Contrasting consequences of podocyte insulin-like growth factor 1 receptor inhibition.

Insulin signaling to the glomerular podocyte via the insulin receptor (IR) is critical for kidney function. In this study we show that near-complete knockout of the closely related insulin-like growth factor 1 receptor (IGF1R) in podocytes is detrimental, resulting in albuminuria in vivo and podocyte cell death in vitro. In contrast, partial podocyte IGF1R knockdown confers protection against doxorubicin-induced podocyte injury. Proteomic analysis of cultured podocytes revealed that while near-complete loss of podocyte IGF1R results in the downregulation of mitochondrial respiratory complex I and DNA damage repair proteins, partial IGF1R inhibition promotes respiratory complex expression. This suggests that altered mitochondrial function and resistance to podocyte stress depends on the level of IGF1R suppression, the latter determining whether receptor inhibition is protective or detrimental. Our work suggests that the partial suppression of podocyte IGF1R could have therapeutic benefits in treating albuminuric kidney disease.

An information theoretic approach to insulin sensing by human kidney podocytes.

Podocytes are key components of the glomerular filtration barrier (GFB). They are insulin-responsive but can become insulin-resistant, causing features of the leading global cause of kidney failure, diabetic nephropathy. Insulin acts via insulin receptors to control activities fundamental to GFB integrity, but the amount of information transferred is unknown. Here we measure this in human podocytes, using information theory-derived statistics that take into account cell-cell variability. High content imaging was used to measure insulin effects on Akt, FOXO and ERK. Mutual Information (MI) and Channel Capacity (CC) were calculated as measures of information transfer. We find that insulin acts via noisy communication channels with more information flow to Akt than to ERK. Information flow estimates were increased by consideration of joint sensing (ERK and Akt) and response trajectory (live cell imaging of FOXO1-clover translocation). Nevertheless, MI values were always <1Bit as most information was lost through signaling. Constitutive PI3K activity is a predominant feature of the system that restricts the proportion of CC engaged by insulin. Negative feedback from Akt supressed this activity and thereby improved insulin sensing, whereas sensing was robust to manipulation of feedforward signaling by inhibiting PI3K, PTEN or PTP1B. The decisions made by individual podocytes dictate GFB integrity, so we suggest that understanding the information on which the decisions are based will improve understanding of diabetic kidney disease and its treatment.

A heterodimeric SNX4--SNX7 SNX-BAR autophagy complex coordinates ATG9A trafficking for efficient autophagosome assembly.

The sorting nexins (SNXs) are a family of peripheral membrane proteins that direct protein trafficking decisions within the endocytic network. Emerging evidence in yeast and mammalian cells implicates a subgroup of SNXs in selective and non-selective forms of autophagy. Using siRNA and CRISPR-Cas9, we demonstrate that the SNX-BAR protein SNX4 is needed for efficient LC3 (also known as MAP1LC3) lipidation and autophagosome assembly in mammalian cells. SNX-BARs exist as homo- and hetero-dimers, and we show that SNX4 forms functional heterodimers with either SNX7 or SNX30 that associate with tubulovesicular endocytic membranes. Detailed image-based analysis during the early stages of autophagosome assembly reveals that SNX4-SNX7 is an autophagy-specific SNX-BAR heterodimer, required for efficient recruitment and/or retention of core autophagy regulators at the nascent isolation membrane. SNX4 partially colocalises with juxtanuclear ATG9A-positive membranes, with our data linking the autophagy defect upon SNX4 disruption to the mis-trafficking and/or retention of ATG9A in the Golgi region. Taken together, our findings show that the SNX4-SNX7 heterodimer coordinates ATG9A trafficking within the endocytic network to establish productive autophagosome assembly sites, thus extending knowledge of SNXs as positive regulators of autophagy.

Enhanced insulin receptor, but not PI3K, signalling protects podocytes from ER stress.

Disruption of the insulin-PI3K-Akt signalling pathway in kidney podocytes causes endoplasmic reticulum (ER) stress, leading to podocyte apoptosis and proteinuria in diabetic nephropathy. We hypothesised that by improving insulin sensitivity we could protect podocytes from ER stress. Here we use established activating transcription factor 6 (ATF6)- and ER stress element (ERSE)-luciferase assays alongside a novel high throughput imaging-based C/EBP homologous protein (CHOP) assay to examine three models of improved insulin sensitivity. We find that by improving insulin sensitivity at the level of the insulin receptor (IR), either by IR over-expression or by knocking down the negative regulator of IR activity, protein tyrosine-phosphatase 1B (PTP1B), podocytes are protected from ER stress caused by fatty acids or diabetic media containing high glucose, high insulin and inflammatory cytokines TNFα and IL-6. However, contrary to this, knockdown of the negative regulator of PI3K-Akt signalling, phosphatase and tensin homolog deleted from chromosome 10 (PTEN), sensitizes podocytes to ER stress and apoptosis, despite increasing Akt phosphorylation. This indicates that protection from ER stress is conferred through not just the PI3K-Akt pathway, and indeed we find that inhibiting the MEK/ERK signalling pathway rescues PTEN knockdown podocytes from ER stress.

Prolonged exposure of mouse and human podocytes to insulin induces insulin resistance through lysosomal and proteasomal degradation of the insulin receptor.

AIMS/HYPOTHESIS: Podocytes are insulin-responsive cells of the glomerular filtration barrier and are key in preventing albuminuria, a hallmark feature of diabetic nephropathy. While there is evidence that a loss of insulin signalling to podocytes is detrimental, the molecular mechanisms underpinning the development of podocyte insulin resistance in diabetes remain unclear. Thus, we aimed to further investigate podocyte insulin responses early in the context of diabetic nephropathy. METHODS: Conditionally immortalised human and mouse podocyte cell lines and glomeruli isolated from db/db DBA/2J mice were studied. Podocyte insulin responses were investigated with western blotting, cellular glucose uptake assays and automated fluorescent imaging of the actin cytoskeleton. Quantitative (q)RT-PCR was employed to investigate changes in mRNA. Human cell lines stably overproducing the insulin receptor (IR) and nephrin were also generated, using lentiviral constructs. RESULTS: Podocytes exposed to a diabetic environment (high glucose, high insulin and the proinflammatory cytokines TNF-α and IL-6) become insulin resistant with respect to glucose uptake and activation of phosphoinositide 3-kinase (PI3K) and mitogen-activated protein kinase (MAPK) signalling. These podocytes lose expression of the IR as a direct consequence of prolonged exposure to high insulin concentrations, which causes an increase in IR protein degradation via a proteasome-dependent and bafilomycin-sensitive pathway. Reintroducing the IR into insulin-resistant human podocytes rescues upstream phosphorylation events, but not glucose uptake. Stable expression of nephrin is also required for the insulin-stimulated glucose uptake response in podocytes and for efficient insulin-stimulated remodelling of the actin cytoskeleton. CONCLUSIONS/INTERPRETATION: Together, these results suggest that IR degradation, caused by high levels of insulin, drives early podocyte insulin resistance, and that both the IR and nephrin are required for full insulin sensitivity of this cell. This could be highly relevant for the development of nephropathy in individuals with type 2 diabetes, who are commonly hyperinsulinaemic in the early phases of their disease.

Autophagy facilitates organelle clearance during differentiation of human erythroblasts: evidence for a role for ATG4 paralogs during autophagosome maturation.

Wholesale depletion of membrane organelles and extrusion of the nucleus are hallmarks of mammalian erythropoiesis. Using quantitative EM and fluorescence imaging we have investigated how autophagy contributes to organelle removal in an ex vivo model of human erythroid differentiation. We found that autophagy is induced at the polychromatic erythroid stage, and that autophagosomes remain abundant until enucleation. This stimulation of autophagy was concomitant with the transcriptional upregulation of many autophagy genes: of note, expression of all ATG8 mammalian paralog family members was stimulated, and increased expression of a subset of ATG4 family members (ATG4A and ATG4D) was also observed. Stable expression of dominant-negative ATG4 cysteine mutants (ATG4B (C74A) ; ATG4D (C144A) ) did not markedly delay or accelerate differentiation of human erythroid cells; however, quantitative EM demonstrated that autophagosomes are assembled less efficiently in ATG4B (C74A) -expressing progenitor cells, and that cells expressing either mutant accumulate enlarged amphisomes that cannot be degraded. The appearance of these hybrid autophagosome/endosome structures correlated with the contraction of the lysosomal compartment, suggesting that the actions of ATG4 family members (particularly ATG4B) are required for the control of autophagosome fusion with late, degradative compartments in differentiating human erythroblasts.

The ins and outs of human reticulocyte maturation: autophagy and the endosome/exosome pathway.

The maturation of reticulocytes into functional erythrocytes is a complex process requiring extensive cytoplasmic and plasma membrane remodeling, cytoskeletal rearrangements and changes to cellular architecture. Autophagy is implicated in the sequential removal of erythroid organelles during erythropoiesis, although how this is regulated during late stages of erythroid differentiation, and the potential contribution of autophagy during reticulocyte maturation, remain unclear. Using an optimized ex vivo differentiation system for human erythropoiesis, we have observed that maturing reticulocytes are characterized by the presence of one or few large vacuolar compartments. These label strongly for glycophorin A (GYPA/GPA) which is internalized from the plasma membrane; however, they also contain organellar remnants (ER, Golgi, mitochondria) and stain strongly for LC3, suggesting that they are endocytic/autophagic hybrid structures. Interestingly, we observed the release of these vacuoles by exocytosis in maturing reticulocytes, and speculate that autophagy is needed to concentrate the final remnants of the reticulocyte endomembrane system in autophagosome/endosome hybrid compartments that are primed to undergo exocytosis.

Maturing reticulocytes internalize plasma membrane in glycophorin A-containing vesicles that fuse with autophagosomes before exocytosis.

The erythrocyte is one of the best characterized human cells. However, studies of the process whereby human reticulocytes mature to erythrocytes have been hampered by the difficulty of obtaining sufficient numbers of cells for analysis. In the present study, we describe an in vitro culture system producing milliliter quantities of functional mature human adult reticulocytes from peripheral blood CD34(+) cells. We show that the final stage of reticulocyte maturation occurs by a previously undescribed mechanism in which large glycophorin A-containing vesicles forming at the cytosolic face of the plasma membrane are internalized and fuse with autophagosomes before expulsion of the autophagosomal contents by exocytosis. Early reticulocyte maturation is characterized by the selective elimination of unwanted plasma membrane proteins (CD71, CD98, and ß1 integrin) through the endosome-exosome pathway. In contrast, late maturation is characterized by the generation of large glycophorin A-decorated vesicles of autophagic origin.

A cryptic mitochondrial targeting motif in Atg4D links caspase cleavage with mitochondrial import and oxidative stress.

The Atg4 cysteine proteases play crucial roles in the processing of Atg8 proteins during autophagy, but their regulation during cellular stress and differentiation remains poorly understood. We have found that two Atg4 family members--Atg4C and Atg4D--contain cryptic mitochondrial targeting sequences immediately downstream of their canonical (DEVD) caspase cleavage sites. Consequently, caspase-cleaved Atg4D (ΔN63 Atg4D) localizes to the mitochondrial matrix when expressed in mammalian cells, where it undergoes further processing to a ~42 kDa mitochondrial form. Interestingly, caspase cleavage is not needed for Atg4D mitochondrial import, because ~42 kDa mitochondrial Atg4D is observed in cells treated with caspase inhibitors and in cells expressing caspase-resistant Atg4D (DEVA(63)). Using HeLa cell lines stably expressing ΔN63 Atg4D, we showed that mitochondrial Atg4D sensitizes cells to cell death in the presence of the mitochondrial uncoupler, CCCP, and that mitochondrial cristae are less extensive in these cells. We further showed that the organization of mitochondrial cristae is altered during the mitochondrial clearance phase in differentiating primary human erythroblasts stably expressing ΔN63 Atg4D, and that these cells have elevated levels of mitochondrial reactive oxygen species (ROS) during late stages of erythropoiesis. Together these data suggest that the import of Atg4D during cellular stress and differentiation may play important roles in the regulation of mitochondrial physiology, ROS, mitophagy and cell viability.

Atg4D at the interface between autophagy and apoptosis.

The Atg4 family of endopeptidases regulates autophagosome biogenesis by priming newly synthesized Atg8 to enable covalent attachment of phosphatidylethanolamine, and by delipidating Atg8 at the lysosomal fusion step. Control of Atg4 activity is therefore crucial, although little is known about how these molecules are regulated in living cells. We have found that one human Atg4 family member (Atg4D) is cleaved at DEVD(63)K by caspase-3 during apoptosis. Importantly, our studies suggest that native Atg4D is enzymatically inactive, but gains GABARAP-L1 priming/delipidation activity following caspase cleavage. Caspase-cleaved Atg4D is also highly cytotoxic; however, toxicity is not due to enhanced autophagy, but is mediated by a putative C-terminal BH3 domain, and is associated with transient recruitment of Atg4D to mitochondria.

Caspase cleavage of Atg4D stimulates GABARAP-L1 processing and triggers mitochondrial targeting and apoptosis.

Autophagy is an important catabolic process with roles in cell survival and cell death. It sequesters cytosol and organelles within double-membrane autophagosomes that deliver their contents to lysosomes for degradation. Autophagosome biogenesis is coordinated by the autophagy-related protein 4 (Atg4) family of C54 endopeptidases (Atg4A-Atg4D). These enzymes prime and then later delipidate the autophagosome marker, Atg8. Here, we show that one family member, Atg4D, is cleaved by caspase-3 in vitro and in apoptotic cells. Atg4D is a poor priming and delipidation enzyme in vitro, but truncated DeltaN63 Atg4D displays increased activity against the Atg8 paralogue, gamma-aminobutyric acid receptor-associated protein-like 1 (GABARAP-L1). In living cells, DeltaN63 Atg4D stimulates the delipidation of GABARAP-L1, whereas siRNA silencing of the gene expressing Atg4D abrogates GABARAP-L1 autophagosome formation and sensitises cells to starvation and staurosporine-induced cell death. Interestingly, Atg4D overexpression induces apoptosis, which is preceded by the caspase-independent recruitment of Atg4D to mitochondria and is facilitated by a putative C-terminal Bcl-2 homology 3 (BH3) domain. Atg4D also acquires affinity for damaged mitochondria in cells treated with hydrogen peroxide. These data suggest that Atg4D is an autophagy regulator that links mitochondrial dysfunction with apoptosis.

A novel role for microtubules in apoptotic chromatin dynamics and cellular fragmentation.

Dramatic changes in cellular dynamics characterise the apoptotic execution phase, culminating in fragmentation into membrane-bound apoptotic bodies. Previous evidence suggests that actin-myosin plays a dominant role in apoptotic cellular remodelling, whereas all other cytoskeletal elements dismantle. We have used fixed cells and live-cell imaging to confirm that interphase microtubules rapidly depolymerise at the start of the execution phase. Around this time, pericentriolar components (pericentrin, ninein and gamma-tubulin) are lost from the centrosomal region. Subsequently, however, extensive non-centrosomal bundles of densely packed, dynamic microtubules rapidly assemble throughout the cytoplasm in all cell lines tested. These microtubules have an important role in the peripheral relocation of chromatin in the dying cell, because nocodazole treatment restricts the dispersal of condensed apoptotic chromatin into surface blebs, and causes the withdrawal of chromatin fragments back towards the cell centre. Importantly, nocodazole and taxol are both potent inhibitors of apoptotic fragmentation in A431 cells, implicating dynamic microtubules in apoptotic body formation. Live-cell-imaging studies indicate that fragmentation is accompanied by the extension of rigid microtubule-rich spikes that project through the cortex of the dying cell. These structures enhance interactions between apoptotic cells and phagocytes in vitro, by providing additional sites for attachment to neighbouring cells.

Is puberty an accelerator of type 1 diabetes in IL6-174CC females?

The pubertal peak in onset of type 1 diabetes occurs earlier in girls than boys. We postulated that this sex difference might be mediated in part by estrogen or by genes regulated by estrogen, such as the interleukin-6 (IL6) gene. Previous studies concerning the role of an estrogen-sensitive single nucleotide polymorphism (SNP) in the IL6 promoter in type 1 diabetes have proved contradictory. We therefore selected a large, genetically homogenous population-based cohort, analyzed by age at onset and sex, to test the hypothesis that the IL6-174G>C SNP affects age at onset of type 1 diabetes in females but not in males. We found that the IL6-174CC genotype was significantly less frequent in females diagnosed after than in those diagnosed before the age of 10 years (19 vs. 13%, P = 0.016). No genotype difference was observed in males stratified for age at onset. Among children diagnosed after age 10, the median age of onset was 11.9 years (intraquartile range 10.7-14.6) in 34 girls homozygous for IL6-174C compared with 13.2 years (11.6-15.4) in 229 girls with other genotypes and 13.5 years (12.0-15.6) in 339 males with any IL6-174 genotype (P = 0.012). These data support the hypothesis that pubertal changes may contribute to accelerated onset of type 1 diabetes in genetically susceptible females. This phenomenon may be orchestrated by the action of estrogen on the IL6 promoter.

Protein interaction mapping: a Drosophila case study.

The Drosophila (fruit fly) model system has been instrumental in our current understanding of human biology, development, and diseases. Here, we used a high-throughput yeast two-hybrid (Y2H)-based technology to screen 102 bait proteins from Drosophila melanogaster, most of them orthologous to human cancer-related and/or signaling proteins, against high-complexity fly cDNA libraries. More than 2300 protein-protein interactions (PPI) were identified, of which 710 are of high confidence. The computation of a reliability score for each protein-protein interaction and the systematic identification of the interacting domain combined with a prediction of structural/functional motifs allow the elaboration of known complexes and the identification of new ones. The full data set can be visualized using a graphical Web interface, the PIMRider (http://pim.hybrigenics.com), and is also accessible in the PSI standard Molecular Interaction data format. Our fly Protein Interaction Map (PIM) is surprisingly different from the one recently proposed by Giot et al. with little overlap between the two data sets. Analysis of the differences in data sets and methods suggests alternative strategies to enhance the accuracy and comprehensiveness of the post-genomic generation of broad-scale protein interaction maps.