Role of ClC-5 in Renal Endocytosis Is Unique among ClC Exchangers and Does Not Require PY-motif-dependent Ubiquitylation

Inactivation of the mainly endosomal 2Cl⁻/H⁺-exchanger ClC-5 severely impairs endocytosis in renal proximal tubules and underlies the human kidney stone disorder Dent''s disease. In heterologous expression systems, interaction of the E3 ubiquitin ligases WWP2 and Nedd4-2 with a “PY-motif” in the cytoplasmic C terminus of ClC-5 stimulates its internalization from the plasma membrane and may influence receptor-mediated endocytosis. We asked whether this interaction is relevant in vivo and generated mice in which the PY-motif was destroyed by a point mutation. Unlike ClC-5 knock-out mice, these knock-in mice displayed neither low molecular weight proteinuria nor hyperphosphaturia, and both receptor-mediated and fluid-phase endocytosis were normal. The abundances and localizations of the endocytic receptor megalin and of the Na⁺-coupled phosphate transporter NaPi-2a (Npt2) were not changed, either. To explore whether the discrepancy in results from heterologous expression studies might be due to heteromerization of ClC-5 with ClC-3 or ClC-4 in vivo, we studied knock-in mice additionally deleted for those related transporters. Disruption of neither ClC-3 nor ClC-4 led to proteinuria or impaired proximal tubular endocytosis by itself, nor in combination with the PY-mutant of ClC-5. Endocytosis of cells lacking ClC-5 was not impaired further when ClC-3 or ClC-4 was additionally deleted. We conclude that ClC-5 is unique among CLC proteins in being crucial for proximal tubular endocytosis and that PY-motif-dependent ubiquitylation of ClC-5 is dispensable for this role.     

In Vitro Functional Analyses of Arrhythmogenic Right Ventricular Cardiomyopathy-Associated Desmoglein-2-Missense Variations

Background

Although numerous sequence variants in desmoglein-2 (DSG2) have been associated with arrhythmogenic right ventricular cardiomyopathy (ARVC), the functional impact of new sequence variations is difficult to estimate.

Methodology/Principal Findings

To test the functional consequences of DSG2-variants, we established an expression system for the extracellular domain and the full-length DSG2 using the human cell line HT1080. We established new tools to investigate ARVC-associated DSG2 variations and compared wild-type proteins and proteins with one of the five selected variations (DSG2-p.R46Q, -p.D154E, -p.D187G, -p.K294E, -p.V392I) with respect to prodomain cleavage, adhesion properties and cellular localisation.

Conclusions/Significance

The ARVC-associated DSG2-p.R46Q variation was predicted to be probably damaging by bioinformatics tools and to concern a conserved proprotein convertase cleavage site. In this study an impaired prodomain cleavage and an influence on the DSG2-properties could be demonstrated for the R46Q-variant leading to the classification of the variant as a potential gain-of-function mutant. In contrast, the variants DSG2-p.K294E and -p.V392I, which have an arguable impact on ARVC pathogenesis and are predicted to be benign, did not show functional differences to the wild-type protein in our study. Notably, the variants DSG2-p.D154E and -p.D187G, which were predicted to be damaging by bioinformatics tools, had no detectable effects on the DSG2 protein properties in our study.     

Stallion spermatozoa selected by single layer centrifugation are capable of fertilization after storage for up to 96 h at 6°C prior to artificial insemination

ABSTRACT: BACKGROUND: One of the challenges faced by equine breeders is ensuring delivery of good quality semen doses for artificial insemination when the mare is due to ovulate. Single Layer Centrifugation (SLC) has been shown to select morphologically normal spermatozoa with intact chromatin and good progressive motility from the rest of the ejaculate, and to prolong the life of these selected spermatozoa in vitro. The objective of the present study was a proof of concept, to determine whether fertilizing ability was retained in SLC-selected spermatozoa during prolonged storage. FINDINGS: Sixteen mares were inseminated with SLC-selected sperm doses that had been cooled and stored at 6°C for 48 h, 72 h or 96 h. Embryos were identified in 11 mares by ultrasound examination 16-18 days after presumed ovulation. CONCLUSION: SLC-selected stallion spermatozoa stored for up to 96 h are capable of fertilization.     

PGC‐1α affects aging‐related changes in muscle and motor function by modulating specific exercise‐mediated changes in old mice

The age‐related impairment in muscle function results in a drastic decline in motor coordination and mobility in elderly individuals. Regular physical activity is the only efficient intervention to prevent and treat this age‐associated degeneration. However, the mechanisms that underlie the therapeutic effect of exercise in this context remain unclear. We assessed whether endurance exercise training in old age is sufficient to affect muscle and motor function. Moreover, as muscle peroxisome proliferator‐activated receptor γ coactivator 1α (PGC‐1α) is a key regulatory hub in endurance exercise adaptation with decreased expression in old muscle, we studied the involvement of PGC‐1α in the therapeutic effect of exercise in aging. Intriguingly, PGC‐1α muscle‐specific knockout and overexpression, respectively, precipitated and alleviated specific aspects of aging‐related deterioration of muscle function in old mice, while other muscle dysfunctions remained unchanged upon PGC‐1α modulation. Surprisingly, we discovered that muscle PGC‐1α was not only involved in improving muscle endurance and mitochondrial remodeling, but also phenocopied endurance exercise training in advanced age by contributing to maintaining balance and motor coordination in old animals. Our data therefore suggest that the benefits of exercise, even when performed at old age, extend beyond skeletal muscle and are at least in part mediated by PGC‐1α.     

Global change‐driven effects on dissolved organic matter composition: Implications for food webs of northern lakes

Northern ecosystems are experiencing some of the most dramatic impacts of global change on Earth. Rising temperatures, hydrological intensification, changes in atmospheric acid deposition and associated acidification recovery, and changes in vegetative cover are resulting in fundamental changes in terrestrial–aquatic biogeochemical linkages. The effects of global change are readily observed in alterations in the supply of dissolved organic matter (DOM)—the messenger between terrestrial and lake ecosystems—with potentially profound effects on the structure and function of lakes. Northern terrestrial ecosystems contain substantial stores of organic matter and filter or funnel DOM, affecting the timing and magnitude of DOM delivery to surface waters. This terrestrial DOM is processed in streams, rivers, and lakes, ultimately shifting its composition, stoichiometry, and bioavailability. Here, we explore the potential consequences of these global change‐driven effects for lake food webs at northern latitudes. Notably, we provide evidence that increased allochthonous DOM supply to lakes is overwhelming increased autochthonous DOM supply that potentially results from earlier ice‐out and a longer growing season. Furthermore, we assess the potential implications of this shift for the nutritional quality of autotrophs in terms of their stoichiometry, fatty acid composition, toxin production, and methylmercury concentration, and therefore, contaminant transfer through the food web. We conclude that global change in northern regions leads not only to reduced primary productivity but also to nutritionally poorer lake food webs, with discernible consequences for the trophic web to fish and humans.     

Einige Feinstrukturbefunde an den Plexus chorioidei von Affen

Zusammenfassung An der lichtmikroskopischen Struktur des plexus chorioideus ventriculi III von Ateles, Cebus, Macaca und Pan fallen deutliche speziesbedingte Unterschiede auf. Bei Macaca wurde die Ultrastruktur der Plexus chorioidei ventriculi III und IV sowie der Plexus chorioidei der Seitenventrikel verglichen. Kuppenzellen überwiegen im elektronen-mikroskopischen Bild der Plexusepithelien des III. und IV. Ventrikels, während unter den Epithelien der Seitenventrikel flachere Zellen dominieren. Diese strukturellen Besonderheiten könnten auf funktionelle Unterschiede in der Liquorbildung hinweisen. Das Problem der extrachorioidalen Liquorproduktion (Curl and Pollay, 1968; Milhorat u. Mitarb., 1971) wird im Zusammenhang mit den regionalen Unterschieden in der Wandstruktur des Aquaeductus cerebri (Sylvii) diskutiert (Merker, 1970). Im Stroma und im Interstitium aller Plexus chorioidei des Rhesusaffen sind makrophagenartige Wanderzellen zu beobachten, deren Strukturbild dem der Epiplexuszellen gleicht; sie werden als ausgewanderte Monozyten gedeutet. Wahrscheinlich gelangen diese Zellen in den Ventrikel und werden dort zu Epiplexuszellen. Einschlüsse vom Typ der Biondi-Körper wurden in den Plexusepithelien der untersuchten Affen nicht beobachtet; die untersuchten Tiere hatten aber noch kein hohes Lebensalter erreicht.

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The fine structure of the choroid plexuses in monkeys

Summary The light microscopic structure of the choroid plexus of the III^rd ventricle was studied in Ateles, Cebus, Macaca and Pan. These choroid plexuses display distinct species differences. The ultrastructure of the choroid plexuses of the III^rd, IV^th and lateral ventricles was compared in Macaca. Cuboidal cells with dome-like protrusions predominate in the epithelium of the choroid plexuses of the III^rd and the IV^th ventricles, while flattened cells are characteristic of the choroid epithelium of the lateral ventricle. These structural peculiarities may reflect functional differences in the production of the cerebrospinal fluid. The problem of extrachoroidal production of the cerebrospinal fluid (Curl and Pollay, 1968; Milhorat et al., 1971) is discussed in connection with regional differences in the structure of the aqueduct wall (Merker, 1970). Macrophage-like cells which morphologically resemble epiplexus cells and which are very abundant within the connective tissue stroma and also between the epithelial cells of all choroid plexuses of the rhesus monkey, are thought to be monocytes. Probably these cells migrate into the ventricle where they become epiplexus cells. Epithelial inclusions of the type of Biondi bodies were not observed in the simian choroid plexuses. However, the animals investigated were not old.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Arbeitskreis Prof. Dr. A. Oksche).     

Metabolism of lutein and zeaxanthin in rhesus monkeys: identification of (3R,6'R)- and (3R,6'S)-3'-dehydro-lutein as common metabolites and comparison to humans

Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3'-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 and 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3'-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6'R)-3'-dehydro-lutein and (3R,6'S)-3'-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These findings were comparable to results obtained with human plasma.