Heparin promotes fibril formation by the N-terminal fragment of amyloidogenic apolipoprotein A-I.

Glycosaminoglycans are known to be associated with extracellular amyloid deposits of various amyloidogenic proteins. In this study, we found that the glycosaminoglycan heparin greatly accelerates the elongation step in fibril formation by the N-terminal 1-83 fragment of human apolipoprotein A-I (apoA-I), especially in the amyloidogenic W50R variant. Using fragment peptides, we demonstrate that heparin significantly promotes ß-transition and fibril formation of the highly amyloidogenic region spanning residues 44-65 and colocalizes with fibrils formed by the W50R variant. These results suggest the possible role of glycosaminoglycans in fibril formation by amyloidogenic apoA-I variants.

Iowa Mutant Apolipoprotein A-I (ApoA-IIowa) Fibrils Target Lysosomes.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-IIowa) is the first mutation that was associated with familial AApoA1 amyloidosis. The N-terminal fragments (amino acid residues 1-83) of apoA-I containing this mutation deposit as amyloid fibrils in patients' tissues and organs, but the mechanisms of cellular degradation and cytotoxicity have not yet been clarified. In this study, we demonstrated degradation of apoA-IIowa fibrils via the autophagy-lysosomal pathway in human embryonic kidney 293 cells. ApoA-IIowa fibrils induced an increase in lysosomal pH and the cytosolic release of the toxic lysosomal protease cathepsin B. The mitochondrial dysfunction caused by apoA-IIowa fibrils depended on cathepsin B and was ameliorated by increasing the degradation of apoA-IIowa fibrils. Thus, although apoA-IIowa fibril transport to lysosomes and fibril degradation in lysosomes may have occurred, the presence of an excess number of apoA-IIowa fibrils, more than the lysosomes could degrade, may be detrimental to cells. Our results thus provide evidence that the target of apoA-IIowa fibrils is lysosomes, and we thereby gained a novel insight into the mechanism of AApoA1 amyloidosis.

Enthalpy-driven interactions with sulfated glycosaminoglycans promote cell membrane penetration of arginine peptides.

The first step of cell membrane penetration of arginine peptides is thought to occur via electrostatic interactions between positive charges of arginine residues and negative charges of sulfated glycosaminoglycans (GAGs) on the cell surface. However, the molecular interaction of arginine peptides with GAG still remains unclear. Here, we compared the interactions of several arginine peptides of Tat, R8, and Rev and their analogues with heparin in relation to the cell membrane penetration efficiency. The high-affinity binding of arginine peptides to heparin was shown to be driven by large favorable enthalpy contributions, possibly reflecting multidentate hydrogen bondings of arginine residues with sulfate groups of heparin. Interestingly, the lysine peptides in which all arginine residues are substituted with lysine residues exhibited negligible binding enthalpy despite of their considerable binding to heparin. In CHO-K1 cells, arginine peptides exhibited a great cell-penetrating ability whereas their corresponding lysine peptides did not penetrate into cells. The degree of cell penetration of arginine peptides markedly decreased by the chlorate treatment of cells which prevents the sulfation of GAG chains. Significantly, the cell penetration efficiency of arginine peptides was found to be correlated with the favorable enthalpy of binding to heparin. These results suggest that the enthalpy-driven strong interaction with sulfated GAGs such as heparan sulfate plays a critical role in the efficient cell membrane penetration of arginine peptides.

CUL2-mediated clearance of misfolded TDP-43 is paradoxically affected by VHL in oligodendrocytes in ALS.

The molecular machinery responsible for cytosolic accumulation of misfolded TDP-43 in amyotrophic lateral sclerosis (ALS) remains elusive. Here we identified a cullin-2 (CUL2) RING complex as a novel ubiquitin ligase for fragmented forms of TDP-43. The von Hippel Lindau protein (VHL), a substrate binding component of the complex, preferentially recognized misfolded TDP-43 at Glu246 in RNA-recognition motif 2. Recombinant full-length TDP-43 was structurally fragile and readily cleaved, suggesting that misfolded TDP-43 is cleared by VHL/CUL2 in a step-wise manner via fragmentation. Surprisingly, excess VHL stabilized and led to inclusion formation of TDP-43, as well as mutant SOD1, at the juxtanuclear protein quality control center. Moreover, TDP-43 knockdown elevated VHL expression in cultured cells, implying an aberrant interaction between VHL and mislocalized TDP-43 in ALS. Finally, cytoplasmic inclusions especially in oligodendrocytes in ALS spinal cords were immunoreactive to both phosphorylated TDP-43 and VHL. Thus, our results suggest that an imbalance in VHL and CUL2 may underlie oligodendrocyte dysfunction in ALS, and highlight CUL2 E3 ligase emerges as a novel therapeutic potential for ALS.

The polyphenol (-)-epigallocatechin-3-gallate prevents apoA-IIowa amyloidosis in vitro and protects human embryonic kidney 293 cells against amyloid cytotoxicity.

INTRODUCTION: Apolipoprotein A-I (apoA-I) amyloidosis is either a non-hereditary form with deposits of wild-type apoA-I proteins in atherosclerotic plaques or a hereditary form with progressive accumulation of mutant apoA-I proteins in different tissues. Several small polyphenolic molecules reportedly inhibited formation of fibrillar assemblies of some amyloidogenic proteins and their cytotoxicity, but small molecules that inhibit apoA-I fibril formation have never been reported. METHODS: Our methods included a thioflavin-T-binding assay, atomic force microscopy and dot blot and cell-based assays. RESULTS: We showed that (-)-epigallocatechin-3-gallate (EGCG), a tea-derived flavanol, inhibited in vitro fibril formation and disaggregated fibrils preformed by the N-terminal 1-83 fragments of wild-type (WT) apoA-I and the G26R point mutation of apoA-I (apoA-IIowa). We eliminated a common structure recognized by the anti-amyloid antibody OC by incubating apoA-IIowa with EGCG or treating apoA-IIowa fibrils with EGCG, which supported the above observation. In addition, EGCG rescued human embryonic kidney 293 cells from cytotoxicity and attenuated production of reactive oxygen species, which were induced by apoA-IIowa fibrils. CONCLUSIONS: Our results support the concept that EGCG inhibits amyloid fibril formation of various amyloidogenic proteins. Thus, EGCG may be a candidate for providing a structure to develop de novo inhibitors for amyloidosis treatment.

K604, a specific acyl­CoA:cholesterol acyltransferase 1 inhibitor, suppresses proliferation of U251­MG glioblastoma cells.

Glioblastoma is the most aggressive type of brain tumor and has a poor prognosis. Increased levels of cholesteryl ester and simultaneous expression of acyl­CoA:cholesterol acyltransferase 1 (ACAT1) in tumor cells indicated that cholesterol esterification is critical to tumor growth. The present study confirmed that human glioblastoma tissues as well as the glioblastoma cell line U251­MG showed significant expression of ACAT1. ACAT1 expression in U251­MG cells increased in a cell proliferation­dependent manner. K604, a selective ACAT1 inhibitor, suppressed the proliferation of U251­MG cells and downregulated the activation of Akt and extracellular signal­regulated kinase in proliferating glioblastoma cells. These results suggested that ACAT1 may be a therapeutic target for the treatment of glioblastoma, with K604 as an effective therapeutic agent.

Cellular interaction and cytotoxicity of the iowa mutation of apolipoprotein A-I (ApoA-IIowa) amyloid mediated by sulfate moieties of heparan sulfate.

The single amino acid mutation G26R in human apolipoprotein A-I (apoA-I) is associated with familial amyloid polyneuropathy III. ApoA-I carrying this mutation (apoA-IIowa) forms amyloid fibrils in vitro. Heparan sulfate (HS) is a glycosaminoglycan that is abundant at the cell surface and in the extracellular matrix. Although HS and its highly sulfated domains are involved in aggregation of amyloid-ß and accumulate in cerebral amyloid plaques of patients with Alzheimer disease and mouse models of this disease, the role of HS in familial amyloid polyneuropathy III has never been addressed. Here, we used cell models to investigate the possible role of HS in the cytotoxicity of apoA-IIowa amyloid. Wild-type CHO cells, but not pgsD-677 cells, an HS-deficient CHO mutant, demonstrated uptake of apoA-IIowa amyloid after incubation with the amyloid. Addition of sulfated glycosaminoglycans to culture media prevented interaction with and cytotoxicity of apoA-IIowa amyloid to CHO cells. Elimination of cell surface HS or inhibition of HS sulfation with chemical reagents interfered with interaction of apoA-IIowa amyloid with CHO cells. We also found that cellular interaction and cytotoxicity of apoA-IIowa amyloid were significantly attenuated in CHO cells that stably expressed the human extracellular endoglucosamine 6-sulfatases HSulf-1 and HSulf-2. Our results thus suggest that cell surface HS mediates cytotoxicity of apoA-IIowa amyloid and that enzymatic remodeling of HS mitigates the cytotoxicity.

CD44-expressing undifferentiated carcinoma with rhabdoid features of the pancreas: molecular analysis of aggressive invasion and metastasis.

Carcinoma with rhabdoid features is a rare malignant tumor with a poor prognosis whose molecular mechanism for aggressive behavior is unclear. We describe an undifferentiated pancreatic carcinoma with rhabdoid features that demonstrated extensive invasion and metastasis. Examination of a 63-year-old man with back pain disclosed a retroperitoneal tumor with multiple metastases. Lymph node biopsy revealed an undifferentiated carcinoma of unknown origin. Intensive chemotherapy was ineffective; the patient died 3 months after initial symptoms. Autopsy showed that the tumor displaced the retroperitoneal space: it diffusely invaded and destroyed the pancreas and duodenum. Histology demonstrated tumor cells with eccentric vesicular nuclei, large nucleoli, juxtanuclear eosinophilic inclusions, and poor cell adhesion. Immunohistochemistry showed that tumor cells expressed cytokeratin and vimentin, and electron microscopy confirmed a perinuclear mass of intermediate fibrils and lipid droplets, which indicated an undifferentiated carcinoma with rhabdoid features. Tumor tissue contained hyaluronan; tumor cells strongly expressed CD44, matrix metalloproteinase-9, hypoxia-inducible factor-1α, hyaluronan synthase 2, and acyl-CoA:cholesterol acyltransferase 1 and had a high Ki-67(+) ratio. Since hyaluronan is a ligand for CD44, formation of CD44-hyaluronan complex on the cell surface activates CD44 and this activation may explain why the tumor manifested aggressive invasion and metastasis throughout the clinical course.

ACAT1-associated Late Endosomes/Lysosomes Significantly Improve Impaired Intracellular Cholesterol Metabolism and the Survival of Niemann-Pick Type C Mice.

We previously demonstrated that macrophages exhibit endoplasmic reticulum fragmentation under cholesterol-rich conditions, which results in the generation of acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1)-associated late endosomes/lysosomes (ACAT1-LE). ACAT1-LE efficiently esterify free cholesterol in loco, even with abnormal egress of free cholesterol from late endosomes. Because impaired free cholesterol transport from late endosomes results in Niemann-Pick type C disease (NPC), the induction of ACAT1-LE is a potential therapeutic intervention for NPC. To examine the effects of ACAT1-LE induction on intracellular cholesterol metabolism, we incubated bone marrow-derived macrophages possessing NPC phenotype (npc1 (-/-)) with methyl-ß-cyclodextrin-cholesterol complex (mßCD-cho), a cholesterol donor. Immunofluorescence confocal microscopy revealed that mßCD-cho treatment of npc1 (-/-) macrophages resulted in significant colocalization of signals from ACAT1 and lysosome-associated membrane protein 2, a late endosome/lysosome marker. npc1 (-/-) macrophages contained significant amounts of free cholesterol with negligible amounts of cholesteryl ester, while wild-type macrophages possessed the same amounts of both cholesterols. mßCD-cho treatment also induced marked restoration of cholesterol esterification activity. mßCD-cho administration in neonate npc1 (-/-) mice improved survival. These results indicate that ACAT1-LE induction in npc1 (-/-) mice corrects impaired intracellular cholesterol metabolism and that restoring cholesterol esterification improves prognosis of npc1 (-/-). These data suggest that ACAT1-LE induction is a potential alternative therapeutic strategy for NPC.

Role of ACAT1-positive late endosomes in macrophages: cholesterol metabolism and therapeutic applications for Niemann-Pick disease type C.

Macrophages in hyperlipidemic conditions accumulate cholesterol esters and develop into foamy transformed macrophages. During this transformation, macrophages demonstrate endoplasmic reticulum fragmentation and consequently produce acyl coenzyme A: cholesterol acyltransferase 1 (ACAT1)-positive late endosomes (ACAT1-LE). ACAT1-LE-positive macrophages effectively esterify modified or native low-density lipoprotein-derived free cholesterol, which results in efficient cholesterol esterification as well as atherosclerotic plaque formation. These macrophages show significant cholesterol ester formation even when free cholesterol egress from late endosomes is impaired, which indicates that free cholesterol is esterified at ACAT1-LE. Genetic blockade of cholesterol egress from late endosomes causes Niemann-Pick disease type C (NPC), an inherited lysosomal storage disease with progressive neurodegeneration. Induction of ACAT1-LE in macrophages with the NPC phenotype led to significant recovery of cholesterol esterification. In addition, in vivo ACAT1-LE induction significantly extended the lifespan of mice with the NPC phenotype. Thus, ACAT1-LE not only regulates intracellular cholesterol metabolism but also ameliorates NPC pathophysiology.

Concomitant accumulation of α-synuclein and TDP-43 in a patient with corticobasal degeneration.

Pathological changes in corticobasal degeneration (CBD) consist of abnormal deposition of the microtubule-associated protein tau. However, the simultaneous accumulation of different misfolded proteins in the brain can be observed in many neurodegenerative diseases with significantly longer disease durations. We encountered a patient with CBD who survived for an extremely long period (18 years) after the diagnosis. We performed an autopsy to elucidate the effect of the longer survival on the pathology of CBD. We observed abnormal aggregation of trans-activating response region DNA-binding protein of 43 kDa (TDP-43) and α-synuclein, as well as phosphorylated tau, in neurons of broader regions of the brain, beyond the amygdala and other limbic areas. We found that phosphorylated tau, α-synuclein, and TDP-43 partially co-existed in the same cellular aggregates. The triple pathologic changes might be related to the longer survival of the patient compared with the typical clinical course of patients with CBD. Further investigations are required to support the hypothesis that tauopathy, synucleinopathy, and TDP-43 proteinopathy might share common pathogenic mechanisms in terms of cross-seeding of the pathologic proteins.

Marked cardiomegaly in a patient with familial amyloidotic polyneuropathy after orthotopic liver transplantation: a case study.

Hepatocyte-derived mutant amyloidogenic transthyretin (ATTR) causes familial amyloidotic polyneuropathy (FAP), for which orthotopic liver transplantation is an established curative treatment. However, some patients with FAP have cardiac amyloidosis after transplantation. Here, we describe a man with an autonomic disorder diagnosed as FAP ATTR Val30Met and marked cardiomegaly after liver transplantation. He underwent orthotopic liver transplantation at 49 years of age and was prescribed prednisolone to prevent graft rejection. Two years later, autonomic dysfunction and severe heart failure gradually developed. He died suddenly at 59. The autopsy revealed marked cardiomegaly (heart weight: 1020 g). Histological and ultrastructural examinations demonstrated massive amyloid deposition and unusual myocardial hypertrophic injury associated with nuclear translocation of the glucocorticoid receptor (GR). No other FAP patients without heart failure showed GR nuclear translocation. GR is a nuclear transcription factor that leads to myocardial hypertrophy, and cumulative prednisolone doses may promote marked cardiomegaly and severe cardiac amyloidosis.

Thrombospondin-1 is highly expressed in desmoplastic components of invasive ductal carcinoma of the breast and associated with lymph node metastasis.

Desmoplastic (scirrhous) invasion and lymph node metastasis are critical for the treatment and prognosis of invasive ductal carcinoma of the breast. Despite being an anti-angiogenic therapeutic candidate, Thrombospondin-1 (TSP-1) promotes invasion and metastasis of some carcinomas. To clarify the effect of TSP-1 on invasion and metastasis, we obtained 101 invasive ductal carcinomas of the breast with axillary lymph node resection. All tumors were histologically divided into two categories, carcinomas with, and those with non- /minimal desmoplastic component. Immunohistochemistry for TSP-1 was performed on all primary tumors and axillary lymph nodes with tumor metastasis. Fifty-four (53.5%) of 101 tumors were recognized as positive for TSP-1 in the cytoplasm of tumor cells. Histological study showed that significantly more cancers with desmoplastic components (46/69, 66.7%) manifested TSP-1 expression than did cancers with no- or minimal (less than 20%) desmoplasia (8/32, 25.0%; p<0.001). Axillary lymph node metastasis was significantly higher in TSP-1-positive- (28/54, 51.9%) than TSP-1-negative cancers (11/47, 23.4%; p<0.005). The present study indicates that tumor cells in the desmoplastic component strongly expressed TSP-1 in invasive ductal carcinoma of the breast and TSP-1 participates in invasion of these tumors. Our findings also suggest that TSP-1 promotes lymph node metastasis and TSP-1 potentially could be a predictive marker for metastasis.

Imprint cytological feature of large cell neuroendocrine carcinoma of the gallbladder: a case report.

Large cell neuroendocrine carcinoma (LCNEC) is a rare poorly differentiated carcinoma with neuroendocrine differentiation showing aggressive clinical behavior. We herein report a case of gallbladder LCNEC, which was difficult to differentiate from poorly differentiated adenocarcinoma. An imprint cytology was very useful for the final diagnosis in this case. A 56-year-old male with left exophthalmos was admitted to the hospital. Radiological examinations revealed the presence of a left gallbladder tumor with orbital metastasis. The histological diagnosis was poorly differentiated adenocarcinoma, and intensive chemoradiotherapy was administered. Unfortunately, the patient died of extensive metastases 36 months after the initial onset of symptoms. An autopsy revealed a tumor mass in the gallbladder associated with multiple liver and peritoneal metastases. Imprint cytology of the main tumor revealed cytological features of LCNEC, and additional histological examinations confirmed this diagnosis. Although performing a histological examination is important for making a final diagnosis, imprint cytology is powerful tool for differential diagnosis of LCNEC, especially in patients with carcinoma with poor differentiation.

Potential association of Helicobacter cinaedi with atrial arrhythmias and atherosclerosis.

Helicobacter cinaedi has been increasingly recognized as an emerging pathogen. Reports of recurrent bacteremia and isolation of H. cinaedi organisms from a patient with myopericarditis led us to postulate that H. cinaedi is associated with chronic inflammatory cardiovascular diseases such as atrial arrhythmias and atherosclerosis. To assess any association of H. cinaedi with atrial arrhythmias, a retrospective case-control study of patients attending Kumamoto University Hospital from 2005 to 2009 was performed. The arrhythmia status of these patients was determined from their electrocardiography and electrophysiological studies. Multiple logistic regression analysis was used to identify independent risk factors. In a comparison of case patients (n= 132) with control subjects (n= 137), H. cinaedi seropositivity was identified as an independent risk factor for atrial arrhythmia (odds ratio, 5.13; 95% confidence interval, 3.0-8.7; P < 0.001). There were no significant differences, however, between these two groups with respect to anti-H. pylori IgG concentrations, anti-Chlamydophila pneumoniae IgG concentrations, and other studied variables. IgG concentrations against H. cinaedi and H. pylori were inversely correlated, which suggests cross-immunity between these two bacteria. Also, to explore any association of H. cinaedi with atherosclerosis, immunohistochemical analysis of atherosclerotic aortic tissues collected post mortem from nine patients was performed. Immunohistochemistry of atherosclerotic aortic tissues from all nine patients detected H. cinaedi antigens inside CD68(+) macrophages. These findings provide the first evidence, to our knowledge, of a possible association of H. cinaedi with atrial arrhythmias and atherosclerosis.

Partial impairment of c-Ret at tyrosine 1062 accelerates age-related hearing loss in mice.

c-Ret has been shown to be crucial for neural development and survival. We have recently shown that complete impairment of tyrosine 1062 (Y1062)-phosphorylation in c-Ret causes congenital hearing loss with neurodegeneration of spiral ganglion neurons (SGNs) in homozygous c-Ret knockin mice (c-Ret-KI(Y1062F/Y1062F)-mice). However, there is no information to link c-Ret and age-related hearing loss. Here we show that partial impairment of Y1062-phosphorylation in c-Ret accelerates age-related hearing loss in heterozygous c-Ret Y1062F knockin mice (c-Ret-KI(Y1062F/+)-mice). In contrast, complete impairment of serine 697 (S697)-phosphorylation in c-Ret did not affect hearing levels in 10-month-old homozygous c-Ret S697A knockin mice (c-Ret-KI(S697A/S697A)-mice). The hearing loss involved late-onset neurodegeneration of spiral ganglion neurons in c-Ret-KI(Y1062F/+)-mice. Morphological abnormalities in inner- and outer-hair cells and the stria vascularis in c-Ret-KI(Y1062F/+)-mice were undetectable. The acceleration of age-related hearing loss in c-Ret-KI(Y1062F/+)-mice was rescued by introducing constitutively activated RET. Thus, our results suggest that c-Ret is a novel age-related hearing loss-related molecule in mice. Our results suggest that these hearing losses partially share a common pathogenesis that is monogenetically caused by a single point mutation (Y1062F) in c-Ret.

Development and characterization of an animal model of severe pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) is a serious pathological phenomenon with poor prognosis, which is associated with morphological as well as hemodynamic alteration of the pulmonary circulation. To establish an animal model mimicking severe human PAH, we combined 2 well-described procedures, i.e. exposure to hypobaric chronic hypoxia and administration of monocrotaline hydrochloride in rats. Compared to a single procedure, the combined procedure induced more severe right ventricle hypertrophy and an increase in right ventricle systolic pressure. Histological examination on the combined procedure model revealed a severe medial hypertrophy as well as occlusive vascular changes of the intra-acinar pulmonary arteries with endothelial lesions. It is noteworthy that severe alterations including concentric neointimal thickening, abnormal endothelial proliferation, plexiform lesions and vascular occlusion with fibrin thrombi were observed in the combined pulmonary hypertension model when exposed to a long period of hypoxia. The present data indicate that a combined treatment of monocrotaline injection and hypobaric chronic hypoxia exposure produces more severe hemodynamic changes and histological alterations. Since human PAH diagnosed in clinical practice is often severe, this combined treatment animal model could be useful to identify relevant therapeutic targets acting on both hemodynamic and structural alterations of the pulmonary circulation.

Suppression of TLR4-mediated inflammatory response by macrophage class A scavenger receptor (CD204).

The class A scavenger receptor (SR-A, CD204), one of the principal receptors expressed on macrophages, has been found to regulate inflammatory response and attenuate septic endotoxemia. However, the detailed mechanism of this process has not yet been well characterized. To clarify the regulative mechanisms of lipopolysaccharide (LPS)-induced macrophage activation by SR-A, we evaluated the activation of Toll-like receptor 4 (TLR4)-mediated signaling molecules in SR-A-deficient (SR-A(-/-)) macrophages. In a septic shock model, the blood levels of tumor necrosis factor (TNF)-α, interleukin (IL)-6 and interferon (IFN)-ß were significantly increased in SR-A(-/-) mice compared to wild-type mice, and elevated nuclear factor kappa B (NFκB) activation was detected in SR-A(-/-) macrophages. SR-A deletion increased the production of pro-inflammatory cytokines, and the phosphorylation of mitogen-activated protein kinase (MAPK) and NFκB in vitro. SR-A deletion also promoted the nuclear translocation of NFκB and IFN regulatory factor (IRF)-3. In addition, a competitive binding assay with acetylated low-density lipoprotein, an SR-A-specific ligand, and anti-SR-A antibody induced significant activation of TLR4-mediated signaling molecules in wild-type macrophages but not in SR-A(-/-) macrophages. These results suggest that SR-A suppresses the macrophage activation by inhibiting the binding of LPS to TLR4 in a competitive manner and it plays a pivotal role in the regulation of the LPS-induced inflammatory response.