EGFR-dependent endocytosis of Wnt9a and Fzd9b promotes ß-catenin signaling during hematopoietic stem cell development in zebrafish.

Cell-to-cell communication through secreted Wnt ligands that bind to members of the Frizzled (Fzd) family of transmembrane receptors is critical for development and homeostasis. Wnt9a signals through Fzd9b, the co-receptor LRP5 or LRP6 (LRP5/6), and the epidermal growth factor receptor (EGFR) to promote early proliferation of zebrafish and human hematopoietic stem cells during development. Here, we developed fluorescently labeled, biologically active Wnt9a and Fzd9b fusion proteins to demonstrate that EGFR-dependent endocytosis of the ligand-receptor complex was required for signaling. In human cells, the Wnt9a-Fzd9b complex was rapidly endocytosed and trafficked through early and late endosomes, lysosomes, and the endoplasmic reticulum. Using small-molecule inhibitors and genetic and knockdown approaches, we found that Wnt9a-Fzd9b endocytosis required EGFR-mediated phosphorylation of the Fzd9b tail, caveolin, and the scaffolding protein EGFR protein substrate 15 (EPS15). LRP5/6 and the downstream signaling component AXIN were required for Wnt9a-Fzd9b signaling but not for endocytosis. Knockdown or loss of EPS15 impaired hematopoietic stem cell development in zebrafish. Other Wnt ligands do not require endocytosis for signaling activity, implying that specific modes of endocytosis and trafficking may represent a method by which Wnt-Fzd specificity is established.

Characterization of a novel fast-growing zebrafish: a new approach to growth hormone transgenesis.

The manipulation of the somatotropic axis, governing growth, has been a focus of numerous transgenic approaches aimed at developing fast-growing fish for research, medicine and aquaculture purposes. However, the excessively high growth hormone (GH) levels in these transgenic fish often result in deformities that impact both fish health and consumer acceptance. In an effort to mitigate these issues and synchronize exogenous GH expression with reproductive processes, we employed a novel transgenic construct driven by a tilapia luteinizing hormone (LH) promoter. This approach was anticipated to induce more localized and lower exogenous GH secretion. In this study, we characterized the growth and reproduction of these transgenic LHp-GH zebrafish using hormonal and physiological parameters. Our findings reveal that LHp-GH fish exhibited accelerated growth in both length and weight, along with a lower feed conversion ratio, indicating more efficient feed utilization, all while maintaining unchanged body proportions. These fish demonstrated higher expression levels of LH and GH in the pituitary and elevated IGF-1 levels in the liver compared to wild-type fish. An examination of reproductive function in LHp-GH fish unveiled lower pituitary LH and FSH contents, smaller follicle diameter in female gonads, and reduced relative fecundity. However, in transgenic males, neither the distribution of spermatogenesis stages nor sperm concentrations differed significantly between the fish lines. These results suggest that coupling exogenous GH expression with endogenous LH expression in females directs resource investment toward somatic growth at the expense of reproductive processes. Consequently, we conclude that incorporating GH under the LH promoter represents a suitable construct for the genetic engineering of commercial fish species, providing accelerated growth while preserving body proportions.

Deficiency of the HGF/Met pathway leads to thyroid dysgenesis by impeding late thyroid expansion.

The mechanisms of bifurcation, a key step in thyroid development, are largely unknown. Here we find three zebrafish lines from a forward genetic screening with similar thyroid dysgenesis phenotypes and identify a stop-gain mutation in hgfa and two missense mutations in met by positional cloning from these zebrafish lines. The elongation of the thyroid primordium along the pharyngeal midline was dramatically disrupted in these zebrafish lines carrying a mutation in hgfa or met. Further studies show that MAPK inhibitor U0126 could mimic thyroid dysgenesis in zebrafish, and the phenotypes are rescued by overexpression of constitutively active MEK or Snail, downstream molecules of the HGF/Met pathway, in thyrocytes. Moreover, HGF promotes thyrocyte migration, which is probably mediated by downregulation of E-cadherin expression. The delayed bifurcation of the thyroid primordium is also observed in thyroid-specific Met knockout mice. Together, our findings reveal that HGF/Met is indispensable for the bifurcation of the thyroid primordium during thyroid development mediated by downregulation of E-cadherin in thyrocytes via MAPK-snail pathway.

Monitoring the Mitochondrial Viscosity Changes During Cuproptosis with Iridium(III) Complex Probe via In Situ Phosphorescence Lifetime Imaging.

Cuproptosis is a novel copper-dependent form of programmed cell death, displaying important regulatory functions in many human diseases, including cancer. However, the relationship between the changes in mitochondrial viscosity, a key factor associated with cellular malfunction, and cuproptosis is still unclear. Herein, we prepared a phosphorescent iridium (Ir) complex probe for precisely monitoring the changes of mitochondrial viscosity during cuprotosis via phosphorescence lifetime imaging. The Ir complex probe possessed microsecond lifetimes (up to 1 µs), which could be easily distinguished from cellular autofluorescence to improve the imaging contrast and sensitivity. Benefiting from the long phosphorescence lifetime, excellent viscosity selectivity, and mitochondrial targeting abilities, the Ir complex probe could monitor the increase in the mitochondrial viscosity during cuproptosis (from 46.8 to 68.9 cP) in a quantitative manner. Moreover, through in situ fluorescence imaging, the Ir complex probe successfully monitored the increase in viscosity in zebrafish treated with lipopolysaccharides or elescolomol-Cu2+, which were well-known cuproptosis inducers. We anticipate that this new Ir complex probe will be a useful tool for in-depth understanding of the biological effects of mitochondrial viscosity during cuproptosis.

The generation and characterization of a transgenic zebrafish line with lens-specific Cre expression.

Purpose: Danio rerio zebrafish constitute a popular model for studying lens development and congenital cataracts. However, the specific deletion of a gene with a Cre/LoxP system in the zebrafish lens is unavailable because of the lack of a lens-Cre-transgenic zebrafish. This study aimed to generate a transgenic zebrafish line in which Cre recombinase was specifically expressed in the lens. Methods: The pTol2 cryaa:Cre-polyA-cryaa:EGFP (enhanced green fluorescent protein) plasmid was constructed and co-injected with Tol2-transposase into one-to-two-cell-stage wild-type (WT) zebrafish embryos. Whole-mount in situ hybridization (ISH), tissue section, hematoxylin and eosin staining, a Western blot, a split-lamp observation, and a grid transmission assay were used to analyze the Cre expression, lens structure, and lens transparency of the transgenic zebrafish. Results: In this study, we generated a transgenic zebrafish line, zTg(cryaa:Cre-cryaa:EGFP), in which Cre recombinase and EGFP were driven by the lens-specific cryaa promoter. zTg(cryaa:Cre-cryaa:EGFP) began to express Cre and EGFP specifically in the lens at the 22 hpf stage, and this ectopic Cre could efficiently and specifically delete the red fluorescent protein (RFP) signal from the lens when zTg(cryaa:Cre-cryaa:EGFP) embryos were injected with the loxP-flanked RFP plasmid. The overexpression of Cre and EGFP did not impair zebrafish development or lens transparency. Accordingly, this zTg(cryaa:Cre-cryaa:EGFP) zebrafish line is a useful tool for gene editing, specifically with zebrafish lenses. Conclusions: We established a zTg(cryaa:Cre-cryaa:EGFP) zebrafish line that can specifically express an active Cre recombinase in lens tissues. This transgenic zebrafish line can be used as a tool to specifically manipulate a gene in zebrafish lenses.

Lectins As Effective Tools in the Study of the Biliary Network and the Parenchymal Architecture of the Zebrafish (Danio rerio) Liver.

Lectins are carbohydrate-binding proteins with specific affinity to glycoconjugates expressed in various tissues. Lectins are of substantial utility as research, histochemical, and diagnostic tools in mammalian systems. Reactivity of 12 commonly used plant-based lectins was studied in zebrafish liver. Four lectins, tomato lectin (TL), wheat germ agglutinin, concanavalin A, and Jacalin showed strong reactivity to hepatic parenchymal structures. Importantly, TL reacted to glycoconjugates within segments of the larval and adult intrahepatic biliary network, from canaliculi to bile ducts. We provide evidence that lectins can serve as important histochemical tools to investigate the structural and functional characteristics of the zebrafish liver.

ß-Estradiol Supplementation Regulates Cholesterol Synthesis Independent of Unsaturated Fat Consumption in Adult Zebrafish.

Obesity is a public health concern resulting in a variety of health complications, including heart disease and insulin resistance. Estrogens have been associated with a reduced risk of obesity, but this relationship remains incompletely understood. We assessed the role of 17ß-estradiol (E2) in mitigating complications associated with obesity by supplementing E2 in the diets of overfed zebrafish. We report that dietary E2 supplementation protects against weight gain and modulates de novo cholesterol synthesis in a sex-specific manner. Our studies lead us to propose a model in which E2 regulates hmgcr expression independently of unsaturated fat consumption. These data can be used to develop sex-specific treatments for obesity-related health conditions.

Chronic exposure to gestodene impairs reproductive system in adult female zebrafish (Daniarerio).

Gestodene (GES) is widely used in human therapy and animal husbandry and is frequently detected in aquatic environments. Although GES adversely affects aquatic organisms at trace levels, its effects on the reproductive biology of fish remain inconclusive. In this study, female zebrafish (Danio rerio) were exposed to environmentally relevant levels of GES for the evaluation of the effects of GES on the reproductive system by using endpoints including gene expression, plasma steroid concentrations, histological and morphological analyses, copulatory behavior, and reproductive output. Adult female zebrafish exposed to environmentally relevant concentrations of GES (4.0, 40.2, and 372.7 ng/L) for 60 d demonstrated stagnant ovarian oocyte development, evidenced by an increase in the percentage of perinuclear and atretic oocytes and a decrease in the percentage of late vitellogenic oocytes. GES-exposed females were less attractive to males and had lower copulatory intimacy than females in control. Consequently, spawning (44.3-49.2 %) and egg fertilization rates (27.9-32.0 %) were decreased. The decreased survival of fertilized eggs and hatching rates were accompanied by increased malformations. These negative effects were associated with abnormal transcriptional levels of gonadal steroid hormones, which were regulated by genes (Hsd17ß3, Hsd11ß2, Hsd20ß, Cyp19a1a, and Cyp11b). Overall, our findings suggest that GES impairs the reproductive system of zebrafish, which may threaten population stability.

Parental exposure to antidepressants has lasting effects on offspring? A case study with zebrafish.

Fish have common neurotransmitter pathways with humans, exhibiting a significant degree of conservation and homology. Thus, exposure to fluoxetine makes fish potentially susceptible to biochemical and physiological changes, similarly to what is observed in humans. Over the years, several studies demonstrated the potential effects of fluoxetine on different fish species and at different levels of biological organization. However, the effects of parental exposure to unexposed offspring remain largely unknown. The consequences of 15-day parental exposure to relevant concentrations of fluoxetine (100 and 1000 ng/L) were assessed on offspring using zebrafish as a model organism. Parental exposure resulted in offspring early hatching, non-inflation of the swimming bladder, increased malformation frequency, decreased heart rate and blood flow, and reduced growth. Additionally, a significant behavioral impairment was also found (reduced startle response, basal locomotor activity, and altered non-associative learning during early stages and a negative geotaxis and scototaxis, reduced thigmotaxis, and anti-social behavior at later life stages). These behavior alterations are consistent with decreased anxiety, a significant increase in the expression of the monoaminergic genes slc6a4a (sert), slc6a3 (dat), slc18a2 (vmat2), mao, tph1a, and th2, and altered levels of monoaminergic neurotransmitters. Alterations in behavior, expression of monoaminergic genes, and neurotransmitter levels persisted until offspring adulthood. Given the high conservation of neuronal pathways between fish and humans, data show the possibility of potential transgenerational and multigenerational effects of pharmaceuticals' exposure. These results reinforce the need for transgenerational and multigenerational studies in fish, under realistic scenarios, to provide realistic insights into the impact of these pharmaceuticals.

Purification, structural characterization, and neuroprotective effect of 3,6-diisobutyl-2,5-piperazinedione from Halomonas pacifica CARE-V15 against okadaic acid-induced neurotoxicity in zebrafish model.

Halomonas pacifica CARE-V15 was isolated from the southeastern coast of India to determine its genome sequence. Secondary metabolite gene clusters were identified using an anti-SMASH server. The concentrated crude ethyl acetate extract was evaluated by GC-MS. The bioactive compound from the crude ethyl acetate extract was fractionated by gel column chromatography. HPLC was used to purify the 3,6-diisobutyl-2,5-piperazinedione (DIP), and the structure was determined using FTIR and NMR spectroscopy. Purified DIP was used in an in silico molecular docking analysis. Purified DIP exhibits a stronger affinity for antioxidant genes like glutathione peroxidase (GPx), glutathione-S-transferase (GST), and glutathione reductase (GSR). Using in silco molecular docking analysis, the protein-ligand binding affinities of GSR (-4.70 kcal/mol), GST (-5.27 kcal/mol), and GPx (-5.37 kcal/mol) were measured. The expression of antioxidant genes were investigated by qRT-PCR. The in vivo reactive oxygen species production, lipid peroxidation, and cell death levels were significantly (p ≤ 0.05) increased in OA-induced group, but all these levels were significantly (p ≤ 0.05) decreased in the purified DIP pretreated group. Purified DIP from halophilic bacteria could thus be a useful treatment for neurological disorders associated with oxidative stress.

Cold-induced FOXO1 nuclear transport aids cold survival and tissue storage.

Cold-induced injuries severely limit opportunities and outcomes of hypothermic therapies and organ preservation, calling for better understanding of cold adaptation. Here, by surveying cold-altered chromatin accessibility and integrated CUT&Tag/RNA-seq analyses in human stem cells, we reveal forkhead box O1 (FOXO1) as a key transcription factor for autonomous cold adaptation. Accordingly, we find a nonconventional, temperature-sensitive FOXO1 transport mechanism involving the nuclear pore complex protein RANBP2, SUMO-modification of transporter proteins Importin-7 and Exportin-1, and a SUMO-interacting motif on FOXO1. Our conclusions are supported by cold survival experiments with human cell models and zebrafish larvae. Promoting FOXO1 nuclear entry by the Exportin-1 inhibitor KPT-330 enhances cold tolerance in pre-diabetic obese mice, and greatly prolongs the shelf-life of human and mouse pancreatic tissues and islets. Transplantation of mouse islets cold-stored for 14 days reestablishes normoglycemia in diabetic mice. Our findings uncover a regulatory network and potential therapeutic targets to boost spontaneous cold adaptation.

Characterization of the zebrafish as a model of ATP-sensitive potassium channel hyperinsulinism.

INTRODUCTION: Congenital hyperinsulinism (HI) is the leading cause of persistent hypoglycemia in infants. Current models to study the most common and severe form of HI resulting from inactivating mutations in the ATP-sensitive potassium channel (KATP) are limited to primary islets from patients and the Sur1 -/- mouse model. Zebrafish exhibit potential as a novel KATPHI model since they express canonical insulin secretion pathway genes and those with identified causative HI mutations. Moreover, zebrafish larvae transparency provides a unique opportunity for in vivo visualization of pancreatic islets. RESEARCH DESIGN AND METHODS: We evaluated zebrafish as a model for KATPHI using a genetically encoded Ca2+ sensor (ins:gCaMP6s) expressed under control of the insulin promoter in beta cells of an abcc8 -/- zebrafish line. RESULTS: We observed significantly higher islet cytosolic Ca2+ in vivo in abcc8 -/- compared with abcc8 +/+ zebrafish larvae. Additionally, abcc8 -/- larval zebrafish had significantly lower whole body glucose and higher whole body insulin levels compared with abcc8 +/+ controls. However, adult abcc8 -/- zebrafish do not show differences in plasma glucose, plasma insulin, or glucose tolerance when compared with abcc8 +/+ zebrafish. CONCLUSIONS: Our results identify that zebrafish larvae, but not adult fish, are a demonstrable novel model for advancement of HI research.

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.

Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Dnttip2 is one of the components of the small subunit (SSU) processome. In yeast, depletion of dnttip2 leads to an inefficient processing of pre-rRNA and a decrease in synthesis of the mature 18S rRNA. However, the biological roles of Dnttip2 in higher organisms are poorly defined. In this study, we demonstrate that dnttip2 is a maternal gene in zebrafish. Depletion of Dnttip2 leads to embryonic lethal with severe digestive organs hypoplasia. The loss of function of Dnttip2 also leads to partial defects in cleavage at the A0-site and E-site during 18S rRNA processing. In conclusion, Dnttip2 is essential for 18S rRNA processing and digestive organ development in zebrafish.

Key HPI axis receptors facilitate light adaptive behavior in larval zebrafish.

The vertebrate stress response (SR) is mediated by the hypothalamic-pituitary-adrenal (HPA) axis and contributes to generating context appropriate physiological and behavioral changes. Although the HPA axis plays vital roles both in stressful and basal conditions, research has focused on the response under stress. To understand broader roles of the HPA axis in a changing environment, we characterized an adaptive behavior of larval zebrafish during ambient illumination changes. Genetic abrogation of glucocorticoid receptor (nr3c1) decreased basal locomotor activity in light and darkness. Some key HPI axis receptors (mc2r [ACTH receptor], nr3c1), but not nr3c2 (mineralocorticoid receptor), were required to adapt to light more efficiently but became dispensable when longer illumination was provided. Such light adaptation was more efficient in dimmer light. Our findings show that the HPI axis contributes to the SR, facilitating the phasic response and maintaining an adapted basal state, and that certain adaptations occur without HPI axis activity.

Alnustone promotes megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway.

OBJECTIVES: Thrombocytopenia is a disease in which the number of platelets in the peripheral blood decreases. It can be caused by multiple genetic factors, and numerous challenges are associated with its treatment. In this study, the effects of alnustone on megakaryocytes and platelets were investigated, with the aim of developing a new therapeutic approach for thrombocytopenia. METHODS: Random forest algorithm was used to establish a drug screening model, and alnustone was identified as a natural active compound that could promote megakaryocyte differentiation. The effect of alnustone on megakaryocyte activity was determined using cell counting kit-8. The effect of alnustone on megakaryocyte differentiation was determined using flow cytometry, Giemsa staining, and phalloidin staining. A mouse model of thrombocytopenia was established by exposing mice to X-rays at 4 Gy and was used to test the bioactivity of alnustone in vivo. The effect of alnustone on platelet production was determined using zebrafish. Network pharmacology was used to predict targets and signaling pathways. Western blotting and immunofluorescence staining determined the expression levels of proteins. RESULTS: Alnustone promoted the differentiation and maturation of megakaryocytes in vitro and restored platelet production in thrombocytopenic mice and zebrafish. Network pharmacology and western blotting showed that alnustone promoted the expression of interleukin-17A and enhanced its interaction with its receptor, and thereby regulated downstream MEK/ERK signaling and promoted megakaryocyte differentiation. CONCLUSIONS: Alnustone can promote megakaryocyte differentiation and platelet production via the interleukin-17A/interleukin-17A receptor/Src/RAC1/MEK/ERK signaling pathway and thus provides a new therapeutic strategy for the treatment of thrombocytopenia.

Discovery of ganoderic acid A (GAA) PROTACs as MDM2 protein degraders for the treatment of breast cancer.

Breast cancer is one of the most common female malignant tumors, with triple-negative breast cancer (TNBC) being the most specific, highly invasive, metastatic and associated with a poor prognosis. Our previous study showed that the natural product ganoderic acid A (GAA) has a certain affinity for MDM2. In this study, two series of novel GAA PROTACs C1-C10 and V1-V10 were designed and synthesized for the treatment of breast cancer. The antitumor activity of these compounds was evaluated against four human tumor cell lines (MCF-7, MDA-MB-231, SJSA-1, and HepG2). Among them, V9 and V10 showed stronger anti-proliferative effects against breast cancer cells, and V10 showed the best selectivity in MDA-MB-231 cells (TNBC), which was 5-fold higher than that of the lead compound GAA. Preliminary structure-activity analysis revealed that V-series GAA PROTACs had better effects than C-series, and the introduction of 2O-4O PEG linkers could significantly improve the antitumor activity. Molecular docking, surface plasmon resonance (SPR), cellular thermal shift assay (CETSA), and Western blot researches showed that both V9 and V10 could bind with MDM2, and degrade the protein through the ubiquitin-proteasome system. Molecular dynamics simulation (MD) revealed that V10 is a bifunctional molecule that can bind to von Hippel-Lindau (VHL) at one end and target MDM2 at the other. In addition, V10 promoted the upregulation of p21 in p53-mutant MDA-MB-231 cells, and induced apoptosis via down-regulation of the bcl-2/bax ratio and the expression of cyclin B1. Finally, in vivo experiments showed that, V10 also exhibited good tumor inhibitory activity in xenografted TNBC zebrafish models, with an inhibition rate of 27.2% at 50 µg/mL. In conclusion, our results suggested that V10 has anti-tumor effects on p53-mutant breast cancer in vitro and in vivo, and may be used as a novel lead compound for the future development of TNBC.

Transcription bodies regulate gene expression by sequestering CDK9.

The localization of transcriptional activity in specialized transcription bodies is a hallmark of gene expression in eukaryotic cells. It remains unclear, however, if and how transcription bodies affect gene expression. Here we disrupted the formation of two prominent endogenous transcription bodies that mark the onset of zygotic transcription in zebrafish embryos and analysed the effect on gene expression using enriched SLAM-seq and live-cell imaging. We find that the disruption of transcription bodies results in the misregulation of hundreds of genes. Here we focus on genes that are upregulated. These genes have accessible chromatin and are poised to be transcribed in the presence of the two transcription bodies, but they do not go into elongation. Live-cell imaging shows that disruption of the two large transcription bodies enables these poised genes to be transcribed in ectopic transcription bodies, suggesting that the large transcription bodies sequester a pause release factor. Supporting this hypothesis, we find that CDK9-the kinase that releases paused polymerase II-is highly enriched in the two large transcription bodies. Overexpression of CDK9 in wild-type embryos results in the formation of ectopic transcription bodies and thus phenocopies the removal of the two large transcription bodies. Taken together, our results show that transcription bodies regulate transcription by sequestering machinery, thereby preventing genes elsewhere in the nucleus from being transcribed.

Mucopolysaccharidosis type II zebrafish model exhibits early impaired proteasomal-mediated degradation of the axon guidance receptor Dcc.

Most of the patients affected by neuronopathic forms of Mucopolysaccharidosis type II (MPS II), a rare lysosomal storage disorder caused by defects in iduronate-2-sulfatase (IDS) activity, exhibit early neurological defects associated with white matter lesions and progressive behavioural abnormalities. While neuronal degeneration has been largely described in experimental models and human patients, more subtle neuronal pathogenic defects remain still underexplored. In this work, we discovered that the axon guidance receptor Deleted in Colorectal Cancer (Dcc) is significantly dysregulated in the brain of ids mutant zebrafish since embryonic stages. In addition, thanks to the establishment of neuronal-enriched primary cell cultures, we identified defective proteasomal degradation as one of the main pathways underlying Dcc upregulation in ids mutant conditions. Furthermore, ids mutant fish-derived primary neurons displayed higher levels of polyubiquitinated proteins and P62, suggesting a wider defect in protein degradation. Finally, we show that ids mutant larvae display an atypical response to anxiety-inducing stimuli, hence mimicking one of the characteristic features of MPS II patients. Our study provides an additional relevant frame to MPS II pathogenesis, supporting the concept that multiple developmental defects concur with early childhood behavioural abnormalities.

Pathogenic TNNI1 variants disrupt sarcomere contractility resulting in hypo- and hypercontractile muscle disease.

Troponin I (TnI) regulates thin filament activation and muscle contraction. Two isoforms, TnI-fast (TNNI2) and TnI-slow (TNNI1), are predominantly expressed in fast- and slow-twitch myofibers, respectively. TNNI2 variants are a rare cause of arthrogryposis, whereas TNNI1 variants have not been conclusively established to cause skeletal myopathy. We identified recessive loss-of-function TNNI1 variants as well as dominant gain-of-function TNNI1 variants as a cause of muscle disease, each with distinct physiological consequences and disease mechanisms. We identified three families with biallelic TNNI1 variants (F1: p.R14H/c.190-9G>A, F2 and F3: homozygous p.R14C), resulting in loss of function, manifesting with early-onset progressive muscle weakness and rod formation on histology. We also identified two families with a dominantly acting heterozygous TNNI1 variant (F4: p.R174Q and F5: p.K176del), resulting in gain of function, manifesting with muscle cramping, myalgias, and rod formation in F5. In zebrafish, TnI proteins with either of the missense variants (p.R14H; p.R174Q) incorporated into thin filaments. Molecular dynamics simulations suggested that the loss-of-function p.R14H variant decouples TnI from TnC, which was supported by functional studies showing a reduced force response of sarcomeres to submaximal [Ca2+] in patient myofibers. This contractile deficit could be reversed by a slow skeletal muscle troponin activator. In contrast, patient myofibers with the gain-of-function p.R174Q variant showed an increased force to submaximal [Ca2+], which was reversed by the small-molecule drug mavacamten. Our findings demonstrated that TNNI1 variants can cause muscle disease with variant-specific pathomechanisms, manifesting as either a hypo- or a hypercontractile phenotype, suggesting rational therapeutic strategies for each mechanism.