Disease Models & Mechanisms最新文献

Peritoneal endometriosis causes pelvic pain and infertility, but the underlying mechanisms related to these symptoms are not fully understood. Endometriosis diagnosis is typically delayed; thus, patient samples are unsuitable to study early endometriosis formation in situ. We generated a 3D co-culture model of early peritoneal endometriosis using patient-derived primary cells, providing unique opportunities to examine endometriotic lesion initiation and progression. The successful assembly of a simple peritoneum layer model comprising a mesothelial monolayer, basement membrane and underlying fibroblasts was achieved by embedding human peritoneal fibroblasts in a Matrigel-collagen I matrix and subsequent seeding with a layer of donor-matched human peritoneal mesothelial cells, while secretion of tissue plasminogen activator demonstrated functional mesothelial physiology. Endometrial epithelial organoids were co-cultured with endometrial stromal cells to form endometrial assembloids mimicking shed endometrial tissue fragments at menstruation, which adhered onto the peritoneal layer model, simulating early endometriotic lesion formation. Our modifiable superficial endometriosis model allows for further refinement to determine the underlying molecular mechanism(s) involved in endometriotic lesion formation.

CHARGE syndrome is a developmental disorder that affects 1 in 10,000 births, and patients exhibit both physical and behavioral characteristics. De novo mutations in CHD7 (chromodomain helicase DNA binding protein 7) cause 67% of CHARGE syndrome cases. CHD7 is a DNA-binding chromatin remodeler with thousands of predicted binding sites in the genome, making it challenging to define molecular pathways linking loss of CHD7 to CHARGE phenotypes. To address this problem, here we used a previously characterized zebrafish CHARGE model to generate transcriptomic and proteomic datasets from larval zebrafish head tissue at two developmental time points. By integrating these datasets with differential expression, pathway, and upstream regulator analyses, we identified multiple consistently dysregulated pathways and defined a set of candidate genes that link loss of chd7 with disease-related phenotypes. Finally, to functionally validate the roles of these genes, CRISPR/Cas9-mediated knockdown of capgb, nefla, or rdh5 phenocopies behavioral defects seen in chd7 mutants. Our data provide a resource for further investigation of molecular mediators of CHD7 and a template to reveal functionally relevant therapeutic targets to alleviate specific aspects of CHARGE syndrome.

Huntington's Disease (HD) is caused by expansion of the polyglutamine stretch in the widely expressed Huntingtin (HTT) protein. HD patients have motor, psychiatric, and cognitive changes due to changes in a variety of neural circuits. Somatostatin-expressing interneurons (SST-INs) can regulate neural circuits largely by inhibiting their target cells. Behaviorally, brain-wide inhibition of SST-INs increased anxiety in mice. Silencing striatal SST-INs caused a decrease in movement in the open field. Mutant HTT (mHTT) expressing mice exhibit abnormal motor, cognitive and psychiatric-like changes as well as electrophysiological changes in a variety of neurons, including striatal SST-INs. However, it is unknown whether cell autonomous expression of mHTT in SST-INs contributes to HD-associated behavioral phenotypes or causes abnormal electrophysiological changes in striatal SST-INs. To address these questions, we reduced mHTT expression in SST-INs throughout the brain of BACHD mice. Our findings show that brain-wide reduction of mHTT in SST-INs rescues anxiety-like behavior in male BACHD mice in the light-dark box, without improving performance in the open field or on the rotarod. Additionally, expression of mHTT in striatal SST-INs cell autonomously drives their increased excitability.

Chronic lymphocytic leukaemia (CLL) cells circulate between the blood, bone marrow (BM), and lymphoid organs, where interactions with the lymph node (LN) microenvironment enhance their survival, proliferation, and drug resistance. Most in vitro models fail to reproduce the spatial and cellular complexity of the LN niche, limiting studies of tissue-specific drug responses. To address this, we developed a 3D LN model using a gelatine scaffold and a clinorotator bioreactor previously validated for a BM system. The scaffold was seeded with human lymphatic fibroblasts and endothelial cells, which deposited extracellular matrix and supported patient-derived CLL cell viability and proliferation. Consistent with in vivo observations, CLL cells within the scaffold downregulated the chemokine receptor CXCR4, further reduced upon proliferative stimulation. Final validation involved treatment with targeted therapies: the Bcl-2 antagonist venetoclax and the BTK inhibitor ibrutinib. Venetoclax treatment revealed greater CLL protection within the LN environment than in BM, whereas ibrutinib's mobilizing effect was comparable. This 3D LN model offers an effective ex vivo platform for studying microenvironment-tumour interactions and tissue-specific drug responses.

Polyphosphates are evolutionarily conserved anionic polymers mediating pleiotropic functions in eukaryotes and prokaryotes, depending on their chain-length. Bacteria typically synthetize long-chains, while human platelets harbour exclusively medium-chains. Polyphosphate-mediated lung and liver-injury have been reported in experimental mouse models but their effects on the kidney remain undefined. Here we assessed kidney histopathology and cytokine levels following intravenous administration of medium-chain (P100) and long-chain (P700) polyphosphates and their synergistic effects with lipopolysaccharides (LPS) in mice. We found that P700 induced albuminuria, renal Kim-1 and Lcn2 transcription, focal renal damage with glomerular microthrombi, tubular degeneration, granular phenotype of slit diaphragm components nephrin and ZO1, and enlarged electron dense vesicles in podocyte cytoplasm indicating lysosome swelling. P700 combined with LPS induced marked multifocal acute tubular necrosis in the cortex, and augmented LPS-induced proinflammatory cytokine levels. No notable effects were seen with P100, indicating that PolyP-mediated kidney injury development is dependent on chain-length. We conclude that long-chain polyphosphates may play a procoagulant role in kidney injury development, by inducing microthrombi characteristic of thrombotic microangiopathy and augmenting cytokine levels under inflammatory conditions.

Tau protein contributes to microtubule stability, which is disrupted in Alzheimer's disease and other Tauopathies. In these diseases, Tau molecules become hyperphosphorylated, misfolded and aggregated, propagating pathology across the brain. Studies dissecting disease mechanisms or screening disease-modifying therapies rely on animal models that unveil pathogenic events in vivo but also take several weeks or months to complete. Here we describe a versatile experimental paradigm that yields results in days and yet offers all the advantages of a genetically tractable in vivo system: the Drosophila wing. Mimicking neurotoxicity, human Tau expression causes cell death in the wing disc leading to quantifiable phenotypes in the adult wing. The neuroprotective peptide NAP ameliorates Tau toxicity in this system, validating it as a cost-effective drug screening tool. Phenocopying adult neurons, Tau toxicity in the wing disc is exacerbated by simulating hyper-phosphorylation and prevented by suppressing aggregation. Additionally, we show that the wing disc can dissect disease mechanisms that underpin clinically relevant Tau variants. Thus, the Drosophila wing offers an in vivo experimental paradigm for fast and efficient exploration of disease mechanism and screening.

Pompe disease (PD) is a rare autosomal recessive disorder caused by acid α-glucosidase (GAA) deficiency, leading to lysosomal glycogen accumulation. Pathogenic GAA variants result in enzyme dysfunction and glycogen storage in cardiac, skeletal, and smooth muscle, as well as in the central nervous system, driving both systemic and neurological manifestations. We previously characterized a transgenic knock-in (KI) mouse carrying the Gaa c.1826dupA variant to 12 weeks of age, showing it recapitulates key biochemical and phenotypic features of PD. Here, we extend this analysis to present a long-term characterization of the Gaa c.1826dupA KI model using physiological, behavioral, biochemical, and histopathological assessments. KI mice exhibited early-onset hypertrophic cardiomyopathy with significant cardiac functional decline, reduced body mass, impaired skeletal muscle strength, locomotion, coordination, and balance. Biochemically, KI mice showed decreased GAA activity and increased lysosomal glycogen accumulation in the heart, diaphragm, gastrocnemius, and brain. Despite these abnormalities, survival did not differ from wild-type mice-a divergence from severe human PD but consistent with other murine models. Collectively, these findings support this KI model as a translational platform for therapeutic evaluation in PD.

Loss of ALX1 gene function causes severe facial clefting and extreme microphthalmia. Previous studies suggest that ALX1 protein function is crucial for patterning the cranial neural crest cell (CNCC)-derived frontonasal mesenchyme, but how ALX1 regulates eye development is not well understood. Here, we show that Alx1 is transiently expressed in the embryonic cranial mesoderm and that Alx1-/- mice exhibit agenesis of extraocular muscles (EOMs) without affecting other muscles. We show that cranial mesoderm-specific Alx1 inactivation resulted in complete EOM agenesis accompanied by failure of activation of the core myogenic regulatory network specifically in, and increased apoptosis of, the EOM progenitor cells. Analysis of mice with temporally induced Alx1 inactivation demonstrated that EOM myogenesis requires Alx1 function before, but not after, formation of the EOM primordium. These data identify ALX1 as a unique and specific upstream regulator of EOM myogenesis, and provide new insights into pathogenic mechanisms underlying ALX1-type frontonasal dysplasia, as well as molecular mechanisms controlling cell fate specification in the early cranial mesoderm.

Neurofibromatosis type 1 (NF1) is a neurogenetic disorder caused by loss of function mutations in the gene neurofibromin 1 (NF1). NF1 encodes neurofibromin, a multifunctional tumor suppressing protein that regulates Ras, cAMP, and dopamine signaling. NF1 predisposes patients to a wide range of symptoms, including peripheral nerve tumors, brain tumors, and cognitive dysfunction. Despite considerable work using animal models to investigate the role of neurofibromin in behavior, translating research into treatment for NF1-associated cognitive dysfunction has not yet been successful. Here, we provide evidence that Cxcr4 chemokine receptor signaling is a regulator of habituation learning and modulator of cAMP-PKA signaling in nf1 mutant larval zebrafish. Combining a small-molecule drug screen and RNAseq analysis, we show that cxcr4b expression is increased in nf1 mutants and that pharmacological inhibition of Cxcr4 with AMD3100 (Plerixafor) improves habituation learning. We further demonstrate that Plerixafor activates cAMP-PKA pathway signaling but has limited effects on Ras-Raf-MEK-ERK pathway signaling in the nf1 mutant brain. CXCR4 was previously identified as a potential therapeutic target for neurofibromin-deficient tumorigenesis. Our results suggest that Cxcr4 signaling also regulates neurofibromin-dependent cognitive function.

The prevalence of hepatocellular carcinoma (HCC) is rising in parallel with increasing obesity and metabolic dysfunction-associated steatohepatitis (MASH). MicroRNAs are key post-transcriptional regulators of gene expression and are attractive targets for HCC therapy. Here we sought to identify and characterize dysregulated microRNAs in MASH-driven HCC (MASH-HCC). We profiled microRNA expression in liver tissue from patients with MASH or MASH-HCC and in zebrafish HCC driven by activated β-catenin (ABC), one of the most commonly mutated oncogenes in MASH-HCC. We found overlap between dysregulated human and zebrafish miRNAs, including miR-21, which was increasingly upregulated from normal liver to MASH to MASH-HCC. We generated transgenic zebrafish that overexpress or sponge miR-21 in hepatocytes. We found that miR-21 overexpression caused larval liver overgrowth and increased HCC, while miR-21 sponge suppressed β-catenin-driven larval liver overgrowth. By performing histologic and lipidomic analysis, we found that overexpression of miR-21, like ABC, suppressed lipid accumulation in response to a high cholesterol diet and increased accumulation of acylcarnitines. Thus miR-21, which is similarly upregulated in human and zebrafish HCC, promotes lipid metabolic changes that may help drive hepatocarcinogenesis.