VHL-dependence of EHHADH Expression in a Human Renal Cell Carcinoma Cell Line

The von Hippel-Lindau tumor suppressor gene (VHL) is mutated in up to 90% of clear cell renal cell carcinoma (ccRCC) cases, thus playing a key role in ccRCC pathogenesis. ccRCC can be classified as a metabolic disease in which alterations in fatty acid metabolism facilitate cancer cell proliferation. Enoyl-CoA hydratase and 3-hydroxyacyl CoA dehydrogenase (EHHADH) is an enzyme involved in peroxisomal fatty acid degradation. It is primarily expressed in renal proximal tubule cells, presumably the origin of ccRCC. Although EHHADH is still a relatively unexplored gene, it is known to be differentially expressed in several tumors. In this study, analysis of several databases revealed that EHHADH expression is downregulated in ccRCC samples compared to healthy kidney samples. Moreover, cell culture experiments were performed to investigate the relationship between EHHADH and VHL at the gene and protein level. qPCR and Western blot analyses using the human ccRCC cell line RCC4 revealed that EHHADH is expressed in a VHL-dependent manner. RCC4 cells reconstituted with VHL show significantly higher EHHADH mRNA and protein levels than VHL-deficient RCC4 control cells. These results indicate that the downregulation of EHHADH in ccRCC reported may be due to the loss of VHL function. This study is the first to molecularly characterize EHHADH, a key enzyme in peroxisomal ß-oxidation, in relation to VHL, suggesting a potential pathogenic interaction that is worthy of further investigation

CGEF-1 regulates mTORC1 signaling during adult longevity and stress response in

The mechanistic target of rapamycin (mTOR) kinase is central to metabolism and growth, and has a conserved role in aging. mTOR functions in two complexes, mTORC1 and mTORC2. In diverse eukaryotes, inhibition of mTORC1 signaling increases lifespan. mTORC1 transduces anabolic signals to stimulate protein synthesis and inhibits autophagy. In this study, we demonstrate that CGEF-1, theC. eleganshomolog of the human guanine nucleotide exchange factor Dbl, is a novel binding partner of RHEB-1 and activator of mTORC1 signaling inC. elegans.cgef-1mutants display prolonged lifespan and enhanced stress resistance. The transcription factors DAF-16/FoxO and SKN-1/Nrf are required for increased longevity and stress tolerance, and induce protective gene expression incgef-1mutants. Genetic evidence indicates thatcgef-1functions in the same pathway withrheb-1, the mTOR kinaselet-363, anddaf-15/Raptor. Whencgef-1is inactivated, phosphorylation of 4E-BP, a central mTORC1 substrate for protein translation is reduced inC. elegans. Moreover, autophagy is increased uponcgef-1and mTORC1 inhibition. In addition, we show that in human cells Dbl associates with Rheb and stimulates mTORC1 downstream targets for protein synthesis suggesting that the function of CGEF-1/Dbl in the mTORC1 signaling pathway is evolutionarily conserved. These findings have important implications for mTOR functions and signaling mechanisms in aging and age-related diseases

SCD5 Regulation by VHL Affects Cell Proliferation and Lipid Homeostasis in ccRCC

Clear cell renal cell carcinoma (ccRCC) is the most common histological subtype of renal cancer, and inactivation of the VHL tumor suppressor gene is found in almost all cases of hereditary and sporadic ccRCCs. CcRCC is associated with the reprogramming of fatty acid metabolism, and stearoyl-CoA desaturases (SCDs) are the main enzymes controlling fatty acid composition in cells. In this study, we report that mRNA and protein expression of the stearoyl-CoA desaturase SCD5 is downregulated in VHL-deficient cell lines. Similarly, in C. elegans vhl-1 mutants, FAT-7/SCD5 activity is repressed, supporting an evolutionary conservation. SCD5 regulation by VHL depends on HIF, and loss of SCD5 promotes cell proliferation and a metabolic shift towards ceramide production. In summary, we identify a novel regulatory function of VHL in relation to SCD5 and fatty acid metabolism, and propose a new mechanism of how loss of VHL may contribute to ccRCC tumor formation and progression

Cell-nonautonomous signaling of FOXO/DAF-16 to the stem cells of Caenorhabditis elegans.

In Caenorhabditis elegans (C. elegans), the promotion of longevity by the transcription factor DAF-16 requires reduced insulin/IGF receptor (IIR) signaling or the ablation of the germline, although the reason for the negative impact of germ cells is unknown. FOXO/DAF-16 activity inhibits germline proliferation in both daf-2 mutants and gld-1 tumors. In contrast to its function as a germline tumor suppressor, we now provide evidence that somatic DAF-16 in the presence of IIR signaling can also result in tumorigenic activity, which counteracts robust lifespan extension. In contrast to the cell-autonomous IIR signaling, which is required for larval germline proliferation, activation of DAF-16 in the hypodermis results in hyperplasia of the germline and disruption of the surrounding basement membrane. SHC-1 adaptor protein and AKT-1 kinase antagonize, whereas AKT-2 and SGK-1 kinases promote, this cell-nonautonomous DAF-16 function. Our data suggest that a functional balance of DAF-16 activities in different tissues determines longevity and reveals a novel, cell-nonautonomous role of FOXO/DAF-16 to affect stem cells

mTORC2-SGK-1 acts in two environmentally responsive pathways with opposing effects on longevity

The nematode worm Caenorhabditis elegans provides a powerful system for elucidating how genetic, metabolic, nutritional, and environmental factors influence aging. The mechanistic target of rapamycin (mTOR) kinase is important in growth, disease, and aging and is present in the mTORC1 and mTORC2 complexes. In diverse eukaryotes, lifespan can be increased by inhibition of mTORC1, which transduces anabolic signals to stimulate protein synthesis and inhibit autophagy. Less is understood about mTORC2, which affects C. elegans lifespan in a complex manner that is influenced by the bacterial food source. mTORC2 regulates C. elegans growth, reproduction, and lipid metabolism by activating the SGK-1 kinase, but current data on SGK-1 and lifespan seem to be conflicting. Here, by analyzing the mTORC2 component Rictor (RICT-1), we show that mTORC2 modulates longevity by activating SGK-1 in two pathways that affect lifespan oppositely. RICT-1/mTORC2 limits longevity by directing SGK-1 to inhibit the stress-response transcription factor SKN-1/Nrf in the intestine. Signals produced by the bacterial food source determine how this pathway affects SKN-1 and lifespan. In addition, RICT-1/mTORC2 functions in neurons in an SGK-1-mediated pathway that increases lifespan at lower temperatures. RICT-1/mTORC2 and SGK-1 therefore oppose or accelerate aging depending upon the context in which they are active. Our findings reconcile data on SGK-1 and aging, show that the bacterial microenvironment influences SKN-1/Nrf, mTORC2 functions, and aging, and identify two longevity-related mTORC2 functions that involve SGK-regulated responses to environmental cues

Functional and spatial analysis of C. elegans SYG-1 and SYG-2, orthologs of the Neph/nephrin cell adhesion module directing selective synaptogenesis.

The assembly of specific synaptic connections represents a prime example of cellular recognition. Members of the Ig superfamily are among the most ancient proteins represented in the genomes of both mammalian and invertebrate organisms, where they constitute a trans-synaptic adhesion system. The correct connectivity patterns of the highly conserved immunoglobulin superfamily proteins nephrin and Neph1 are crucial for the assembly of functional neuronal circuits and the formation of the kidney slit diaphragm, a synapse-like structure forming the filtration barrier. Here, we utilize the nematode C. elegans model for studying the requirements of synaptic specificity mediated by nephrin-Neph proteins. In C. elegans, the nephrin/Neph1 orthologs SYG-2 and SYG-1 form intercellular contacts strictly in trans between epithelial guidepost cells and neurons specifying the localization of synapses. We demonstrate a functional conservation between mammalian nephrin and SYG-2. Expression of nephrin effectively compensated loss of syg-2 function in C. elegans and restored defective synaptic connectivity further establishing the C. elegans system as a valuable model for slit diaphragm proteins. Next, we investigated the effect of SYG-1 and SYG-2 trans homodimerization respectively. Strikingly, synapse assembly could be induced by homophilic SYG-1 but not SYG-2 binding indicating a critical role of SYG-1 intracellular signalling for morphogenetic events and pointing toward the dynamic and stochastic nature of extra- and intracellular nephrin-Neph interactions to generate reproducible patterns of synaptic connectivity

Eculizumab as a treatment for C3 glomerulopathy: a single-center retrospective study

Abstract Background C3 Glomerulopathy (C3G) is a rare glomerular disease caused by dysregulation of the complement pathway. Based on its pathophysiology, treatment with the monoclonal antibody eculizumab targeting complement C5 may be a therapeutic option. Due to the rarity of the disease, observational data on the clinical response to eculizumab treatment is scarce. Methods Fourteen patients (8 female, 57%) treated for C3 glomerulopathy at the medical center of the University of Freiburg between 2013 and 2022 were included. Subjects underwent biopsy before enrollment. Histopathology, clinical data, and response to eculizumab treatment were analyzed. Key parameters to determine the primary outcome were changes of estimated glomerular filtration rate (eGFR) over time. Positive outcome was defined as > 30% increase, stable outcome as ±30%, negative outcome as decrease > 30% of eGFR. Results Eleven patients (78.8%) were treated with eculizumab, three received standard of care (SoC, 27.2%). Median follow-up time was 68 months (IQR: 45–98 months). Median eculizumab treatment duration was 10 months (IQR 5–46 months). After eculizumab treatment, five patients showed a stable outcome, six patients showed a negative outcome. Among patients receiving SoC, one patient showed a stable outcome, two patients showed a negative outcome. Conclusions The benefit of eculizumab in chronic progressive C3 glomerulopathy is limited

Defects in gonadal integrity.

<p>n: Numbers of examined animals.</p>#<p>: Animals were raised at 15°C until examination.</p><p>P values were relative to:</p>a<p>: N2;</p>b<p>: <i>Is[daf-16::gfp]</i>;</p>c<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i>;</p>d<p>: <i>Is[daf-16::gfp]</i>(L4440);</p>e<p>: <i>shc-1(ok198)</i>;</p>f<p>: <i>shc-1(ok198)</i>(L4440);</p>g<p>: <i>shc-1(ok198);akt-1(ok525)</i>^{<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#nt115" target="_blank">#</a>};</p>h<p>: <i>akt-1(ok525)</i>;</p>i<p>: <i>shc-1(ok198);akt-1(ok525)</i>;</p>j<p>: <i>shc-1(ok198);daf-18(e1375);akt-1(ok525)</i>;</p>k<p>: <i>shc-1(ok198);akt-1(ok525)</i>;</p>l<p>: <i>shc-1(ok198);Is[daf-16::gfp]</i> at 15°C.</p

Active DAF-16 derogates integrity of the gonadal basement membrane.

<p>(A) Knock-down of <i>daf-16</i> by RNAi suppressed the early lethality of shc<i>-1(ok198);Is[daf-16::gfp]</i> animals. Animals died within the first five days of adulthood: <i>shc-1(ok198);Is[daf-16::gfp](L4440)</i> as control: 45.7%, <i>shc-1(ok198);Is[daf-16::gfp]+daf-16</i> RNAi: 1.4%. (B) <i>daf-16</i> mutation suppresses the defects in gonad integrity of <i>shc-1</i> mutant animals. (C) Transgenic expression of a constitutively nuclearly located <i>daf-16(4A)::gfp</i> results in disruption of the gonad in <i>shc-1(−)</i>, but not in wild type animals (See also <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen.1002836.s007" target="_blank">Figure S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>). (D) SHC-1 does not affect the subcellular localization of DAF-16(4A)::GFP. In both wild type and <i>shc-1(ok198)</i> animals DAF-16(4A)::GFP is constitutively located in the nucleus. The mean lifespan and statistical analyses in this figure are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t001" target="_blank">Table 1</a>. <i>byEx</i> represents extrachromosomal transgenic alleles which are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>. Mean values and statistic analysis of defect in gonadal integrity are summarized in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t002" target="_blank">Table 2</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002836#pgen-1002836-t003" target="_blank">Table 3</a>. (E) SHC-1 does not affect expression level of transgenic <i>daf-16(4A)::gfp</i>. Columns represent pooled normalised values of three independent experiments plus standard deviation (SD). Mann Whitney test. Normalized GFP intensity: <i>byEx797</i>: 1.00±2.5 (n = 31); <i>shc-1;byEx797</i>: 1.00±2.1 (n = 33), P = 0.9670; <i>byEx798</i>: 1.00±0.19 (n = 37); <i>shc-1;byEx798</i>:1.06±0.22 (n = 48), P = 0.1591.</p