Summary Gradual changes in steady-state levels of beta amyloid peptides (Aβ) in brain are considered an initial step in the amyloid cascade hypothesis of Alzheimer's disease. Aβ is a product of the secretase cleavage of amyloid precursor protein (APP). There is evidence that the membrane lipid environment may modulate secretase activity and alters its function. Cleavage of APP strongly depends on membrane properties. Since Aβ perturbs cell membrane fluidity, the cell membrane may be the location where the neurotoxic cascade of Aβ is initiated. Therefore, we tested effects of oligomeric Aβ on membrane fluidity of whole living cells, the impact of exogenous and cellular Aβ on the processing of APP and the role of GM-1 ganglioside. We present evidence that oligoAβ(1-40) stimulates the amyloidogenic processing of APP by reducing membrane fluidity and complexing with GM-1 ganglioside. This dynamic action of Aβ may start a vicious circle, where endogenous Aβ stimulates its own production. Based on our novel findings, we propose that oligoAβ(1-40) accelerates the proteolytic cleavage of APP by decreasing membrane fluidity.
PDX-positive cells are lost in the urine in disease states that require podocyte regeneration and are a useful non-invasive marker for glomerular disease activity. These cells are possibly derived from the parietal epithelial layer.
Defects in podocyte signaling are the basis of many inherited glomerular diseases leading to glomerulosclerosis. CD2-associated protein (CD2AP) is highly expressed in podocytes and is considered to play an important role in the maintenance of the glomerular slit diaphragm. Mice deficient for CD2AP (CD2AP ؊/؊ ) appear normal at birth but develop a rapid onset nephrotic syndrome at 3 weeks of age. We demonstrate that impaired intracellular signaling with subsequent podocyte damage is the reason for this delayed podocyte injury in CD2AP ؊/؊ mice. We document that CD2AP deficiency in podocytes leads to diminished signal initiation and termination of signaling pathways mediated by receptor tyrosine kinases (RTKs). In addition, we demonstrate that CIN85, a paralog of CD2AP, is involved in termination of RTK signaling in podocytes. CIN85 protein expression is increased in CD2AP ؊/؊ podocytes in vitro. Stimulation of CD2AP؊/؊ podocytes with various growth factors, including insulin-like growth factor 1, vascular endothelial growth factor, and fibroblast growth factor, resulted in a significantly decreased phosphatidylinositol 3-kinase/AKT and ERK signaling response. Moreover, increased CIN85 protein is detectable in podocytes in diseased CD2AP ؊/؊ mice, leading to decreased base-line activation of ERK and decreased phosphorylation after growth factor stimulation in vivo. Because repression of CIN85 protein leads to a restored RTK signaling response, our results support an important role of CD2AP/ CIN85 protein balance in the normal signaling response of podocytes. Phosphatidylinositol 3-kinase (PI3K)2 and Ras/ERK mitogen-activated protein kinase signaling pathways are key factors for determining the specificity of cellular responses, including cell proliferation, cell differentiation, and cell survival (1, 2). We and others have previously demonstrated that the PI3K/AKT signaling response plays an important role for podocyte survival in particular in the presence of active transforming growth factor  (3, 4). We recently demonstrated that the PI3K/AKT response is directly targeted by cytokine cross-talk and subsequently influences the cellular outcome (5). The adaptor molecules CD2-associated protein (CD2AP) and CIN85 belong to a family of adaptor molecules that selectively control the spatial and temporal assembly of multiprotein complexes that transmit intracellular signals. For both molecules various interaction partners have been described placing them at the center of regulatory pathways involving signaling (6, 7), cytoskeletal arrangement (8, 9), vesicular trafficking (10), and endocytosis (11,12). In this study we demonstrate that CD2AP and CIN85 contribute to the balance of RTK signaling in podocytes. The anatomical localization of the podocyte exposes this highly specialized cell type to a variety of cellular stressors like stretch force, reactive oxygen species, osmotic milieu changes, cytokines and chemokines, filtrated toxins, and waste products. Therefore, this location requires the cells to have a highly e...
Podocyte apoptosis initiates progressive glomerulosclerosis in TGF-1 transgenic and CD2AP-knockout (CD2AP؊/؊) mice. It was previously shown that in both mouse models, activation of the TGF- pathway is the key event during development of podocyte apoptosis. Furthermore, CD2AP is an important modifier of TGF--induced survival signaling via activation of the phosphoinositol 3-kinase/AKT signaling pathway. This article presents IGF-binding protein-3 (IGFBP-3) as a new modulator of apoptosis and survival signaling in glomerular podocytes. High expression of IGFBP-3 protein in the urine of diseased CD2AP؊/؊ mice was discovered, and IGFBP-3 expression in glomerular podocytes and parietal cells was detected. IGFBP-3 can induce changes in podocyte actin cytoskeleton, leads to apoptosis in cultured murine podocytes, and can enhance TGF-1-induced apoptosis in vitro. For studying this process on a molecular level, proapoptotic p38 mitogen-activated protein kinase pathways and antiapoptotic phosphoinositol 3-kinase/AKT pathways were examined in cultured murine podocytes. It was found that IGFBP-3 increments the level of TGF-1-induced phosphorylated p38 mitogen-activated protein kinase and decreases the phosphorylation of antiapoptotic AKT. This effect is specific for the co-stimulation of IGFBP-3 with TGF-1 because a combination of IGFBP-3 with bone morphogenic protein-7 (BMP-7), another member of the TGF- superfamily, results in apoptosis opposing signaling effects with a strong increase of phosphorylated AKT and subsequent functional effects. These results demonstrate that the IGF/IGFBP axis plays an important role in the development of podocyte apoptosis by modulation of TGF- and BMP-7-induced pro-and antiapoptotic signals.
There is keen interest in the role of the isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) in protein prenylation and cell function in Alzheimer's disease (AD). We recently reported elevated FPP and GGPP brain levels and increased gene expression of FPP synthase (FPPS) and GGPP synthase (GGPPS) in the frontal cortex of AD patients. Cholesterol levels and gene expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were similar in AD and control samples, suggesting that homeostasis of FPP and GGPP but not cholesterol is specifically targeted in brain tissue of AD patients (Neurobiol Dis 2009 35:251-257). In the present study, it was determined if cellular levels of FPP, GGPP, and cholesterol affect beta-amyloid (Abeta) abundance in SH-SY5Y cells, expressing human APP695. Cells were treated with different inhibitors of the mevalonate/isoprenoid/cholesterol pathway. FPP, GGPP, cholesterol, and Abeta(1-40) levels were determined, and activities of farnesyltransferase and geranylgeranyltransferase I were measured. Inhibitors of different branches of the mevalonate/isoprenoid/cholesterol pathway as expected reduced cholesterol and isoprenoid levels in neuroblastoma cells. Abeta(1-40) levels were selectively reduced by cholesterol synthesis inhibitors but not by inhibitors of protein isoprenylation, indicating that changes in cholesterol levels per se and not isoprenoid levels account for the observed modifications in Abeta production.
Progressive tubulointerstitial fibrosis is the common end point leading to end-stage renal disease in experimental and clinical settings. Since the peptide hormone leptin is involved not only in the regulation of obesity but also in the regulation of inflammation and fibrosis, we tested the hypothesis whether leptin deficiency has an impact on tubulointerstitial fibrosis in mice. Leptin-deficient (ob/ob) and leptin receptor-deficient mice (db/db) were exposed to 14 days of unilateral ureteral obstruction (UUO). The degree of fibrosis and inflammation was compared with that in sham-operated mice by performing immunohistochemistry, quantitative PCR, and Western blotting. We found that tubulointerstitial fibrosis was significantly reduced in the obstructed kidneys of ob/ob compared with db/db mice or control mice. Detailed analysis of infiltrating inflammatory cells by immunohistochemistry revealed a significant reduction of CD4(+) cells at 14 days after UUO in both ob/ob and db/db mice. In contrast, we could not detect significant differences in CD8(+) cells and macrophage content. Transforming growth factor (TGF)-beta mRNA levels, TGF-beta-induced Smad-2/3 activation, and the upregulation of downstream target genes were significantly reduced in ob/ob mice. In addition, we demonstrated that leptin could enhance TGF-beta signaling in normal rat kidney fibroblasts in vitro. We conclude that leptin can serve as a cofactor of TGF-beta activation and thus plays an important role in renal tubulointerstitial fibrosis. Therefore, selective blockade of the leptin axis might provide a therapeutic possibility to prevent or delay fibrotic kidney disease.
The glomerular microenvironment is influenced by circulating growth factors that are filtered from the blood stream and pass the glomerular filtration barrier. In this study, we wanted to explore the role of IGF-binding proteins (IGFBPs) in two diseases that concern podocytes. We analyzed glomerular expression and urinary excretion of IGFBP-1, -2, and -3 in patients with focal segmental glomerulosclerosis (FSGS) or minimal change disease (MCD). We found that patients with active FSGS excrete high amounts of podocalyxin positive cells as well as IGFBP-1 and -3. In human podocytes, we can induce mRNA expression of IGFBP-3 in response to TGF-beta and in human microvascular endothelial cells expression of IGFBP-1 and -3 in response to TGF-beta and Bradykinin. We conclude that the local expression of IGFBPs in podocytes and endothelial cells might contribute to the pathogenesis of glomerular disease and that IGFBP-1 and -3 are potential non-invasive markers of FSGS.
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