Severe elastolysis in hereditary gelsolin (AGel) amyloidosis.

AGel amyloidosis is a dominantly inherited systemic amyloidosis caused by mutations p.D214N or p.D214Y resulting in gelsolin amyloid (AGel) formation. AGel accumulates extracellularly in many tissues and alongside elastic fibres. AGel deposition associates with elastic fibre degradation leading to severe clinical manifestations, such as cutis laxa and angiopathic complications. We analysed elastic fibre pathology in dermal and vascular tissue and plasma samples from 35 patients with AGel amyloidosis and 40 control subjects by transmission electron microscopy, immunohistochemistry and ELISA methods. To clarify the pathomechanism(s) of AGel-related elastolysis, we studied the roles of MMP-2, -7, -9, -12 and -14, TIMP-1 and TGFß. We found massive accumulation of amyloid fibrils along elastic fibres as well as fragmentation and loss of elastic fibres in all dermal and vascular samples of AGel patients. Fibrils of distinct types formed fibrous matrix. The degradation pattern of elastic fibres in AGel patients was different from the age-related degradation in controls. The elastin of elastic fibres in AGel patients was strongly decreased compared to controls. MMP-9 was expressed at lower and TGFß at higher levels in AGel patients than in controls. The accumulation of amyloid fibrils with severe elastolysis characterises both dermal and vascular derangement in AGel amyloidosis.

Gelsolin amyloid angiopathy causes severe disruption of the arterial wall.

Hereditary gelsolin amyloidosis (HGA) is a dominantly inherited systemic disease reported worldwide. HGA is characterized by ophthalmological, neurological, and dermatological manifestations. AGel amyloid accumulates at basal lamina of epithelial and muscle cells, thus amyloid angiopathy is encountered in nearly every organ. HGA patients have cardiovascular, hemorrhagic, and potentially vascularly induced neurological problems. To clarify pathomechanisms of AGel angiopathy, we performed histological, immunohistochemical, and electron microscopic analyses on facial temporal artery branches from 8 HGA patients and 13 control subjects. We demonstrate major pathological changes in arteries: disruption of the tunica media, disorganization of vascular smooth muscle cells, and accumulation of AGel fibrils in arterial walls, where they associate with the lamina elastica interna, which becomes fragmented and diminished. We also provide evidence of abnormal accumulation and localization of collagen types I and III and an increase of collagen type I degradation product in the tunica media. Vascular smooth muscle cells appear to be morphologically and semi-quantitatively normal, only their basal lamina is often thickened. In conclusion, angiopathy in HGA results in severe disruption of arterial walls, characterized by prominent AGel deposition, collagen derangement and severe elastolysis, and it may be responsible for several, particularly hemorrhagic, disease manifestations in HGA.

Ezrin is down-regulated in diabetic kidney glomeruli and regulates actin reorganization and glucose uptake via GLUT1 in cultured podocytes.

Diabetic nephropathy is a complication of diabetes and a major cause of end-stage renal disease. To characterize the early pathophysiological mechanisms leading to glomerular podocyte injury in diabetic nephropathy, we performed quantitative proteomic profiling of glomeruli isolated from rats with streptozotocin-induced diabetes and controls. Fluorescence-based two-dimensional difference gel electrophoresis, coupled with mass spectrometry, identified 29 differentially expressed spots, including actin-binding protein ezrin and its interaction partner, NHERF2, which were down-regulated in the streptozotocin group. Knockdown of ezrin by siRNA in cultured podocytes increased glucose uptake compared with control siRNA-transfected cells, apparently by increasing translocation of glucose transporter GLUT1 to the plasma membrane. Knockdown of ezrin also induced actin remodeling under basal conditions, but reduced insulin-stimulated actin reorganization. Ezrin-dependent actin remodeling involved cofilin-1 that is essential for the turnover and reorganization of actin filaments. Phosphorylated, inactive cofilin-1 was up-regulated in diabetic glomeruli, suggesting altered actin dynamics. Furthermore, IHC analysis revealed reduced expression of ezrin in the podocytes of patients with diabetes. Our findings suggest that ezrin may play a role in the development of the renal complication in diabetes by regulating transport of glucose and organization of the actin cytoskeleton in podocytes.

Agonist occupancy is essential for forward trafficking of AMPA receptors.

Regulated trafficking of AMPA receptors to cell surface and to synapses is an important determinant of neuronal excitability. In the present study, we have addressed the role of agonist binding and desensitization in the early trafficking of glutamate receptor-D (GluR-D) AMPA receptors. Analysis of point-mutated GluR-D receptors, via electrophysiology and immunofluorescence, revealed that agonist-binding activity is essential for efficient delivery to cell surface in transfected cell lines and in neurons. Cotransfection with stargazin could fully rescue the surface expression of nonbinding mutant receptors in cell lines, indicating that stargazin is able to interact with and promote exit of AMPA receptors from endoplasmic reticulum (ER) independently of agonist binding. Secretion of separately expressed ligand-binding domain constructs showed a similar dependency of agonist binding to that observed with full-length GluR-D, supporting the idea that glutamate-induced closure of the binding site cleft is registered by ER quality control as a necessary priming step for transport competence. In contrast to agonist binding, the ability of the receptor to undergo desensitization had only a minor influence on trafficking. Our results are consistent with the hypothesis that AMPA receptors are synthesized as intrinsically unstable molecules, which require glutamate binding for structural stability and for transport-competence.