Essential role of Alix in regulating cardiomyocyte exosome biogenesis under physiological and stress conditions.

BACKGROUND: Exosomes released by cardiomyocytes are essential mediators of intercellular communications within the heart, and various exosomal proteins and miRNAs are associated with cardiovascular diseases. However, whether the endosomal sorting complex required for transport (ESCRT) and its key component Alix is required for exosome biogenesis within cardiomyocyte remains poorly understood. METHODS: Super-resolution imaging was performed to investigate the subcellular location of Alix and multivesicular body (MVB) in primary cardiomyocytes. Cardiomyocyte-specific Alix-knockout mice were generated using AAV9/CRISPR/Cas9-mediated in vivo gene editing. A stable Alix-knockdown H9c2 cardiomyocyte line was constructed through lentiviral-mediated delivery of short hairpin RNA. In order to determine the role of Alix in controlling exosome biogenesis, exosomes from cardiomyocyte-specific Alix-knockout mice plasma and Alix-knockdown H9c2 culture medium were isolated and examined by western blot, NTA analysis and transmission electron microscopy. Biochemical and immunofluorescence analysis were performed to determine the role of ESCRT machinery in regulating MVB formation. Lastly, transverse aortic constriction (TAC)-induced cardiac pressure overload model was established to further explore the role of Alix-mediated exosome biogenesis under stress conditions. RESULTS: A significant proportion of Alix localized to the MVB membrane within cardiomyocytes. Genetic deletion of Alix in murine heart resulted in a reduction of plasma exosome content without affecting cardiac structure or contractile function. Consistently, the downregulation of Alix in H9c2 cardiomyocyte line also suppressed the biogenesis of exosomes. We found the defective ESCRT machinery and suppressed MVB formation upon Alix depletion caused compromised exosome biogenesis. Remarkably, TAC-induced cardiac pressure overload led to increased Alix, MVB levels, and elevated plasma exosome content, which could be totally abolished by Alix deletion. CONCLUSION: These results establish Alix as an essential and stress-sensitive regulator of cardiac exosome biogenesis and the findings may yield valuable therapeutic implications.

Lgl1 deficiency disrupts hippocampal development and impairs cognitive performance in mice.

Cellular polarity is crucial for brain development and morphogenesis. Lethal giant larvae 1 (Lgl1) plays a crucial role in the establishment of cell polarity from Drosophila to mammalian cells. Previous studies have found the importance of Lgl1 in the development of cerebellar, olfactory bulb, and cerebral cortex. However, the role of Lgl1 in hippocampal development during the embryonic stage and function in adult mice is still unknown. In our study, we created Lgl1-deficient hippocampus mice by using Emx1-Cre mice. Histological analysis showed that the Emx1-Lgl1-/- mice exhibited reduced size of the hippocampus with severe malformations of hippocampal cytoarchitecture. These defects mainly originated from the disrupted hippocampal neuroepithelium, including increased cell proliferation, abnormal interkinetic nuclear migration, reduced differentiation, increased apoptosis, gradual disruption of adherens junctions, and abnormal neuronal migration. The radial glial scaffold was disorganized in the Lgl1-deficient hippocampus. Thus, Lgl1 plays a distinct role in hippocampal neurogenesis. In addition, the Emx1-Lgl1-/- mice displayed impaired behavioral performance in the Morris water maze and fear conditioning test.

The p38 MAPK Inhibitor SB203580 Abrogates Tumor Necrosis Factor-Induced Proliferative Expansion of Mouse CD4+Foxp3+ Regulatory T Cells.

There is now compelling evidence that tumor necrosis factor (TNF) preferentially activates and expands CD4+Foxp3+ regulatory T cells (Tregs) through TNF receptor type II (TNFR2). However, it remains unclear which signaling transduction pathway(s) of TNFR2 is required for the stimulation of Tregs. Previously, it was shown that the interaction of TNF-TNFR2 resulted in the activation of a number of signaling pathways, including p38 MAPK, NF-κB, in T cells. We thus examined the role of p38 MAPK and NF-κB in TNF-mediated activation of Tregs, by using specific small molecule inhibitors. The results show that treatment with specific p38 MAPK inhibitor SB203580, rather than NF-κB inhibitors (Sulfasalazine and Bay 11-7082), abrogated TNF-induced expansion of Tregs in vitro. Furthermore, upregulation of TNFR2 and Foxp3 expression in Tregs by TNF was also markedly inhibited by SB203580. The proliferative expansion and the upregulation of TNFR2 expression on Tregs in LPS-treated mice were mediated by TNF-TNFR2 interaction, as shown by our previous study. The expansion of Tregs in LPS-treated mice were also markedly inhibited by in vivo treatment with SB203580. Taken together, our data clearly indicate that the activation of p38 MAPK is attributable to TNF/TNFR2-mediated activation and proliferative expansion of Tregs. Our results also suggest that targeting of p38 MAPK by pharmacological agent may represent a novel strategy to up- or downregulation of Treg activity for therapeutic purposes.

Trio gene is required for mouse learning ability.

Trio is a guanine nucleotide exchange factor with multiple guanine nucleotide exchange factor domains. Trio regulates cytoskeleton dynamics and actin remodeling and is involved in cell migration and axonal guidance in neuronal development. The null allele of the Trio gene led to embryonic lethality, and Trio null embryos displayed aberrant organization in several regions of the brain at E18.5, including hippocampus. Nestin-Trio-/- mice, in which the Trio gene was deleted specifically in the neuronal system by the Nestin-Cre system, displayed severe phenotypes, including low survival rate, ataxia and multiple developmental defects of the cerebellum. All Nestin-Trio-/- mice died before reaching adulthood, which hinders research on Trio gene function in adult mice. Thus, we generated EMX1-Trio-/- mice by crossing Trio-floxed mice with EMX1-Cre mice in which Cre is expressed in the brain cortex and hippocampus. EMX1-Trio-/- mice can survive to adulthood. Trio gene deletion results in smaller brains, an abnormal hippocampus and disordered granule cells in the dentate gyrus (DG) and cornu ammonis (CA). Behavior tests showed that Trio deletion interfered with the hippocampal-dependent spatial learning in the mice, suggesting that Trio plays critical roles in the learning ability of adult mice. We conclude that the Trio gene regulates the neuronal development of the hippocampus and that it affects the intelligence of adult mice.

Decreased expression of elastin and lysyl oxidase family genes in urogenital tissues of aging mice.

AIM: This study aims to investigate the expression levels of elastin and lysyl oxidase (LOX) family members in the urogenital tissues of natural aging mice and accelerated ovarian aging mice. METHOD: Uteri, vaginas and bladders were harvested from 18-month-old female mice and accelerated ovarian aging mice developed by chemotherapeutic agents. Untreated 3-month-old female mice were used as controls. The expression levels of elastin and LOX family members were determined by real-time polymerase chain reaction and western blot. RESULTS: Compared with untreated young female mice, the expression of elastin and LOX family members significantly decreased both in natural aging mice and accelerated ovarian aging mice. CONCLUSION: Aging is a high-risk factor for pelvic floor disorders. The failure of elastic fiber synthesis and assembly due to the decline in expression levels of elastin and LOX family members during aging may explain the molecular mechanism causing pelvic floor disorders.