Comparative Proteome Research in a Zebrafish Model for Vanishing White Matter Disease

Vanishing white matter (VWM) disease is a genetic leukodystrophy leading to severe neurological disease and early death. VWM is caused by bi-allelic mutations in any of the five genes encoding the subunits of the eukaryotic translation factor 2B (EIF2B). Previous studies have attempted to investigate the molecular mechanism of VWN by constructing models for each subunit of EIF2B that causes VWM disease. The underlying molecular mechanisms of the way in which mutations in EIF2B3 result in VWM are largely unknown. Based on our recent results, we generated an eif2b3 knockout (eif2b3^−/−) zebrafish model and performed quantitative proteomic analysis between the wild-type (WT) and eif2b3^−/− zebrafish, and identified 25 differentially expressed proteins. Four proteins were significantly upregulated, and 21 proteins were significantly downregulated in eif2b3^−/− zebrafish compared to WT. Lon protease and the neutral amino acid transporter SLC1A4 were significantly increased in eif2b3^−/− zebrafish, and crystallin proteins were significantly decreased. The differential expression of proteins was confirmed by the evaluation of mRNA levels in eif2b3^−/− zebrafish, using whole-mount in situ hybridization analysis. This study identified proteins which candidates as key regulators of the progression of VWN disease, using quantitative proteomic analysis in the first EIF2B3 animal model of VWN disease.     

An Engineered sgsh Mutant Zebrafish Recapitulates Molecular and Behavioural Pathobiology of Sanfilippo Syndrome A/MPS IIIA

Mucopolysaccharidosis IIIA (MPS IIIA, Sanfilippo syndrome type A), a paediatric neurological lysosomal storage disease, is caused by impaired function of the enzyme N-sulfoglucosamine sulfohydrolase (SGSH) resulting in impaired catabolism of heparan sulfate glycosaminoglycan (HS GAG) and its accumulation in tissues. MPS IIIA represents a significant proportion of childhood dementias. This condition generally leads to patient death in the teenage years, yet no effective therapy exists for MPS IIIA and a complete understanding of the mechanisms of MPS IIIA pathogenesis is lacking. Here, we employ targeted CRISPR/Cas9 mutagenesis to generate a model of MPS IIIA in the zebrafish, a model organism with strong genetic tractability and amenity for high-throughput screening. The sgsh^Δex5−6 zebrafish mutant exhibits a complete absence of Sgsh enzymatic activity, leading to progressive accumulation of HS degradation products with age. sgsh^Δex5−6 zebrafish faithfully recapitulate diverse CNS-specific features of MPS IIIA, including neuronal lysosomal overabundance, complex behavioural phenotypes, and profound, lifelong neuroinflammation. We further demonstrate that neuroinflammation in sgsh^Δex5−6 zebrafish is largely dependent on interleukin-1β and can be attenuated via the pharmacological inhibition of Caspase-1, which partially rescues behavioural abnormalities in sgsh^Δex5−6 mutant larvae in a context-dependent manner. We expect the sgsh^Δex5−6 zebrafish mutant to be a valuable resource in gaining a better understanding of MPS IIIA pathobiology towards the development of timely and effective therapeutic interventions.     

The Amino Acid Transporter Mct10/Tat1 Is Important to Maintain the TSH Receptor at Its Canonical Basolateral Localization and Assures Regular Turnover of Thyroid Follicle Cells in Male Mice

Cathepsin K-mediated thyroglobulin proteolysis contributes to thyroid hormone (TH) liberation, while TH transporters like Mct8 and Mct10 ensure TH release from thyroid follicles into the blood circulation. Thus, thyroid stimulating hormone (TSH) released upon TH demand binds to TSH receptors of thyrocytes, where it triggers Gα_q-mediated short-term effects like cathepsin-mediated thyroglobulin utilization, and Gα_s-mediated long-term signaling responses like thyroglobulin biosynthesis and thyrocyte proliferation. As reported recently, mice lacking Mct8 and Mct10 on a cathepsin K-deficient background exhibit excessive thyroglobulin proteolysis hinting towards altered TSH receptor signaling. Indeed, a combination of canonical basolateral and non-canonical vesicular TSH receptor localization was observed in Ctsk^−/−/Mct8^−/y/Mct10^−/− mice, which implies prolonged Gα_s-mediated signaling since endo-lysosomal down-regulation of the TSH receptor was not detected. Inspection of single knockout genotypes revealed that the TSH receptor localizes basolaterally in Ctsk^−/− and Mct8^−/y mice, whereas its localization is restricted to vesicles in Mct10^−/− thyrocytes. The additional lack of cathepsin K reverses this effect, because Ctsk^−/−/Mct10^−/− mice display TSH receptors basolaterally, thereby indicating that cathepsin K and Mct10 contribute to TSH receptor homeostasis by maintaining its canonical localization in thyrocytes. Moreover, Mct10^−/− mice displayed reduced numbers of dead thyrocytes, while their thyroid gland morphology was comparable to wild-type controls. In contrast, Mct8^−/y, Mct8^−/y/Mct10^−/−, and Ctsk^−/−/Mct8^−/y/Mct10^−/− mice showed enlarged thyroid follicles and increased cell death, indicating that Mct8 deficiency results in altered thyroid morphology. We conclude that vesicular TSH receptor localization does not result in different thyroid tissue architecture; however, Mct10 deficiency possibly modulates TSH receptor signaling for regulating thyrocyte survival.     

Minocycline Counteracts Ectopic Calcification in a Murine Model of Pseudoxanthoma Elasticum: A Proof-of-Concept Study

Pseudoxanthoma elasticum (PXE) is an intractable Mendelian disease characterized by ectopic calcification in skin, eyes and blood vessels. Recently, increased activation of the DNA damage response (DDR) was shown to be involved in PXE pathogenesis, while the DDR/PARP1 inhibitor minocycline was found to attenuate aberrant mineralization in PXE cells and zebrafish. In this proof-of-concept study, we evaluated the anticalcifying properties of minocycline in Abcc6^−/− mice, an established mammalian PXE model. Abcc6^−/− mice received oral minocycline supplementation (40 mg/kg/day) from 12 to 36 weeks of age and were compared to untreated Abcc6^−/− and Abcc6^+/+ siblings. Ectopic calcification was evaluated using X-ray microtomography with three-dimensional reconstruction of calcium deposits in muzzle skin and Yasue’s calcium staining. Immunohistochemistry for the key DDR marker H2AX was also performed. Following minocycline treatment, ectopic calcification in Abcc6^−/− mice was significantly reduced (−43.4%, p < 0.0001) compared to untreated Abcc6^−/− littermates. H2AX immunostaining revealed activation of the DDR at sites of aberrant mineralization in untreated Abcc6^−/− animals. In conclusion, we validated the anticalcifying effect of minocycline in Abcc6^−/− mice for the first time. Considering its favorable safety profile in humans and low cost as a generic drug, minocycline may be a promising therapeutic compound for PXE patients.     

Conditional Loss of the Exocyst Component Exoc5 in Retinal Pigment Epithelium (RPE) Results in RPE Dysfunction,Photoreceptor Cell Degeneration,and Decreased Visual Function

To characterize the mechanisms by which the highly conserved exocyst trafficking complex regulates eye physiology in zebrafish and mice, we focused on Exoc5 (also known as sec10), a central exocyst component. We analyzed both exoc5 zebrafish mutants and retinal pigmented epithelium (RPE)-specific Exoc5 knockout mice. Exoc5 is present in both the non-pigmented epithelium of the ciliary body and in the RPE. In this study, we set out to establish an animal model to study the mechanisms underlying the ocular phenotype and to establish if loss of visual function is induced by postnatal RPE Exoc5-deficiency. Exoc5^−/− zebrafish had smaller eyes, with decreased number of melanocytes in the RPE and shorter photoreceptor outer segments. At 3.5 days post-fertilization, loss of rod and cone opsins were observed in zebrafish exoc5 mutants. Mice with postnatal RPE-specific loss of Exoc5 showed retinal thinning associated with compromised visual function and loss of visual photoreceptor pigments. Abnormal levels of RPE65 together with a reduced c-wave amplitude indicate a dysfunctional RPE. The retinal phenotype in Exoc5^−/− mice was present at 20 weeks, but was more pronounced at 27 weeks, indicating progressive disease phenotype. We previously showed that the exocyst is necessary for photoreceptor ciliogenesis and retinal development. Here, we report that exoc5 mutant zebrafish and mice with RPE-specific genetic ablation of Exoc5 develop abnormal RPE pigmentation, resulting in retinal cell dystrophy and loss of visual pigments associated with compromised vision. Together, these data suggest that exocyst-mediated signaling in the RPE is required for RPE structure and function, indirectly leading to photoreceptor degeneration.     

BMP3 Affects Cortical and Trabecular Long Bone Development in Mice

Bone morphogenetic proteins (BMPs) have a major role in tissue development. BMP3 is synthesized in osteocytes and mature osteoblasts and has an antagonistic effect on other BMPs in bone tissue. The main aim of this study was to fully characterize cortical bone and trabecular bone of long bones in both male and female Bmp3^−/− mice. To investigate the effect of Bmp3 from birth to maturity, we compared Bmp3^−/− mice with wild-type littermates at the following stages of postnatal development: 1 day (P0), 2 weeks (P14), 8 weeks and 16 weeks of age. Bmp3 deletion was confirmed using X-gal staining in P0 animals. Cartilage and bone tissue were examined in P14 animals using Alcian Blue/Alizarin Red staining. Detailed long bone analysis was performed in 8-week-old and 16-week-old animals using micro-CT. The Bmp3 reporter signal was localized in bone tissue, hair follicles, and lungs. Bone mineralization at 2 weeks of age was increased in long bones of Bmp3^−/− mice. Bmp3 deletion was shown to affect the skeleton until adulthood, where increased cortical and trabecular bone parameters were found in young and adult mice of both sexes, while delayed mineralization of the epiphyseal growth plate was found in adult Bmp3^−/− mice.     

EGFR Transgene Stimulates Spontaneous Formation of MCF7 Breast Cancer Cells Spheroids with Partly Loss of HER3 Receptor

Multicellular spheroids with 3D cell–cell interactions are a useful model to simulate the growth conditions of cancer. There is evidence that in tumor spheroids, the expression of various essential molecules is changed compared to the adherent form of cell cultures. These changes include growth factor receptors and ABC transporters and result in the enhanced invasiveness of the cells and drug resistance. It is known that breast adenocarcinoma MCF7 cells can spontaneously form 3D spheroids and such spheroids are characterized by high expression of EGFR/HER2, while the natural phenotype of MCF7 cells is EGFR^low/HER2^low. Therefore, it was interesting to reveal if high epidermal growth factor receptor (EGFR) expression is sufficient for the conversion of adherent MCF7 to spheroids. In this study, an MCF7 cell line with high expression of EGFR was engineered using the retroviral transduction method. These MCF7-EGFR cells assembled in spheroids very quickly and grew predominantly as a 3D suspension culture with no special plates, scaffolds, growth supplements, or exogenous matrixes. These spheroids were characterized by a rounded shape with a well-defined external border and 100 µM median diameter. The sphere-forming ability of MCF7-EGFR cells was up to 5 times stronger than in MCF7^wt cells. Thus, high EGFR expression was the initiation factor of conversion of adherent MCF7^wt cells to spheroids. MCF7-EGFR spheroids were enriched by the cells with a cancer stem cell (CSC) phenotype CD24^−/low/CD44⁻ in comparison with parental MCF7^wt cells and MCF7-EGFR adhesive cells. We suppose that these properties of MCF7-EGFR spheroids originate from the typical features of parental MCF7 cells. We showed the decreasing of HER3 receptors in MCF7-EGFR spheroids compared to that in MCF^wt and in adherent MCF7-EGFR cells, and the same decrease was observed in the MCF7^wt spheroids growing under the growth factors stimulation. To summarize, the expression of EGFR transgene in MCF7 cells stimulates rapid spheroids formation; these spheroids are enriched by CSC-like CD24⁻/CD44⁻ cells, they partly lose HER3 receptors, and are characterized by a lower potency in drug resistance pomp activation compared to MCF7^wt. These MCF7-EGFR spheroids are a useful cancer model for the development of anticancer drugs, including EGFR-targeted therapeutics.     

Dnmt3aa but Not Dnmt3ab Is Required for Maintenance of Gametogenesis in Nile Tilapia (Oreochromis niloticus)

Dnmt3a, a de novo methyltransferase, is essential for mammalian germ line DNA methylation. Only one Dnmt3a is identified in mammals, and homozygous mutants of Dnmt3a are lethal, while two Dnmt3a paralogs, dnmt3aa and dnmt3ab, are identified in teleosts due to the third round of genome duplication, and homozygous mutants of dnmt3aa and dnmt3ab are viable in zebrafish. The expression patterns and roles of dnmt3aa and dnmt3ab in gonadal development remain poorly understood in teleosts. In this study, we elucidated the precise expression patterns of dnmt3aa and dnmt3ab in tilapia gonads. Dnmt3aa was highly expressed in oogonia, phase I and II oocytes and granulosa cells in ovaries and spermatogonia and spermatocytes in testes, while dnmt3ab was mainly expressed in ovarian granulosa cells and testicular spermatocytes. The mutation of dnmt3aa and dnmt3ab was achieved by CRISPR/Cas9 in tilapia. Lower gonadosomatic index (GSI), increased apoptosis of oocytes and spermatocytes and significantly reduced sperm quality were observed in dnmt3aa^−/− mutants, while normal gonadal development was observed in dnmt3ab^−/− mutants. Consistently, the expression of apoptotic genes was significantly increased in dnmt3aa^−/− mutants. In addition, the 5-methylcytosine (5-mC) level in dnmt3aa^−/− gonads was decreased significantly, compared with that of dnmt3ab^−/− and wild type (WT) gonads. Taken together, our results suggest that dnmt3aa, not dnmt3ab, plays important roles in maintaining gametogenesis in teleosts.     

Acetylsalicylic Acid Reduces Passive Aortic Wall Stiffness and Cardiovascular Remodelling in a Mouse Model of Advanced Atherosclerosis

Acetylsalicylic acid (ASA) is widely used in secondary prevention of cardiovascular (CV) disease, mainly because of its antithrombotic effects. Here, we investigated whether ASA can prevent the progression of vessel wall remodelling, atherosclerosis, and CV complications in apolipoprotein E deficient (ApoE^−/−) mice, a model of stable atherosclerosis, and in ApoE^−/− mice with a mutation in the fibrillin-1 gene (Fbn1^C1039G+/−), which is a model of elastic fibre fragmentation, accompanied by exacerbated unstable atherosclerosis. Female ApoE^−/− and ApoE^−/−Fbn1^C1039G+/− mice were fed a Western diet (WD). At 10 weeks of WD, the mice were randomly divided into four groups, receiving either ASA 5 mg/kg/day in the drinking water (ApoE^−/− (n = 14), ApoE^−/−Fbn1^C1039G+/− (n = 19)) or plain drinking water (ApoE^−/− (n = 15), ApoE^−/−Fbn1^C1039G+/− (n = 21)) for 15 weeks. ApoE^−/−Fbn1^C1039G+/− mice showed an increased neutrophil–lymphocyte ratio (NLR) compared to ApoE^−/− mice, and this effect was normalised by ASA. In the proximal ascending aorta wall, ASA-treated ApoE^−/−Fbn1^C1039G+/− mice showed less p-SMAD2/3 positive nuclei, a lower collagen percentage and an increased elastin/collagen ratio, consistent with the values measured in ApoE^−/− mice. ASA did not affect plaque progression, incidence of myocardial infarction and survival of ApoE^−/−Fbn1^C1039G+/− mice, but systolic blood pressure, cardiac fibrosis and hypertrophy were reduced. In conclusion, ASA normalises the NLR, passive wall stiffness and cardiac remodelling in ApoE^−/−Fbn1^C1039G+/− mice to levels observed in ApoE^−/− mice, indicating additional therapeutic benefits of ASA beyond its classical use.     

Male-Dependent Promotion of Colitis in 129 Rag2−/− Mice Co-Infected with Helicobacter pylori and Helicobacter hepaticus

The prevalence of gastric Helicobacter pylori (Hp) infection is ~50% of the world population. However, how Hp infection influences inflammatory bowel disease in humans is not fully defined. In this study, we examined whether co-infection with Hp influenced Helicobacter hepaticus (Hh)–induced intestinal pathology in Rag2^−/− mice. Rag2^−/− mice of both sexes were infected with Hh, of which a subgroup was followed by infection with Hp two weeks later. Co-infected males, but not females, had significantly higher total colitis index scores in the colon at both 10 and 21 weeks post-Hh infection (WPI) and developed more severe dysplasia at 21 WPI compared with mono-Hh males. There were no significant differences in colonization levels of gastric Hp and colonic Hh between sexes or time-points. In addition, mRNA levels of colonic Il-1β, Ifnγ, Tnfα, Il-17A, Il-17F, Il-18, and Il-23, which play important roles in the development and function of proinflammatory innate lymphoid cell groups 1 and 3, were significantly up-regulated in the dually infected males compared with mono-Hh males at 21 WPI. These data suggest that concomitant Hp infection enhances the inflammatory responses in the colon of-Hh-infected Rag2^−/− males, which results in more severe colitis and dysplasia.     

Effects of Melatonin on the Proliferation and Apoptosis of Sheep Granulosa Cells under Thermal Stress

The cross-talk between oocyte and somatic cells plays a crucial role in the regulation of follicular development and oocyte maturation. As a result, granulosa cell apoptosis causes follicular atresia. In this study, sheep granulosa cells were cultured under thermal stress to induce apoptosis, and melatonin (MT) was examined to evaluate its potential effects on heat-induced granulosa cell injury. The results demonstrated that the Colony Forming Efficiency (CFE) of granulosa cells was significantly decreased (heat 19.70% ± 1.29% vs. control 26.96% ± 1.81%, p < 0.05) and the apoptosis rate was significantly increased (heat 56.16% ± 13.95%vs. control 22.80% ± 12.16%, p < 0.05) in granulosa cells with thermal stress compared with the control group. Melatonin (10⁻⁷ M) remarkably reduced the negative effects caused by thermal stress in the granulosa cells. This reduction was indicated by the improved CFE and decreased apoptotic rate of these cells. The beneficial effects of melatonin on thermal stressed granulosa cells were not inhibited by its membrane receptor antagonist luzindole. A mechanistic exploration indicated that melatonin (10⁻⁷ M) down-regulated p53 and up-regulated Bcl-2 and LHR gene expression of granulosa cells under thermal stress. This study provides evidence for the molecular mechanisms of the protective effects of melatonin on granulosa cells during thermal stress.     

Retinal Degeneration and Microglial Dynamics in Mature Progranulin-Deficient Mice

Progranulin (PGRN) is a secreted glycoprotein that regulates numerous cellular processes. The role of PGRN as a regulator of lysosomes has recently received attention. The purpose of this study was to characterize the retinal phenotype in mature PGRN knockout (Grn^−/−) mice. The a-wave amplitude of scotopic electroretinogram and outer nuclear thickness were significantly reduced at 6 months of age in Grn^−/− mice compared to wild-type (Grn^+/+) mice. In Grn^−/− mice, retinal microglial cells accumulated on the retinal pigment epithelium (RPE) apical layer, and the number of infiltrated microglia and white fundus lesions between 2 and 6 months of age showed a close affinity. In Grn^+/+ mice, PGRN was located in the retina, while the strongest PGRN signals were detected in the RPE-choroid. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid were demonstrated. Our data suggest that the subretinal translocation of microglia is a characteristic phenotype in the retina of mature PGRN knockout mice. The different effects of PGRN deficiency on the expression of lysosomal proteins between the retina and RPE-choroid might modulate microglial dynamics in PGRN knockout mice.