce bradycardia by targeting the heart pacemaker channel Hcn4 via chemical (ivabradine) and genetic (hcn4 mutant) methods. Bradycardia results in reduced brain vessel diameter and mural cell (pericyte and vascular smooth muscle cell) number. Endothelial cells are the first responders in sensing changes in blood flow, and we show that signalling through the canonical endothelial-autonomous mechanosensitive pathway (Piezo1, Mek5, Erk5, Klf2) is reduced in bradycardia. To identify the ligand-receptor combination that transmits signals to developing mural cells, we show that expression of the Notch ligand jagged2b is decreased in the brain of both hcn4 and klf2 mutants. jag2b knockdown reduces mural cell numbers in brain vessels. Restoring jag2b levels increases mural cell numbers in both wildtype and hcn4 mutants. Our work connects bradycardia, mechanosensitive signaling and mural cell recruitment demonstrating that mural cell numbers can be increased in bradycardia by restoring Notch signalling via upregulating endothelial Jag2b. SummaryBradycardia models show reduced blood flow, Piezo1-klf2-jag2b-notch3 mechanosensing and mural cell recruitment to developing brain vasculature. Restoration of jag2, an endogenous endothelial cell ligand, rescues mural cell numbers in bradycardia mutants.

RationaleChronic obstructive pulmonary disease (COPD) represents a leading cause of global morbidity and mortality. Genome-wide association studies (GWAS) have implicated numerous genetic variants in lung function impairment, yet confidently identifying the underlying genes and pathways, and translating these findings into mechanistic insight, remains a significant challenge. ObjectivesTo leverage the genetic amenability and high-throughput screening capability of Drosophila melanogaster to determine the role of candidate causal genes in epithelial cell homeostasis. MethodsWe performed a loss-of-function analysis of 60 prioritised lung function candidate causal genes implicated from GWAS in two distinct epithelia: the dorsal thorax and trachea. ResultsWe identified 57/60 tested candidate genes that alter at least one aspect of epithelial morphology and behaviour upon knockdown. With a focus on junctional integrity, cell delamination and tissue growth, we identified 11 genes for further study: Sec6, RpS26, pAbp, Arf102f, Riok1, Sra-1, Inpp5e, CG31759, ssh, eIF6 and Rtf1. Further characterisation found a significant reduction in junctional E-Cadherin levels following Arf102F, Rtf1, RioK1 and Sra-1 knockdown. Following a secondary screen in the Drosophila tracheal system for priority candidates, Sec6 and RpS26 were associated with significant airway defects and a reduction in larval body size. 8/11 priority genes exhibited differential lung gene expression between controls and patients with COPD. ConclusionsThese data demonstrate the amenability of Drosophila melanogaster to perform in vivo functional analyses of candidate causal genes at scale. Initial findings implicate several genes in epithelial homeostasis and integrity, providing new mechanistic understanding and potential therapeutic targets for COPD. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=164 SRC="FIGDIR/small/731340v1_ufig1.gif" ALT="Figure 1"> View larger version (44K): org.highwire.dtl.DTLVardef@dbff6dorg.highwire.dtl.DTLVardef@15e84f0org.highwire.dtl.DTLVardef@69d97aorg.highwire.dtl.DTLVardef@144f47e_HPS_FORMAT_FIGEXP M_FIG C_FIG

Transcriptional regulators and chromatin remodellers are among the most important risk gene categories across the genetic landscape of neurodevelopmental disorders (NDDs). The zinc finger and homeodomain transcription factor ADNP is prominently associated with Helsmoortel-Van der Aa Syndrome (HVDAS), which is characterized by intellectual disability and autism spectrum disorder. Heterozygous frameshifting mutations account for the majority of HVDAS mutations, but it remains unclear how HVDAS mutations affect ADNP dosage, and how dosage in turn relates to neurodevelopmental and behavioral phenotypes. Here, we compared an allelic series of Adnp cKOs and germline heterozygotes. Using a conditional allele, we first deleted Adnp throughout the neural tube using Nestin-Cre. At E15.5, cKO brains exhibited altered upper-layer neuron production. However, AdnpNestincKOs exhibited perinatal lethality, precluding further behavioral characterization. Next, we compared germline heterozygotes (gHets) versus cKOs generated using the Emx1-Cre driver. We found that AdnpDel/+and AdnpL822fs6/+ gHets exhibited cortical hypoplasia that was quantitatively identical, albeit less severe in comparison to AdnpEmxcKOs. In behavioral testing, AdnpEmx cKOs accordingly exhibited the strongest phenotypes, including hallmarks of elevated anxiety. However, both AdnpEmx cKOs and AdnpL822fs6/+ gHets exhibited remarkably similar sex-specific deficits in learning during Morris Water Maze testing. Taken together, our work suggests that cortical growth and learning represent core phenotypes shared across Adnp mutant models irrespective of dosage. Moreover, since Adnp mutant phenotypes closely correspond with our prior findings in Chd4 mutants, our results collectively suggest that Adnp regulates behavior via the ChAHP chromatin remodelling complex.

Membrane Contact sites (MCS) have emerged as physiologically relevant zones that coordinate inter-organelle communication and cellular function. VAPB, an ER-resident MCS tethering protein, plays a central role in regulating MCSs through its numerous protein interactors, thereby influencing cellular homeostasis. A pathogenic missense VAPBP56S mutation causes familial Amyotrophic Lateral Sclerosis 8 (ALS8) in humans, with progressive degeneration of motor neurons. The precise mechanisms underlying the motor neurodegeneration remain poorly understood. In this study, we examine lipid imbalance in the brain of a Drosophila model of ALS8 (VAPBP58S). Specifically, we find that lipid homeostasis is disrupted in an age-dependent manner. Strikingly, cholesterol esters and sphingolipids show an age-dependent increase, while cholesterol shows a decrease. Intriguingly, from a cellular perspective, despite the accumulation of triacylglycerols (TAGs) in the brains of VAPBP58S animals, the increased neutral lipid species do not correlate with lipid droplets (LDs), which are fewer in density and smaller in size. Lipid imbalance and progressive motor dysfunction in VAPBP58S animals can be reversed by expressing VAPBWT, suggesting a relationship between VAPB activity and lipid flux. To uncover VAPBs role in lipid homeostasis, we modulate VAPB activity in neurons and glia to dissect out tissue-specific roles. We find that both cell types contribute to lipid homeostasis in differential ways. In glia, LD flux is strongly dependent on VAPB activity, a dependence further recapitulated in cultured human cell lines, suggesting evolutionary conservation of the regulatory mechanism. Thus, we hypothesise that lipid dysregulation constitutes a critical pathogenic feature of ALS8, with the VAPBP56S allele disrupting lipid homeostasis in the neuro-glial axis. Summary StatementThe ER-membrane tethering protein VAPB regulates lipid homeostasis

Genetic uniqueness of the tumor microenvironment significantly influences cancer growth, survival, and response to therapy, independent of the cancer cells intrinsic properties or the adaptive immune system. Using genetically distinct Rag1-/- mouse models, this study shows that different strains exhibit varied tumor growth kinetics and survival outcomes when xenografted with identical leukemic and solid tumor cell lines. This study further highlights the critical role of the myeloid immune compartment and shows that disrupting both lymphoid and myeloid systems alters cancer progression. These results also reveal that the tumor microenvironment can permanently alter cancer cell phenotypes and significantly affect chemotherapy efficacy, as seen with Cisplatins varying effects across strains. These findings underscore the importance of considering genetic background in preclinical cancer models, suggesting that reliance upon a single mouse strain may lead to incomplete conclusions about cancer biology and treatment efficacy. SUMMARY STATEMENTPre-clinical xenograft mammalian models are used to study human diseases. Here we report that the genetic uniqueness of the tumor microenvironment, independent of the immune system, can determine the fate of cancer progression, survival, and therapy response.

Hereditary hemorrhagic telangiectasia (HHT) is a rare vascular disorder caused by pathogenic variants in members of the BMP9/ALK1 signaling hub. In the present study we show that, regardless of whether the alterations are caused by reduced BMP9/ALK1 signaling (pathogenic variants in the ENG or ALK1 genes) or by overactivation of this pathway (such as the SMAD6 pathogenic variants), all are associated with increased endothelial cell (EC) proliferation and high levels of ERK MAPK activation in patient biopsies. We reproduced this phenotype in vitro in ECs lacking SMAD6 or after SMAD1 knockdown using siRNA. Loss of SMAD6 leads to dysregulation of the Notch pathway, with downregulation of phosphatases and consequent overstimulation of ERK. In normal ECs, BMP9 and Notch signaling inhibit ERK activity by upregulating PPP1R3C, a regulatory subunit of the PP1 phosphatase. Notably, BMP9-mediated inhibition of ERK is abolished when cells are transfected with siRNA targeting PPP1R3C. ERK hyperactivation was also observed in an HHT2 mouse model (ALK1-2loxP;Cdh5-CreERT2). Loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine; these injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation involved in HHT pathogenesis, suggesting that ERK inhibition may represent a promising therapeutic strategy for these patients. Translational PerspectiveHereditary hemorrhagic telangiectasia (HHTs) is a rare vascular disorder caused by mutations in members of the BMP9/ALK1 signaling hub. In the present study we show that all different forms of HHTs are associated with increased endothelial cell (EC) proliferation, which correlates with high levels of ERK activation in patient biopsies. ERK hyperactivation is also observed in an HHT2 mouse model in which loss of both ALK1 alleles in adult mice leads to vascular failure and hemorrhages in the lung and intestine. These injuries are significantly reduced by treatment with the MEK/ERK inhibitor selumetinib. Overall, our work identifies a key role for ERK activation in HHT pathogenesis, suggesting that ERK/MEK inhibitors may represent a promising therapeutic strategy for these patients.

IntroductionThe quality of preclinical studies for rare diseases, such as Duchenne muscular dystrophy (DMD), relies on the availability of comprehensive natural disease history data. In addition to the classic BL10-mdx mouse, in recent years, the D2-mdx model has increasingly been used as an alternative model due to its reportedly more severely impaired phenotype. To improve our understanding of disease progression in these two DMD models, we conducted a comprehensive natural history study. Materials and MethodsThis involved a cross-sectional analysis of key in vivo and ex vivo outcome measures performed in two independent laboratories, using the same study setup in compliance with TREAT-NMD Standard Operating Procedures (SOPs), while also taking advantage of site-specific expertise. Globally, largely comparable results were obtained across the two study sites. ResultsBody composition showed pronounced differences between the strains, with BL10-mdx mice displaying a hypertrophic and D2-mdx mice displaying an atrophic phenotype. Dystrophic mice of each strain exhibited significant alterations of disease-relevant indices related to muscle functionality and integrity, mostly worsening with age, in comparison to their wildtypes. Cardiac function was affected earlier and more severely in D2-mdx mice. DiscussionNotably, for some parameters, genetic-background related differences were observed, emphasizing the need to include control groups with matching genetic backgrounds in experimental designs. ConclusionsCollectively, our natural history study provides benchmark data for these two mdx mouse strains to guide model selection for preclinical DMD studies, allowing accurate data interpretation. HighlightsO_LIDistinct body composition phenotypes: BL10-mdx mice exhibit pseudohypertrophy while D2-mdx mice display pronounced atrophy. C_LIO_LIEarlier cardiac dysfunction in D2-mdx: D2-mdx mice develop reduced ejection fraction and stroke volume from 28 weeks, while BL10-mdx only at 52 weeks. C_LIO_LIGenetic background-dependent variations: Intrinsic deficits in wildtype D2 mice demonstrate that genetic background influences outcome measures independent of dystrophic pathology. C_LIO_LIComparable ex vivo muscle physiology: Despite divergent in vivo phenotypes, isolated muscle contractile parameters show similar impairment in both dystrophic models. C_LIO_LIMulti-site standardized validation: Cross-sectional study at two independent laboratories following harmonized TREAT-NMD Standard Operating Procedures. C_LI

Niemann-Pick disease type C (NPC) is a rare autosomal recessive neurodegenerative lysosomal storage disease caused by pathogenic variants in NPC1 or NPC2. Sudden death can occur due to seizures, but cardiac involvement has not been well defined. We performed 12-lead electrocardiograms (ECG) in 14 adult NPC patients (8 male, 6 female). Cardiac structure and function were examined in Npc1-/- adult mouse hearts, alongside wild-type controls. Glycosphingolipid accumulation was quantified by high-performance liquid chromatography, fibrosis and collagen deposition were quantified using Massons Trichrome (M&T) and Picrosirius Red (PR) staining. Whole-heart morphology, including chamber size and wall thickness, was assessed. Ex vivo ECG recordings assessed conduction abnormalities and arrhythmias. RNA-seq transcriptomics characterised molecular pathways altered in Npc1-/- hearts. 8/14 patients showed ECG abnormalities including abnormal QRS transitions (N=8), increased QRS amplitude (N=4), fascicular block (N=2), and abnormal T wave inversion (N=1). 13 patients also had transthoracic echocardiograms identifying mildly impaired LV systolic function (N=2) and increased wall thickness/LV mass (N=4). In Npc1-/- mice, age-related glycosphingolipid accumulation was associated with pronounced ventricular fibrotic remodelling. There was a significant increase in stained connective tissue area and connective tissue to cardiac tissue ratio in both MT and PR staining. ECG from Langendorff-perfused Npc1-/- hearts showed QT prolongation and atrioventricular conduction abnormalities under isoprenaline stress. Transcriptomics revealed major changes in Npc1-/- hearts, consistent with histological fibrosis and linking NPC to inflammation-driven remodelling and arrhythmogenesis. These findings support routine cardiac screening in NPC patients and highlight the need for further studies to improve management and treatment.

The tunicate Ciona robusta offers a tractable non-vertebrate chordate model for probing gene function via tissue-specific, CRISPR/Cas9-mediated mutagenesis in F0. Building on Arcadia Sciences Zoogle platform, which identifies and ranks orthologs of human genes from various non-traditional model organisms, we carried out a pilot project to probe the developmental roles of three notochord- and endoderm-expressed candidate orthologs of human disease genes (Fcho, Pgm3, and Nckap1) alongside a fourth gene (Plastin) implicated in papilla cell elongation. This preprint compiles and updates a series of research project milestones previously posted episodically on Zenodo. Here we summarize the full results and our conclusion about this pilot project. Using CRISPR/Cas9, we found that tissue-specific knockout of Pgm3 and, to a lesser extent, Fcho caused significant defects in larval tail elongation. Separately, CRISPR knockout of Plastin, an actin-bundling gene expressed throughout the sensory-adhesive papillae of the larva, caused a subtle reduction in papilla cell elongation when combined as a duoble knockout with another actin-bundling protein-encoding gene, Villin. These results identify Pgm3 as the most promising candidate for further development as a Ciona-based model of human disease and demonstrate the utility of tissue-specific CRISPR screening for prioritizing candidate disease gene orthologs identified through comparative genomics platforms like Zoogle.