Role of TRPC3 in Right Ventricular Dilatation under Chronic Intermittent Hypoxia in 129/SvEv Mice.

Patients with obstructive sleep apnea (OSA) exhibit a high prevalence of pulmonary hypertension and right ventricular (RV) hypertrophy. However, the exact molecule responsible for the pathogenesis remains unknown. Given the resistance to RV dilation observed in transient receptor potential canonical 3(Trpc3)-/- mice during a pulmonary hypertension model induced by phenylephrine (PE), we hypothesized that TRPC3 also plays a role in chronic intermittent hypoxia (CIH) conditions, which lead to RV dilation and dysfunction. To test this, we established an OSA mouse model using 8- to 12-week-old 129/SvEv wild-type and Trpc3-/- mice in a customized breeding chamber that simulated sleep and oxygen cycles. Functional parameters of the RV were evaluated through analysis of cardiac cine magnetic resonance images, while histopathological examinations were conducted on cardiomyocytes and pulmonary vessels. Following exposure to 4 weeks of CIH, Trpc3-/- mice exhibited significant RV dysfunction, characterized by decreased ejection fraction, increased end-diastole RV wall thickness, and elevated expression of pathological cardiac markers. In addition, reactive oxygen species (ROS) signaling and the endothelin system were markedly increased solely in the hearts of CIH-exposed Trpc3-/- mice. Notably, no significant differences in pulmonary vessel thickness or the endothelin system were observed in the lungs of wild-type (WT) and Trpc3-/- mice subjected to 4 weeks of CIH. In conclusion, our findings suggest that TRPC3 serves as a regulator of RV resistance in response to pressure from the pulmonary vasculature, as evidenced by the high susceptibility to RV dilation in Trpc3-/- mice without notable changes in pulmonary vasculature under CIH conditions.

Functional and Phenotypic Characterisations of Common Syngeneic Tumour Cell Lines as Estrogen Receptor-Positive Breast Cancer Models.

Estrogen receptor-positive breast cancers (ER+ BCas) are the most common form of BCa and are increasing in incidence, largely due to changes in reproductive practices in recent decades. Tamoxifen is prescribed as a component of standard-of-care endocrine therapy for the treatment and prevention of ER+ BCa. However, it is poorly tolerated, leading to low uptake of the drug in the preventative setting. Alternative therapies and preventatives for ER+ BCa are needed but development is hampered due to a paucity of syngeneic ER+ preclinical mouse models that allow pre-clinical experimentation in immunocompetent mice. Two ER-positive models, J110 and SSM3, have been reported in addition to other tumour models occasionally shown to express ER (for example 4T1.2, 67NR, EO771, D2.0R and D2A1). Here, we have assessed ER expression and protein levels in seven mouse mammary tumour cell lines and their corresponding tumours, in addition to their cellular composition, tamoxifen sensitivity and molecular phenotype. By immunohistochemical assessment, SSM3 and, to a lesser extent, 67NR cells are ER+. Using flow cytometry and transcript expression we show that SSM3 cells are luminal in nature, whilst D2.0R and J110 cells are stromal/basal. The remainder are also stromal/basal in nature; displaying a stromal or basal Epcam/CD49f FACS phenotype and stromal and basal gene expression signatures are overrepresented in their transcript profile. Consistent with a luminal identity for SSM3 cells, they also show sensitivity to tamoxifen in vitro and in vivo. In conclusion, the data indicate that the SSM3 syngeneic cell line is the only definitively ER+ mouse mammary tumour cell line widely available for pre-clinical research.

Differential patterns of opioid and dopamine D1 receptor antagonism on nutritive and non-nutritive sweetener intakes in C57BL/6:129 hybrid mice relative to inbred C57BL/6 and 129 mice.

Opioid and dopamine (DA) D1 receptor antagonists differentially reduce nutritive and non-nutritive sweetener intakes in inbred mouse strains. Sucrose intake was more effectively reduced by naltrexone in C57BL/6 (B6) mice relative to 129P3 (129) mice, but more effectively reduced by SCH23390 in 129 mice relative to B6 mice. Opioid and DA D1 antagonists differentially reduced saccharin intakes in B6 mice relative to other strains. Given these differential patterns in sweetener intake in B6 and 129 mice, the present study examined whether systemic naltrexone (0.01-5 mg/kg) and SCH23390 (50-1600 nmol/kg) reduced intakes of 10 % sucrose or 0.2 % saccharin solutions over a 120 min time course in first-generation hybrid mice (B6:129) of B6 and 129 parents and reduced low-nutritive sweetener intakes in 129 mice. Naltrexone (5 mg/kg) significantly reduced 10 % sucrose intake in B6:129 hybrid mice more like that of 129 than B6 mice. In contrast, SCH23390 (400-1600 nmol/kg) reduced 10 % sucrose intake in B6:129 hybrid mice more effectively than that observed in B6 or 129 parental strains. Because 129 mice consumed relatively low amounts of 0.2 % saccharin, they were tested with a more attractive low-nutritive solution containing 0.2 % saccharin and 2 % sucrose. Naltrexone failed to reduce saccharin intake in B6:129 hybrid mice but suppressed saccharin+sucrose intake in 129 mice more like that observed in B6 mice. SCH23390 similarly inhibited saccharin or saccharin+sucrose intakes in hybrid B6:129, 129, and B6 mice with B6 mice more resistant to the lowest SCH23390 dose. Thus, whereas sucrose intake in B6:129 hybrid mice exhibited similar sensitivity to opioid and to a lesser degree DA D1 antagonism to their 129, but not B6 parents, opioid and DA D1 mediation of low- and non-nutritive sweet intake produced unique profiles among B6:129 hybrid and B6 and 129 strains which does not support a simple heritability explanation.

The Lack of Natural IgM Increases Susceptibility and Impairs Anti-Vi Polysaccharide IgG Responses in a Mouse Model of Typhoid.

Circulating IgM present in the body prior to any apparent Ag exposure is referred to as natural IgM. Natural IgM provides protective immunity against a variety of pathogens. Salmonella enterica serovar Typhi (S. Typhi) is the causative agent of typhoid fever in humans. Because mice are not permissive to S. Typhi infection, we employed a murine model of typhoid using S. enterica serovar Typhimurium expressing the Vi polysaccharide (ViPS) of S. Typhi (S. Typhimurium strain RC60) to evaluate the role of natural IgM in pathogenesis. We found that natural mouse IgM binds to S. Typhi and S. Typhimurium. The severity of S. Typhimurium infection in mice is dependent on presence of the natural resistance-associated macrophage protein 1 (Nramp1) allele; therefore, we infected mice deficient in secreted form of IgM (sIgM) on either a Nramp1-resistant (129S) or -susceptible (C57BL/6J) background. We found that the lack of natural IgM results in a significantly increased susceptibility and an exaggerated liver pathology regardless of the route of infection or the Nramp1 allele. Reconstitution of sIgM-/- mice with normal mouse serum or purified polyclonal IgM restored the resistance to that of sIgM+/+ mice. Furthermore, immunization of sIgM-/- mice with heat-killed S. Typhi induced a significantly reduced anti-ViPS IgG and complement-dependent bactericidal activity against S. Typhi in vitro, compared with that of sIgM+/+ mice. These findings indicate that natural IgM is an important factor in reducing the typhoid severity and inducing an optimal anti-ViPS IgG response to vaccination.

Differences in Diurnal Rhythm of Rod Outer Segment Renewal between 129T2/SvEmsJ and C57BL/6J Mice.

In all mammalian species tested to date, rod photoreceptor outer segment renewal is a circadian process synchronized by light with a burst of outer segment fragment (POS) shedding and POS phagocytosis by the adjacent retinal pigment epithelium (RPE) every morning at light onset. Recent reports show that RPE phagocytosis also increases shortly after dark onset in C57BL/6 (C57) mice. Genetic differences between C57 mice and 129T2/SvEmsJ (129) mice may affect regulation of outer segment renewal. Here, we used quantitative methods to directly compare outer segment renewal in C57 and 129 mouse retina. Quantification of rhodopsin-positive phagosomes in the RPE showed that in 129 mice, rod POS phagocytosis after light onset was significantly increased compared to C57 mice, but that 129 mice did not show a second peak after dark onset. Cone POS phagosome content of RPE cells did not differ by mouse strain with higher phagosome numbers after light than after dark. We further quantified externalization of the "eat me" signal phosphatidylserine by outer segment tips, which precedes POS phagocytosis. Live imaging of retina ex vivo showed that rod outer segments extended PS exposure in both strains but that frequency of outer segments with exposed PS after light onset was lower in C57 than in 129 retina. Taken together, 129 mice lacked a burst of rod outer segment renewal after dark onset. The increases in rod outer segment renewal after light and after dark onset in C57 mice were attenuated compared to the peak after light onset in 129 mice, suggesting an impairment in rhythmicity in C57 mice.

Dusp26 phosphatase regulates mitochondrial respiration and oxidative stress and protects neuronal cell death.

The dual specificity protein phosphatases (Dusps) control dephosphorylation of mitogen-activated protein kinases (MAPKs) as well as other substrates. Here, we report that Dusp26, which is highly expressed in neuroblastoma cells and primary neurons is targeted to the mitochondrial outer membrane via its NH2-terminal mitochondrial targeting sequence. Loss of Dusp26 has a significant impact on mitochondrial function that is associated with increased levels of reactive oxygen species (ROS), reduction in ATP generation, reduction in mitochondria motility and release of mitochondrial HtrA2 protease into the cytoplasm. The mitochondrial dysregulation in dusp26-deficient neuroblastoma cells leads to the inhibition of cell proliferation and cell death. In vivo, Dusp26 is highly expressed in neurons in different brain regions, including cortex and midbrain (MB). Ablation of Dusp26 in mouse model leads to dopaminergic (DA) neuronal cell loss in the substantia nigra par compacta (SNpc), inflammatory response in MB and striatum, and phenotypes that are normally associated with Neurodegenerative diseases. Consistent with the data from our mouse model, Dusp26 expressing cells are significantly reduced in the SNpc of Parkinson's Disease patients. The underlying mechanism of DA neuronal death is that loss of Dusp26 in neurons increases mitochondrial ROS and concurrent activation of MAPK/p38 signaling pathway and inflammatory response. Our results suggest that regulation of mitochondrial-associated protein phosphorylation is essential for the maintenance of mitochondrial homeostasis and dysregulation of this process may contribute to the initiation and development of neurodegenerative diseases.

Generation of mice for evaluating endogenous p16Ink4a protein expression.

The cyclin-dependent kinase inhibitor p16Ink4a plays a central role in cellular senescence in vitro. Although previous studies suggested cellular senescence is integrated in the systemic mechanisms of organismal aging, the localization and the dynamics of p16Ink4a in tissues remain poorly understood, which hinders uncovering the role of p16Ink4a under the in vivo context. One of the reasons is due to the lack of reliable reagents; as we also demonstrate here that commonly used antibodies raised against human p16INK4A barely recognize its murine ortholog. Here we generated a mouse model, in which the endogenous p16Ink4a is HA-tagged at its N-terminus, to explore the protein expression of p16Ink4a at the organismal level. p16Ink4a was induced at the protein level along the course of senescence in primary embryonic fibroblasts derived from the mice, consistently to its transcriptional level. Remarkably, however, p16Ink4a was not detected in the tissues of the mice exposed to pro-senescence conditions including genotoxic stress and activation of oncogenic signaling pathways, indicating that there is only subtle p16Ink4a proteins induced. These results in our mouse model highlight the need for caution in evaluating p16Ink4a protein expression in vivo.

Lack of ZnT8 protects pancreatic islets from hypoxia- and cytokine-induced cell death.

Pancreatic ß-cells depend on the well-balanced regulation of cytosolic zinc concentrations, providing sufficient zinc ions for the processing and storage of insulin, but avoiding toxic effects. The zinc transporter ZnT8, encoded by SLC30A8,is a key player regarding islet cell zinc homeostasis, and polymorphisms in this gene are associated with altered type 2 diabetes susceptibility in man. The objective of this study was to investigate the role of ZnT8 and zinc in situations of cellular stress as hypoxia or inflammation. Isolated islets of WT and global ZnT8-/- mice were exposed to hypoxia or cytokines and cell death was measured. To explore the role of changing intracellular Zn2+ concentrations, WT islets were exposed to different zinc concentrations using zinc chloride or the zinc chelator N,N,N',N'-tetrakis(2-pyridinylmethyl)-1,2-ethanediamine (TPEN). Hypoxia or cytokine (TNF-α, IFN-γ, IL1-ß) treatment induced islet cell death, but to a lesser extent in islets from ZnT8-/- mice, which were shown to have a reduced zinc content. Similarly, chelation of zinc with TPEN reduced cell death in WT islets treated with hypoxia or cytokines, whereas increased zinc concentrations aggravated the effects of these stressors. This study demonstrates a reduced rate of cell death in islets from ZnT8-/- mice as compared to WT islets when exposed to two distinct cellular stressors, hypoxia or cytotoxic cytokines. This protection from cell death is, in part, mediated by a reduced zinc content in islet cells of ZnT8-/- mice. These findings may be relevant for altered diabetes burden in carriers of risk SLC30A8 alleles in man.

Fine-tuning of MEK signaling is pivotal for limiting B and T cell activation.

MEK1 and MEK2, the only known activators of ERK, are attractive therapeutic candidates for both cancer and autoimmune diseases. However, how MEK signaling finely regulates immune cell activation is only partially understood. To address this question, we specifically delete Mek1 in hematopoietic cells in the Mek2 null background. Characterization of an allelic series of Mek mutants reveals the presence of distinct degrees of spontaneous B cell activation, which are inversely proportional to the levels of MEK proteins and ERK activation. While Mek1 and Mek2 null mutants have a normal lifespan, 1Mek1 and 1Mek2 mutants retaining only one functional Mek1 or Mek2 allele in hematopoietic cell lineages die from glomerulonephritis and lymphoproliferative disorders, respectively. This establishes that the fine-tuning of the ERK/MAPK pathway is critical to regulate B and T cell activation and function and that each MEK isoform plays distinct roles during lymphocyte activation and disease development.

Microtubule-associated protein tau in murine kidney: role in podocyte architecture.

Tau is a cytoskeletal protein that is expressed mainly in neurons and is involved in several cellular processes, such as microtubule stabilization, axonal maintenance, and transport. Altered tau metabolism is related to different tauopathies being the most important Alzheimer's disease where aberrant hyperphosphorylated and aggregated tau is found in the central nervous system. Here, we have analyzed that function in kidney by using tau knockout mice generated by integrating GFP-encoding cDNA into exon 1 of MAPT (here referred to as TauGFP/GFP). IVIS Lumina from PerkinElmer demonstrated GFP expression in the kidney. We then demonstrated by qPCR that the main tau isoform in the kidney is Tau4R. The GFP reporter allowed us to demonstrate that tau is found in the glomeruli of the renal cortex, and specifically in podocytes. This was further confirmed by immunohistochemistry. TauGFP/GFP mice present a podocyte cytoskeleton more dynamic as they contain higher levels of detyrosinated tubulin than wild-type mice. In addition, transmission electron microscopy studies demonstrated glomerular damage with a decrease in urinary creatinine. Our results prove that tau has an important role in kidney metabolism under normal physiological conditions.

Regulation of gene expression by the APP family in the adult cerebral cortex.

Amyloid precursor protein (APP) is associated with both familial and sporadic forms of Alzheimer's disease. APP has two homologs, amyloid precursor-like protein 1 and 2 (APLP1 and APLP2), and they have functional redundancy. APP intracellular c-terminal domain (AICD), produced by sequential α- or ß- and γ-secretase cleavages, is thought to control gene expression, similarly as the ICD of Notch. To investigate the role of APP family in transcriptional regulation, we examined gene expression changes in the cerebral cortex of APP/APLP1/APLP2 conditional triple knockout (cTKO) mice, in which APP family members are selectively inactivated in excitatory neurons of the postnatal forebrain. Of the 12 previously reported AICD target genes, only Nep and Npas4 mRNA levels were significantly reduced in the cerebral cortex of cTKO mice, compared to littermate controls. We further examined global transcriptional changes by RNA-seq and identified 189 and 274 differentially expressed genes in the neocortex and hippocampus, respectively, of cTKO mice relative to controls. Gene Ontology analysis indicated that these genes are involved in a variety of cellular functions, including extracellular organization, learning and memory, and ion channels. Thus, inactivation of APP family alters transcriptional profiles of the cerebral cortex and affects wide-ranging molecular pathways.

Long-term effects of pharmacological inhibition of anaplastic lymphoma kinase in neurofibromatosis 1 mutant mice.

Neurofibromatosis type 1 (NF1) is associated with behavioral alterations and cognitive impairments. There is a genetic interaction between NF1 and the receptor tyrosine kinase Alk. Short-term pharmacological Alk inhibition, with a compound FDA-approved for cancer starting 10 days prior to cognitive testing, was shown to improve cognitive performance of NF1 heterozygous (HET) mice. However, effects of long-term Alk inhibition on behavioral cognitive performance are not known. Therefore, in the study described below we determine the effects of prolonged pharmacological Alk inhibition for 24 weeks on behavioral and cognitive performance of NF1 HET mice. As these studies have the ultimate objective of developing a treatment for humans with neurofibromatosis and acceptable side effects in the context of cancer are not acceptable in the context of long-term treatment of patients with neurofibromatosis, we included additional behavioral tests of anxiety-like and depressive-like behaviors as well. Long-term effects of Alk inhibition had genotype-dependent effects, consistent with a specific interaction between Alk and NF1. Beneficial effects of long-term Alk inhibition in NF1 HET mice included rescue of impairments in object recognition in NF1 HET males and females, and improved cognitive performance of NF1 HET males and females in the water maze test. In contrast, long-term Alk inhibition had detrimental effects in WT mice not seen after short-term treatments. As longer treatments are translationally more relevant for NF1 patients, these data highlight the important to assess long-term effects of drugs, especially of repurposed drugs used originally as part of cancer therapy.

IL-1 receptor signaling in podocytes limits susceptibility to glomerular damage.

Interleukin (IL)-1 receptor type 1 (IL-1R1) activation triggers a proinflammatory signaling cascade that can exacerbate kidney injury. However, the functions of podocyte IL-1R1 in glomerular disease remain unclear. To study the role of IL-1R1 signaling in podocytes, we selectively ablated podocyte IL-1R1 in mice (PKO mice). We then subjected PKO mice and wild-type controls to two glomerular injury models: nephrotoxic serum (NTS)- and adriamycin-induced nephropathy. Surprisingly, we found that IL-1R1 activation in podocytes limited albuminuria and podocyte injury during NTS- and adriamycin-induced nephropathy. Moreover, deletion of IL-1R1 in podocytes drove podocyte apoptosis and glomerular injury through diminishing Akt activation. Activation of Akt signaling abrogated the differences in albuminuria and podocyte injury between wild-type and PKO mice during NTS. Thus, IL-1R1 signaling in podocytes limits susceptibility to glomerular injury via an Akt-dependent signaling pathway. These data identify an unexpected protective role for IL-1R1 signaling in podocytes in the pathogenesis of glomerular disease.NEW & NOTEWORTHY The present study establishes that activation of the receptor for interleukin-1 limits susceptibility to damage to the kidney glomerulus in preclinical mouse models by stimulating Akt signaling cascades inside the podocyte.

Transient receptor potential cation channel 6 contributes to kidney injury induced by diabetes and hypertension.

Diabetes mellitus (DM) and hypertension (HTN) are major risk factors for chronic kidney injury, together accounting for >70% of end-stage renal disease. In this study, we assessed whether DM and HTN interact synergistically to promote kidney dysfunction and whether transient receptor potential cation channel 6 (TRPC6) contributes to this synergism. In wild-type (WT; B6/129s background) and TRPC6 knockout (KO) mice, DM was induced by streptozotocin injection to increase fasting glucose levels to 250-350 mg/dL. HTN was induced by aorta constriction (AC) between the renal arteries. AC increased blood pressure (BP) by ∼25 mmHg in the right kidney (above AC), whereas BP in the left kidney (below AC) returned to near normal after 8 wk, with both kidneys exposed to the same levels of blood glucose, circulating hormones, and neural influences. Kidneys of WT mice exposed to DM or HTN alone had only mild glomerular injury and urinary albumin excretion. In contrast, WT kidneys exposed to DM plus HTN (WT-DM + AC mice) for 8 wk had much greater increases in albumin excretion and histological injury. Marked increased apoptosis was also observed in the right kidneys of WT-DM + AC mice. In contrast, in TRPC6 KO mice with DM + AC, right kidneys exposed to the same levels of high BP and high glucose had lower albumin excretion and less glomerular damage and apoptotic cell injury compared with right kidneys of WT-DM + AC mice. Our results suggest that TRPC6 may contribute to the interaction of DM and HTN to promote kidney dysfunction and apoptotic cell injury.NEW & NOTEWORTHY A major new finding of this study is that the combination of moderate diabetes and hypertension promoted marked renal dysfunction, albuminuria, and apoptotic cell injury, and that these effects were greatly ameliorated by transient receptor potential cation channel 6 deficiency. These results suggest that transient receptor potential cation channel 6 may play an important role in contributing to the interaction of diabetes and hypertension to promote kidney injury.

Functional Analysis Identifies Damaging CHEK2 Missense Variants Associated with Increased Cancer Risk.

Heterozygous carriers of germline loss-of-function variants in the tumor suppressor gene checkpoint kinase 2 (CHEK2) are at an increased risk for developing breast and other cancers. While truncating variants in CHEK2 are known to be pathogenic, the interpretation of missense variants of uncertain significance (VUS) is challenging. Consequently, many VUS remain unclassified both functionally and clinically. Here we describe a mouse embryonic stem (mES) cell-based system to quantitatively determine the functional impact of 50 missense VUS in human CHEK2. By assessing the activity of human CHK2 to phosphorylate one of its main targets, Kap1, in Chek2 knockout mES cells, 31 missense VUS in CHEK2 were found to impair protein function to a similar extent as truncating variants, while 9 CHEK2 missense VUS resulted in intermediate functional defects. Mechanistically, most VUS impaired CHK2 kinase function by causing protein instability or by impairing activation through (auto)phosphorylation. Quantitative results showed that the degree of CHK2 kinase dysfunction correlates with an increased risk for breast cancer. Both damaging CHEK2 variants as a group [OR 2.23; 95% confidence interval (CI), 1.62-3.07; P < 0.0001] and intermediate variants (OR 1.63; 95% CI, 1.21-2.20; P = 0.0014) were associated with an increased breast cancer risk, while functional variants did not show this association (OR 1.13; 95% CI, 0.87-1.46; P = 0.378). Finally, a damaging VUS in CHEK2, c.486A>G/p.D162G, was also identified, which cosegregated with familial prostate cancer. Altogether, these functional assays efficiently and reliably identified VUS in CHEK2 that associate with cancer. SIGNIFICANCE: Quantitative assessment of the functional consequences of CHEK2 variants of uncertain significance identifies damaging variants associated with increased cancer risk, which may aid in the clinical management of patients and carriers.

HDAC2 Facilitates Pancreatic Cancer Metastasis.

The mortality of patients with pancreatic ductal adenocarcinoma (PDAC) is strongly associated with metastasis, a multistep process that is incompletely understood in this disease. Although genetic drivers of PDAC metastasis have not been defined, transcriptional and epigenetic rewiring can contribute to the metastatic process. The epigenetic eraser histone deacetylase 2 (HDAC2) has been connected to less differentiated PDAC, but the function of HDAC2 in PDAC has not been comprehensively evaluated. Using genetically defined models, we show that HDAC2 is a cellular fitness factor that controls cell cycle in vitro and metastasis in vivo, particularly in undifferentiated, mesenchymal PDAC cells. Unbiased expression profiling detected a core set of HDAC2-regulated genes. HDAC2 controlled expression of several prosurvival receptor tyrosine kinases connected to mesenchymal PDAC, including PDGFRα, PDGFRß, and EGFR. The HDAC2-maintained program disabled the tumor-suppressive arm of the TGFß pathway, explaining impaired metastasis formation of HDAC2-deficient PDAC. These data identify HDAC2 as a tractable player in the PDAC metastatic cascade. The complexity of the function of epigenetic regulators like HDAC2 implicates that an increased understanding of these proteins is needed for implementation of effective epigenetic therapies. SIGNIFICANCE: HDAC2 has a context-specific role in undifferentiated PDAC and the capacity to disseminate systemically, implicating HDAC2 as targetable protein to prevent metastasis.

Metformin improves relevant disease parameters in an autosomal dominant polycystic kidney disease mouse model.

Autosomal dominant polycystic kidney disease (ADPKD), caused by mutations in the polycystin 1 (PKD1) or polycystin 2 genes, presents with progressive development of kidney cysts and eventual end-stage kidney disease with limited treatment options. Previous work has shown that metformin reduces cyst growth in rapid ADPKD mouse models via inhibition of cystic fibrosis transmembrane conductance regulator-mediated fluid secretion, mammalian target of rapamycin, and cAMP pathways. The present study importantly tested the effectiveness of metformin as a therapy for ADPKD in a more clinically relevant Pkd1RC/RC mouse model, homozygous for the R3277C knockin point mutation in the Pkd1 gene. This mutation causes ADPKD in humans. Pkd1RC/RC male and female mice, which have a slow progression to end-stage kidney disease, received metformin (300 mg/kg/day in drinking water vs. water alone) from 3 to 9 or 12 mo of age. As previously reported, Pkd1RC/RC females had a more severe disease phenotype as compared with males. Metformin treatment reduced the ratio of total kidney weight-to-body weight relative to age-matched and sex-matched untreated controls at both 9 and 12 mo and reduced the cystic index in females at 9 mo. Metformin also increased glomerular filtration rate, lowered systolic blood pressure, improved anemia, and lowered blood urea nitrogen levels relative to controls in both sexes. Moreover, metformin reduced gene expression of key inflammatory markers and both gene and protein expression of kidney injury marker-1 and cyclin-dependent kinase-1 versus untreated controls. Altogether, these findings suggest several beneficial effects of metformin in this highly relevant slowly progressive ADPKD mouse model, which may help inform new ADPKD therapies in patients.NEW & NOTEWORTHY Metformin treatment improved ADPKD disease severity in a relevant, slowly progressive ADPKD mouse model that recapitulates a PKD-associated PKD1 mutation. Relative to controls, metformin reduced kidney weight/body weight, cystic index and BUN levels, while improving GFR, blood pressure and anemia. Metformin also reduced key inflammatory and injury markers, along with cell proliferation markers. These findings suggest several beneficial effects of metformin in this ADPKD mouse model, which may help inform new ADPKD therapies in patients.

Mitochondrial bioenergetics of astrocytes in Fragile X syndrome: new perspectives on culture conditions and sex effects.

Fragile X syndrome (FXS) is a genetic disorder that is characterized by a range of cognitive and behavioral deficits, including mild-moderate intellectual disability. The disease is characterized by an X-linked mutation of the Fmr1 gene, which causes silencing of the gene coding for fragile X mental retardation protein (FMRP), a translational regulator integral for neurodevelopment. Mitochondrial dysfunction has been recently associated with FXS, with reports of increases in oxidative stress markers, reactive oxygen species, and lipid peroxidation being present in the brain tissue. Astrocytes, a prominent glial cell within the central nervous system (CNS), play a large role in regulating oxidative homeostasis within the developing brain and dysregulation of astrocyte redox balance in FXS, which may contribute to oxidative stress. Astrocyte function and mitochondrial bioenergetics are significantly influenced by oxygen availability and circulating sex hormones; yet, these parameters are rarely considered during in vitro experimentation. Given that the brain normally develops in a range of hypoxic conditions and FXS is a sex-linked genetic disorder, we investigated how different oxygen levels (normoxic vs. hypoxic) and biological sex affected mitochondrial bioenergetics of astrocytes in FXS. Our results demonstrate that both mitochondrial respiration capacity and reactive oxygen species emission are altered with Fmr1 deletion in astrocytes and these changes were dependent upon both sexual dimorphism and oxygen availability.

The endocannabinoid system impacts seizures in a mouse model of Dravet syndrome.

Dravet syndrome is a catastrophic childhood epilepsy with multiple seizure types that are refractory to treatment. The endocannabinoid system regulates neuronal excitability so a deficit in endocannabinoid signaling could lead to hyperexcitability and seizures. Thus, we sought to determine whether a deficiency in the endocannabinoid system might contribute to seizure phenotypes in a mouse model of Dravet syndrome and whether enhancing endocannabinoid tone is anticonvulsant. Scn1a+/- mice model the clinical features of Dravet syndrome: hyperthermia-induced seizures, spontaneous seizures and reduced survival. We examined whether Scn1a+/- mice exhibit deficits in the endocannabinoid system by measuring brain cannabinoid receptor expression and endocannabinoid concentrations. Next, we determined whether pharmacologically enhanced endocannabinoid tone was anticonvulsant in Scn1a+/- mice. We used GAT229, a positive allosteric modulator of the cannabinoid (CB1) receptor, and ABX-1431, a compound that inhibits the degradation of the endocannabinoid 2-arachidonoylglycerol (2-AG). The Scn1a+/- phenotype is strain-dependent with mice on a 129S6/SvEvTac (129) genetic background having no overt phenotype and those on an F1 (129S6/SvEvTac x C57BL/6J) background exhibiting a severe epilepsy phenotype. We observed lower brain cannabinoid CB1 receptor expression in the seizure-susceptible F1 compared to seizure-resistant 129 strain, suggesting an endocannabinoid deficiency might contribute to seizure susceptibility. GAT229 and ABX-1431 were anticonvulsant against hyperthermia-induced seizures. However, subchronic ABX1431 treatment increased spontaneous seizure frequency despite reducing seizure severity. Cnr1 is a putative genetic modifier of epilepsy in the Scn1a+/- mouse model of Dravet syndrome. Compounds that increase endocannabinoid tone could be developed as novel treatments for Dravet syndrome.

MG53 E3 Ligase-Dead Mutant Protects Diabetic Hearts From Acute Ischemic/Reperfusion Injury and Ameliorates Diet-Induced Cardiometabolic Damage.

Cardiometabolic diseases, including diabetes and its cardiovascular complications, are the global leading causes of death, highlighting a major unmet medical need. Over the past decade, mitsugumin 53 (MG53), also called TRIM72, has emerged as a powerful agent for myocardial membrane repair and cardioprotection, but its therapeutic value is complicated by its E3 ligase activity, which mediates metabolic disorders. Here, we show that an E3 ligase-dead mutant, MG53-C14A, retains its cardioprotective function without causing metabolic adverse effects. When administered in normal animals, both the recombinant human wild-type MG53 protein (rhMG53-WT) and its E3 ligase-dead mutant (rhMG53-C14A) protected the heart equally from myocardial infarction and ischemia/reperfusion (I/R) injury. However, in diabetic db/db mice, rhMG53-WT treatment markedly aggravated hyperglycemia, cardiac I/R injury, and mortality, whereas acute and chronic treatment with rhMG53-C14A still effectively ameliorated I/R-induced myocardial injury and mortality or diabetic cardiomyopathy, respectively, without metabolic adverse effects. Furthermore, knock-in of MG53-C14A protected the mice from high-fat diet-induced metabolic disorders and cardiac damage. Thus, the E3 ligase-dead mutant MG53-C14A not only protects the heart from acute myocardial injury but also counteracts metabolic stress, providing a potentially important therapy for the treatment of acute myocardial injury in metabolic disorders, including diabetes and obesity.