C. pneumoniae, H. pylori, C. neoformans, B. burgdorferri, P. gingivalis) or immune defence. Such pathogens promote beta-amyloid deposition and tau phosphorylation and may thus be causative agents, whose effects are conditioned by genes. The antimicrobial effects of beta-amyloid, the localisation of APP/gamma-secretase in immunocompetent dendritic cells, and gamma secretase cleavage of numerous pathogen receptors suggest that this network is concerned with pathogen disposal, effects which may be abrogated by the presence of beta-amyloid autoantibodies in the elderly. These autoantibodies, as well as those to nerve growth factor and tau, also observed in Alzheimer's disease, may well be antibodies to pathogens, due to homology between human autoantigens and pathogen proteins. NGF or tau antibodies promote beta-amyloid deposition, neurofibrillary tangles, or cholinergic neuronal loss, and, with other autoantibodies, such as anti-ATPase, are potential agents of destruction, whose formation is dictated by sequence homology between pathogen and human proteins, and thus by pathogen strain and human genes. Pathogen elimination in the ageing population and removal of culpable autoantibodies might reduce the incidence and offer hope for a cure in this affliction.Alzheimer's disease susceptibility genes, APP and gamma-secretase, are involved in the herpes simplex life cycle, and that of other suspect pathogens ( Many of the environmental risk factors associated with Alzheimer's disease, including infectious agents . A ll models  (see htt details ). These Several studies have implicated the herpes simplex virus in the aetiology of Alzheimer's disease –13. Virahttp://www.polygenicpathways.co.uk/alzenvrisk.htm) and include HHV-6, Chlamydia pneumoniae, Helicobacter pylori, periodontal pathogens involved in gum disease [Borrelia burgdorferi, and Cryptococcus neoformans. HIV-1 is also able to provoke dementia with Alzheimer's disease pathology [H. pylori eradication has been reported to improve performance and increase lifespan in Alzheimer's disease patients [C. neoformans infection [C. Pneumoniae, H. pylori and the periodontal pathogen, P. Gingivalis, have also been implicated in atherosclerosis [C. neoformans infection in rabbits induces an increase in neutrophil superoxide production, plasma lipid peroxidation, and an increase in inflammatory cells, forerunners of atherosclerosis [per se, to induce cerebral beta-amyloid deposition in animal models (see above).Along with herpes simplex, a number of other pathogens have been implicated in Alzheimer's disease and its associated pathologies. The viral, bacterial, spirochete, and fungal pathogens implicated in dementia or Alzheimer's disease are referenced at , phosphatidylinositol-binding clathrin assembly protein (PICALM) and complement receptor 1 (CR1) as well as the ATP cassette transporter ABCA7, Bridging integrator BIN1, a CD2-associated protein (CD2AP), CD33, ephrin A1 (EPHA1), and a membrane-spanning 4-domains, subfamily A (MS4A) cluster recently honed down to MS4A2, although other genes within this cluster may also be relevant [Genomewide association studies (GWAS) have now identified a subset of genes which, along with APOE4 , contribrelevant , 31.APP and gamma secretase, and previous GWAS results are majoritarily involved in pathogen entry and defence, particularly in relation to herpes simplex, but also to other relevant pathogens, and in the immune network. This suggests that genes, pathogens, and the immune system act together to cause Alzheimer's disease, and that a focus on pathogen detection and elimination should be a priority in the ageing at risk population.As discussed below, the major Alzheimer's disease genes implicated by the recent GWAS data, as well as http://www.genome.gov/gwastudies/ [http://www.polygenicpathways.co.uk/alzenvrisk.htm. The genes returned from very large sample sets  include ABCA7, APOE, BIN1, CD2AP, CD33, CLU, CR1, EPHA1, MS4A2, MS4A4A, MS4A4E, MS4A6A, and PICALM whose properties in relation to diverse pathogens were identified by literature survey. While it is recognised that such genes, particularly APOE, ABCA7, CR1, and clusterin, which are involved in lipoprotein function and/or amyloid processing (see below), may exert effects on other relevant branches of Alzheimer's disease pathophysiology, the focus of this paper is on pathogens and the immune system, which appear to be the common factors integrating this network. Throughout the text, these and other genes implicated in Alzheimer's disease from the GWAS and pre-GWAS era are highlighted in bold and appended to the various processes in which they are involved  Herpes simplex binding proteins, and key interactors, currently numbering over 450, are stocked and referenced at http://www.polygenicpathways.co.uk/herpeshost.html. KEGG pathway analysis of this interactome is provided at http://www.polygenicpathways.co.uk//HERPESKEGG.htm. Expression data are provided in http://www.biogps.gnf.org/, which provides general gene information and mRNA expression profiles for most human genes, based on custom arrays from 79 human issues [tau (NP_001116538.2) were identified using the BepiPred server http://www.cbs.dtu.dk/services/BepiPred/ [Borrelia burgdorferi, C. neoformans, Helicobacter pylori, herpes viruses HSV-1, HSV-2, HHV-6, and the cytomegalovirus (HHV-5)) using the NCBI BLAST server (Protein versus protein: BlastP) [The genes identified in a number of recent genomewide association studies are available at the GWAS repository at the National Human Genome Research Institute studies/  and, alon issues . PredictepiPred/  and thei BlastP) .Complement receptor 1 (highly expressed in myeloid CD33+ cells (bone marrow) http://www.biogps.org/#goto=genereport&id=1378/) is a receptor for herpes simplex, adenovirus 5, the influenza virus and HIV-1, as well as for a number of other pathogens, including P. gingivalis, C. neoformans, Streptococcus pneumoniae, Staphylococcus aureus, and the malaria parasite, Plasmodium falciparum [Clusterin, predominantly expressed in brain, liver, and testis, (http://www.biogps.org/#goto=genereport&id=1191/) is a ligand for the lipoprotein receptor, megalin (LRP2) that is involved in beta-amyloid clearance, and also a complement inhibitor that prevents the formation of the membrane attack complex, a channel that is inserted into pathogen membranes, killing them by lysis [CR1 ligand, C3 and its derivatives and to CD59, a further inhibitor of the formation of the complement membrane attack complex (see review) [C. pneumoniae interacts with this pathway by binding to properdin (CFP), a protein that stabilises the complement C3 and C5 convertase and contributes to the formation of the membrane attack complex [C. neoformans [H. pylori, and the complement pathway is involved in bactericidal effects against this pathogen [P. gingivalis also uses complement receptor 3 ) for entry [C. neoformans [C. pneumoniae entry in human coronary artery endothelial cells [P. gingivalis although the pathogen has devised an elegant escape strategy involving digestion of complement components C3, C4, and C5 by bacterial secreted proteases, known as gingipains [lciparum  and is alciparum . Clusterby lysis . This coby lysis , 47. The review) . C. pneu complex . CD59 is complex . Complemoformans  and crypoformans . Complempathogen . P. gingor entry , and heror entry  as does oformans , while Ial cells . This maal cells . T. C3 aCD2AP, activates T cell receptor signalling resulting in the secretion of interleukins  and granulocyte macrophage colony stimulating factor (CSF2) [The complement inhibitor CD59 is also a ligand for CD2, and CD59 activation of this receptor, presumably involving r (CSF2) , 61.ABCA7 plays a role in the complement-mediated activation of phagocytosis in macrophages. Complement component C1q, which binds to IgM or IgG complexed antigens (relevant to most pathogens), binds to macrophage calreticulin and LRP1 and C1q binding to macrophages markedly increased the expression of both LRP1 and ABCA7, effects which enhance the phagocytic abilities of macrophages [CR1, an effect involved in the immune clearance of opsonised pathogens [rophages . C1q alsathogens . C1q alsathogens .ADRB1, ADRB2, BIN1, CAV1, CD2AP, CLU, DNM2, HLA-A, HSPA1B, LDLR, NTRK1, PICALM) and either recycled or tagged for destruction by the ubiquitin/proteasome system  or by lysosomes . Early endosomes receive traffic from the cell surface, which is transferred to late endosomes for traffic to lysosomes. Late endosomes also receive traffic from the trans-Golgi network used to synthesise proteins and from phagocytic pathways . Endosomal traffic moves along the microtubule  or actin/myosin  networks via dynein/dynactin  or kinesin , related motors and Rho GTPases, and vacuolar sorting proteins  inter alia [Mammalian surface receptors are endocytosed, via clathrin-dependent or independent processes , and recruiting the clathrin and adaptor protein 2 (AP-2) to the plasma membrane. The AP-2 complex is a heterotetramer consisting of permutations of two large adaptins ) or beta (AP2B1), a medium adaptin , and a small adaptin (sigma AP2S1). PICALM controls the endocytosis of the cation-independent mannose-6-phosphate IGF2 receptor (IGF2R) [C. pneumoniae for cellular entry [Helicobacter pylori VacA cytotoxin [clusterin. PICALM also binds to a nuclear exportin crm-1 (XPO1) used by the herpes simplex virus during its life cycle [ (IGF2R) , one use (IGF2R)  and by Car entry . IGF2R iytotoxin . The manfe cycle .SORL1, and the APP cleaving beta-secretase BACE2, while GGA2 binds to the beta-secretases BACE1 and BACE2,  SORL1 and the prolyl-isomerase PIN1.Gamma-adaptins  bind to clathrins and mannose-6-phosphate receptors and regulate protein traffic between the Golgi network and the lysosome and the sorting of mannose-6-phosphate receptors (IGF2R and M6PR) at the trans-Golgi network . This neCD2AP, primarily expressed in dendritic cells and B lymphoblasts http://www.biogps.org/#goto=genereport&id=23607/, is a scaffolding molecule that regulates the actin cytoskeleton and is primarily associated with the T-lymphocyte marker protein CD2. CD2 stimulates T cell activation and is involved in the creation of contacts between antigen presenting cells and T cells , effects mediated via CD2AP and clathrin [CD2AP is also involved in the entry of the helicobacter vacuolating toxin VacA and connects the actin cytoskeleton to early endosomes containing VacA [P. gingivalis [clathrin . CD2AP iing VacA . CD2 is ngivalis .CD2AP also binds to the actin-bonding protein, cortactin (CTTN), a protein that is exploited by several bacteria , fungi (Candida Albicans), and viruses (Vaccinia) enabling them to modify the actin cytoskeleton, which they use for transport [CD2AP has not been specifically associated with herpes simplex, although the actin cytoskeleton is exploited by this and many other viruses [ransport –79. CD2A viruses .CD2AP also associated with E-Cadherin, (CDH1) [H. pylori toxin CagA [Helicobacter pylori protein HtRA allowing the pathogen to invade the intracellular compartment [C. pneumoniae infection of human brain microvascular endothelial cells, contributing to vascular permeability changes and atherosclerosis [, (CDH1) . The ect, (CDH1) . E-Cadhexin CagA  and is apartment . CDH1 anclerosis .BIN1), also known as amphiphysin 2, is primarily expressed in the pineal and skeletal muscle, or otherwise ubiquitously http://www.biogps.org/#goto=genereport&id=274/. It is also involved in the clathrin-mediated endocytosis machinery [DNM2 [BIN1 also participates in phagocytosis in macrophages and is associated, but only transiently, with early phagosomes; however, it is retained on vacuoles containing Chlamydia pneumoniae, an effect that reduces the ability of the macrophage system to kill the bacteria via nitric oxide generation. Macrophages expressing a dominant negative BIN1 internalise C. pneumoniae, but do not allow their killing [H. pylori protein CagA [Bridging integrator 1 , ITGB4,CD33, mainly expressed in myeloid cells, monocytes, and dendritic cells (http://www.biogps.org/#goto=genereport&id=945/), is a member of the sialic acid binding Immunoglobulin g-like lectin (SIGLEC) family. CD33-related SIGLECs regulate adaptive immune responses and are also important as macrophage pattern recognition receptors for sialylated pathogens, including enveloped viruses [CD33 binds to alpha2-3- or alpha2-6-linked sialic acids (N-acetyl neuraminic acid) [C. neoformans, is involved in fungal adhesion to macrophages [B. burgdorferi [Helicobacter pylori adhesins also bind to this particular form of sialic acid [P. gingivalis [ic acid) . These pic acid) . N-acetyrophages , and is gdorferi , while Hlic acid , 100 as ngivalis .BIN1, as well as its relationship to the clathrin mediated endocytosis machinery, also regulates the expression of indoleamine 2,3-dioxygenase (IDO1), an enzyme that catalyzes the first rate-limiting step in tryptophan metabolism to N-formyl-kynurenine [T. gondii, and to a number of viruses, including herpes simplex and other herpes viruses [GRIN2B, GRIN3A). Plasma tryptophan levels are also lower in the ageing population and in Alzheimer's disease, a pattern accompanied by immune activation, and by increased concentrations of quinolinic acid [nurenine . IDO1 upnurenine . This ID viruses . IDO1 pr viruses   receptor, which is involved in allergic responses in which allergens bound to receptor bound IgE result in the activation of allergic mediators such as histamine [C. pneumoniae, H. pylori, and B. burgdorferi [C. neoformans infection [MS4A4A, MS4A4E, and MS4A6A) are also structurally related to the immunoglobulin E receptor and to CD20 (MS4A1) and also regulate B cell and T cell proliferation and/or differentiation [istamine . Mice imistamine . High leistamine  and humaistamine  and alsogdorferi –115. IgEnfection . Other mntiation , 118.EPHA1 is an ephrin receptor, primarily expressed in the liver and otherwise ubiquitously (http://www.biogps.org/#goto=genereport&id=2041/). Only three protein/protein interactions for EPHA1 are reported in the NCBI gene interaction section, including its ligand EFNA1, the anaplastic lymphoma receptor tyrosine kinase (ALK), and a SMAD-specific E3 ubiquitin protein ligase 2 (SMURF2). EFNA1 is one of several proteins identified as being important in the entry of C. pneumoniae into human coronary artery endothelial cells [TGF beta receptors, to which it binds [Clusterin is a ligand for TGF beta receptors (TGFBR1/TGFBR2) [TGF beta signalling exerts immunosuppressive effects and inhibits host immunosurveillance and the recruitment of immunocompetent cells by chemokines [http://www.biogps.org/#goto=genereport&id=238/). It plays a role in neural development, and its expression decreases with age [al cells . SMURF2 al cells  and playit binds . Cluster/TGFBR2) . TGF betemokines . ALK is with age . ALK is with age . Its keyABCA7 is an ATP-binding cassette transporter, predominantly localised in the pineal gland and cells of the immune network . The lipoproteins APOA1 and APOE are substrates for ABCA7, and in cultured HEK-293 cells, plasma membrane-situated ABCA7 increases the efflux of phosphatidylcholine and sphingomyelin efflux to APOA1 and APOE, with no effect on cholesterol efflux [APOE, but not to lipid free APOE, is increased by ABCA7 expression in HEK-293 cells [C. neoformans infection. Sphingomyelin is a receptor for the Helicobacter toxin VacA [C. pneumoniae-infected cells [C. neoformans infection [l efflux . However93 cells . Sphingo93 cells . Herpes 93 cells . Inhibit93 cells  and alsoxin VacA  and is aed cells . Phosphaed cells . Phosphanfection .ABCA7 expression increases the extracellular surface deposition of ceramide (derived from sphingomyelin) [C. pneumoniae inclusions, an effect that may play a role in the antiapoptotic effects of this bacterium [APOA1 exerts antiviral effects against herpes simplex and inhibits viral entry into cells as well as viral-induced cell fusion and intercellular spread [ABCA7 is expressed intracellularly and does not participate in cholesterol or phospholipid efflux, instead playing a role in the phagocytosis of apoptotic cells, an important general defence mechanism against invading pathogens [omyelin) . Ceramidomyelin) . Ceramidacterium . APOA1 er spread . In macrathogens , 138.APOE4 allele facilitates the entry and transmission of herpes simplex in mice models [APOE is also involved in hepatitis C, HIV-1, and herpes simplex infectivity [APOE4 facilitates the binding of C. pneumoniae elementary bodies to host cells [Possession of the e models . In man,ectivity –143, andst cells .APOE mRNA is primarily expressed in the liver, adipocytes; kidney and brain, with very low expression in the peripheral immune network (http://www.biogps.org/#goto=genereport&id=348/) but nevertheless plays an important role in the immune system. For example, the presence of the APOE4 allele is associated with an enhanced macrophage inflammatory response, and cytokine responses to the intracerebral injection of lipopolysaccharide are increased in APOE4 transgenic mice, which also exhibit increased microglial activation. The anti-inflammatory effects of 17-beta-oeastradiol on microglia are also reduced in such animals [CRP) levels are also decreased in APOE4 carriers [CRP is an acute phase protein that binds to phosphocholine on dead or dying cells and on bacteria, subsequently activating the complement pathway [Klebsiella pneumoniae) or endotoxaemia is also decreased in APOE knockout mice [ animals , 146. C-carriers –147. CRP pathway . Resistaout mice .Helicobacter pylori, influenza, C. pneumoniae or P. gingivalis) in APOE knockout mice [Helicobacter pylori is able to promote atherosclerosis in heterozygous APOE (+/−) LDLR (+/−) mice, which is associated with an immune response to the bacterial heat shock protein hsp60 [In addition, atherosclerosis is induced or worsened by infection with a number of relevant pathogens . PVRL2 is a receptor for herpes simplex (HSV-1 and HSV-2) [TOMM40, a receptor for certain chlamydial species [RFC3 is part of a complex necessary for human DNA polymerase activity, a process exploited by many viruses including herpes simplex, whose virion component ICP34.5 binds to proliferating cell nuclear antigen (PCNA), an RFC3 binding partner and also a cofactor for DNA polymerase [ZNF224 is a transcriptional repressor binding to the protein arginine methyltransferase, PRMT5 [Prior to the very large GWAS collaboration, several other genes had been identified in smaller genomewide studies (d HSV-2) , and the species . The replymerase . ZNF224 e, PRMT5 . Proteine, PRMT5 .DISC1 is a component of the microtubule-associated dynein motor complex used in viral traffic [TTLL7  also regulates tubulin phosphorylation [CELF2  is a member of the APOBEC1 cytidine deaminase mRNA editing complex that also controls herpes simplex viral replication [GAB2 is a member of the GRB2-associated binding protein family which act as adapter hubs transmitting signalling via cytokine and growth factor receptors, and T- and B-cell antigen receptors (definition from NCBI gene), while PAX2 inhibits the expression of the antimicrobial peptide beta defensin (DEFB1) [H. pylori or chlamydial infections [C. neoformans and other pathogens [MTHD1L (methylenetetrahydrofolate dehydrogenase (NADP+ dependent) 1-like) is involved in the mitochondrial synthesis of tetrahydrofolate which in turn is important in the de novo synthesis of purines and thymidylate and in the regeneration of methionine from homocysteine (definition from NCBI gene). Many pathogens, including herpes simplex, express thymidylate kinases, which are important for viral replication and a target for acyclovir [C. pneumoniae IgG immunoreactivity in carotid artery atherosclerosis [H. pylori infection in the context of atherosclerosis [APOC1 is a component of high-density lipoprotein: herpes simplex is present in all lipoprotein blood fractions in blood  and the lipid component of these lipoproteins binds to viral glycoprotein B [APOA1, APOA4, APOA5, APOC1, APOC2, APOC3, APOC4, APOD, and APOE). No immediately apparent pathogen-relevant interactions were found for FAM113B , PCDH11X (which is ubiquitously expressed http://www.biogps.org/#goto=genereport&id=27328/), or SASH1  although the pathogen/immune theme is clearly carried through, particularly in relation to herpes simplex, in this second rank of Alzheimer's disease susceptibility genes. traffic ; TTLL7 (rylation  and can lication . GAB2 is (DEFB1) , a gene  (DEFB1) , as wellfections , 169, alathogens . MTHD1L cyclovir . Hyperhoclerosis  and is aclerosis . The aporotein B  . This is primarily mediated via lipoprotein receptors. A2M, or APOE-bound Aβ, is cleared by the lipoprotein receptor LRP1, while LRP2  clears clusterin-bound Aβ. LRP8 is a receptor for both APOE and clusterin. APOA1 is also involved in beta-amyloid clearance via its transporter ABCA1. The role of ABCA7 has not been examined, although APOA1 is also a ligand for this transporter (see above). The Varicella Zoster and herpes simplex glycoprotein E binding protein, insulin-degrading enzyme, are also involved in beta-amyloid degradation, as is caspase-3 which is activated by the herpes simplex viral US3 kinase. The HSV-1 binding protein, complement C3 is also a ligand for LRP1 and LRP8, both of which play a role in C3 cellular uptake. Beta amyloid in the bloodstream is processed by its binding to complement C3, which subsequently binds to complement receptor 1 on erythrocytes. The effects above are referenced in a recent review [t review .APP, a procedure necessary for the subsequent cleavage of APP and the generation of beta-amyloid [PICALM knockdown does not [APP, is clathrin dependent [LRP1, LRP2, LRP8, LDLR, VLDLR) [Clathrin-dependent endocytosis is also involved in the internalisation and recycling of neuronal -amyloid , 176, an-amyloid , but not-amyloid . Howeverdoes not . The accependent . Clathri, VLDLR) , all of , VLDLR) .ABCA7 plays a role in beta-amyloid secretion, which is increased in Chinese hamster ovary cells expressing APP and ABCA7. This was related to an effect on APP intracellular retention, rather than on secretase-mediated proteolysis of APP [s of APP .Gamma-secretase cleaves APP, and other gamma-secretase substrates also play key roles in APP processing (ADAM10) [LRP1, LDLR, VLDLR), and other processes relevant to Alzheimer's disease, for example, NOTCH signalling [(ADAM10) , lipid agnalling .CD2AP, CD33, and EPHA1 or for MS4A-related proteins, although such are not precluded.No other immediately apparent relationships with beta-amyloid could be found by literature survey for helicobacter, chlamydiae, P. gingivalis, and C. neoformans [http://www.polygenicpathways.co.uk/herpeshost.html for herpes simplex). Tau (MAPT) stabilises microtubules by interacting with tubulins and promoting microtubule assembly [tau is phosphorylated, by any of several kinases, microtubules become disorganised. Tau hyperphosphorylation and neurofibrillary tangles are among the core pathologies of Alzheimer's disease [tau  suggesting that tau phosphorylation could be a direct result of a viral kinase [Many pathogens, including herpes simplex, oformans –188, useassembly . When ta disease  and can  disease . In relal kinase .APP plays a key role in the herpes simplex life cycle and is involved in its intracellular transport [ransport , an efferansport , 195.β antibodies [TLR2, TLR4), which are also involved in beta-amyloid clearance [Beta-amyloid is an antimicrobial peptide with broad spectrum activity against a variety of yeasts and bacteria, effects that were attenuated by anti-Atibodies , Beta-amtibodies . Beta-amlearance , 199. Thlearance . As immuGamma secretase is constituted of four components: the presenilins (PSEN1 or PSEN2), anterior pharynx-defective-1 (APH1A), the Presenilin enhancer-2 (PSENEN), and nicastrin (NCSTN) [PSEN1  http://www.biogps.org/#goto=genereport&id=5663/; dendritic cells and natural killer cells for PSENENhttp://www.biogps.org/#goto=genereport&id=55851/, dendritic cells and myeloid cells for nicastrin http://www.biogps.org/#goto=genereport&id=23385/ and B cells, dendritic cells, natural killer cells, and myeloid cells for APH1A. The substrate, APP, is the only gene in this set that appears to be preferentially distributed in brain compartments, but, as with gamma-secretase components, it is also highly expressed in dendritic cells of the immune system (http://www.biogps.org/#goto=genereport&id=351/). The primary function of such cells is to process antigens and present them to B cells and T cells. They scout for and recognise pathogens via the agency of numerous pattern recognition receptors, for example, Toll receptors , or viral DNA sensors, expressed on their surface [ (NCSTN) . While a surface , 203.APP, gamma secretase is involved in the proteolysis of at least three herpes simplex receptors, nectin 1 alpha (PVRL1) [gamma secretase substrate, is an HIV-1 and papillomavirus receptor [As well as cleaving  (PVRL1) , and syn (PVRL1) . SDC1 is (PVRL1) –209, whireceptor , 211.gamma secretase substrates  [Several other nd Saura ) also fund Saura , CD44, aoformans , CD46, aoformans : CD46 isngivalis . Desmoglngivalis , while rnd VLDLR , 218. LDreceptor . NOTCH1 receptor , 221, whreceptor . The lowreceptor , Ephrin receptor  and sialreceptor , 226 andlomannan . Fractallomannan , while tlomannan : phosphalomannan . Dystroglomannan , 232 andathogen) .gamma secretase, and the concentration of both APP and gamma-secretase components in dendritic cells suggest that a major function of this key group, implicated in both familial and late onset Alzheimer's disease, is dedicated to pathogen defence, and that increased beta-amyloid generation is primarily a defence mechanism to rid the body (and brain) of invading pathogens: This scenario is supported by the ability of herpes simplex, C. pneumoniae, and B. burgdorferi to increase beta-amyloid deposition [ The antimicrobial and immunostimulant effects of beta-amyloid, the cleavage of a number of pathogen receptors by position –236. Oneposition .Viruses and bacteria express proteins containing short contiguous amino acid stretches  that are identical to those in human proteins: these pathogen/human consensi number in millions and concern all human proteins –241.FCER1G) in the case of the rhinovirus, or the SARS coronavirus, or via the tripartite motif protein, TRIM21, in the case of adenoviruses, where they are able to activate an intracellular immune attack. It would appear that the cellular entry of antibody laden viruses is diverted from their usually preferred receptors towards those used by antibodies [MS4A family. Fc gamma receptors are localised in microglia and astrocytes in the brain and their expression is upregulated by blood brain barrier disruption [http://www.biogps.org/#goto=genereport&id=6737/).Autoantibodies, which are observed in many, if not most human diseases, are often regarded as an epiphenomenon of little consequence. However, they can traverse the blood brain barrier   was predicted as immunogenic (B cell epitope), as defined by the server set cutoff index of 0.35. For the analysis in tau protein with an immunogenicity index >2.5 were examined for homology. For other proteins (NGF and beta-amyloid), the analysis concerned immunogenic regions above the cutoff value of 0.35.Almost the entire length of the All pathogens express proteins with homology to each autoantigen, specifically within predicted B-cell epitope regions of the human autoantigen , suggestCHRNA7, that displace its ligand alpha-bungarotoxin [AGER) [S100B, have also been reported [As well as autoantibodies to these three proteins, a number of autoantibodies targeting highly relevant proteins have been reported in Alzheimer's disease. These have functional effects on their target proteins and include antibodies that block the activity of ATP synthase, induce apoptosis, and increase the cellular uptake of high density lipoprotein , 267, anarotoxin : autoantn [AGER) , to the reported .many different birds when it alights on dark trees covered with soot pollution, while darker melanised forms are selectively targeted on lighter coloured trees [several causes to do so. The causes can hide in plain sight, as epidemiological studies, as applied to human diseases, could conclude that the birds are not killing the moths, as they are always present, on both sets of trees, in both genetic conditions, whether the moths are alive or dead (c.f. the ubiquitous C. neoformans and many other common pathogens). Many of the pathogens implicated in Alzheimer's disease , and several other risk factors  are able to promote cerebral beta-amyloid deposition in animal models, without the aid of any gene variant. Subsets of susceptibility genes can be related to each of these amyloidogenic pathways [APP, beta amyloid, and gamma secretase, seem intimately concerned with pathogen life cycles and defence and the immune network. This suggests that the diverse microbial risk factors as well as other dietary and environmental factors implicated in Alzheimer's disease are in fact causative agents, whose deleterious effects are conditioned by susceptibility genes. As the environmental risk factors are amenable to therapy, while the susceptibility gene products have so far proved largely resistant, this suggests numerous ways with which to tackle the problem of Alzheimer's disease.Using a classical example from the field of population genetics and Darwinism, the light coloured genes of the peppered moth favour its selective predation by ed trees . The colpathways . In the APOE knockout mice, while the viral load is much increased in APOE4 transgenic mice, compared to APOE3 mice, suggesting that APOE4 favours the establishment of a greater number of latent viruses in cerebral tissue [NTRK1 [GSK3B, NTRK1, NTRK2, PIK3R1, and SOS2). Vitamin A supplementation in rats increases the cerebral levels of both NGF and BDNF [ESR1) [APOE4, the isoform least able to transport the vitamin A precursor retinyl palmitate, A2M ABCA1 ALB ALDH2 APOA1 CHD4 CLU CYP46A1 ESR1 GSTM1 GSTP1 HSPG2 KLF5 LIPA LPL LRAT LRP2 LRPAP1 MEF2A NPAS2 NR1H2 PARP1 PIN1 POU2F1 PPARA PPARG RXRA THRA TTR UBQLN1 VDR and many others controlled by retinoid receptor element  . High leF [ESR1) , 283. Viiewed in ).IL6 [TNF [IL6 plasma and CSF levels have been reported to be increased in Alzheimer's disease and the secretion of IL6 from monocytes is increased [IL6 plasma levels are raised by infection with C. pneumoniae [Helicobacter pylori [IL6 production in monocytes is stimulated by C. neoformans [COMT, CYP19A1, HSD11B1). Cortisol levels are increased in the ageing population and in Alzheimer's disease [ADRB1) [PTGS2), celecoxib and indomethacin block viral reactivation and nonsteroidal anti-inflammatory use has been associated with a lower incidence of Alzheimer's disease. Lysophosphatidic acid is also able to reactivate the virus [LPAR5). Hypoxia also increases the replication of herpes simplex [NOS2 are also more susceptible to herpes virus infection, and reactivation occurs is stimulated by caspase 3 activation (CASP3) [The virus can also be reactivated by heat , IL6 [28IL6 , or TNF IL6 [TNF . IL6 plancreased –289. IL6eumoniae  or Helicr pylori , and IL6oformans . Stress-oformans   , 298. Gl [ADRB1) : cyclooxhe virus  (LPAR5). simplex : transie simplex , and the simplex . Vitamin simplex . Low Vit simplex . Low vit simplex . Mice de (CASP3) .Thus, fever or cytokine release induced by diverse infections might be expected to reactivate herpes simplex as would several of the conditions associated with Alzheimer's disease .These pathogens form a very small proportion of an extensive human microbiome comprising of trillions of bacteria, viruses, and other pathogens, whose influence on diseases is increasingly recognised . IndividThe principles discussed here may apply to many other, if not most, human diseases.Statins , fish coABCA1, ABCA2, ABCA7, ABCG1, ACADS, ALDH2, APP, APOA1, APOA4, APOA5, APOC1-4, APOE, CH25H, CYP46A1, DHCR24, FDPS, HMGCR, HMGCS2, HSD11B1, LIPC, LRP1, LRP2, LRP8, LDLR, LIPA, LPL, OLR1, PPARA, PPARG, PTPLA, VLDLR, NPC1, NPC2, SOAT1, SREBF1), homocysteine, methionine, and folate metabolism  and inflammatory pathways . As with the risk factors, the success of such protective agents is likely to be determined by genes and other confounding factors. In this genomic era, affordable whole genome sequencing will soon be achieved, ushering in an age of more effective treatments tailored to individual genetic profiles. Alzheimer's disease is clearly multifactorial with many related genetic and environmental risk factors, several underlying pathologies, and several available protective strategies. A recent study has also shown that many other seemingly benign factors  as well as high blood pressure and diabetes, constituting a frailty index, combine to markedly increase both the incidence and severity of Alzheimer's disease [As with the risk factors, protective agents can be linked to genetic and pathological pathways involved in cholesterol and lipid function  would also be very informative as selective autoantibody removal via affinity dialysis might well be expected to influence the severity and progression of Alzheimer's disease.B. burgdorferi, several periodontal pathogen treponemas species were detected in brain samples and the pathological features of Alzheimer's disease were reproduced by infection in vitro [Since the submission of this paper, Miklossy has reported a highly significant association between spirochete infection and Alzheimer's disease. As well as in vitro . In addiin vitro , 321. Th


Insulin signaling in central nervous system (CNS) has emerged as a novel field of research since decreased brain insulin levels and/or signaling were associated to impaired learning, memory, and age-related neurodegenerative diseases. Thus, besides its well-known role in longevity, insulin may constitute a promising therapy against diabetes- and age-related neurodegenerative disorders. More interestingly, insulin has been also faced as the potential missing link between diabetes and aging in CNS, with Alzheimer's disease (AD) considered as the “brain-type diabetes.” In fact, brain insulin has been shown to regulate both peripheral and central glucose metabolism, neurotransmission, learning, and memory and to be neuroprotective. And a future challenge will be to unravel the complex interactions between aging and diabetes, which, we believe, will allow the development of efficient preventive and therapeutic strategies to overcome age-related diseases and to prolong human “healthy” longevity. Herewith, we aim to integrate the metabolic, neuromodulatory, and neuroprotective roles of insulin in two age-related pathologies: diabetes and AD, both in terms of intracellular signaling and potential therapeutic approach. Almost all cell types are responsive to insulin. However, liver, muscle, and adipose tissue are the most sensitive to the hormone , renderiUntil three decades ago, insulin was considered only as a peripheral hormone, unable to cross the blood-brain barrier (BBB) and to affect the central nervous system (CNS) –5. Howevβ-cells and is transported by CSF into the brain [Insulin present in adult CNS is primarily derived from pancreatic he brain –5, 9, 10he brain . Thus, ihe brain . Moreovehe brain . Alternahe brain , 14. De novo insulin synthesis in brain has been proposed as an alternative source of insulin in CNS. This hypothesis has been supported by the detection of preproinsulin I and II mRNA in rat fetal brain and cultured neurons and also by insulin immunoreactivity in neuronal endoplasmic reticulum, Golgi apparatus, cytoplasm, axon, dendrites, and synapses [+- and Ca2+-induced membrane depolarization [synapses –21. Addisynapses , its presynapses , 18, thesynapses , and thesynapses  further rization . More sprization . Thus, irization . Similarrization , 27, 28.Taken this together, the next obvious question regards insulin and IR physiological role(s) in brain. Herewith, we will first analyse brain IR-mediated signaling pathways and the pivotal role of insulin in brain  glucose metabolism, synaptic transmission, memory/learning, and neuroprotection. Then, we will focus preferentially on the protective effect of insulin against diabetes and its long-term complications in CNS, aging/longevity, and age-related neurodegenerative disorders ). Finally, we will briefly discuss the pros and cons of the potential therapeutic interest of insulin in diabetes- and age-related neurodegeneration. In fact, from the evidences presented herein, insulin appears to be a naturally occurring hormone essential for normal CNS function.Once in brain, insulin rapidly binds to IR, which are highly abundant (but selectively distributed) throughout CNS , 26, 29,α (120–135 kDa) and two β (95 kDa) subunits [Drosophila homolog of IR and mollusc IR-related protein, suggesting that their features have been highly conserved throughout evolution [: a “peripheral-” like type (with lower density in glia cells) and a neuron-specific type (highly expressed in neurons) [α subunits of the neuronal IR or IGF-1R, insulin (or IGF-1) promotes autophosphorylation of the β subunit at Tyr residues 1158, 1162, and 1163, triggering its intrinsic Tyr kinase activity [β (GSK-3β) and the Ras/Raf-1/extracellular signal-regulated kinases  [IR and IGF-1R are tetrameric glycoproteins that belong to the receptor tyrosine (Tyr) kinase superfamily, composed of two α 10–135 kDasubunits –40. Braiα 1–135 kDa subunits , 42. Besneurons) . Due to neurons) . Once boactivity –46 and pactivity , 46. As  ERK1/2) , 40, 46.After binding of the p85 SH2 domain to the phosphorylated Tyr of active IRS, PI3K becomes active and p110 inhibition is relieved, allowing translocation of active PI3K to plasma membrane and subsequent formation of PI-3, 4, 5-trisphosphate, and PI-3,4-bisphosphate. Then, these molecules bind to and recruit downstream signaling proteins containing pleckstrin homology domains. Amongst them, the serine (Ser)/threonine (Thr) kinase Akt can be recruited to the plasma membrane , 46–48 aβ, a Ser/Thr protein kinase that becomes inactive upon Akt-mediated phosphorylation at the N-terminal Ser9, initiating multiple physiologic effects [β is negatively regulated also by PKC and c-AMP-dependent protein kinase (PKA)  [β can be targeted to different intracellular locations  to easily access its substrates [β (Ser9 dephosphorylated) appears mostly in nuclei, mitochondria, and membrane lipid rafts , while inactive GSK-3β is mostly cytosolic [β can be also activated by phosphorylation at Tyr216, and, although this mechanism remains unclear, changes in intracellular Ca2+ or Fyn (a member of the Src Tyr kinase family) appear to be involved [β, occurring after PI3K inhibition, has been described to prevent apoptosis, while overexpression of a constitutively active GSK-3β resulted in PC12 cell death [β levels, after transient activation of PI3K/Akt signalling, prevented apoptosis induced by hydrogen peroxide (H2O2) in neural stem cells. Moreover, we previously showed that insulin prevented neuronal IR/IGF-1R inactivation upon oxidative stress and, by inhibiting GSK-3β (via PI-3K/Akt activation), insulin stimulated the synthesis of proteins involved in neuronal antioxidant defence, glucose metabolism, and antiapoptotic mechanisms [β-induced apoptosis remain unclear, decreased CREB-mediated expression of Bcl-2 [Of relevance in this signaling pathway is the GSK-3 effects , 50–53. r IGF-1) , 55. Depbstrates : active ytosolic . Besidesinvolved , 59. Thell death –62. Intell death  describechanisms . Althougof Bcl-2 , 66, mitof Bcl-2 –70, phosof Bcl-2 . β, IGF-1-induced activation of Akt also phosphorylates and inhibits FoxO3, which is retained in cytoplasm by the 14-3-3 protein. As a result, nuclear translocation of FoxO3 is blocked, as well as its subsequent deleterious targeting of nuclear genes, thus promoting hippocampal and cerebellar granule neuronal survival [κB (NF-κB) either directly [κB (IκB) phosphorylation at Ser32  [κB activation has been shown to increase Cu/Zn -superoxide dismutase (SOD) expression and MnSOD levels after H2O2 and amyloid beta (Aβ) treatment, protecting against oxidative stress and apoptosis in PC12 and NT2 cells, respectively [Besides GSK-3survival , 71, 72.survival . Akt maydirectly , 75 or vulation) , 77. Impectively . Alternaectively .m), intracellular ATP levels, NAD(P)H redox state, and hexokinase activity, that is, improves adult neuronal glucose metabolism and axonal outgrowth [Another target for insulin-induced PI-3K/Akt pathway is CREB, which, upon phosphorylation at Ser133, enhances mitochondrial membrane potential  binds to IR and Grb2 is attracted to bind to SHC via its SH2 domains, thus activating the ERK1/2 signaling pathway. SH3 domains are other Grb2 domains that interact with guanine nucleotide exchange son of sevenless (SOS) protein, stimulating the exchange of GDP for GTP at Ras, which becomes active and then recruits the Ser/Thr kinase Raf. Subsequent activation of MEK  leads to phosphorylation (and activation) of ERK1/2 on Thr and Tyr residues , 81–83, D-aspartate (NMDA) receptor-mediated excitotoxicity [Interestingly, ERK1/2 activation was originally described to play an antiapoptotic role in neurons via phosphorylation of Bad at Ser112 , 85. Contoxicity .Traditionally, insulin signaling through PI3K-dependent pathway was considered as functionally separated from ERK- or MAPK-dependent signals, with PI3K controlling metabolism, while the mitogenic role was related with ERK1/2 . HoweverBesides PI3K/Akt and ERK1/2 signaling pathways, insulin has also been described to protect against neuronal and non-neuronal apoptosis via activation of MAPK signaling , 94 , thus decreasing body weight [The most well-known insulin effect is the regulation of peripheral glucose transport and metabolism , 114. Hoy weight , 45, 116y weight , 45, 117y weight , 119. Thy weight .Although traditionally brain glucose metabolism was considered essentially insulin insensitive , some evβ-adrenoreceptors and glucose extrusion from glial glycogen stores, namely, in astrocytes [An alternative pathway for insulin to provide energy for neurons involves inhibition of neuronal norepinephrine uptake, with subsequent activation of glial trocytes , 120. Astrocytes .Taken together, these evidences suggest that any deleterious interference in the cross-talk between insulin and neuronal glucose metabolism may impair ATP synthesis and culminate in neuronal apoptosis . In thisSome authors hypothesized that brain insulin may play other roles apart from glucose metabolism, based on the heterogenous distribution of IR in brain , the poo2+ influx and reinforcing synaptic communication between neurons [Interestingly, these results appear to correlate with high IR expression in hypothalamus and limbic system  , 15, 115 neurons , and als neurons , 154. Th neurons . Additio neurons , 156. Ap neurons , 156, thβ (proapoptotic), and transcription factor of the Forkhead box 1  [Regarding this issue, we and others showed that insulin or IGF-1 attenuated both retinal and brain neuronal apoptosis induced by damaging conditions  , 157–162tivated) , 165. Intivated) . Surpristivated)  describetivated)  failed ttivated) , 168.Independently of the underlying signaling pathway, insulin has been increasingly shown to play a neuroprotective role against several damaging conditions, including oxidative stress, and to mitigate neuronal apoptotic death , 157. SiDiabetes mellitus, one of the most common metabolic disorders, is a major disorder of insulin regulation. Diabetes has reached epidemic proportions in western countries, and, according to World Health Organization estimates, it will affect ~300 million people worldwide in 2025, rendering diabetes an important public health concern in 21st century . This isA consequence of diabetes, mostly of type 2 diabetes, is insulin resistance and chronic peripheral hyperinsulinemia, accompanied by downregulated insulin transport into the brain and its subsequent deprivation from insulin beneficial effects . Indeed,β peptide (Aβ) [In previous studies, we described that, despite the apparent protection against oxidative stress in type 2 diabetic Goto-Kakizaki (GK) rat brain synaptosomes (probably due to higher plasma and brain vitamin E levels) , they haide (Aβ) , and weride (Aβ)  or insulide (Aβ) . This reide (Aβ) –181.•) production) may inhibit mitochondrial complexes III and IV and ATP synthase, either by nitrosylation or protein thiol oxidation [m, opening of mitochondrial permeability transition pore (mPTP), and cell death [Although the underlying mechanisms of diabetes-induced mitochondrial dysfunction are not completely understood, it has been proposed that increased mitochondrial nitric oxide synthase (NOS) activity  formation, glucose autoxidation, mitochondrial dysfunction, endoplasmic reticulum stress, and impaired antioxidant defences , 184, cuBesides brain glucose metabolism and oxidative stress, diabetes may also impair neurotransmission. Indeed, glutamate affinity for AMPA, but not NMDA receptors, was shown to be decreased in type 1 diabetic STZ rats . Others A receptors upon insulin treatment might protect against the cytotoxic effects of released excitatory amino acids. Importantly, the damaging effects of diabetes on neurotransmission may be potentiated by impairment of Ca2+ uptake to the endoplasmic reticulum and/or mitochondria, further exacerbating neuronal excitotoxicity [As an antidiabetes therapy, insulin has been the best studied and more efficient pharmacological compound mainly used in the treatment of type 1 diabetes. Nevertheless, it has been also increasingly used in the treatment of type 2 diabetes noncontrolled by diet, exercise or, oral antidiabetic agents, and its use has been shown to decrease microvascular complications and mortality , 191. Intoxicity . β) against brain oxidative stress and mitochondrial dysfunction [From the abovementioned, it seems that only diabetes-related hyperglycemia account for the long-term damage and that, by normalizing blood glucose levels, insulin might protect against those injuries. Nevertheless, it has been also increasingly demonstrated that recurrent hypoglycemic episodes (the most common side-effect of insulin therapy) may adversely affect diabetic CNS , 172, refunction , 194, thfunction . This sufunction , thus lifunction . Under tdb/db mice brain. Surprisingly, SHIP2 inhibition ameliorated hippocampal synaptic plasticity and memory formation. Conversely, when SHIP2 was overexpressed in mice, Akt-mediated IR/IGF-1R signaling was impaired, the neuroprotection by insulin/IGF-1 was attenuated, and increased neuronal apoptosis and impaired memory were also reported [As described before, diabetes has been widely associated with slowly progressive end-organ damage in brain , 195, rereported . Interestingly, insulin treatment has been described to prevent biochemical and pathological indices of peripheral sensory neuropathy in animal models of type 1 diabetes at doses that did not impact on hyperglycemia , suggestAging can be defined as a time-dependent loss of fitness that begins after the organism attains its maximum reproductive competence , that isA parallel antiaging paradigm has been increasingly proposed, in which the effect of genetic changes or caloric restriction in insulin signaling pathway might slow the rate of living, thus decreasing metabolism and oxidative stress –214. ThiMore recently, it has been suggested that epigenetic factors modulated by aging  impose a metabolic (redox) shift towards an increased reliance on glycolysis , probably mediated by age- and sedentarism-related insulin resistance (conditions that require lower metabolic demands). Accordingly, oxidized Kelch-like ECH-associated protein 1 (Keap-1) may block NF-E2-related factor (Nrf) release to signal mitochondrial biogenesis, thus decreasing mitochondrial synthesis and forcing cells to rely mostly on glycolysis . As a coAge-associated changes in IR signaling may also arise from increased cholesterol levels and decreased membrane fluidity  or from modification of IR internalization, reexpression, or degradation by the proteasome . NoteworDrosophila, mice, and man [γ-2 (PPARγ-2) gene (two well-known insulin sensitizers) in centenarians and long-lived men, whose type 2 diabetes incidence was also dramatically decreased [Drosophila genes that encode IR or IRS homologues were shown to extend lifespan of female fruit flies [Undissociated from aging is the longevity, which can be defined as the property of approaching the species-specific maximum lifespan, that is, the oldest observed age of death in the species . Rather  and man , 221–225ecreased . Additioecreased . In thisecreased . Null muit flies . Similarit flies , and oveit flies . Accordiit flies . Surprisit flies , suggestit flies .clk-1 mutant worms, showing reduced respiratory, developmental and behavioural rates, but longer lifespan. Such phenomena may be related with the fact that CLK-1 is essential for ubiquinone synthesis, an important component of the mitochondrial electron respiratory chain [Drosophila mutants with lower expression of electron transfer chain components in adult neurons [Studies in centenarian people and caloric restriction in rodents and nonhuman primates suggested that prerequisites for longevity include  decreased fasting glucose and oxidative stress , 231. Inry chain . Similar neurons  and in a neurons . In this regard, type 2 diabetes appears to be, at least partially, a model for premature aging. This idea has been further confirmed by a decrease of cellular replicative senescence in diabetic subjects . Thus, oTaken together, if (1) aging is frequently related with sedentarism and (2) the organism does not require extra demands for energy, then it can survive longer on the lower energy levels resulting from glycolysis. This can occur by downregulating the mitochondrial electron transport chain components and the activities of several redox-sensitive transcription factors, enzymes, transporters, and signaling proteins , as has been widely described . Howeverβ  by the β- and γ-secretases) [β1−40 and Aβ1−42) [β [β accumulation is also a common process in normal aged human, but its massive deposition in AD [βPP, impaired AβPP proteolytic processing, and/or mutations in the genes encoding for AβPP or presenilins [AD is a complex and common neurodegenerative disease that afflicted 26.6 million people worldwide in 2006, a number that can quadruplicate by 2050 . Clinicaretases) . The mosAβ1−42) . Althoug1−42) [β , 179. Imon in AD  may resuβPP gene in chromosome 21 [per se is a disease, and we must bear in mind that the boundaries between late-onset AD and “normal” aging are not absolute. This may be due to (1) increasing evidences from epidemiological, immunohistochemical, and molecular genetics studies suggesting a heterogenous etiology for AD [post mortem morphological and histological brain analysis [Only 5% (or less) of all AD cases have an early onset, autosomal dominant familial origin, probably due to missense mutations in presenilin (PS) genes 1 or 2 in chromosomes 14 and 1, or in the Aosome 21 . Additioosome 21 . On the osome 21 , 232. Aly for AD  and (2) analysis . Indeed,analysis . Therefoβ was shown to directly induce overproduction of reactive oxygen and nitrogen species and neurotoxicity [β-associated decreased plasma antioxidant defences , as described by several authors [β neurotoxicity in human and rat cortical and hippocampal neurons and delayed disease progression in AD patients [Drosophila) has been suggested to constitute an age-related stress-resistance mechanism in AD brains [Although the exact pathophysiologic mechanisms underlying signal transduction abnormalities and neurodegeneration in AD brain remain unknown, oxidative stress and metabolic dysfunction appear to be involved , 243. Intoxicity . This wa authors –248. Add authors , 250. Sipatients . MoreoveD brains . Taken together, these data unequivocally demonstrate the involvement of oxidative stress in AD and suggest a potential therapeutic role for antioxidant supplementation , 252. Hoα-ketoglutarate dehydrogenase) were decreased in AD brains and cells exposed to Aβ [Although the underlying causes of reduced metabolism in AD are not completely understood, it has been reported that atrophy of cerebral vasculature (the major metabolic exchange surface of brain), a decrement in brain glucose transport activity, or even impaired insulin signaling might play a crucial role . Indeed,ed to Aβ –264. Mored to Aβ  observedpost mortem brain tissue [One of the most common changes in electron transport chain underlying mitochondrial dysfunction in AD is decreased cytochrome oxidase activity, described in such distinct AD human tissues as platelets , 267 andn tissue , 269. Fun tissue , 271. Van tissue  also repn tissue , 273. ε4 (APOε4) allele further supports this hypothesis [β-cell loss in both diseases [Noteworthy, among dementia-type disorders, AD has been increasingly associated with type 2 diabetes , 275. Inpothesis , 27, 278pothesis , and thidiseases .β oligomers were shown to interfere with IR function, probably due to a loss of these receptors at dendrites and their increased expression in the cell soma [More interestingly, a recent proposal refers that AD can be an “insulin-resistant brain state” , or evenell soma , despiteell soma . Accordiell soma  suggesteβ levels [β inhibited Aβ production [β levels, as in Tg2576 AD transgenic mice [βPP cleavage and Aβ levels in brain. Similar results were obtained upon selective neuronal disruption of IGF-1R in Tg2576 mice [β accumulation by promoting its transport into CNS via Aβ-binding carrier proteins  [β degradation via insulin-degrading enzyme (IDE). This metalloprotease, that also catabolizes insulin and IGF-1, can be competitively inhibited by insulin resistance [β degradation, increasing its neurotoxicity and promoting AD [β clearance in hippocampal neurons [Concerning increased fasting plasma insulin, decreased CSF insulin levels, and/or decreased CSF/plasma insulin ratio in AD patients , 175, thβ levels , 274, duoduction  and its oduction , 40. Howoduction  and its nic mice . More renic mice  reported576 mice . Alternaalbumin) , 290–292sistance , 278, imoting AD , 46, 278oting AD , 46, tha neurons . β, ERK1/2, and Cdk-5 [β inhibition blocked specific phosphorylation of tau protein in PC12 cells. Moreover, in diabetic animals treated with insulin, a complete prevention of tau protein hyperphosphorylation was reported, probably resulting from reestablishment of brain insulin signaling [Insulin and IGF-1 were also described to modulate both physiological and abnormal neuronal tau protein phosphorylation, in a process involving Akt, GSK-3nd Cdk-5 , 40, 44.nd Cdk-5  observedignaling . This isignaling  and IRS-ignaling . Interesignaling  and insuignaling  were designaling , 296 andignaling , insulinignaling . From all the above-mentioned evidences, we believe that a valuable therapeutic window for insulin against diabetes- and age-related neurodegenerative disorders  may exist. Under these conditions, an inadequate trophic support to brain may occur due to lower insulin/IGF-1 levels and/or damaged IR/IGF-1R-mediated signaling, affecting gene transcription and culminating in neurodegeneration/death, cognitive dysfunction, and, ultimately, in long-term complications (in case of diabetes) and/or in AD (and other dementia) . Thus, tβ for IDE, increasing extracellular Aβ accumulation  [1 (DAQB1) that does not competitively inhibit IDE and modulates IR [β and subsequent Aβ production and tau protein hyperphosphorylation (as well as increased Aβ clearance via stimulation of transthyretin and IDE), another strategy could be the improvement of insulin signaling [β inhibitors could be attractive, but it might also impair several vital physiological targets of this kinase or have no impact on other critical components of the neurodegenerative cascade [However, chronically high insulin levels in brain can be deleterious , due to  of IDE) , 46. A t of IDE) , 303. Otlates IR , (2) theignaling , 46, 218per se or on external, lifestyle factors remains a matter of debate. In this regard, one of the main challenges for the next decades will be to unravel the complex interactions between aging and diabetes that underlie insulin resistance, allowing the development of more efficient preventive and therapeutic strategies to overcome age-related neurodegenerative diseases.In the last three decades, brain insulin signaling has faced a novel and increased interest in neuroscience research, either in its signaling pathways and/or as a promising therapy against diabetes and age-related neurodegenerative disorders . From the first studies recognizing the abundance of insulin and IR in brain to its potential involvement in numerous neurodegenerative diseases associated with diabetes (particularly type 2 diabetes) and aging mediated less than 20 years. Indeed, in 1999, Halter described that elderly people have impaired insulin sensitivity, which might account for by the slight age-related increase in fasting glucose levels and the delay in return to normal glucose levels after an oral glucose tolerance test. However, more than 10 years later, the elucidation on whether the origin of this insulin resistant state relies on aging β production (and its increased clearance) and tau protein hyperphosphorylation, two well-known hallmarks of the pathology.Concerning AD, a recent hypothesis points towards the idea that AD is the “brain-type diabetes.” This is supported by decreased number or binding capacity of brain IR in both AD patients and mouse models, increased risk for type 2 diabetes in AD patients (and vice versa), and the accumulation of hyperphosphorylated tau in the CNS of IRS-2-disrupted mice, a model of type 2 diabetes. Thus, restoring insulin levels and/or its receptor-mediated signaling cascades (without affecting blood glucose levels) constitute a potentially interesting therapeutic strategy against AD, due to the inhibition of AMore recent studies focused on the importance of insulin/IR signaling in increased longevity. However, the results remain highly controversial. In summary, in CNS, rather than just an “acquired” peripheral hormone, insulin appears to be a naturally occurring peptide of the outmost importance.


Postoperative radiation therapy, 5000 cGy, was delivered, synchronous with triweekly CMF. After radiation therapy, two courses of HDCT with CBDCA, IFX, VP-16, were given, with hematological growth factors. After a median follow-up of 81 months, all patients were evaluable for toxicity and response. Most important toxicity were grade 3 skin reaction and grade 4 hematological in 3% and 31% of patients, respectively. Pathological complete response was observed in 25% of patients receiving preoperative chemotherapy. Treatment failures were as follows: eight visceral, four contralateral breast cancer, four locoregional, and one leukemia. Five-year progression-free survival and overall survival rate were 78% and 91%, respectively. Induction chemotherapy, followed by chemoradiation therapy and HDCT, provides a prolonged disease-free period and a significant increase in overall survival in TNBC, with an acceptable toxicity profile.Anthracyclines (A) and taxanes (T) are standard first-line chemotherapy agents for patients with advanced breast cancer. Platinum analogues have also shown activity in the triple-negative breast cancer (TNBC) histology, but clinical data are limited. Here we report the long-term follow-up of a phase II study on TNBC treated with a combined modality therapy, including induction with AT, cyclophosphamide, methotrexate, and 5-fluorouracil (CMF) with concurrent radiation therapy, and a dose-dense consolidation chemotherapy (HDCT) with carboplatin (CBDCA), ifosfamide (IFX), etoposide (VP-16). Patients' median age was 44 years, with 73% premenopausal. Epirubicin 75 mg/m Basic knowledge on mechanisms causing breast cancer progression has driven significant progresses in its treatment, with the introduction of more sophisticated hormonal and targeted therapies. Unfortunately triple-negative breast cancer (TNBC) is a biological entity that lacks estrogen receptors (ER), progesterone receptors (PGR), and human epidermal growth factor receptor 2 (HER2) . TheAt the time our study was undertaken, there was a limited experience in the treatment of breast cancer with platinum analogues , 20. Nev3, absolute neutrophil count >1500/mm3, hemoglobin level >9.0 g/dL, platelets >100,000/mm3, serum bilirubin <1.5 mg/dL, aspartate aminotransferase, and alanine aminotransferase <4 times the upper limit of normal and normal cardiac and renal functions . Patients with additional malignancies, other than curatively treated skin and cervical cancer or with active cardiovascular disease, were excluded. The protocol was approved by the Ethical Committee of the Civilian Hospital of Avezzano, Italy, and of the other participating institutions, and written informed consent was obtained from each patient.The study recruited biopsy-proven, previously untreated patients with large (T2–T4) ER negative (ER−), PGR negative (PGR−), HER2-negative breast cancer, aged 18–70 years, with an Eastern Cooperative Oncology Group (ECOG) performance status <2, and a life expectancy of at least 12 weeks. The patients, not pregnant or lactating, had to have an adequate hematological reserve and hepatic and renal function, documented by a WBC count >3000/mmA complete staging workup was carried out, including medical history, physical examination, electrocardiogram, and bidimensional echocardiography, mammography, chest X-ray, liver ultrasonography, and radionuclide bone scan. A computed tomography or magnetic resonance imaging of the brain was also carried out in case of suggestion of brain involvement. Baseline laboratory studies included complete blood counts (CBCs), liver and renal function tests, estradiol, progesterone, follicle-stimulating hormone (FSH), luteinizing hormone (LH), carcino-embryonic antigen (CEA), and carbohydrate antigen (CA 15-3). A core biopsy of the breast tumor was also performed along with immunohistochemical assessment of hormone receptors, Ki-67, HER2. In case of HER2 (+), FISH was performed to determine HER2 positivity. Tumors were classified as ER+ or PGR+ if staining was present in >1% or more of tumor nuclei. The Ki-67 cut point of 13% was used to designate a tumor as high proliferation.2 and docetaxel 75 mg/m2, both given over 2 hours on day 1. Premedication with dexamethasone at a dose of 8 mg was given 12, 6, and 1 hour before docetaxel administration and then twice a day for 4 days after chemotherapy. Breast-sparing surgery when feasible, or modified radical mastectomy together with standard level I and II axillary lymph node dissection, was carried out 3 weeks after the end of chemotherapy for patients with T4 or inflammatory tumors and as initial treatment to the 58 patients with T2–T3 tumors. XRT was delivered to the 26 patients with radical mastectomy and to the 44 patients with quadrantectomy at the dose of 5000 cGy, 200 cGy/fraction, 5 fractions/week, to the chest wall after mastectomy or to the residual breast after breast-conserving surgery, and at the apex of the axilla and supraclavicular lymph nodes. A boost of 1000 cGy was delivered to the tumor bed. XRT was given synchronous with six courses of triweekly cyclophosphamide 600 mg/m2, methotrexate 40 mg/m2, 5-fluorouracil 600 mg/m2 (CMF). One month after the end of chemoradiation therapy, two courses of dose-dense chemotherapy (HDCT) with CBDCA, area under the concentration curve (AUC) = 7, VP-16 400 mg/m2, ifosfamide, and uromitexan 6000 mg/m2, were delivered, over 3 days, supported by glycosilated recombinant granulocyte colony-stimulating factor (G-CSF) 300 μg/day. Chemotherapy was delivered at full doses if absolute neutrophil count (ANC) >1500 mm3 and platelets >100,000 mm3. For ANC between 1000 and 1499 mm3, and/or platelets between 75,000 and 99,000 mm3, chemotherapy was administered at doses reduced to 50%. In presence of lower ANC or platelet values, treatment was omitted.An intensive psychological support that improved adherence to treatment was implemented by one of the authors (C. O. C. R.). Fifty-one (73%) of the 70 patients were premenopausal: they received a leuteinizing hormoneion . The sixToxicity was evaluated according to the National Cancer Institute—Common Toxicity Criteria version 3. A CBC and chemistry were checked weekly in all patients and daily in case of grade 4 leukopenia. A bidimensional echocardiogram was carried out at completion of treatment and every 6 months thereafter for 3 years. After chemotherapy, tumor response was assessed according to standard World Health Organization criteria . Medical2 contingency table analysis. Statistical analysis was performed with SAS statistical software .The number of patients required for the study was calculated according to a Simon minimax design . The firFrom July 2001 to April 2007, 70 consecutive cases with triple-negative large breast cancer were entered in the study at the Civilian Hospital of Avezzano, at the University of L'Aquila and at the Foundation “Carlo Ferri”. All the patients completed the planned protocol. Analysis was performed on the basis of intention to treat. After a median follow-up of 81 months (minimum 24.2 months), 53 patients (76%) were progression free and 62 patients (89%) were alive. Median PFS was not reached yet, and 5-year PFS and OS rate were 78% and 91%, respectively, while 10-year PFS and OS rate were 64% and 83%, respectively  and 2. O9/L in 21 patients, with only three episodes of febrile neutropenia. Seven patients showed grade 3–4 thrombocytopenia, but no platelet transfusion was required.Thirty-seven (53%) patients showed grade 3–4 neutropenia. Neutrophils fell below 0.5 × 10Alopecia was almost universal. Grade 3 skin reaction was observed in two patients during radiation therapy synchronous with chemotherapy. Among the other non-hematological side-effects, emesis, fatigue, loss of appetite, and mucositis were the most common. In particular, 70% of patients complained of nausea/vomiting, but only in few cases was it severe. Half of patient patients suffered from grade 1 diarrhea. Severe stomatitis was observed in five (7%) patients, while no episodes of grade 3 peripheral neuropathy were observed. Musculoskeletal symptoms like transient arthralgias and myalgias occurred in a total of 32 (45%) patients, but they were severe in only two cases, and generally responded well to acetominofen. In the majority of cases, these symptoms were related to G-CSF administration.Cardiac toxicity was almost absent. Only five patients developed a decline in left ventricular ejection fraction (LVEF) by >10%, but this remained asymptomatic and no treatment was required. Finally, seven episodes of transient increase of fivefold of the aspartate aminotransferase (AST) and alanine aminotransferase (ALT) serum levels were observed, without any clinical sign of liver dysfunction.Recent progress in medical science has led to a sophisticated classification of breast carcinomas, based on variations in gene expression patterns derived from cDNA microarrays. This classification, distinguishing breast cancers into basal type (triple negative), luminal, and HER2/neu, has been correlated with clinical outcome and response to therapy –5. HowevOther strategies have been adopted to improve the clinical outcome of patients with TNBC. In recent years several studies have emphasized the role of vascular endothelial growth factor (VEGF) as a key mediator of angiogenesis. Bevacizumab (Avastin), the best-known anti-angiogenic agent, is a humanized monoclonal antibody that binds to VEGF and prevents it from interacting with vascular endothelial cells , 32. BevWe and others have previously shown that two courses of CBDCA, CTX, and VP16 as consolidation therapy provide significant survival benefits to patients with basal-like TNBC , 34. GluCollectively, it is common opinion that mastectomy renders radiation therapy unnecessary unless the tumor is 5 cm or larger, its margins are irregular with signs of invasion through basal membranes of adjacent normal mammary tissue, or if there is nodal metastasis. Tseng et al., however, suggested that adjuvant radiation in all patients with metaplastic breast cancer may lead to improved OS . Due to In conclusion, in our cohort of patients with TNBC our multipronged approach involving induction chemotherapy, followed by chemoradiation therapy and by HDCT provides a prolonged disease-free period and a significant increase in OS, with an acceptable toxicity profile. We anticipate that our results will prompt the application of our treatment strategy in the clinical management of TNBC.


Poly(ADP-ribosyl) polymerases (PARPs) have traditionally been linked to chromosome maintenance and DNA repair. Recent findings identify PARPs as key modulators of metabolism through their influence on SIRT1 activity, hinting to a possible role of PARPs as longevity regulators. Defects in energy metabolism, and, more specifically, mitochondrial function are intimately linked to the general decline in physiological functions during ageing . While g+ as a cosubstrate, which is converted into nicotinamide (NAM) by the respective enzymatic activities [+ to form polymers of ADP-ribose units onto acceptor proteins, cleaving off NAM. Cellular poly(ADP-ribose) (PAR) formation is dramatically stimulated upon DNA damage, and, as a result, NAD+ levels plummet when DNA is damaged [Recent observations from our lab , 4 can ctivities . PARP activities ). The re damaged , 7. Whil damaged , 9. The + levels [m of SIRT1 for NAD+ is around the physiological concentration range of NAD+, implying that in diverse circumstances, NAD+ can rate-limit SIRT1 activity [SIRT1, the first mammalian member of the sirtuin family identified, is a NAD+-depedent deacetylase that regulates diverse aspects of global metabolism by regulating the acetylation status and activity of a number of metabolic enzymes and transcriptional regulators . Notable+ levels . The Km activity .+ sensor that translates changes of the metabolic/redox state of the cell into adaptive transcriptional responses [Due to this particular characteristic, SIRT1 has been proposed to act as an intracellular NADesponses . Furtheresponses .+ as a common cosubstrate has in the past led to propose a possible interrelationship between PARP and SIRT1 activities [+ content [+ is not immediate, probably because PARP-1 activity is very low in cells/tissues in basal conditions, and it requires a protracted inhibition of PARP-1 to promote significant effects on NAD+ accumulation. This enhanced NAD+ content then activates SIRT1, leading to the deacetylation of key targets that induce mitochondrial biogenesis [2O2, PARP-1 is activated and SIRT1 activity is robustly reduced, as PARP-1 activation limits NAD+ bioavailability [+ levels and SIRT1 activity [+ availability.The use of NADtivities . In two tivities , 4. The  content . This efogenesis . In contogenesis . Converslability , 7, 15. activity . Collectactivity , these o+ levels in the cellular models [+ levels, was attributed to a higher expression level of SIRT1 [+ availability (PARP-1) and by regulating SIRT1 expression at the transcriptional level (PARP-2)  [+ homeostasis are, however, not clear [Our results obtained in the PARP-1 and PARP-2 knockout mice would also predict that higher PARP-1 gene dosage would compromise metabolic fitness. In line with the predictions from our studies, mice overexpressing hPARP-1 were heavier than their wildtype littermates , an effeain used , 26, 27.se (Nnt) , 29, proot clear , 31. FinDespite these novel and tantalizing findings, the many remaining open questions clearly illustrate that, half a century after their initial discovery , PARPs s


Numerous antimicrobial peptides (AMPs) from marine fish have been identified, isolated and characterized. These peptides act as host defense molecules that exert antimicrobial effects by targeting the lipopolysaccharide (LPS) of Gram-negative bacteria. The LPS-AMP interactions are driven by the biophysical properties of AMPs. In this review, therefore, we will focus on the physiochemical properties of AMPs; that is, the contributions made by their sequences, net charge, hydrophobicity and amphipathicity to their mechanism of action. Moreover, the interactions between LPS and fish AMPs and the structure of fish AMPs with LPS bound will also be discussed. A better understanding of the biophysical properties will be useful in the design of AMPs effective against septic shock and multidrug-resistant bacterial strains, including those that commonly produce wound infections. Recent research has shown that certain marine peptides possess antimicrobial, antifungal, antiviral, anticoagulant and/or antifreeze properties; indeed, the number of antimicrobial peptides (AMPs) isolated from marine organisms and which are effective against pathogenic bacteria continues to grow. These AMPs are found in a wide range of phyla, including Crustacea, Cnidaria, Mollusca and Porifera, as well as in a number of fish species ,2,3,4,5.Tachypleus tridentatus and Limulus polyphemus [L setiferus [P. monodom [F. chinensis [M. japonicas [L. vannamei [F. paulensis, L. schmitti [L. styrirostris [P. monodon based on findings from the expressed sequence tag database [ALFs were initially isolated from hemocytes of the horseshoe crabs (chelicerates) lyphemus ,7 and welyphemus ,8. Crustetiferus , P. mono monodom , F. chinhinensis , M. japoaponicas ,13, L. vvannamei ,15, F. pschmitti  and L. sirostris . In addidatabase . Many ofdatabase . In maridatabase ,21, espedatabase ,28,29,30LPS is the major component of the outer membrane of Gram-negative marine bacteria. Part of the function of LPS is to protect marine bacteria from harmful environmental factors. A major component of LPS is lipid A, also known as endotoxin. Lipid A molecules are the main pathogens responsible for septic shock in both fish and humans. When Gram-negative marine bacteria enter the body of a marine fish, Lipid A triggers the innate immune system via Toll-like receptors, which activates inflammatory signaling pathways that lead to septic shock. ALFs from marine fish are able to bind and neutralize lipid A, thereby preventing the inflammatory signaling ,32. ThesScylla tranquebarica ALF (StALF) was isolated from hemocytes from the mud crab Scylla tranquebarica, while Portunus pelagicus ALF (PpALF2) was isolated from the blue swimmer crab Portunus pelagicus. Comparison of their sequences with those of previously characterized ALFs showed that both contain an LPS-binding domain [2 and StALF contain 12.4% lysine (Lys) and also contain 5.2% and 6.2% arginine (Arg), respectively. In addition, they contain substantial percentages of hydrophobic amino acids. Like other ALF molecules, PpALF2 and StALF exhibit a repeat pattern of cationic and hydrophobic amino acids within a disulfide loop, which suggests they share the same functional domain. Previous studies also reported that alternating hydrophilic and hydrophobic amino acids are characteristic of molecules with antimicrobial and LPS binding activities [The ALF isoform g domain . The seqtivities ,35,36, ativities . Moreovetivities . N-terminal region include 10 hydrophobic residues and 17 positively charged amino acids. Similarly, the 35 residues of the StALF N-terminal region contain 10 highly hydrophobic amino acids and 20 positively charged amino acids. The LPS binding domain is formed within a 22-residue disulfide loop . The sequences of the loops are similar with the PpALF2 loop containing seven positively charged amino acids and a tryptophan (Trp) and the StALF loop containing nine positively charged amino acids and a Trp. The identical residues include C1, H2, R5, K6, P7, K8, F9, R10, K11, F12, K13, L14, Y15, H16, E17, G18, K19, F20, W21 and C22. However, residues 3 and 4 differ between the two loops: FF in PpALF vs. IR in StALF. Based on this difference in the LPS binding domain, the two molecules differ with respect to their molecular weights, net cationicity and percent hydrophobicity , it contains a 24-amino acids LPS binding site (TCHIRRKPKFRKFKLYHEGKFWCP) with a disulfide-bonded loop region spanning residues 54–77 of the protein. Moreover, a synthetic 24-amino acid peptide consisting of the residues in the predicted LPS binding region exhibits bactericidal activity towards several species [N-terminal region of the protein [An ALF from the mud crab  library . Designa species . The ALF protein . This giScylla paramamosain have been produced [Pichia pastris. Comparative analysis of the recombinant proteins (rSP-ALF1 and rSp-ALF2) indicates the presence of a putative LPS binding loop, and synthetic peptide fragments of the LPS binding domain ((sSP-ALF1 (Acetylated-HTCHFRRRPKVRKFKLYHEGKFWCPG-aminated) and sSp-ALF2 (Acetylated-HTCHIRRRPKFRKFKLYHEGKFWCPG-aminated) as well as the recombinant proteins (rSP-ALF1 and rSp-ALF2), themselves, exhibit strong bactericidal activity against Gram-positive and Gram-negative bacteria [The full-length cDNAs encoding SpALF1 (JQ069030) and SpALF2 (JQ06931) from the mud crab produced   has been cloned, expressed with a Histag in E. coli, and characterized [Scylla paramamosain and about 33%–53% amino acid sequence identify with other known ALFs. Expressed and purified recombinant SsALF exhibits both LPS neutralization and antimicrobial activity that involves bacterial membrane disruption [cterized . Sequencsruption . MoreovePortunus trituberculatus ALF (PtALF) from the swimming crab Portunus trituberculatus was first identified in a hemocyte cDNA library [Vibrio alginolyticus, PtALF mRNA is ubiquitously expressed in muscle, stomach, heart, gill, hepatopancreas and hemocytes, suggesting it play an essential role in the animal’s innate immune response [Scylla serrata, S. paramamosain, P. leniusculus and P. monodon, respectively [. It is therefore likely that PtALF will share the anti-LPS and antibacterial activities of other ALFs [ library . Howeverresponse . PtALF aresponse  , was identified in an eyestalk cDNA library [V. alginolyticus into hemocytes, again indicating PtALF6 is significantly involved in the elimination of invasive pathogens [N-terminal and two α-helices at the C-terminal, including a hairpin loop formed by a disulfide bond (C27 and C48) between strands 2 and 3 of the anti-parallel β-sheet [Another ALF from  library . Like otathogens . Interesathogens . The LPS β-sheet .Penaeus monodon: Group A includes Penaeus monodon ALF1 (ALFPm1) and Penaeus monodon ALF2 (ALFPm2), while group B contains Penaeus monodon ALF3-5 (ALFPm3-5) [N-terminal region than the C-terminal, and a conserved disulfide loop contains a positively charged cluster as the putative LPS-binding domain [P. monodon. Earlier findings that ALFPm3 exerts its antibacterial effects by binding to LPS, which is why it is known as an ALF [Vibrio harveyi cells, acting through a membrane disruption mechanism [P. monodom [V. harveyi infection, which is indicative of the important function of ALFs against bacterial invasion [Through nucleotide sequence analysis, two groups of ALFs have been derived from different genomic loci from the black tiger shrimp LFPm3-5) . Nearly g domain . Of the g domain , is the s an ALF ; howevers an ALF . Furtherechanism , and is  monodom . Tissue invasion . ALFPm3 invasion . Interesinvasion .Biophysical techniques such as SPR indicate that rALFPm3 binds lipid A of LPS and OM-174, the water-soluble analogue of lipid A . HoweverThe ALFPm3 protein #35–51 contains part of a putative LPS-binding region (KPYLKRFQVYYKGRMWC) and shows some antimicrobial activity, but does not contain a disulfide bond. On the other hand, the synthetic LPS binding peptide clearly shows less antimicrobial activity towards Gram-negative bacteria than intact rALFPm3. It is noteworthy that reduction of the disulfide bond in the LPS-binding region of ALFPm3 protein #35–51 does not completely eliminate its antimicrobial activity; its effect on LPS binding activity is not yet known .The amino acid composition of the putative LPS-binding domains of ALFPms differs somewhat. There are clear differences in the total numbers of positively charged amino acid residues (Arg and Lys) among the ALFPms. ALFPm1 (CRYSQRPSFYRWELYFNGRMWC) and ALFPm2 (CRYSQRPSFYRWELYFNGRMWC) contain four Arg, while ALFPm3 (CKFTVKPYLKRFQVYYKGRMWC), ALFPm4 (CKFTVKPYLKRFQVYYKGRMWC) and ALFPm6 (CSFNVTPKFKRWQLYFRGRMWC) contain two Lys and two Arg. Moreover, the LPS binding domain of ALFPm1, 2 and 6 contains two Trp residues, while those of ALFPm3 and 4 have only one Trp residue. Consequently, the degree of amphipathicity varies among ALFPms which is mainly based on the balance of the sizes of their cationicity and hydrophobicity .Marsupenaeus japonicus ALF (MjALF)) was isolated from the kuruma prawn Marsupenaeus japonicus. The deduced amino acid sequence of MjALF1 has 42% homology with the Japanese horseshoe crab T. tridentatus ALF, and Clustal W alignment showed 36.6% homology with L-ALF from the Atlantic horseshoe crab L. polyphemus [in vivo [cDNA encoding an ALF-like peptide  and MjALF2 (CRYSQRPTFYRWELYFRGSMWC) share identical amino acids at C1, P7, F9, W12, L14, F16, G18, M20, W21 and C22. In addition, synthetic peptides containing amino acids C30 to C51, corresponding to the LPS-binding domainFenneropenaeus chinensis (ALFFc) was cloned from hemocytes from the Chinese fleshy prawn Fenneropenaeus chinensis. Multiple alignments showed the amino acid sequence of ALFFc shares 56% homology with the sequences of ALFs from Tachypleus tridentatus and L. polyhemus. ALFFc contains a disulfide loop (CKFTVKPYIKRFQLYYKGRMWC) that binds LPS and neutralizes its septic effects [V. anguillarum infection [The full-length cDNA encoding ALF  effects ,58,59. Anfection   have been identified in the giant freshwater prawn Macrobrachium rosenbergii. Of them, MrALF5 (CSFQVKPRIKRWELYFRGTMWC), MrALF6 CIYKRTGYFYKWELHYKAEVRC and MrALF7 (CTYNMRPFFKNWKLYYSASVIC) have been characterized [E. coli challenge. By contrast, MrALF5-7 gene expression is upregulated by Vibrio or white spot syndrome virus challenge, suggesting MrALF5-7 may participate in the immune response to the bacteria and virus. Importantly, MrALF5-7 contain a LPS-binding domain with two conserved Cys residues and eight conserved hydrophobic amino acids; however, their amino acid compositions, balance of net charge and hydrophobicity and degree of amphipathicity all differ from one another (Multiple isoforms of cterized . MrALF5  another .Homarus americarius1 (ALFHa1) and ALF Homarus americarius2 (ALFHa2) were identified from the American lobster Homarus americarius [N-terminal signal peptide and Cys residues participating in a disulfide bridge. The gene expression of ALFHa1 was increased by Vibrio fluvialis in the gill, hemocytes and hepatopancreas, whereas expression of ALFHa2 was not significantly affected by Vibrio in any of the three tissues tested [in vitro assay conditions. ALF ricarius . The seqs tested . The LPSMacrobrachium olfersi (ALFMolf) was identified in hemocytes from the freshwater prawn Macrobrachium olfersi [The sequence of an ALF  olfersi . The ami olfersi   was identified in the Chinese mitten crab Eriocheir sinensis [E. coli, and the recombinant EsALF exhibited bactericidal activity towards a spectrum of bacterial strains that was similar to recombinant ALFs from P. monodon [T. tridentatus [Vibrio anguillarum infection. The sequence of the LPS binding domain in EsALF (CNTRVMPTIKKFELYFRGRVWC) contains several cationic (three Arg and two Lys) and hydrophobic residues for LPS binding activity (The cDNA encoding sinensis . The mat monodon  and T. tdentatus . EsALF mactivity .Tachypleus tridentatus and Limulus polyphemus. The sequence between amino acids 31 and 52 (CRKPTFRRLKWKIKFKFKC) comprises the LBS binding domain [in vivo [The Limulus Anti-LPS factor was identified in hemocytes from the marine chelicerates g domain . CLP19 i[in vivo . It has [in vivo , and to [in vivo . In addi[in vivo . The ord[in vivo , which i[in vivo ,55  and Lys16 is in a C-terminal longer helix (residues Lys16-Ser28). Residues Pro13, Leu14 and Phe 15 form a short loop between the short helix and the longer helix. Other residues, Phe2-Ala4 and Ser29-Glu33, fall into extended conformations in the LPS micelle [S. aureus (MRSA) infection in mice with skin injuries. GE33 reduced the amounts of two pro-inflammatory mediators, TNF-α and IL-6, released during wound healing, thereby promoting the wound healing process [Pardaxin is an amphipathic polypeptide composed of 33 amino acid residues (GFFALIPKIISSPLFKTLLSAVGSALSSSGGQE) which was identified in the pacific peacock sole fish avoninus  and the rmoratus   with LPS was shown to contain a disulfide-stabilized β-hairpin. Another study of the NMR structure showed that a tachyplesin-analog (KWFRVYRGIYRRR-NH2) was stabilized in LPS micelles through the interaction between the aromatic ring of W2 and the side chain of the nonpolar amino acid V5 and the cationic side chain of R11 [Anti-LPS tachyplesin was isolated from the hemocytes of the horseshoe crab . Using Nn of R11 . Apart fn of R11 , while tn of R11 . It is nn of R11 .N-terminal and more hydrophobic residues at their C-terminal. The positively charged Lys and Arg residues contribute to electrostatic interactions with the diphosphoryl head groups of the LPS, while the hydrophobic residues most likely interact with the hydrophobic acyl chains of the LPS though hydrophobic interactions. Another study also explained that the extra two Lys residues at the N-terminus of the peptide likely increase the peptide’s LPS-neutralizing activity [Sushi 1  and Sushi 3  were derived from the LPS-binding domain of an LPS-sensitive serine protease, Factor C, from the horseshoe crab ,77 Figu. Both peactivity . 2) [Glyptocephalus cynoglossus [The NRC-16 peptide (GWKKWLRKGAKHLGQAAIK-NH2)  is a truoglossus . NRC-16 oglossus . This peoglossus , which ioglossus ,83,84,85in vivo, and to protect against sepsis in animal models [The cationic ALFs with various potentials to form amphipathic α-helices, β-sheet and loop are rich in cationic and hydrophobic side chains. Particularly, the LPS binding domain of ALFs mostly adopt β-sheet with amphipathic loop. The other ALFs (NRC-16 and pardaxin) adopted amphipathic α-helices with LPS. This clearly explains that the cationic and hydrophobic residues, hydrophobicity, amphipathic property and secondary structures are needed for the LPS binding domain of the ALFs. Moreover, some of the ALFs discussed in this review bind LPS and exhibit both anti-LPS and antimicrobial activity, but are not sufficient to neutralize LPS-induced macrophage activation. Others were capable of binding to pathogens and killing bacteria, exhibiting both LPS sequestering and neutralization, though their mechanism of action is not yet known. It is believed that AMPs  are able to tightly bind LPS and break its aggregates ,87, whicl models . Arabidopsis thaliana hydathodes by Xanthomonas campestris, a Gram-negative bacterium that is a common plant pathogen [LPS also contributes directly and indirectly to bacterial biofilm formation ,90 and ipathogen .in vitro and in vivo. In addition, these peptides exhibit very low cytotoxicity under physiological conditions, making SALPs promising candidates for application as therapeutic agents for the prevention and treatment of septic shock [It is clear that ALFs are essential components of innate immunity in fish, contributing to the first line of defense against a wide range of pathogens. AMPs and ALFs are capable of binding and neutralizing LPS and promoting angiogenesis and wound healing ,97. It hic shock .Because of their antimicrobial, antibiofilm, anti-inflammatory, and pro-angiogenesis and wound healing activities, there is growing interest in the therapeutic potential of ALFs. It is anticipated that the design of new AMPs based on the LPS binding domain of ALFs will enable development of novel synthetic peptides with antimicrobial, anti-inflammatory and anti-biofilm activities in a wound environment.


The steroidogenic acute regulatory (StAR) protein-related lipid transfer (START) domain proteins constitute a family of evolutionarily conserved and widely expressed proteins that have been implicated in lipid transport, metabolism, and signaling. The 15 well-characterized mammalian START domain-containing proteins are grouped into six subfamilies. The START domain containing 7 mRNA encodes StarD7, a member of the StarD2/phosphatidylcholine transfer protein (PCTP) subfamily, which was first identified as a gene overexpressed in a choriocarcinoma cell line. Recent studies show that the StarD7 protein facilitates the delivery of phosphatidylcholine to the mitochondria. This review summarizes the latest advances in StarD7 research, focusing on the structural and biochemical features, protein-lipid interactions, and mechanisms that regulate StarD7 expression. The implications of the role of StarD7 in cell proliferation, migration, and differentiation are also discussed. Lipids are currently recognized as versatile and dynamic regulators of various cellular processes such as growth, development, survival, intracellular signaling, and membrane trafficking. Lipid alterations are present in most human diseases such as cardiovascular disease, obesity, metabolic defects at birth, cancer, Alzheimer’s disease, and preeclampsia. The transport of lipids and proteins between organelles is a crucial event in the organization of different cellular compartments. This process is mediated by vesicular transport through the fusion of vesicles to an acceptor compartment or by monomeric transport among different organelles . MonomerIn this review, we summarize the current state of knowledge in StarD7 research, focusing on the molecular characteristics, protein-lipid interactions, and mechanisms that regulate StarD7 expression.N-terminal region. Our group provided the first evidence of the presence of the StarD7 protein in human trophoblast cells, revealing a unique specific band of approximately 34 kDa, which suggested that the predicted StarD7 34.7 kDa peptide and not the 43.1 kDa peptide is synthesized in human trophoblastic tissues [The StarD7, located on the short arm of chromosome 2p12-2p11.2, comprises 8 exons spanning 23.96 kb . StarD7  tissues . StarD7  tissues . Recentl tissues . They pr tissues .Two ubiquitination sites at lysine 137 and lysine 339 of StarD7 (NP_064536.2) were identified by affinity capture and mass spectrometry . In addiFar-ultraviolet circular dichroism spectroscopy shows that the StarD7 spectrum has a minimum at 218 nm, which is typical of a predominantly β-sheet conformation . SecondaIn summary, current data support that StarD7 is synthesized as a short-lived precursor protein of 43.1 kDa and is processed to a 34.7 kDa mature protein that can undergo posttranslational modifications that modulate its functionality.Several studies have shown that StarD7 interacts, binds, and transports lipid molecules. An earlier study demonstrated that StarD7 is a surface-active protein, since it forms stable monolayers by adsorption at the air-buffer interface . StarD7 The mechanism of StarD7 interaction with the lipid-membrane interface has been elucidated by fluorescence dequenching of small unilamellar vesicles (donor liposomes) labeled with 2--1-hexadecanoyl-sn-glycero-3-phosphocholine (BODIPY-FL-C5-HPC) by dilution with nonlabeled large unilamellar vesicles (acceptor liposomes) . This apin vitro ELISA binding assays that the StarD7 protein can bind cardiolipin and PS but not ceramide or phosphatidylinositol (unpublished results). In addition, fluorescence resonance energy transfer-based assays have shown that StarD7-I has a much higher preference for phosphatidylcholine (PC) than for PS, PE, or SM (approximately 5% of its PC-transfer activity) [in vitro differentiating cytotrophoblast cells, supporting that StarD7 plays a role in the delivery of lipids to the plasma membrane [et al., it can be proposed that StarD7 contributes to lipid intermembrane exchange by promoting transient hemifusion [In addition to the StarD7-lipid interaction, our group demonstrated by ctivity) . A compactivity) . In linemembrane . More remembrane . In addimembrane ,2,26,35.mifusion . TogetheHuman StarD7 orthologous genes have been annotated in the Ensembl and GenBank databases in different genomes, suggesting a conserved physiological function. In addition, the StarD7 protein has been predicted in most animal phyla, vertebrates, and invertebrates as well as in plants, which underscores its functional role.Semiquantitative RT-PCR assays in a series of tumor cell lines have shown that StarD7 has widespread expression, predominantly in trophoblast-derived JEG-3, JAR, and HTR8-SVneo cells, hepatocellular carcinoma HepG2 cells, and colorectal adenocarcinoma HT29 and Caco-2 cells. Low StarD7 transcript levels are found in human cervix adenocarcinoma HeLa cells, human breast adenocarcinoma MCF7 cells, human lung adenocarcinoma A549 cells, human melanoma SK-MEL-31 cells, human acute myelocytic leukemia K-562 cells, and human promyelocytic leukemia HL-60 cells . Westernet al.[in vivo by chromatin immunoprecipitation (ChIP) assays. Taken together, these studies strongly indicate that Wnt signaling regulates StarD7 transcription in JEG-3 cells through the canonical pathway [The first evidence of the regulation of StarD7 by β-catenin/T Cell-Specific Transcription Factor 4 (TCF4) was given by Lee et al., who repet al.. Genes eet al.–58. In let al.–63. β-Caet al.. Site-di pathway   motif. However, disruption of this binding site led to an approximately 30% decrease in StarD7 reporter activity, suggesting that SF-1 regulates StarD7 expression through diverse promoter interactions in addition to this -element .There is increasing evidence to show that the nuclear hormone receptor and canonical Wnt pathway interact at different levels to regulate cell growth, proliferation, differentiation, apoptosis, and metastatic potential in various tissues . β-CatenCollectively, these findings indicate that β-catenin acts in conjunction with SF-1 to activate the StarD7 promoter . The autIn addition to intracellular lipid transport, START proteins may play various other key biological roles. StarD2/PCTP is involved in energy substrate utilization ; StarD12In vitro trophoblast fusion and differentiation are accompanied by a significant increase in ABCG2 expression [ABCG2 is a member of the ABC protein superfamily of multidrug efflux transporters . ABCG2 ipression , whereaspression . Low exppression ,90. TherStarD7 knockdown causes an increase in β-human chorionic gonadotropin (βhCG) mRNA expression as well as protein synthesis and secretion . There win vitro syncytialization [Although downregulation of StarD7 induces differentiation of JEG-3 cells, StarD7 expression increases in normal cytotrophoblast cells undergoing spontaneous lization . Thus, ilization . Second,lization , which plization . In contlization ,93. ThesStarD7 is a member of the START domain superfamily, which is involved in various physiological processes such as lipid transfer, metabolism, and modulation of signaling pathways. Since its discovery, the biochemical characteristics and lipid interaction properties of StarD7 have been described. The expression of StarD7 has been shown in several human cell lines and in some human and mouse tissues. Genome wide analysis has shown that the StarD7 transcript is altered in various conditions such as metabolic state, inflammation processes, cancer and behavior. The regulation of StarD7 expression in trophoblast cell lines by Wnt/β-catenin and SF-1 has also been reported.An important question that remains unanswered is whether the biological functions of StarD7 are related only to phosphatidylcholine delivery to mitochondria or can be extended to other intermembrane lipid transfer activities.Despite the well-established role of StarD7 in modulating trophoblast cell proliferation, migration, and differentiation, the mechanisms that control these processes remain largely unknown. Elucidating these mechanisms is important to understand several pathologies that are associated with pregnancy, such as recurrent miscarriage, gestational diabetes, intrauterine growth retardation and preeclampsia, in which trophoblast cell proliferation, migration, and differentiation are altered.