Cell biology
Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in PKD1 or PKD2, which encode polycystin-1 (PC1) and polycystin-2 (PC2) respectively. These proteins are thought to form a signaling complex that can flux cations including calcium. One of the earliest symptoms in ADPKD is a decline in the concentrating ability of the kidneys, occurring prior to cyst formation. We reasoned that hyperosmolality stimulates the polycystin complex, and that the loss of this function impairs water reabsorption. We found that hyperosmolality resulted in the phosphorylation of a microtubule associated protein 4 (MAP4) in a PC1-dependent manner which then elicited ER-localized PC2 calcium signals. ER-localized PC2 hyperosmotic calcium signals were required for trafficking of the water channel aquaporin (AQP2). Pre-cystic PC1-KO and PC2-KO murine kidneys had cytosolic localized AQP2, and diluted urine compared to their respective controls. Kidney tissue sections from ADPKD patients showed decreased AQP2 apical membrane localization in cystic and non-cystic tubules. Our study demonstrates that osmolality is a physiological stimulus of the polycystin complex, and loss of polycystin osmosensing results in impaired water reabsorption via AQP2. This likely contributes to the declined concentrating ability of the kidneys and high circulating vasopressin levels in ADPKD patients.
Authors
Karla M. Márquez-Nogueras, Ryne M. Knutila, Virdjinija Vuchkovska, Charlie Yang, Patricia Outeda, Darren P. Wallace, Ivana Y. Kuo
Abstract
Autophagy is a recycling pathway in which damaged proteins, protein aggregates, and organelles are delivered to lysosomes for degradation. Autophagy insufficiency is thought to contribute to osteoporosis. Accordingly, autophagy elimination from the osteoblast lineage reduces bone formation and bone mass. However, whether increasing autophagy would benefit bone health is unknown. Here, we increased expression of endogenous transcription factor EB gene (Tfeb) in osteoblast lineage cells in vivo via CRISPR activation (TfebCRa mice). Elevated Tfeb stimulated autophagy and lysosomal biogenesis in osteoblasts. TfebCRa mice displayed a robust increase in femoral and vertebral cortical thickness at 4.5 months of age. Increases in cortical thickness was due to increased periosteal bone formation. Tfeb elevation also increased femoral trabecular bone volume. These changes increased bone strength of TfebCRa mice. Female TfebCRa mice displayed a progressive increase in bone mass and at 12 months of age had high cortical thickness and trabecular bone volume. Increased vertebral trabecular bone volume was due to elevated bone formation. Osteoblastic cultures showed that Tfeb elevation increased proliferation and mineral deposition. Overall, these results demonstrate TFEB-driven stimulation of autophagy in osteoblast lineage cells is associated with increased bone formation and strength and may represent an effective approach to combat osteoporosis.
Authors
Alicen James, James A. Hendrixson, Ilham Kadhim, Adriana Marques-Carvalho, Jacob Laster, Julie Crawford, Jeff Thostenson, Visanu Wanchai, Amy Y. Sato, Intawat Nookaew, Jinhu Xiong, Maria Almeida, Melda Onal
Abstract
Secondary lymphedema is characterized by fibrosis and impaired lymphatic function. Although TGF-β is a key regulator of fibrosis in this disease, the cellular mechanisms regulating this process remain unknown. Epithelial–mesenchymal transition (EMT), a mechanism by which TGF-β induces fibrosis in other skin diseases, is characterized by loss of epithelial cell markers and cellular polarity, upregulation of fibrotic gene expression, and gain of migratory capacity. Using clinical lymphedema biopsy specimens and animal models, we show that keratinocytes in the basal layer of the epidermis undergo EMT in lymphedematous skin, migrate into the dermis, and contribute to dermal fibrosis. In vitro studies using cultured primary human keratinocytes treated with lymphatic fluid from the affected limbs of patients with secondary lymphedema resulted in a TGF-β–mediated increased expression of EMT markers. We show for the first time that EMT is activated by TGF-β in secondary lymphedema and that this process plays an important role in regulating skin fibrosis in this disease.
Authors
Hyeung Ju Park, Jinyeon Shin, Ananta Sarker, Mark G. Klang, Elyn Riedel, Michelle Coriddi, Joseph H. Dayan, Sarit Pal, Babak J. Mehrara, Raghu P. Kataru
Abstract
Heterozygosity for missense mutations in one of 3 seemingly redundant calmodulin (CALM)-encoding genes can cause life-threatening arrhythmias, suggesting that small fractions of mutant CALM protein suffice to cause a severe phenotype. However, the exact molar ratios of wildtype to mutant CALM protein in calmodulinopathy hearts remain unknown. The aim of the present study was to quantitate mutant versus wildtype CALM transcript and protein levels in hearts of knock-in mice harboring the p.N98S mutation in the Calm1 gene. We found that the transcripts from the mutant Calm1 allele were the least abundantly expressed Calm transcripts in both hetero- and homozygous mutant hearts, while mutant hearts accumulate high levels of N98S-CALM protein in a Calm1N98S allele dosage-dependent manner, exceeding those of wildtype CALM protein. We further show that the severity of the electrophysiological phenotype incrementally increases with the graded increase in the mutant-to-wildtype CALM protein expression ratio seen in homozygous versus heterozygous mutant mice. We finally show a decrease in N98S-CALM protein degradation, suggesting that mutant CALM stabilization contributed to its enrichment in the heart. Our results support what we believe to be a novel mechanism by which a mutation in a single Calm gene can give rise to a severe phenotype.
Authors
Wen-Chin Tsai, Chiu-Fen Yang, Shu-Yu Lin, Suh-Yuen Liang, Wei-Chung Tsai, Shuai Guo, Xiaochun Li, Susan Ofner, Kai-Chien Yang, Tzu-Ching Meng, Peng-Sheng Chen, Michael Rubart
Abstract
More than one third of patients with glioblastoma experience tumour progression during adjuvant therapy. In this study, we performed a high-throughput drug repurposing screen of FDA-approved agents capable of crossing the blood-brain barrier that to find agents to counteract acquired or inherent glioma cell resistance to temozolomide-associated cytotoxicity. We identified the cholesterol processing inhibitor, lomitapide, as a potential chemosensitizer in glioblastoma. In vitro treatment of temozolomide-resistant glioblastoma cells with lomitapide resulted in decreased intracellular ubiquinone levels and sensitized cells to temozolomide-induced ferroptosis. Concomitant treatment with lomitapide and temozolomide (TMZ) prolonged survival and delayed tumour recurrence in a mouse glioblastoma model, compared to treatment with TMZ alone. Our data identified lomitapide as a potential adjunct for treatment of temozolomide-resistant glioblastoma.
Authors
Alyona Ivanova, Taylor M. Wilson, Kimia Ghannad-Zadeh, Esmond Tse, Robert Flick, Megan Wu, Sunit Das
N6-methyladenosine (m6A) dysregulation contributes to network excitability in temporal lobe epilepsy
Abstract
Analogous to DNA methylation and protein phosphorylation, it is now well understood that RNA is also subject to extensive processing and modification. N6-methyladenosine (m6A) is the most abundant internal RNA modification and regulates RNA fate in several ways, including stability and translational efficiency. The role of m6A in both experimental and human epilepsy remains unknown. Here, we used transcriptome-wide m6A arrays to obtain a detailed analysis of the hippocampal m6A-ome from both mouse and human epilepsy samples. We combined this with human proteomic analyses and show that epileptic tissue displays disrupted metabolic and autophagic pathways that may be directly linked to m6A processing. Specifically, our results suggest that m6A levels inversely correlate with protein pathway activation. Finally, we show that elevated levels of m6A decrease seizure susceptibility and severity in mice. Together, our findings indicate that m6A represents an additional layer of gene regulation complexity in epilepsy and may contribute to the pathomechanisms that drive the development and maintenance of hyperexcitable brain networks.
Authors
Justine Mathoux, Marc-Michel Wilson, Sujithra Srinivas, Gabrielle Litovskich, Leticia Villalba Benito, Cindy Tran, Jaideep Kesavan, Aileen Harnett, Theresa Auer, Amaya Sanz-Rodriguez, Mohammad Kh. A.E. Alkhayyat, Mairéad Sullivan, Zining Liu, Yifan Huang, Austin Lacey, Norman Delanty, Jane Cryan, Francesca M. Brett, Michael A. Farrell, Donncha F. O’Brien, Pablo M. Casillas-Espinosa, Eva M. Jimenez-Mateos, Jeffrey C. Glennon, Mary Canavan, David C. Henshall, Gary P. Brennan
Chronic integrated stress response causes dysregulated cholesterol synthesis in white matter disease
Abstract
Maladaptive integrated stress response (ISR) activation is observed in human diseases of the brain. Genetic mutations of eIF2B, a critical mediator of protein synthesis, cause chronic pathway activation resulting in a leukodystrophy but the precise mechanism is unknown. We generated N208Y eIF2Bα mice and found that this metabolite binding mutation leads to destabilization of eIF2Bα, a systemic ISR, and neonatal lethality. 2BAct, an eIF2B activator, rescued lethality and significantly extended the lifespan of this severe model, underscoring its therapeutic potential in pediatric disease. Continuous treatment was required for survival, as withdrawal led to ISR induction in all tissues and rapid deterioration, thereby providing a model to assess the impact of the ISR in vivo by tuning drug availability. Single nuclei RNA-sequencing of the CNS identified astrocytes, oligodendrocytes, and ependymal cells as the cell types most susceptible to eIF2B dysfunction and revealed dysfunctional maturation of oligodendrocytes. Moreover, ISR activation decreased cholesterol biosynthesis, a process critical for myelin formation and maintenance. As such, persistent ISR engagement may contribute to pathology in other demyelinating diseases.
Authors
Karin Lin, Nina Ly, Rejani B. Kunjamma, Ngoc Vu, Bryan King, Holly M. Robb, Eric G. Mohler, Janani Sridar, Qi Hao, José Zavala-Solorio, Chunlian Zhang, Varahram Shahryari, Nick van Bruggen, Caitlin F. Connelly, Bryson D. Bennett, James J. Lee, Carmela Sidrauski
Abstract
Autophagy is a catabolic quality control pathway that has been linked to neurodegenerative disease, atherosclerosis and ageing, and can be modified by nutrient availability in preclinical models. Consequently, there is immense public interest in stimulating autophagy in people. However, progress has been hampered by the lack of techniques to measure human autophagy. As a result, several key concepts in the field, including nutritional modulation of autophagy, have yet to be validated in humans. We conducted a single arm pre-post study in 42 healthy individuals, to assess whether an acute nutritional intervention could modify autophagy in humans. Two blood samples were collected per participant: after a 12 h overnight fast and 1 h post-consumption of a high protein meal. Autophagy turnover was assessed using a physiologically relevant measure of autophagic flux in peripheral blood mononuclear cells. A lysosomal inhibitor was added directly to whole blood, with the resulting build-up of autophagy marker LC3B-II designated as flux, and measured quantitatively via ELISA. Notably, consumption of a high protein meal had no impact on autophagy, with no differences between overnight fasting and postprandial autophagic flux. We observed sexual dimorphism in autophagy, with females having higher autophagic flux compared to males (p = 0.0031). Exploratory analyses revealed sex-specific correlations between autophagy, insulin and glucose signalling. Importantly, our findings show that an acute nutritional intervention (overnight fasting followed by consumption of a protein-rich meal) does not change autophagic flux in humans, highlighting the need to conduct further autophagy studies in humans.
Authors
Sanjna Singh, Célia Fourrier, Kathryn J. Hattersley, Leanne K. Hein, Jemima Gore, Alexis Martin, Linh V.P. Dang, Barbara King, Rachael A. Protzman, Paul J. Trim, Leonie K. Heilbronn, Julien Bensalem, Timothy J. Sargeant
Abstract
Regulatory T cells (Tregs) are known to play critical roles in tissue repair via provision of growth factors, such as amphiregulin (Areg). Areg-producing Tregs have previously been difficult to study because of an inability to isolate live Areg-producing cells. In this report, we created a reporter mouse to detect Areg expression in live cells (AregThy1.1). We employed influenza A and bleomycin models of lung damage to sort Areg-producing and non-Areg-producing Tregs for transcriptomic analyses. Single-cell RNA-Seq revealed distinct subpopulations of Tregs and allowed transcriptomic comparisons of damage-induced populations. Single-cell TCR sequencing showed that Treg clonal expansion was biased toward Areg-producing Tregs and largely occurred within damage-induced subgroups. Gene module analysis revealed functional divergence of Tregs into immunosuppression-oriented and tissue repair–oriented groups, leading to identification of candidate receptors for induction of repair activity in Tregs. We tested these using an ex vivo assay for Treg-mediated tissue repair, identifying 4-1BB agonism as a mechanism for reparative activity induction. Overall, we demonstrate that the AregThy1.1 mouse is a promising tool for investigating tissue repair activity in leukocytes.
Authors
Lucas F. Loffredo, Katherine A. Kaiser, Adam Kornberg, Samhita Rao, Kenia de los Santos-Alexis, Arnold Han, Nicholas Arpaia
Abstract
Asthma is characterized by exacerbated response to triggers such as allergen. While pulmonary neuroendocrine cells (PNECs), a rare population of airway epithelial cells, are essential for amplifying allergen-induced asthma response, how PNECs are regulated to achieve this role remains poorly understood. Here we show that in the adult mouse airway, inactivation of achaete-scute-like protein 1 gene in PNECs led to loss of these cells. Intriguingly, exposure of these mutants to house dust mites (HDM), a common allergen, led to reappearance of PNECs. Similarly, exposure of wild-type mice to HDM led to PNEC hyperplasia, a result of proliferation of existing PNECs and transdifferentiation from club cells. Single-cell RNA-Seq experiments revealed PNEC heterogeneity, including the emergence of an allergen-induced PNEC subtype. Notch signaling was downregulated in HDM-treated airway, and treatment with Notch agonist prevented PNEC hyperplasia. These findings together suggest that HDM-induced PNEC hyperplasia may contribute to exacerbated asthma response.
Authors
Estelle Kim, Brian K. Wells, Hannah Indralingam, Yujuan Su, Jamie Verheyden, Xin Sun
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