Secretion Of Amyloid Research Articles

Serum Amyloid P Secreted by Bone Marrow Adipocytes Drives Skeletal Amyloidosis.

The accumulation of amyloid fibrils has been identified in tissues outside the brain, yet little is understood about the formation of extracerebral amyloidosis and its impact on the aging process of these organs. Here, we demonstrate that both transgenic mice modeling Alzheimer's disease (AD) and naturally aging mice exhibit accumulated senescent bone marrow adipocytes (BMAds), accompanied by amyloid deposits surrounding the BMAds. Senescent BMAds acquire a secretory phenotype, resulting in a marked increase in the secretion of serum amyloid P component (SAP), also known as pentraxin 2 (PTX2). SAP/PTX2 colocalizes with amyloid deposits around senescent BMAds in vivo and is sufficient to promote the formation of insoluble amyloid deposits from soluble Aβ peptides in in vitro and ex vivo 3D BMAd-based culture experiments. Additionally, Combined treatment with SAP/PTX2 and Aβ peptides promotes osteoclastogenesis but inhibits osteoblastogenesis of the precursor cells. Transplantation of senescent BMAds into the bone marrow cavity of healthy young mice is sufficient to induce bone loss. Finally, pharmacological depletion of SAP/PTX2 from aged mice abolishes bone marrow amyloid deposition and effectively rescues the low bone mass phenotype. Thus, senescent BMAds, through the secretion of SAP/PTX2, contribute to the age-associated development of skeletal amyloidosis and resultant bone deficits.

Hepatocytes coordinate immune evasion in cancer via release of serum amyloid A proteins.

T cell infiltration into tumors is a favorable prognostic feature, but most solid tumors lack productive T cell responses. Mechanisms that coordinate T cell exclusion are incompletely understood. Here we identify hepatocyte activation via interleukin-6/STAT3 and secretion of serum amyloid A (SAA) proteins 1 and 2 as important regulators of T cell surveillance of extrahepatic tumors. Loss of STAT3 in hepatocytes or SAA remodeled the tumor microenvironment with infiltration by CD8+ T cells, while interleukin-6 overexpression in hepatocytes and SAA signaling via Toll-like receptor 2 reduced the number of intratumoral dendritic cells and, in doing so, inhibited T cell tumor infiltration. Genetic ablation of SAA enhanced survival after tumor resection in a T cell-dependent manner. Likewise, in individuals with pancreatic ductal adenocarcinoma, long-term survivors after surgery demonstrated lower serum SAA levels than short-term survivors. Taken together, these data define a fundamental link between liver and tumor immunobiology wherein hepatocytes govern productive T cell surveillance in cancer.

Serum amyloid A contributes to radiation-induced lung injury by activating macrophages through FPR2/Rac1/NF-κB pathway.

Patients who receive thoracic radiotherapy may suffer from radiation-induced lung injury, but the treatment options are limited as the underlying mechanisms are unclear. Using a mouse model of right thorax irradiation with fractionated doses of X-rays for three consecutive days (8 Gy/per day), this study found that the thoracic irradiation (Th-IR) induced tissue injury with aberrant infiltration of macrophages, and it significantly increased the secretion of TNF-α, IL-1β, IL-6, TGF-β1 and serum amyloid A (SAA) in mice. Interestingly, SAA could activate macrophages and then induce epithelial-mesenchymal transition (EMT) of lung epithelial cells and fibrosis progression in lung tissue. Mechanistically, SAA enhanced the transient binding of FPR2 to Rac1 protein and further activated NF-κB signaling pathway in macrophages. Inhibition of FPR2 significantly reduced pulmonary fibrosis induced by SAA administration in mice. In addition, cimetidine could reduce the level of SAA release after irradiation and attenuate the lung injury induced by SAA or Th-IR. In conclusion, our results demonstrated that SAA activated macrophages via FPR2/Rac1/NF-κB pathway and might contribute to the Th-IR induced lung injury, which may provide a new strategy to attenuate radiation-induced adverse effects during radiotherapy.

Fecal Microbiota Transplantation: A Microbiome Modulation Technique for Alzheimer's Disease.

Alzheimer’s disease (AD) is the most common form of dementia and the fifth leading cause of death among the elderly. AD involves parts of the brain that can lead to progressive memory loss and impaired language skills and cognitive thinking, affecting one’s ability to carry out daily activities. Aging, bad dietary habits, family history, as well as altered gut microbiota composition may play a role in the pathogenesis of AD. Although the association between the imbalance of gut microbiota and AD is still difficult to determine, it has been suggested that dysbiosis can lead to the increased secretion of lipopolysaccharides and amyloid, which may impair the permeability of the intestine and the blood-brain barrier. Moreover, it can progress the process of neuroinflammation, amyloid-beta formation, and ultimately neuronal death. Microbiota-targeted interventions such as personalized diet, probiotics, or fecal microbiota transplantation (FMT) might represent a potential therapeutic option for AD. This review article discusses the procedure of FMT and its possible side effects on the recipient’s body. In addition, we review the role of FMT in the context of its application in various nervous system-related disorders (AD, Parkinson’s disease, multiple sclerosis).

Serum Health Biomarkers in African and Asian Elephants: Value Ranges and Clinical Values Indicative of the Immune Response.

Simple SummaryBiomarkers are biological molecules found in the blood or other fluids or tissues that can indicate normal or abnormal processes or disease. Developing tools to measure biomarkers that indicate immune function and establishing concentrations observed within a species is an important first step in their use for managing health and understanding disease processes. Here we report assays, observed value ranges, and concentrations during illness or injury for seven immune biomarkers measured in the serum of African and Asian elephants under human care. Concentrations were variable in both clinical and non-clinical samples, but all seven biomarkers were elevated in at least one case and most increased in response to routine vaccination in a single Asian elephant. These tools provide an exciting avenue for monitoring health status and helping diagnose and treat health problems in wildlife species, like elephants.Serum biomarkers indicative of inflammation and disease can provide useful information regarding host immune processes, responses to treatment and prognosis. The aims of this study were to assess the use of commercially available anti-equine reagents for the quantification of cytokines (tumor necrosis factor-alpha (TNF-α), interferon-gamma (IFN-γ), interleukins (IL) 2, 6, and 10) in African (Loxodonta africana, n = 125) and Asian (Elephas maximus, n = 104) elephants, and alongside previously validated anti-human reagents for acute-phase proteins (serum amyloid A and haptoglobin), calculate species-specific biomarker value ranges. In addition, we used opportunistically collected samples to investigate the concentrations of each biomarker during identified clinical cases of illness or injury, as a first step to understanding what biomarkers may be useful to managing elephant health. Immune biomarkers were each elevated above the calculated species-specific value ranges in at least one clinical case, but due to variability in both clinical and non-clinical samples, only serum amyloid A was significantly higher in clinical compared to non-clinical paired samples, with tendencies for higher TNF-α and IL-10. We also detected increased secretion of serum amyloid A and all five cytokines following routine vaccination of a single Asian elephant, indicating that these biomarkers can be beneficial for studying normal immune processes as well as pathology. This study indicates that assays developed with commercial reagents can be used to quantify health biomarkers in wildlife species and identifies several that warrant further investigation to elucidate immune responses to various pathologies.

Motifs and interface amino acid-mediated regulation of amyloid biogenesis in microbes to humans: potential targets for intervention.

Amyloids are linked to many debilitating diseases in mammals. Some organisms produce amyloids that have a functional role in the maintenance of their biological processes. Microbes utilize functional bacterial amyloids (FuBA) for pathogenicity and infections. Amyloid biogenesis is regulated differentially in various systems to avoid its toxic accumulation. A familiar feature in the process of amyloid biogenesis from humans to microbes is its regulation by protein-protein interactions (PPI). The spatial arrangement of amino acid residues in proteins generates topologies like flat interface and linear motif, which participate in protein interactions. Motifs and interface residue-mediated interactions have a direct or an indirect impact on amyloid secretion and assembly. Some motifs undergo post-translational modifications (PTM), which effects interactions and dynamics of the amyloid biogenesis cascade. Interaction-induced local changes stimulate global conformational transitions in the PPI complex, which indirectly affects amyloid formation. Perturbation of such motifs and interface residues results in amyloid abolishment. Interface residues, motifs and their respective interactive protein partners could serve as potential targets for intervention to inhibit amyloid biogenesis.

AMPK activation does not enhance autophagy in neurons in contrast to MTORC1 inhibition: different impact on β-amyloid clearance

ABSTRACT The physiological AKT-MTORC1 and AMPK signaling pathways are considered key nodes in the regulation of anabolism-catabolism, and particularly of macroautophagy/autophagy. Indeed, it is reported that these are altered processes in neurodegenerative proteinopathies such as Alzheimer disease (AD), mainly characterized by deposits of β-amyloid (Aβ) and hyperphosphorylated MAPT. These accumulations disrupt the optimal neuronal proteostasis, and hence, the recovery/enhancement of autophagy has been proposed as a therapeutic approach against these proteinopathies. The purpose of the present study was to characterize the modulation of autophagy by MTORC1 and AMPK signaling pathways in the highly specialized neurons, as well as their repercussions on Aβ production. Using a double transgenic mice model of AD, we demonstrated that MTORC1 inhibition, either in vivo or ex vivo (primary neuronal cultures), was able to reduce amyloid secretion through moderate autophagy induction in neurons. The pharmacological prevention of autophagy in neurons augmented the Aβ secretion and reversed the effect of rapamycin, confirming the anti-amyloidogenic effects of autophagy in neurons. Inhibition of AMPK with compound C generated the expected decrease in autophagy induction, though surprisingly did not increase the Aβ secretion. In contrast, increased activity of AMPK with metformin, AICAR, 2DG, or by gene overexpression did not enhance autophagy but had different effects on Aβ secretion: whereas metformin and 2DG diminished the secreted Aβ levels, AICAR and PRKAA1/AMPK gene overexpression increased them. We conclude that AMPK has a significantly different role in primary neurons than in other reported cells, lacking a direct effect on autophagy-dependent amyloidosis. Abbreviations: 2DG: 2-deoxy-D-glucose; Aβ: β-amyloid; ACACA: acetyl-CoA carboxylase alpha; ACTB: actin beta; AD: Alzheimer disease; AICAR: 5-aminoimidazole-4-carboxamide-1-β-riboside; AKT: AKT kinases group (AKT1 [AKT serine/threonine kinase 1], AKT2 and AKT3); AMPK: adenosine 5‘-monophosphate (AMP)-activated protein kinase; APP: amyloid beta precursor protein; APP/PSEN1: B6.Cg-Tg (APPSwe, PSEN1dE9) 85Dbo/J; ATG: autophagy related; ATP: adenosine triphosphate; BafA1: bafilomycin A1; CA: constitutively active; CGN: cerebellar granule neuron; CoC/compound C: dorsommorphin dihydrochloride; ELISA: enzyme-linked immunosorbent assay; GAPDH: glyceraldehyde-3-phosphate dehydrogenase; GFP: green fluorescent protein; Gmax: GlutaMAX™; IN1: PIK3C3/VPS34-IN1; KI: kinase-inactive; MAP1LC3B/LC3: microtubule associated protein 1 light chain 3; MAPT/TAU: microtubule associated protein tau; Metf: metformin; MRT: MRT68921; MTORC1: mechanistic target of rapamycin kinase complex 1; NBR1: NBR1 autophagy cargo receptor; PRKAA: 5ʹ-AMP-activated protein kinase catalytic subunit alpha; PtdIns3K: phosphatidylinositol 3-kinase; Rapa: rapamycin; RPS6KB1/S6K: ribosomal protein S6 (RPS6) kinase polypeptide 1; SCR: scramble; SQSTM1/p62: sequestosome 1; ULK1/2: unc-51 like autophagy activating kinase 1/2; WT: wild type.

Artificial Inclusion Bodies for Clinical Development

Bacterial inclusion bodies (IBs) are mechanically stable protein particles in the microscale, which behave as robust, slow‐protein‐releasing amyloids. Upon exposure to cultured cells or upon subcutaneous or intratumor injection, these protein materials secrete functional IB polypeptides, functionally mimicking the endocrine release of peptide hormones from secretory amyloid granules. Being appealing as delivery systems for prolonged protein drug release, the development of IBs toward clinical applications is, however, severely constrained by their bacterial origin and by the undefined and protein‐to‐protein, batch‐to‐batch variable composition. In this context, the de novo fabrication of artificial IBs (ArtIBs) by simple, cell‐free physicochemical methods, using pure components at defined amounts is proposed here. By this, the resulting functional protein microparticles are intriguing, chemically defined biomimetic materials that replicate relevant functionalities of natural IBs, including mammalian cell penetration and local or remote release of functional ArtIB‐forming protein. In default of severe regulatory issues, the concept of ArtIBs is proposed as a novel exploitable category of biomaterials for biotechnological and biomedical applications, resulting from simple fabrication and envisaging soft developmental routes to clinics.

Calcilytic NPS 2143 Reduces Amyloid Secretion and Increases sAβPPα Release from PSEN1 Mutant iPSC-Derived Neurons.

Despite numerous efforts and studies over the last three decades, Alzheimer’s disease (AD) remains a disorder not fully understood and incurable so far. Development of induced pluripotent stem cell (iPSC) technology to obtain terminally differentiated neurons from adult somatic cells revolutionized the study of AD, providing a powerful tool for modelling the disease and for screening candidate drugs. Indeed, iPSC reprogramming allowed generation of neurons from both sporadic and familial AD patients with the promise to recapitulate the early pathological mechanisms in vitro and to identify novel targets. Interestingly, NPS 2143, a negative allosteric modulator of the calcium sensing receptor, has been indicated as a possible therapeutic for AD. In the present study, we assessed the potential of our iPSC-based familial AD cellular model as a platform for drug testing. We found that iPSC-derived neurons respond to treatment with γ-secretase inhibitor, modifying the physiological amyloid-β protein precursor (AβPP) processing and amyloid-β (Aβ) secretion. Moreover, we demonstrated the expression of calcium sensing receptor (CaSR) protein in human neurons derived from healthy and familial AD subjects. Finally, we showed that calcilytic NPS 2143 induced a changing of Aβ and sAβPPα secreted into conditioned media and modulation of CaSR and PSEN1 expression at the plasma membrane of AD neurons. Overall, our findings suggest that NPS 2143 affects important AD processes in a relevant in vitro system of familial AD.

NF-E2-related factor 2 activation boosts antioxidant defenses and ameliorates inflammatory and amyloid properties in human Presenilin-1 mutated Alzheimer's disease astrocytes.

Alzheimer's disease (AD) is a common dementia affecting a vast number of individuals and significantly impairing quality of life. Despite extensive research in animal models and numerous promising treatment trials, there is still no curative treatment for AD. Astrocytes, the most common cell type of the central nervous system, have been shown to play a role in the major AD pathologies, including accumulation of amyloid plaques, neuroinflammation, and oxidative stress. Here, we show that inflammatory stimulation leads to metabolic activation of human astrocytes and reduces amyloid secretion. On the other hand, the activation of oxidative metabolism leads to increased reactive oxygen species production especially in AD astrocytes. While healthy astrocytes increase glutathione (GSH) release to protect the cells, Presenilin‐1‐mutated AD patient astrocytes do not. Thus, chronic inflammation is likely to induce oxidative damage in AD astrocytes. Activation of NRF2, the major regulator of cellular antioxidant defenses, encoded by the NFE2L2 gene, poses several beneficial effects on AD astrocytes. We report here that the activation of NRF2 pathway reduces amyloid secretion, normalizes cytokine release, and increases GSH secretion in AD astrocytes. NRF2 induction also activates the metabolism of astrocytes and increases the utilization of glycolysis. Taken together, targeting NRF2 in astrocytes could be a potent therapeutic strategy in AD.

NEURONAL APOE MODULATES A SPORADIC ALZHEIMER’S DISEASE PHENOTYPE IN PATIENT-DERIVED INDUCED NEURONS

The APOE-ε4 allele is the strongest genetic risk factor for sporadic Alzheimer's disease (sAD) and may be enriched in other clinically relevant populations. While APOE is predominantly expressed by astrocytes in the central nervous system, neuronal expression of APOE is of increasing interest in age-related cognitive impairment, neurological injury, and neurodegeneration. The human and mouse APOE promoter sequences are dissimilar, and contain distinct regions that control expression in a species specific, cell-type specific manner. APOE mRNA and protein is detectable in primary human neuronal cultures in a manner suggestive of de novo protein synthesis. However, neuronal expression of APOE has not been linked to neurotoxicity in a human system. We reprogrammed induced pluripotent stem cells (iPSCs) from three unrelated sAD patients with the ε3/ε4 genotype, used CRISPR/Cas9 gene editing to generate ε3/ε3 isogenic lines, and differentiated them into pure cultures of glutamatergic neurons with no detectable glia. APOE is synthesized and secreted by human induced neurons. Genetic correction of ε4 to ε3 reduces tau phosphorylation and phosphoactivation of a specific kinase. Moreover, ε4 is associated with altered amyloid secretion, and exacerbates the neurotoxic effects of a calcium ionophore. APOE is expressed in human glia-free neuronal culture system, and APOE isoform alters sAD-associated molecular pathways. More detailed mechanistic studies are required to better understand this phenomenon. However, the net effect is that endogenous expression of APOE-ε4 weakens neurons and contributes to the sAD cellular phenotype independently of, but likely cooperatively with, glial APOE.

The FapF Amyloid Secretion Transporter Possesses an Atypical Asymmetric Coiled Coil

Gram-negative bacteria possess specialized biogenesis machineries that facilitate the export of amyloid subunits, the fibers of which are key components of their biofilm matrix. The secretion of bacterial functional amyloid requires a specialized outer-membrane protein channel through which unfolded amyloid substrates are translocated. We previously reported the crystal structure of the membrane-spanning domain of the amyloid subunit transporter FapF from Pseudomonas. However, the structure of the periplasmic domain, which is essential for amyloid transport, is yet to be determined. Here, we present the crystal structure of the N-terminal periplasmic domain at 1.8-Å resolution. This domain forms a novel asymmetric trimeric coiled coil that possesses a single buried tyrosine residue as well as an extensive hydrogen-bonding network within a glutamine layer. This new structural insight allows us to understand this newly described functional amyloid secretion system in greater detail.

Dihydroceramide Desaturase 1 Inhibitors Reduce Amyloid-β Levels in Primary Neurons from an Alzheimer’s Disease Transgenic Model

The induction of autophagy has recently been explored as a promising therapeutic strategy to combat Alzheimer's disease. Among many other factors, there is evidence that ceramides/dihydroceramides act as mediators of autophagy, although the exact mechanisms underlying such effects are poorly understood. Here, we describe how two dihydroceramide desaturase inhibitors (XM461 and XM462) trigger autophagy and reduce amyloid secretion by neurons. Neurons isolated from wild-type and APP/PS1 transgenic mice were exposed to the two dihydroceramide desaturase inhibitors to assess their effect on these cell's protein and lipid profiles. Both dihydroceramide desaturase inhibitors increased the autophagic vesicles in wild-type neurons, reflected as an increase in LC3-II, and this was correlated with the accumulation of dihydroceramides and dihydrosphingomyelins. Exposing APP/PS1 transgenic neurons to these inhibitors also produced a 50% reduction in amyloid secretion and/or production. The lipidomic defects triggered by these dihydroceramide desaturase inhibitors were correlated with a loss of S6K activity, witnessed by the changes in S6 phosphorylation, which strongly suggested a reduction of mTORC1 activity. The data obtained strongly suggest that dihydroceramide desaturase 1 activity may modulate autophagy and mTORC1 activity in neurons, inhibiting amyloid secretion and S6K activity. As such, it is tantalizing to propose that dihydroceramide desaturase 1 may be an important therapeutic target to combat amyloidosis.

The role of astrocytes in amyloid production and Alzheimer's disease.

Alzheimer's disease (AD) is marked by the presence of extracellular amyloid beta (Aβ) plaques, intracellular neurofibrillary tangles (NFTs) and gliosis, activated glial cells, in the brain. It is thought that Aβ plaques trigger NFT formation, neuronal cell death, neuroinflammation and gliosis and, ultimately, cognitive impairment. There are increased numbers of reactive astrocytes in AD, which surround amyloid plaques and secrete proinflammatory factors and can phagocytize and break down Aβ. It was thought that neuronal cells were the major source of Aβ. However, mounting evidence suggests that astrocytes may play an additional role in AD by secreting significant quantities of Aβ and contributing to overall amyloid burden in the brain. Astrocytes are the most numerous cell type in the brain, and therefore even minor quantities of amyloid secretion from individual astrocytes could prove to be substantial when taken across the whole brain. Reactive astrocytes have increased levels of the three necessary components for Aβ production: amyloid precursor protein, β-secretase (BACE1) and γ-secretase. The identification of environmental factors, such as neuroinflammation, that promote astrocytic Aβ production, could redefine how we think about developing therapeutics for AD.

Abstract IA09: The commensal microbiota as instructors and arbiters of homeostatic and pathogenic immune responses

Abstract The vertebrate intestinal tract is colonized by hundreds of species of bacteria that outnumber the total cells in the host, yet must be compartmentalized and tolerated to prevent invasive growth and harmful inflammatory responses. A key function of commensal microbes is to contribute to the adaptive immune repertoire and to diverse lymphocyte effector functions. T cell responses against non-invasive commensals, as exemplified by responses elicited by the segmented filamentous bacteria (SFB) and Helicobacter hepaticus, contribute to shaping the repertoire of effector/memory and regulatory T cells. SFB adhere to the epithelium in the terminal ileum of mice and induce differentiation of Th17 cells that normally protect the mucosal barrier, but contribute to autoimmune disease in susceptible mice. H. hepaticus colonizes the large intestine and induces Treg cells in wild type mice and inflammatory Th17 cells in IL-10-deficient mice. How T cells elicited by commensal bacteria can influence autoimmunity is a central question that remains unsolved. We are studying the antigenic specificity of microbiota-induced T cells and the mechanisms by which their functions are acquired upon interaction with distinct commensal species. We find that induction of SFB-specific Th17 cells and H. hepaticus-specific Treg cells requires priming and polarization of antigen-specific RORgt-expressing CD4+ T cells (also expressing Foxp3 for Tregs) in the draining lymph nodes. Induction of IL-17 in lamina propria Th17 cells occurs only in regions with adjacent SFB colonization. Acquisition of Th17 effector functions requires the local activation of IL-22-producing innate lymphoid cells (ILC3). These, in turn, act on intestinal epithelial cells to induce secretion of serum amyloid A proteins, SAA1 and SAA2, that activate primed RORgt+ T cells. Expression of the Th17 cell effector program requires formation of a complex of RORgt with a DEAD-box RNA helicase and a long noncoding RNA that, together, confer transcriptional activity at target loci. Requirements for induction of Treg versus Th17 cells will be discussed, as will be the potential role of SAAs in inflammation. These studies in mice are not only relevant for human autoimmune diseases, many of which have Th17 cell involvement, but may also provide insights into how commensal microbe-specific T cell responses could be harnessed for mucosal vaccination and cancer immunotherapy. Indeed, it has recently become evident that the microbiota plays an important role in patient responses to cancer chemotherapy and checkpoint immunotherapy. A better understanding of how individual or communities of bacteria (or commensal viruses and fungi) regulate tumor-specific T cell responses may pave the way for microbiota-based cancer therapies. Citation Format: Teruyuki Sano, Mo Xu, Lin Wu, June-Tong Lee, Wendy Huang, Yi Yang, Gretchen Diehl, Dan R. Littman. The commensal microbiota as instructors and arbiters of homeostatic and pathogenic immune responses [abstract]. In: Proceedings of the Second CRI-CIMT-EATI-AACR International Cancer Immunotherapy Conference: Translating Science into Survival; 2016 Sept 25-28; New York, NY. Philadelphia (PA): AACR; Cancer Immunol Res 2016;4(11 Suppl):Abstract nr IA09.

Single-Cell Detection of Secreted Aβ and sAPPα from Human IPSC-Derived Neurons and Astrocytes.

We have established a technology that, for the first time, detects secreted analytes from single human neurons and astrocytes. We examine secretion of the Alzheimer's disease-relevant factors amyloid β (Aβ) and soluble amyloid precursor protein-alpha (sAPPα) and present novel findings that could not have been observed without a single-cell analytical platform. First, we identify a previously unappreciated subpopulation that secretes high levels of Aβ in the absence of detectable sAPPα. Further, we show that multiple cell types secrete high levels of Aβ and sAPPα, but cells expressing GABAergic neuronal markers are overrepresented. Finally, we show that astrocytes are competent to secrete high levels of Aβ and therefore may be a significant contributor to Aβ accumulation in the brain.

Selective astrocytic endothelin-1 overexpression contributes to dementia associated with ischemic stroke by exaggerating astrocyte-derived amyloid secretion.

Endothelin-1 (ET-1) is synthesized by endothelial cells and astrocytes in stroke and in brains of Alzheimer's disease patients. Our transgenic mice with ET-1 overexpression in the endothelial cells (TET-1) showed more severe blood-brain barrier (BBB) breakdown, neuronal apoptosis, and glial reactivity after 2-hour transient middle cerebral artery occlusion (tMCAO) with 22-hour reperfusion and more severe cognitive deficits after 30 minutes tMCAO with 5 months reperfusion. However, the role of astrocytic ET-1 in contributing to poststroke cognitive deficits after tMCAO is largely unknown. Therefore, GET-1 mice were challenged with tMCAO to determine its effect on neurologic and cognitive deficit. The GET-1 mice transiently displayed a sensorimotor deficit after reperfusion that recovered shortly, then more severe deficit in spatial learning and memory was observed at 3 months after ischemia compared with that of the controls. Upregulation of TNF-α, cleaved caspase-3, and Thioflavin-S-positive aggregates was observed in the ipsilateral hemispheres of the GET-1 brains as early as 3 days after ischemia. In an in vitro study, ET-1 overexpressing astrocytic cells showed amyloid secretion after hypoxia/ischemia insult, which activated endothelin A (ETA) and endothelin B (ETB) receptors in a PI3K/AKT-dependent manner, suggesting role of astrocytic ET-1 in dementia associated with stroke by astrocyte-derived amyloid production.

Structure of the nonameric bacterial amyloid secretion channel.

Various strains of bacteria are able to produce a unique class of functional amyloids termed curli, which are critical for biofilm formation, host cell adhesion, and colonization of inert surfaces. Curli are secreted via the type VIII bacterial secretion system, and they share biochemical and structural characteristics with amyloid fibers that have been implicated in deleterious disease in humans. Here, we report the crystal structure of Escherichia coli CsgG, which is an essential lipoprotein component of the type VIII secretion system and which forms a secretion channel in the bacterial outer membrane for transporting curli subunits. CsgG forms a crown-shaped, symmetric nonameric channel that spans the outer membrane via a 36-strand β-barrel, with each subunit contributing four β-strands. This nonameric complex contains a central channel with a pore located at the middle. The eyelet of the pore is ∼12 Å in diameter and is lined with three stacked nine-residue rings consisting of Tyr-66, Asn-70, or Phe-71. Our structure-based functional studies suggest that Tyr-66 and Phe-71 residues function as gatekeepers for the selective secretion of curli subunits. Our study describes in detail, to our knowledge, the first core structure of the type VIII bacterial secretion machinery. Importantly, our structural analysis suggests that the curli subunits are secreted via CsgG across the bacterial outer membrane in an unfolded form.

Structural and mechanistic insights into the bacterial amyloid secretion channel CsgG

Curli are functional amyloid fibres that constitute the major protein component of the extracellular matrix in pellicle biofilms formed by Bacteroidetes and Proteobacteria (predominantly of the α and γ classes). They provide a fitness advantage in pathogenic strains and induce a strong pro-inflammatory response during bacteraemia. Curli formation requires a dedicated protein secretion machinery comprising the outer membrane lipoprotein CsgG and two soluble accessory proteins, CsgE and CsgF. Here we report the X-ray structure of Escherichia coli CsgG in a non-lipidated, soluble form as well as in its native membrane-extracted conformation. CsgG forms an oligomeric transport complex composed of nine anticodon-binding-domain-like units that give rise to a 36-stranded β-barrel that traverses the bilayer and is connected to a cage-like vestibule in the periplasm. The transmembrane and periplasmic domains are separated by a 0.9-nm channel constriction composed of three stacked concentric phenylalanine, asparagine and tyrosine rings that may guide the extended polypeptide substrate through the secretion pore. The specificity factor CsgE forms a nonameric adaptor that binds and closes off the periplasmic face of the secretion channel, creating a 24,000 Å(3) pre-constriction chamber. Our structural, functional and electrophysiological analyses imply that CsgG is an ungated, non-selective protein secretion channel that is expected to employ a diffusion-based, entropy-driven transport mechanism.

Sphingosine kinases modulate the secretion of amyloid β precursor protein from SH-SY5Y neuroblastoma cells: the role of α-synuclein.

Sphingosine kinases (SphK 1&2) are involved in the regulation of cell survival, differentiation and neurotransmitter secretion. Current data suggest potential links between sphingolipid signalling, α-synuclein (ASN) and Alzheimer's disease (AD). Our aim was to investigate the possible role of SphKs and ASN in the regulation of the production and secretion of the amyloid β precursor protein (APP). We have previously shown that ASN intensified the secretion and toxicity of amyloid β (Aβ) to the point where it caused cell death. Our current results show that APP, the precursor protein for Aβ, is also influenced by ASN. The stable overexpression of wtASN in SH-SY5Y cells caused a three-fold, significant increase of the cellular APP level. This suggests that the influence of ASN on Aβ metabolism may actually occur at the level of APP protein rather than only through the changes of its cleavage into Aβ. To elucidate the mechanisms of APP modulation the cells were exposed to S1P and an SphK inhibitor (SKI). 72 h S1P treatment at 5 µM caused a nearly 50% reduction of the cellular APP signal. S1P also caused a tendency towards higher APP secretion, though the results were insignificant. The inhibition of SphKs decreased medium APP levels in a dose-dependent manner, reaching significance at 5 µM SKI with a correspondingly elevated intracellular level. Thus, it is reasonable to expect that in fact the influence of SphK activity on APP might be pro-secretory. This would also be in agreement with numerous articles on SphK-dependent secretion in the literature. The chronic nature of AD further suggests that subtle alterations in APP metabolism could have the potential to drive important changes in brain condition.