The Mediator CDK8-Cyclin C complex modulates Dpp signaling in Drosophila by stimulating Mad-dependent transcription.

Dysregulation of CDK8 (Cyclin-Dependent Kinase 8) and its regulatory partner CycC (Cyclin C), two subunits of the conserved Mediator (MED) complex, have been linked to diverse human diseases such as cancer. Thus, it is essential to understand the regulatory network modulating the CDK8-CycC complex in both normal development and tumorigenesis. To identify upstream regulators or downstream effectors of CDK8, we performed a dominant modifier genetic screen in Drosophila based on the defects in vein patterning caused by specific depletion or overexpression of CDK8 or CycC in developing wing imaginal discs. We identified 26 genomic loci whose haploinsufficiency can modify these CDK8- or CycC-specific phenotypes. Further analysis of two overlapping deficiency lines and mutant alleles led us to identify genetic interactions between the CDK8-CycC pair and the components of the Decapentaplegic (Dpp, the Drosophila homolog of TGFß, or Transforming Growth Factor-ß) signaling pathway. We observed that CDK8-CycC positively regulates transcription activated by Mad (Mothers against dpp), the primary transcription factor downstream of the Dpp/TGFß signaling pathway. CDK8 can directly interact with Mad in vitro through the linker region between the DNA-binding MH1 (Mad homology 1) domain and the carboxy terminal MH2 (Mad homology 2) transactivation domain. Besides CDK8 and CycC, further analyses of other subunits of the MED complex have revealed six additional subunits that are required for Mad-dependent transcription in the wing discs: Med12, Med13, Med15, Med23, Med24, and Med31. Furthermore, our analyses confirmed the positive roles of CDK9 and Yorkie in regulating Mad-dependent gene expression in vivo. These results suggest that CDK8 and CycC, together with a few other subunits of the MED complex, may coordinate with other transcription cofactors in regulating Mad-dependent transcription during wing development in Drosophila.

Cortical perfusion as assessed with contrast-enhanced ultrasound is lower in patients with chronic kidney disease than in healthy subjects but increases under low salt conditions.

BACKGROUND: Disturbances in renal microcirculation play an important role in the pathophysiology of chronic kidney disease (CKD), but the lack of easy accessible techniques hampers our understanding of the regulation of the renal microcirculation in humans. We assessed whether contrast-enhanced ultrasound (CEUS) can identify differences in cortical perfusion and alterations induced by different dietary salt intakes in CKD patients and controls. METHODS: Participants underwent CEUS twice: once after 5 days of high-salt (HS) intake, and again after 5 days of low salt (LS) diet. Sonovue® (0.015 mL/kg/min) was perfused as contrast agent and four consecutive destruction-reperfusion sequences were analysed per visit. The primary outcome measure was the (change in) mean perfusion index (PI) of the renal cortex. RESULTS: Forty healthy volunteers (mean age ± standard deviation 50 ± 8 years) and 18 CKD Stages 2-4 patients [aged 55 ± 11 years, estimated glomerular filtration rate (eGFR) 54 ± 28 mL/min/1.73 m2] were included and underwent CEUS without side effects. Under HS conditions, cortical PI was significantly lower in CKD patients [1618 ± 1352 versus 3176 ± 2278 arbitrary units (a.u) in controls, P = 0.034]. Under LS, renal PI increased in CKD patients (with +1098 to 2716 ± 1540 a.u., P = 0.048), whereas PI remained stable in controls. In the continuous analysis, PI correlated with eGFR (Spearman's r = 0.54, P = 0.005) but not with age, sex, blood pressure or aldosterone levels. CONCLUSIONS: CEUS identified important reductions in cortical micro-perfusion in patients with moderate CKD. Lowering salt intake increased perfusion in CKD patients, but not in controls, underlining the benefits of an LS diet in CKD patients. Whether a low PI is an early sign of kidney damage and predicts renal function decline needs further study.

Parallel roles of transcription factors dFOXO and FER2 in the development and maintenance of dopaminergic neurons.

Forkhead box (FOXO) proteins are evolutionarily conserved, stress-responsive transcription factors (TFs) that can promote or counteract cell death. Mutations in FOXO genes are implicated in numerous pathologies, including age-dependent neurodegenerative disorders, such as Parkinson's disease (PD). However, the complex regulation and downstream mechanisms of FOXOs present a challenge in understanding their roles in the pathogenesis of PD. Here, we investigate the involvement of FOXO in the death of dopaminergic (DA) neurons, the key pathological feature of PD, in Drosophila. We show that dFOXO null mutants exhibit a selective loss of DA neurons in the subgroup crucial for locomotion, the protocerebral anterior medial (PAM) cluster, during development as well as in adulthood. PAM neuron-targeted adult-restricted knockdown demonstrates that dFOXO in adult PAM neurons tissue-autonomously promotes neuronal survival during aging. We further show that dFOXO and the bHLH-TF 48-related-2 (FER2) act in parallel to protect PAM neurons from different forms of cellular stress. Remarkably, however, dFOXO and FER2 share common downstream processes leading to the regulation of autophagy and mitochondrial morphology. Thus, overexpression of one can rescue the loss of function of the other. These results indicate a role of dFOXO in neuroprotection and highlight the notion that multiple genetic and environmental factors interact to increase the risk of DA neuron degeneration and the development of PD.

An interaction screen identifies headcase as a regulator of large-scale pruning.

Large-scale pruning, the removal of long neuronal processes, is deployed widely within the developing nervous system and is essential for proper circuit formation. In Drosophila the dendrites of the class IV dendritic arborization sensory neuron ddaC undergo large-scale pruning by local degeneration controlled by the steroid hormone ecdysone. The molecular mechanisms that control such events are largely unknown. To identify new molecules that orchestrate this developmental degeneration, we performed a genetic interaction screen. Our approach combines the strength of Drosophila forward genetics with detailed in vivo imaging of ddaC neurons. This screen allowed us to identify headcase (hdc) as a new gene involved in dendrite pruning. hdc is evolutionarily conserved, but the protein's function is unknown. Here we show that hdc is expressed just before metamorphosis in sensory neurons that undergo remodeling. hdc is required in a cell-autonomous manner to control dendrite severing, the first phase of pruning. Our epistasis experiments with known regulators of dendrite pruning reveal hdc as a founding member of a new pathway downstream of ecdysone signaling.

Maintenance of mitochondrial integrity in midbrain dopaminergic neurons governed by a conserved developmental transcription factor.

Progressive degeneration of dopaminergic (DA) neurons in the substantia nigra is a hallmark of Parkinson's disease (PD). Dysregulation of developmental transcription factors is implicated in dopaminergic neurodegeneration, but the underlying molecular mechanisms remain largely unknown. Drosophila Fer2 is a prime example of a developmental transcription factor required for the birth and maintenance of midbrain DA neurons. Using an approach combining ChIP-seq, RNA-seq, and genetic epistasis experiments with PD-linked genes, here we demonstrate that Fer2 controls a transcriptional network to maintain mitochondrial structure and function, and thus confers dopaminergic neuroprotection against genetic and oxidative insults. We further show that conditional ablation of Nato3, a mouse homolog of Fer2, in differentiated DA neurons causes mitochondrial abnormalities and locomotor impairments in aged mice. Our results reveal the essential and conserved role of Fer2 homologs in the mitochondrial maintenance of midbrain DA neurons, opening new perspectives for modeling and treating PD.

Short-term changes in dietary sodium intake influence sweat sodium concentration and muscle sodium content in healthy individuals.

OBJECTIVE: There is increasing evidence that sodium can be stored in the skin and muscles without being osmotically active, yet whether acute changes in dietary sodium intake alter sweat and muscle sodium content has not been investigated previously. METHODS: In a cross-over design, we assessed muscle sodium content by Na-MRI in 38 healthy normotensive volunteers (aged 33.5 ±â€Š11.1 years, 76.3% women) after 5 days of high-sodium diet (6 g of salt added to their normal diet) and 5 days of a low-sodium diet. In a subgroup of 18 participants (72.2% women) we conducted quantitative pilocarpine iontophoretic sweat collections and measured the sodium concentration in sweat. Plasma aldosterone and plasma renin activity levels were measured in all participants. RESULTS: Under high-sodium diet conditions urinary sodium excretion, muscle sodium content and sweat sodium concentration all increased significantly. Muscle sodium content (rm = 0.47, P = 0.03) and sodium sweat concentration (rm = 0.72, P < 0.001) correlated positively with salt intake as estimated by 24-h urine sodium excretion. Age, sex or the phase of the menstrual cycle did not influence muscle or sweat sodium concentrations or their changes. In contrast, plasma aldosterone levels were negatively associated with both muscle sodium (rs = -0.42, P = 0.0001) and sweat sodium content (rs = -0.52, P = 0.002). Plasma renin activity correlated negatively with sweat sodium (rs = -0.43, P = 0.012) and muscle sodium levels (rs = -0.42, P < 0.001). CONCLUSION: Muscle and sweat sodium concentrations are significantly higher on a high-salt intake in healthy male and female individuals, suggesting that muscle and sweat play a role in regulating sodium balance in humans.

Acute and Chronic Effects of SGLT2 Inhibitor Empagliflozin on Renal Oxygenation and Blood Pressure Control in Nondiabetic Normotensive Subjects: A Randomized, Placebo-Controlled Trial.

Background The sodium/glucose cotransporter 2 inhibitor empagliflozin has cardiorenal protective properties through mechanisms beyond glucose control. In this study we assessed whether empagliflozin modifies renal oxygenation as a possible mechanism of renal protection, and determined the metabolic, renal, and hemodynamic effects of empagliflozin in nondiabetic subjects. Methods and Results In this double-blind, randomized, placebo-controlled study, 45 healthy volunteers underwent blood and urine sampling, renal ultrasound, and blood-oxygenation-level-dependent magnetic resonance imaging before and 180 minutes after administration of 10 mg empagliflozin (n=30) or placebo (n=15). These examinations were repeated after 1 month of daily intake. Cortical and medullary renal oxygenation were not affected by the acute or chronic administration of empagliflozin, as determined by 148 renal blood-oxygenation-level-dependent magnetic resonance imaging examinations. Empagliflozin increased glucosuria (24-hour glucosuria at 1 month: +50.1±16.3 g). The acute decrease in proximal sodium reabsorption, as determined by endogenous fractional excretion of lithium (-34.6% versus placebo), was compensated at 1 month by a rise in plasma renin activity (+28.6%) and aldosterone (+55.7%). The 24-hour systolic and diastolic ambulatory blood pressures decreased significantly after 1 month of empagliflozin administration (-5.1 and -2.0 mm Hg, respectively). Serum uric acid levels decreased (-28.4%), hemoglobin increased (+1.7%), and erythropoietin remained the same. Conclusions Empagliflozin has a rapid and significant effect on tubular function, with sustained glucosuria and transient natriuresis in nondiabetic normotensive subjects. These effects favor blood pressure reduction. No acute or sustained changes were found in renal cortical or medullary tissue oxygenation. It remains to be determined whether this is the case in nondiabetic or diabetic patients with congestive heart failure or kidney disease. REGISTRATION: URL: https://www.clini​caltr​ials.gov; Unique identifier: NCT03093103.

An ESCRT module is required for neuron pruning.

Neural circuits are refined by both functional and structural changes. Structural remodeling by large-scale pruning occurs where relatively long neuronal branches are cut away from their parent neuron and removed by local degeneration. Until now, the molecular mechanisms executing such branch severing events have remained poorly understood. Here, we reveal a role for the Endosomal Sorting Complex Required for Transport (ESCRT) machinery during neuronal remodeling. Our data show that a specific ESCRT pruning module, including members of the ESCRT-I and ESCRT-III complexes, but not ESCRT-0 or ESCRT-II, are required for the neurite scission event during pruning. Furthermore we show that this ESCRT module requires a direct, in vivo, interaction between Shrub/CHMP4B and the accessory protein Myopic/HD-PTP.

Distinct roles for Mediator Cdk8 module subunits in Drosophila development.

Mediator (MED) is a conserved multisubunit complex bridging transcriptional activators and repressors to the general RNA polymerase II initiation machinery. In yeast, MED is organized in three core modules and a separable 'Cdk8 module' consisting of the cyclin-dependent kinase Cdk8, its partner CycC, Med12 and Med13. This regulatory module, specifically required for cellular adaptation to environmental cues, is thought to act through the Cdk8 kinase activity. Here we have investigated the functions of the four Cdk8 module subunits in the metazoan model Drosophila. Physical interactions detected among the four fly subunits provide support for a structurally conserved Cdk8 module. We analyzed the in vivo functions of this module using null mutants for Cdk8, CycC, Med12 and Med13. Each gene is required for the viability of the organism but not of the cell. Cdk8-CycC and Med12-Med13 act as pairs, which share some functions but also have distinct roles in developmental gene regulation. These data reveal functional attributes of the Cdk8 module, apart from its regulated kinase activity, that may contribute to the diversification of genetic programs.