Mucosal Tolerance Induced by an Immunodominant Peptide from Rat α3(IV)NC1 in Established Experimental Autoimmune Glomerulonephritis

Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in Wistar Kyoto (WKY) rats by immunization with the noncollagenous domain of the α 3 chain of type IV collagen, α3(IV)NC1. Recent studies have identified an immunodominant peptide, pCol (24-38), from the N-terminus of rat α3(IV)NC1; this peptide contains the major B- and T-cell epitopes in EAG and can induce crescentic nephritis. In this study, we investigated the mechanisms of mucosal tolerance in EAG by examining the effects of the nasal administration of this peptide after the onset of disease. A dose-dependent effect was observed: a dose of 300 μg had no effect, a dose of 1000 μg resulted in a moderate reduction in EAG severity, and a dose of 3000 μg produced a marked reduction in EAG severity accompanied by diminished antigen-specific, T-cell proliferative responses. These results demonstrate that mucosal tolerance in EAG can be induced by nasal administration of an immunodominant peptide from the N-terminus of α3(IV)NC1 and should be of value in designing new therapeutic strategies for patients with Goodpasture’s disease and other autoimmune disorders.Goodpasture’s, or anti-glomerular basement membrane (GBM), disease is an autoimmune disorder characterized by rapidly progressive glomerulonephritis and lung hemorrhage.^1,2 The disease is caused by autoimmunity to a component of the GBM, the non–collagenous domain of the α3 chain of type IV collagen, α3(IV)NC1.^3,4 Epitope mapping studies have localized the immunodominant region for antibody binding to the amino terminal of the α3(IV)NC1 molecule.^5,6 Experimental autoimmune glomerulonephritis (EAG), an animal model of Goodpasture’s disease, can be induced in susceptible strains of rats and mice by immunization with GBM^7,8,9 or with recombinant α3(IV)NC1.^10,11,12 This results in the development of circulating and deposited anti-GBM antibodies, with focal necrotizing crescentic glomerulonephritis and lung hemorrhage. EAG shares many features with the human disease, in that the renal and lung pathology are very similar,¹³ and the anti-GBM antibodies show the same specificity for the main target antigen, α3(IV)NC1.^10,11,12There is now compelling evidence for the role of both humoral and cell-mediated immunity in the pathogenesis of EAG. The pathogenic role of anti-GBM antibodies has been demonstrated in a variety of passive transfer studies.^9,14,15,16 Transfer of disease has been demonstrated using antibodies pooled from the serum of nephritic mice,⁹ antibodies purified from the urine of nephritic rats,¹⁴ monoclonal antibodies derived from rats with EAG,¹⁵ and antibodies eluted from the kidney of nephritic rats.¹⁶ In the latter study, it was shown that deposited anti-GBM antibody has a higher functional affinity for GBM than circulating antibody.The pathogenic role of T cells in EAG has also been demonstrated in several studies. T cells have been shown to be present in the glomeruli of animals with EAG,^11,13 to proliferate in response to α3(IV)NC1,^12,17 and to transfer disease to naive recipients.^9,18 Glomerular T cells from rats with EAG show restricted T-cell receptor CDR3 spectratypes, demonstrating that they are an oligoclonal antigen-driven population.¹⁹ Anti-T-cell immunotherapy has been shown to be effective in preventing or ameliorating disease.^20,21,22,23 Anti-CD4 mAb therapy is effective in the prevention of EAG,²⁰ anti-CD8 mAb therapy is effective in both prevention and treatment of established disease,²¹ and inhibition of T-cell co-stimulation by blockade of either the CD28-B7 pathway²² or the CD154-CD40 pathway²³ has been shown to reduce the severity of glomerulonephritis.Further evidence supporting the role of T-cell-mediated cellular immunity in the pathogenesis of EAG is documented in recent studies demonstrating that synthetic peptides derived from α3(IV)NC1 can induce glomerulonephritis in WKY rats.^24,25,26,27 Recent studies from our group have identified a 15-mer immunodominant peptide, pCol, (24-38) from the N-terminus of rat α3(IV)NC1, which contains the major B- and T-cell epitopes in EAG, and which can induce crescentic nephritis.²⁴ Previous studies by Luo and colleagues²⁵ showed that a 24-mer synthetic peptide, pCol, (28-51) from the N-terminus of α3(IV)NC1 was capable of inducing glomerulonephritis, although this was mild and inconsistent, whereas Wu and colleagues²⁶ showed that a 13-mer peptide, pCol, (28-40) containing a T-cell epitope from α3(IV)NC1, induced severe crescentic glomerulonephritis. In further characterization of this T-cell epitope, it was shown that only three residues were critical for disease induction.²⁷ In addition, it has been reported that peptides containing the T-cell epitope pCol (28-40) not only induced severe glomerulonephritis, but also triggered a diversified anti-GBM antibody response through B-cell epitope spreading, suggesting that the autoantibody response to GBM antigens could be induced by a single nephritogenic T-cell epitope.^28,29,30Mucosal tolerance is a phenomenon whereby peripheral immunological tolerance may be induced by the mucosal administration of autoantigens.^{31,32,33,34,35} The inhibitory effect of orally or nasally administered autoantigens, or immunodominant peptides, has been widely reported in experimental models of autoimmune disease in rodents, including encephalomyelitis,^36,37,38 arthritis,^39,40,41 myasthenia gravis,^42,43 interstitial nephritis,⁴⁴ and glomerulonephritis.^9,45,46 In several of these studies, it has been shown that nasal administration of lower doses of antigen than those given orally has been effective in inducing mucosal tolerance,^36,40,42 and in treating established disease.^37,38,43 Our previous work in EAG has shown that both oral administration of GBM antigen⁴⁵ and nasal administration of recombinant α3(IV)NC1⁴⁶ are effective in preventing the development of crescentic nephritis. However, neither of these studies demonstrated successful treatment of established EAG, which would clearly be more applicable to patients with glomerulonephritis.In the present study, we have examined the effect of nasal administration of an immunodominant peptide, pCol, (24-38) from α3(IV)NC1 after the onset of disease in EAG. We show for the first time that mucosal tolerance and reduction in glomerular injury in established EAG can be induced by nasal administration of an immunodominant peptide. This work may lead to new antigen-specific treatment strategies for patients with anti-GBM disease and other autoimmune disorders.     

Phenolic Compounds Prevent Alzheimer’s Pathology through Different Effects on the Amyloid-β Aggregation Pathway

Inhibition of amyloid-β (Aβ) aggregation is an attractive therapeutic strategy for Alzheimer’s disease (AD). Certain phenolic compounds have been reported to have anti-Aβ aggregation effects in vitro. This study systematically investigated the effects of phenolic compounds on AD model transgenic mice (Tg2576). Mice were fed five phenolic compounds (curcumin, ferulic acid, myricetin, nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA)) for 10 months from the age of 5 months. Immunohistochemically, in both the NDGA- and RA-treated groups, Aβ deposition was significantly decreased in the brain (P < 0.05). In the RA-treated group, the level of Tris-buffered saline (TBS)-soluble Aβ monomers was increased (P < 0.01), whereas that of oligomers, as probed with the A11 antibody (A11-positive oligomers), was decreased (P < 0.001). However, in the NDGA-treated group, the abundance of A11-positive oligomers was increased (P < 0.05) without any change in the levels of TBS-soluble or TBS-insoluble Aβ. In the curcumin- and myricetin-treated groups, changes in the Aβ profile were similar to those in the RA-treated group, but Aβ plaque deposition was not significantly decreased. In the ferulic acid-treated group, there was no significant difference in the Aβ profile. These results showed that oral administration of phenolic compounds prevented the development of AD pathology by affecting different Aβ aggregation pathways in vivo. Clinical trials with these compounds are necessary to confirm the anti-AD effects and safety in humans.Alzheimer’s disease (AD) is the most common form of dementia, resulting in deterioration of cognitive function and behavioral changes.¹ One of the pathological hallmarks of AD is extracellular deposits of aggregated amyloid-β protein (Aβ) in the brain parenchyma (senile plaques) and cerebral blood vessels (cerebral amyloid angiopathy (CAA)).¹ Deposition of high levels of fibrillar Aβ in the AD brain is associated with loss of synapses, impairment of neuronal functions, and loss of neurons.^2,3,4,5 Aβ was sequenced from meningeal vessels and senile plaques of AD patients and individuals with Down’s syndrome.^6,7,8 The subsequent cloning of the gene encoding the β-amyloid precursor protein and its localization to chromosome 21,^9,10,11,12 coupled with the earlier recognition that trisomy 21 (Down’s syndrome) invariably leads to the neuropathology of AD,¹³ set the stage for the proposal that Aβ accumulation is the primary event in AD pathogenesis. In addition, certain mutations associated with familial AD have been identified within or near the Aβ region of the coding sequence of gene of the amyloid precursor proteins,^14,15 presenilin-1 and presenilin-2,¹⁶ which alter amyloid precursor protein metabolism through a direct effect on γ-secretase.^17,18 These findings set the stage for the proposal that Aβ aggregation is the primary event in AD pathogenesis and leading to the proposal that anti-Aβ aggregation is a strategy for AD therapy.^19,20 Furthermore, there have been recent reports^{21,22,23,24,25} that Aβ fibrils are not the only toxic form of Aβ for developing AD, and smaller species of aggregated Aβ, Aβ oligomers, may represent the primary toxic species in AD. Therefore, it is necessary to consider the inhibition of Aβ oligomer formation as well as Aβ fibrils for the treatment of AD.²⁶To date, it has been reported that various compounds inhibit the formation and extension of Aβ fibrils, as well as destabilizing Aβ fibrils in vitro.^{19,20,27,28,29,30,31,32,33,34,35,36} Among the reported compounds, several phenolic compounds, such as wine-related polyphenols (myricetin (Myr), morin, and tannic acid, and so on), curcumin (Cur), ferulic acid (FA), nordihydroguaiaretic acid (NDGA), and rosmarinic acid (RA) had especially strong anti-Aβ aggregation effects in vitro. Furthermore, it was shown recently that a commercially available grape seed polyphenolic extract, MegaNatural-Az, inhibited fibril formation, protofibril formation, and oligomerization of Aβ.³⁷ Moreover, MegaNatural-Az also reduced cerebral amyloid deposition as well as attenuating AD-type cognitive deterioration using transgenic mice.³⁸ In addition to these studies by the current authors, several other researchers have reported similar effects of phenolic compounds.^{26,39,40,41,42,43,44} First, Cur decreased cerebral Aβ plaque burden in vivo,^{39,40,41,42,44} and inhibited the formation of Aβ oligomers in vitro.^26,39 Second, epigallocatechin gallate efficiently inhibited fibril and oligomer formation of Aβ.⁴³ However, a very recent in vitro study²⁶ reported that Cur, Myr, and NDGA inhibited the formation of Aβ oligomers, but Cur and NDGA promoted the formation of Aβ fibrils. This indicated that the effects of these phenolic compounds on Aβ aggregation remain controversial. These different results may reflect different experimental conditions in these studies. To resolve this problem, a systematic in vivo study is required; however, few reports on the effects of phenolic compounds on Aβ aggregation in vivo have been published so far, except for reports about Cur.^{39,40,41,42,44}To elucidate the inhibitory effects of phenolic compounds on Aβ aggregation in vivo, several phenolic compounds, including Cur, FA, Myr, NDGA, and RA, were fed to AD model mice, and the cerebral plaque burden and formation of Aβ oligomers were compared systematically.     

A Protein Kinase Cδ-Dependent Protein Kinase D Pathway Modulates ERK1/2 and JNK1/2 Phosphorylation and Bim-Associated Apoptosis by Asbestos

Inhalation of asbestos and oxidant-generating pollutants causes injury and compensatory proliferation of lung epithelium, but the signaling mechanisms that lead to these responses are unclear. We hypothesized that a protein kinase (PK)Cδ-dependent PKD pathway was able to regulate downstream mitogen-activated protein kinases, affecting pro- and anti-apoptotic responses to asbestos. Elevated levels of phosphorylated PKD (p-PKD) were observed in distal bronchiolar epithelial cells of mice inhaling asbestos. In contrast, PKCδ−/− mice showed significantly lower levels of p-PKD in lung homogenates and in situ after asbestos inhalation. In a murine lung epithelial cell line, asbestos caused significant increases in the phosphorylation of PKCδ-dependent PKD, ERK1/2, and JNK1/2/c-Jun that occurred with decreases in the BH3-only pro-apoptotic protein, Bim. Silencing of PKCδ, PKD, and use of small molecule inhibitors linked the ERK1/2 pathway to the prevention of Bim-associated apoptosis as well as the JNK1/2/c-Jun pathway to the induction of apoptosis. Our studies are the first to show that asbestos induces PKD phosphorylation in lung epithelial cells both in vivo and in vitro. PKCδ-dependent PKD phosphorylation by asbestos is causally linked to a cellular pathway that involves the phosphorylation of both ERK1/2 and JNK1/2, which play opposing roles in the apoptotic response induced by asbestos.Asbestos is a group of naturally occurring mineral fibers that are linked to the development of lung cancer, mesothelioma, and pleural and pulmonary fibrosis, ie, asbestosis.^1,2 The mechanisms leading to asbestos-related diseases are still unclear, but oxidative stress due to phagocytosis of longer fibers, iron-driven generation of oxidants from fiber surfaces, and depletion of cellular antioxidants are linked to cell injury and inflammation.^3,4,5,6Bronchiolar and alveolar type II epithelial cells, which first encounter asbestos fibers after inhalation, are key cell types in asbestos-associated inflammation and fibroproliferation.² Initial cell reactions to asbestos include epithelial cell injury, ie, apoptosis and necrosis,^5,6 which may lead to compensatory cell proliferation^7,8 and the production of inflammatory and fibrogenic cytokines.^8,9,10 Asbestos-induced signaling mechanisms governing these cell responses appear to involve a broad variety of cascades including the mitogen-activated protein kinases (MAPK),^3,7,11,12 nuclear factor-κB (NF-κB),^9,13,14 and the protein kinase (PK)C^10,12,15,16 and A families.¹⁷A critical signaling protein involved in asbestos signaling is PKCδ, which is known to be activated in bronchiolar and alveolar epithelial cells in vivo and in vitro^10,12,16 via increased formation of diacylglycerol.¹⁸ We have shown that PKCδ governs apoptosis via an oxidant-dependent mitochondrial pathway after exposure of lung epithelial cells to asbestos fibers.¹⁶ Recent studies comparing PKCδ +/+ and PKCδ −/− mice also reveal an important role of PKCδ in metalloproteinase expression as well as cytokine production in vitro and in vivo.^10,15 A variety of other studies also link PKCδ to either pro-apoptotic or anti-apoptotic events depending on the stimulus and cell type.^19,20In this study, we focused on PKD as a potential link between PKCδ, activation of MAPKs and downstream repercussions such as expression of fos/jun proto-oncogenes and apoptosis in asbestos-exposed lung epithelium. PKD is a serine/threonine protein kinase classified as a subfamily of the Ca²⁺/calmodulin-dependent kinase superfamily.²¹ PKD1, which includes mouse PKD and its human homolog PKCμ, is the most extensively studied PKD.²² The other two members of this family include PKD2²³ and PKD3, (originally PKCν).²⁴ Conserved regions of PKDs include a phosphorylation-dependent catalytic domain, a pleckstrin-homology domain that inhibits the catalytic activity, and cysteine-rich motifs that recruit PKD to the plasma membrane. PKCδ is proposed to interact with the pleckstrin-homology domain of PKD, transphosphorylating its activation loop at Ser744 and Ser748, and leading to PKD activation.²⁵ In addition, PKD can be activated through the Src-Abl pathway by tyrosine phosphorylation of Tyr463 (T463) in the pleckstrin-homology domain after oxidative stress,²⁶ as well as by caspase-mediated proteolytic cleavage 27 and by bone morphogenetic protein 2.²⁸ Downstream targets of PKD signaling include several important signaling molecules such as ERK1/2, JNK1/2, and NF-κB,^21,26,29,30 but how these affect functional ramifications of carcinogens, such as asbestos, are unclear.The BH3-only protein, Bim, is a pro-apoptotic member of the Bcl-2 family that links stress-induced signals to the core apoptotic machinery.^31,32 There are three different splice variants of the Bim gene encoding short, long, and extra-long Bim proteins (BimS, BimL, and BimEL).³³ BimS-induced apoptosis requires mitochondrial localization but not interaction with anti-apoptosis proteins,³⁴ whereas BimL is bound to microtubules and is less cytotoxic.³⁵ Disruption of BimL binding to microtubules via JNK-dependent phosphorylation can cause its redistribution to the mitochondria and induction of pro-apoptotic machinery.³⁶ BimEL is post-translationally regulated by ERK1/2, which promotes its phosphorylation and rapid dissociation from Mcl-1 and Bcl-x(L)³⁷ and proteasomal degradation.³⁸We reveal here that PKD is involved in multiple signaling events after asbestos inhalation and in vitro. Specifically, PKD is a downstream effector of PKCδ and modulates phosphorylation of both ERK1/2 and JNK1/2 in lung epithelial cells after asbestos exposure. Our data also suggest that PKD inhibits apoptosis through an ERK1/2-mediated destabilization of the pro-apoptotic BH3-only protein, BimEL. The fact that PKD is an important signaling molecule in MAPK signaling and survival after cell injury by asbestos may have important therapeutic implications in asbestos-related diseases.     

ICAM-1 Is Necessary for Epithelial Recruitment of γδ T Cells and Efficient Corneal Wound Healing

Wound healing and inflammation are both significantly reduced in mice that lack γδ T cells. Here, the role of epithelial intercellular adhesion molecule-1 (ICAM-1) in γδ T cell migration in corneal wound healing was assessed. Wild-type mice had an approximate fivefold increase in epithelial γδ T cells at 24 hours after epithelial abrasion. ICAM-1^−/− mice had 50.9% (P < 0.01) fewer γδ T cells resident in unwounded corneal epithelium, which failed to increase in response to epithelial abrasion. Anti-ICAM-1 blocking antibody in wild-type mice reduced epithelial γδ T cells to a number comparable to that of ICAM-1^−/− mice, and mice deficient in lymphocyte function-associated antigen-1 (CD11a/CD18), a principal leukocyte receptor for ICAM-1, exhibited a 48% reduction (P < 0.01) in peak epithelial γδ T cells. Re-epithelialization and epithelial cell division were both significantly reduced (∼50% at 18 hours, P < 0.01) after abrasion in ICAM-1^−/− mice versus wild-type, and at 96 hours, recovery of epithelial thickness was only 66% (P < 0.01) of wild-type. ICAM-1 expression by corneal epithelium in response to epithelial abrasion appears to be critical for accumulation of γδ T cells in the epithelium, and deficiency of ICAM-1 significantly delays wound healing. Since γδ T cells are necessary for efficient epithelial wound healing, ICAM-1 may contribute to wound healing by facilitating γδ T cell migration into the corneal epithelium.Intercellular adhesion molecule-1 (ICAM-1, CD54)¹ is a conserved member of the immunoglobulin supergene family² and is expressed by many cell types in response to stimuli such as cytokines,^3,4 and oxidative and physical stress.^5,6 It has been extensively studied in the context of adhesion and transmigration of leukocytes through endothelium⁷ and epithelium,^8,9 and it also serves as an adhesive ligand for leukocyte-mediated cytotoxic activity.^9,10,11 ICAM-1 is recognized by members of the β2 (CD18) integrin family, especially lymphocyte function-associated antigen (LFA)-1 (CD11a/CD18),¹² and this adhesion is critical to many of the migratory and cytotoxic events in which ICAM-1 participates.^7,10,11 ICAM-1 also functions as a signaling molecule, dependent on its cytoplasmic tail interacting with cytoskeletal elements.⁷ This capability influences functions such as leukocyte transendothelial migration⁷ and vascular permeability.¹³Of importance to the current study is the fact that ICAM-1 can be expressed by corneal epithelial cells and limbal vessel endothelial cells.^{14,15,16,17,18,19,20,21} It appears to be expressed in conditions associated with inflammation, but its role in this context is poorly understood, especially its expression by the epithelial cells. Using a murine model of central corneal epithelial abrasion, we observed ICAM-1 on corneal epithelial cells in the periphery of the cornea, a region not directly injured by the abrasion.¹⁴ Since migration and division of these cells account for wound closure and re-establishment of full thickness epithelium necessary for healing,^22,23 it was of interest to determine whether ICAM-1 is necessary for these processes. To this end we studied wound healing in mice that do not express ICAM-1.^24,25As a part of this evaluation, we focused attention on γδ T cells. We observed in earlier studies that epithelial expression of ICAM-1 occurred at a time when γδ T cells increased within the corneal epithelium,^14,26 and that γδ T cell-deficient mice exhibited poor corneal wound healing. Since these leukocytes express LFA-1,²⁷ and LFA-1/ICAM-1 interactions support adhesion of human lymphocytes to human epithelial cells expressing ICAM-1,^20,27 it seemed possible that γδ T cell accumulation in the epithelium after corneal abrasion would be influenced by the absence of ICAM-1.     

Differential Interferon Responses Enhance Viral Epitope Generation by Myocardial Immunoproteasomes in Murine Enterovirus Myocarditis

Murine models of coxsackievirus B3 (CVB3)-induced myocarditis mimic the divergent human disease course of cardiotropic viral infection, with host-specific outcomes ranging from complete recovery in resistant mice to chronic disease in susceptible hosts. To identify susceptibility factors that modulate the course of viral myocarditis, we show that type-I interferon (IFN) responses are considerably impaired in acute CVB3-induced myocarditis in susceptible mice, which have been linked to immunoproteasome (IP) formation. Here we report that in concurrence with distinctive type-I IFN kinetics, myocardial IP formation peaked early after infection in resistant mice and was postponed with maximum IP expression concomitant to massive inflammation and predominant type-II IFN responses in susceptible mice. IP activity is linked to a strong enhancement of antigenic viral peptide presentation. To investigate the impact of myocardial IPs in CVB3-induced myocarditis, we identified two novel CVB3 T cell epitopes, virus capsid protein 2 [285-293] and polymerase 3D [2170-2177]. Analysis of myocardial IPs in CVB3-induced myocarditis revealed that myocardial IP expression resulted in efficient epitope generation. As opposed to the susceptible host, myocardial IP expression at early stages of disease corresponded to enhanced CVB3 epitope generation in the hearts of resistant mice. We propose that this process may precondition the infected heart for adaptive immune responses. In conclusion, type-I IFN-induced myocardial IP activity at early stages coincides with less severe disease manifestation in CVB3-induced myocarditis.Myocarditis is often induced by cardiotropic viruses: in about 20% of patients, viral myocarditis leads to its sequela dilated cardiomyopathy, which is linked to chronic inflammation and persistence of cardiotropic viruses.^1,2,3,4 Dilated cardiomyopathy is the most common cause of heart failure in young patients and appears to be a major cause of sudden unexpected death in this cohort. Enteroviruses, including group-B coxsackieviruses, have been linked to the development of myocarditis and dilated cardiomyopathy associated with adverse prognosis.^5,6 Well-established murine models of coxsackievirus B3 (CVB3) myocarditis mimic the human disease progress and are valuable in delineating the underlying mechanisms that determine the divergent courses of myocarditis^7,8,9,10: resistant C57BL/6 mice eliminate the virus following mild acute myocarditis; no chronic inflammation is detected. In contrast, major histocompatibility complex (MHC)-matched A.BY/SnJ mice develop severe acute infection and ongoing chronic myocarditis, thus conferring susceptibility to chronic disease.^7,9Host responses to viral infection trigger the release of interferons (IFNs). IFNs of the α/β subtype are assigned to type I IFNs, whereas IFN-γ is the only type II IFN. IFNs exert numerous antiviral effects in innate and adaptive immunity.¹¹ Although type I IFN-receptor-deficiency was not associated with a dramatic effect on early viral replication in the heart, type I IFN signaling was found to be essential for the prevention of early death due to CVB3-infection.¹² The extraordinary impact of type I IFNs was substantiated in a recent study illustrating acute fulminant infection and chronic disease progression in IFN-β deficient mice.¹³ Deletion of type II IFN receptors was not associated with enhanced mortality in CVB3-infection.¹² IFN-γ responses were shown to be protective in cellular immunity in CVB3-infection.⁹ In addition, expression of IFN-γ conferred protection in enterovirus myocarditis, which may be linked to the activation of nitric oxide-mediated antiviral activity of macrophages.^14,15 Thus, both type I and type II IFN are active in CVB3- myocarditis.One downstream effect of IFN signaling is the induction of immunoproteasome (IP) formation in the target organ of the immune response. Particularly IFN-γ was shown to induce IP expression.^16,17,18 Efficient generation of viral epitopes that stimulate CD8⁺ T cells strongly relies on host-cell IP and, in addition, protein degradation by proteasomes is also essential in the regulation of inflammatory and stress responses, cell cyclus, and apoptosis control.¹⁹ The 20S proteasome as the catalytic core of the proteasome resembles a cylinder-shaped structure of stacked heptameric rings formed by either α or β subunits. The proteolytic function of the so-called standard proteasome is restricted to the β1, β2, and β5 subunit.²⁰ Three alternative catalytic subunits, the so-called immunosubunits β1i, β2i, and β5i, which are incorporated into 20S proteasomes, thus forming IP with altered catalytic characteristics, are expressed on cytokine stimulation.^21,22 It is highly notable that IP activity is linked to a strong enhancement of antigenic viral peptide presentation.^{23,24,25,26,27}Cardiac proteasomes contribute to the modulation of cardiac function in health and disease.²⁸ However, apart from the reported observation that IPs are expressed in the myocardium in acute CVB3 myocarditis, their functional impact has not been studied so far.¹⁰ The present study focuses on IFN-induced myocardial IP activity in CVB3 myocarditis.     

Up-Regulation of Soluble Axl and Mer Receptor Tyrosine Kinases Negatively Correlates with Gas6 in Established Multiple Sclerosis Lesions

Multiple sclerosis is a disease that is characterized by inflammation, demyelination, and axonal damage; it ultimately forms gliotic scars and lesions that severely compromise the function of the central nervous system. Evidence has shown previously that altered growth factor receptor signaling contributes to lesion formation, impedes recovery, and plays a role in disease progression. Growth arrest-specific protein 6 (Gas6), the ligand for the TAM receptor tyrosine kinase family, consisting of Tyro3, Axl, and Mer, is important for cell growth, survival, and clearance of debris. In this study, we show that levels of membrane-bound Mer (205 kd), soluble Mer (∼150 kd), and soluble Axl (80 kd) were all significantly elevated in homogenates from established multiple sclerosis lesions comprised of both chronic active and chronic silent lesions. Whereas in normal tissue Gas6 positively correlated with soluble Axl and Mer, there was a negative correlation between Gas6 and soluble Axl and Mer in established multiple sclerosis lesions. In addition, increased levels of soluble Axl and Mer were associated with increased levels of mature ADAM17, mature ADAM10, and Furin, proteins that are associated with Axl and Mer solubilization. Soluble Axl and Mer are both known to act as decoy receptors and block Gas6 binding to membrane-bound receptors. These data suggest that in multiple sclerosis lesions, dysregulation of protective Gas6 receptor signaling may prolong lesion activity.Multiple sclerosis (MS) is a debilitating white matter disease of the central nervous system (CNS). Although much of the evidence from animal models and MS suggests it to be an autoimmune disorder mediated by TH-1 type T cells,¹ other possible causes include genetic and environmental factors, antibody-dependent cytotoxicity, and bacterial and viral infections that may mediate altered protein expression resulting in inflammation, axonal and oligodendrocyte damage, demyelination and CNS scarring.² Growth and survival factors that protect against axonal and oligodendrocyte damage or loss, and dampen the inflammatory response are actively being pursued for MS therapy.^2,3,4,5,6 One growth factor associated with oligodendrocyte maturation, survival and dampening the immune response is growth-arrest specific protein 6 (Gas6). Gas6 is a secreted protein that is widely expressed in the central and peripheral nervous systems by endothelial cells and neurons, and is involved in numerous physiological and pathological functions including cell growth, survival and apoptosis.^{7,8,9,10,11,12} Gas6 binds and activates the TAM family of receptor tyrosine kinases consisting of Tyro3 (Rse/Dtk/Sky), Axl (Ufo), and Mer (Eyk).^{8,11,13,14,15} Many cell types express all three receptors and receptor activation can result from homophilic and heterophilic interactions.^16,17 Axl contains the major and minor Gas6 binding groove. Only the minor groove is conserved in Tyro3 and Mer and as a result, response to Gas6 is mediated in a concentration-dependent manner; Gas6 binding affinity is Axl>Tyro3>Mer.¹⁸We previously reported mRNA expression of Axl, Tyro3, and Mer receptors on human fetal oligodendrocytes and the ability of Gas6 to promote oligodendrocyte survival in vitro by activating Axl, resulting in Axl directly and indirectly recruiting phosphatidylinositol 3 kinase and activating the Akt pathway.^19,20 Moreover, we have shown that Gas6/Axl signaling through the Akt pathway can protect oligodendrocytes from tumor necrosis factor α (TNFα)-induced apoptosis.²¹ Down-regulation or deletion of Axl, even in the presence of Gas6, results in loss of protection against TNFα.²²During the relapse phase of relapsing-remitting MS, serum TNFα levels and TNFα mRNA are elevated.^23,24 TNFα is one of the major cytokines expressed in MS lesions.²⁵ TNFα is cleaved to its mature, soluble, secretable form by the matrix metalloproteinase (MMP) TNFα converting enzyme, also known as ADAM17.^26,27,28 MMPs, including ADAM17 and ADAM10 are involved in normal processes such as wound repair and tissue remodeling and are associated with disease states, including MS.^{29,30,31,32,33,34,35,36} Expression of ADAM17 is observed in acute and chronic active MS lesions, primarily in perivascular cuffs and cells morphologically resembling lymphocytes.³⁷ ADAM17 up-regulation in cerebrospinal fluid of MS patients is associated with inflammation and increased soluble TNFα.^37,38 ADAM10 is constitutively expressed on astrocytes in normal appearing white matter and on astrocytes and perivascular macrophages in MS lesions.^38,39,40,41 ADAM10 cleaves Axl, and ADAM17 cleaves Axl and Mer. Cleaved forms of receptors can result in internalization of the receptor and transport off the membrane for recycling. Cleavage can also result in shortened, soluble forms that act as a decoy to regulate the level of a growth factor at the membrane.^41,42Soluble forms of Axl and Mer can reduce the number of viable receptors for Gas6 binding and act as a decoy by sequestering Gas6 extracellularly; potentially a normal mechanism of receptor activation regulation.^40,41 During inflammation, soluble Mer has been reported to inhibit macrophage clearance of apoptotic cells.^41,43 Also, soluble Axl blocked the protective effect of membrane-bound Axl by inhibiting Gas6 induced tyrosine phosphorylation of Axl.⁴⁴ The binding of Gas6 to TAM receptors acts as an inhibitor of inflammation by inhibiting Toll-like receptor and cytokine receptor cascades.⁴⁴ Up-regulation of Axl and its subsequent interaction with interferon α and β receptors results in the expression of cytokine and Toll-like receptor inhibitors.^44,45 Thus, loss of Gas6 signaling, along with dysregulation of the balance between Gas6, Axl and Mer by increased extracellular levels of soluble Axl and Mer, might detrimentally impact the nervous system, especially in established (chronic active and chronic silent) lesions associated with MS. Chronic active MS lesions are characterized by ongoing demyelination, astrogliosis, macrophage and lymphocyte infiltration, astroglial hypertrophy, and oligodendrocyte hyperplasia.⁴⁶ Chronic silent MS lesions are characterized by the absence of actively infiltrating and inflammatory cells, oligodendrocyte loss and no evidence of ongoing demyelination.^46,47,48In this study, we investigated in chronic active and chronic silent MS lesions whether increased expression of soluble Axl and Mer was associated with increased expression of the MMPs ADAM17 and ADAM10, similar to previous studies that showed an association between increased ADAM17 and ADAM10 with TNFα in the CNS of MS patients.^37,38 We also investigated whether in lesions increased soluble Axl and Mer was associated with decreased Gas6, resulting in loss of the beneficial effects from activating membrane-bound Axl and Mer receptors.     

Dual Roles of CD40 on Microbial Containment and the Development of Immunopathology in Response to Persistent Fungal Infection in the Lung

Persistent pulmonary infection with Cryptococcus neoformans in C57BL/6 mice results in chronic inflammation that is characterized by an injurious Th2 immune response. In this study, we performed a comparative analysis of cryptococcal infection in wild-type versus CD40-deficient mice (in a C57BL/6 genetic background) to define two important roles of CD40 in the modulation of fungal clearance as well as Th2-mediated immunopathology. First, CD40 promoted microanatomic containment of the organism within the lung tissue. This protective effect was associated with: i) a late reduction in fungal burden within the lung; ii) a late accumulation of lung leukocytes, including macrophages, CD4⁺ T cells, and CD8+ T cells; iii) both early and late production of tumor necrosis factor-α and interferon-γ by lung leukocytes; and iv) early IFN-γ production at the site of T cell priming in the regional lymph nodes. In the absence of CD40, systemic cryptococcal dissemination was increased, and mice died of central nervous system infection. Second, CD40 promoted pathological changes in the airways, including intraluminal mucus production and subepithelial collagen deposition, but did not alter eosinophil recruitment or the alternative activation of lung macrophages. Collectively, these results demonstrate that CD40 helps limit progressive cryptococcal growth in the lung and protects against lethal central nervous system dissemination. CD40 also promotes some, but not all, elements of Th2-mediated immunopathology in response to persistent fungal infection in the lung.CD40, a 48-kDa type I transmembrane protein and member of the tumor necrosis factor receptor family, is a well-described costimulatory molecule expressed on B cells, dendritic cells (DC), macrophages, basophils, and platelets as well as nonhematopoietic cells including fibroblasts, epithelial, and endothelial cells. The ligand for CD40, known as CD154 or CD40L, is a type II transmembrane protein member of the tumor necrosis factor (TNF) superfamily expressed primarily by activated T cells, B cells, and platelets.^1,2,3 CD40 can be induced on DC, monocytes, and macrophages under inflammatory conditions.^4,5 Signaling via the CD40/CD40L pathway exerts numerous biological effects including: i) increased cytokine expression (especially TNF-α and Th1 cytokines interleukin (IL)-12 and interferon (IFN)-α) and nitric oxide production; ii) upregulation of additional costimulatory molecules (CD80 and CD86) on antigen-presenting cells (APC); iii) enhanced cell survival (particularly of B and T cells, DC, and endothelial cells); iv) Ig isotype switching; and v) somatic hypermutation of Ig.^1,4,5The CD40/CD40L signaling pathway contributes to adaptive Th1 immune responses required to clear Leishmanisa spp.,^6,7,8 Trypanosoma spp.,^6,7,8,9 Shistosoma mansoini,¹⁰ and the fungi Candida albicans¹¹ and Pneumocystis spp.¹² The enhanced production of IFN-γ, TNF-α, and nitric oxide associated with CD40/CD40L signaling is thought to be responsible for this protective effect. However, other studies have suggest that CD40/CD40L signaling is not required for successful host defense against Listeria monocytogenes,^13,14 Toxoplasma gondi,¹⁵ lymphocytic choriomeningitis virus,^16,17 or the fungus Hisoplasma capsulatum.^18,19 In models of Mycobacterium spp. infection, CD40 appears dispensable for clearance of an i.v. infection,^20,21 but essential for clearing the organism in response to aerosolized infection in the lungs.^22,23 Thus, the role of CD40 in antimicrobial host defense varies and depends not only on the specific pathogen but also on the primary site of infection.Cryptococcus neoformans, an opportunistic fungal pathogen acquired through inhalation, causes significant morbidity and mortality primarily in patients with AIDS, lymphoid or hematological malignancies, or patients receiving immunosuppressive therapy secondary to autoimmune disease or organ transplantation.^24,25 Infection in non-immunocompromised patients has been reported.^26,27,28 Murine models of cryptococcal infection in CBA/J or BALB/c mice demonstrate that development of a Th1 antigen-specific immune response characterized by IFN-γ production and classical activation of macrophages is required to eradicate the organism.^{29,30,31,32,33,34,35,36,37,38,39,40} In contrast, a model of persistent cryptococcal infection has been developed using C57BL/6 mice;^{41,42,43,44,45,46,47} this model reflects many features observed in humans diagnosed with allergic bronchopulmonary mycosis.⁴⁸ Specifically, these mice fail to clear the organism from the lung and develop characteristic Th2-mediated immunopathology including: i) tissue eosinophilia; ii) airway hyperreactivity, mucus production, and fibrosis; and iii) alternative macrophage activation associated with YM1 crystal deposition.The molecular mechanisms responsible for the immunopathologic response associated with persistent cryptococcal infection are not clearly defined. These features are abrogated in the absence of IL-4,⁴⁵ whereas more severe Th2-mediated lung injury occurs in the absence of IFN-γ.^29,41 TNF-α exerts a protective effect by enhancing IFN- γ production and the subsequent classical activation of lung macrophages.^31,35,49,50 Lymphocytes are critical mediators of this Th2 response as the pathological features of chronic cryptococcal infection are substantially diminished in CD4 T cell-depleted mice despite no change in fungal clearance.⁴²Although interactions between CD4 T cells and APC are critical determinants of T cell polarization in response to cryptococcal lung infection,^{49,51,52,53,54,55} the contribution of specific costimulatory molecules including the CD40/CD40L signaling pathway has not been fully elucidated. In vitro studies suggest that activation of the CD40/CD40L pathway in response to Cryptococcus promotes IFN-γ production by T cells and TNF-α, and nitric oxide (NO) production by monocytes.⁵⁶ In the absence of CD40L, primary pulmonary infection with a weakly virulent strain of C. neoformans was associated with impaired fungal clearance; however, measurements of immune function at the site of infection in the lung or evidence of systemic fungal dissemination were not evaluated.⁵⁷ The potential to target CD40 therapeutically is highlighted by studies showing that treatment of mice with disseminated or intracerebral cryptococcal infection with an agonist antibody to CD40 in combination with IL-2 improves survival.^58,59 In this study, we used gene-targeted CD40-deficient mice (on a C57BL/6 genetic background), a clinically relevant model, and assessments of immune function and histopathology in the lung to identify two unique roles for the CD40-signaling pathway in response to persistent cryptococcal lung infection.     

Integrin-Mediated Transforming Growth Factor-β Activation,a Potential Therapeutic Target in Fibrogenic Disorders

A subset of integrins function as cell surface receptors for the profibrotic cytokine transforming growth factor-β (TGF-β). TGF-β is expressed in an inactive or latent form, and activation of TGF-β is a major mechanism that regulates TGF-β function. Indeed, important TGF-β activation mechanisms involve several of the TGF-β binding integrins. Knockout mice suggest essential roles for integrin-mediated TGF-β activation in vessel and craniofacial morphogenesis during development and in immune homeostasis and the fibrotic wound healing response in the adult. Amplification of integrin-mediated TGF-β activation in fibrotic disorders and data from preclinical models suggest that integrins may therefore represent novel targets for antifibrotic therapies.The multifunctional cytokine transforming growth factor-β (TGF-β) plays major roles in the biology of immune, endothelial, epithelial, and mesenchymal cells during development and adult life in invertebrate and vertebrate species.^1,2 In mammals, these functions are mediated by three isoforms, TGF-β1, 2, and 3, which are each widely expressed.³ All three isoforms interact with the same cell surface receptors (TGFBR2 and ALK5) and signal through the same intracellular signaling pathways, which involve either canonical (ie, SMADs) or noncanonical (ie, MAPK, JUN, PI3K, PP2A, Rho, PAR6) signaling effectors.^4,5 The canonical TGF-β signaling pathway, whereby TGF-β signaling is propagated from the TGF-β receptor apparatus through phosphorylation of cytoplasmic SMAD-2/3, complex formation with SMAD-4, nuclear translocation of the SMAD-2/3/4 complex, and binding to SMAD response elements located in the promoter regions of many genes involved in the fibrogenic response, has been the most intensively studied.⁶ However, despite having similar signaling partners, each isoform serves individual biological functions, perhaps due to differences in binding affinity to TGF-β receptors, activation mechanism, signaling intensity or duration, or spatial and/or temporal distribution.⁷Knockout and conditional deletion models of TGF-β isoforms, receptors, and signaling mediators, as well as function-blocking reagents targeting all TGF-β isoforms, have revealed essential roles for TGF-β in T-cell, cardiac, lung, vascular, and palate development.^{8,9,10,11,12,13,14,15} For instance, mice deficient in TGF-β1 either die in utero owing to defects in yolk sac vasculogenesis or are born and survive into adult life but develop severe multiorgan autoimmunity.¹² Genetic deletion of TGF-β signaling mediators has shown an essential role for Smad2 in early patterning and mesodermal formation,^16,17 and mice lacking Smad3 are viable and fertile, but exhibit limb malformations,¹⁸ immune dysregulation, colitis,¹⁹ colon carcinomas,²⁰ and alveolar enlargement.²¹In adult tissues, the TGF-β pathway is thought to regulate the dynamic interactions among immune, mesenchymal, and epithelial cells to maintain homeostasis in response to environmental stress.²² The normal homeostatic pathways mediated by TGF-β are perturbed in response in chronic repetitive injury. In cases of injury, TGF-β becomes a major profibrogenic cytokine, delaying epithelial wound healing by inhibiting epithelial proliferation and migration and promoting apoptosis and expanding the mesenchymal compartment by inducing fibroblast recruitment, fibroblast contractility, and extracellular matrix deposition.²³ Indeed, intratracheal transfer of adenoviral recombinant TGF-β1 to the rodent lung dramatically increases fibroblast accumulation and expression of type I and type III collagen around airways and in the pulmonary interstitium,^24,25 and neutralizing anti-TGF-β antibodies can block experimental bleomycin or radiation-induced pulmonary fibrosis.^26,27 Increased activity of the TGF-β pathway has also been implicated in fibrotic lung disease, glomerulosclerosis, and restenosis of cardiac vessels.^23,28,29,30 Most TGF-β-mediated pathological changes appear to be attributed to the TGF-β1 isoform.³¹The complexity of TGF-β1 function in humans is illuminated by hereditary disorders with generalized or cell type-specific enhancement or deficiency in either TGF-β1 itself or its signaling effectors. Mutations that increase the activity of the TGF-β pathway lead to defects in bone metabolism (ie, Camurati-Engelmann disease) and in connective tissue (ie, Marfan syndrome), and in aortic aneurysms (ie, Loeys-Dietz syndrome), whereas mutations that lead to decreased activity of the TGF-β pathway correlate with cancer occurrence and prognosis.³² The role of TGF-β as a tumor suppressor in cancer is not straightforward, however, because TGF-β can also enhance tumor growth and metastasis, perhaps through its roles in immune suppression, cell invasion, epithelial-mesenchymal transition, or angiogenesis.^19,33,34,35Despite the multiple essential functions of TGF-β, a single dose or short-term administration of a pan-TGF-β neutralizing antibody is reportedly well tolerated at doses that inhibit organ fibrosis or experimental carcinoma cell growth and metastasis, with no reported side effects in adult mice and rats. This treatment has shown therapeutic efficacy in inhibiting experimental fibrosis.^{27,28,36,37,38,39,40} Because of these promising results, single-dose phase I/II clinical trials using neutralizing pan-TGF-β antibodies have been performed or are ongoing for metastatic renal cell carcinoma, melanoma, focal segmental glomerulosclerosis, and idiopathic pulmonary fibrosis (Genzyme Corporation, http://www.genzymeclinicalresearch.com, last accessed August 27, 2009). However, it is likely that long-term global inhibition of TGF-β will have undesirable side effects, because targeted deletion of TGF-β signaling in various cell types may lead to accelerated atherosclerosis, autoimmunity, or carcinoma development.^9,12,41 Clearly, careful targeting of the TGF-β pathway to minimize systemic effects is a highly desirable goal.     

Amyloid-Peptide Vaccinations Reduce β-Amyloid Plaques but Exacerbate Vascular Deposition and Inflammation in the Retina of Alzheimer’s Transgenic Mice

Alzheimer’s disease (AD) is pathologically characterized by accumulation of β-amyloid (Aβ) protein deposits and/or neurofibrillary tangles in association with progressive cognitive deficits. Although numerous studies have demonstrated a relationship between brain pathology and AD progression, the Alzheimer’s pathological hallmarks have not been found in the AD retina. A recent report showed Aβ plaques in the retinas of APPswe/PS1ΔE9 transgenic mice. We now report the detection of Aβ plaques with increased retinal microvascular deposition of Aβ and neuroinflammation in Tg2576 mouse retinas. The majority of Aβ-immunoreactive plaques were detected from the ganglion cell layer to the inner plexiform layer, and some plaques were observed in the outer nuclear layer, photoreceptor outer segment, and optic nerve. Hyperphosphorylated tau was labeled in the corresponding areas of the Aβ plaques in adjacent sections. Although Aβ vaccinations reduced retinal Aβ deposits, there was a marked increase in retinal microvascular Aβ deposition as well as local neuroinflammation manifested by microglial infiltration and astrogliosis linked with disruption of the retinal organization. These results provide evidence to support further investigation of the use of retinal imaging to diagnose AD and to monitor disease activity.Cerebral abnormalities including neuronal loss, neurofibrillary tangles, senile plaques with aggregated β-amyloid protein (Aβ) deposits, microvascular deposition of Aβ, and inflammation are well-known pathological hallmarks of Alzheimer’s disease (AD).^1,2,3 Despite the controversial evidence about the contribution of Aβ to the development of AD-related cognitive deficits, accumulation of toxic, aggregated forms of Aβ plays a crucial role in the pathogenesis of familial types of AD.^4,5 Overexpression of amyloid precursor protein (APP) in trisomy 21, altered APP processing resulting from mutations in APP, presenilin 1 (PS1), or 2 (PS2), and, as-of-yet unidentified other familial AD, related mutations, lead to Aβ deposition and Aβ plaques in the brain as well as cognitive abnormalities.^6,7 Therefore, to understand the molecular basis of amyloid protein deposition and to detect Aβ plaques in brain, parenchyma ante-mortem are currently among the most active areas of research in AD.Besides cognitive abnormalities, patients with AD commonly complain of visual anomalies, in particular, related to color vision,^8,9 spatial contrast sensitivity,¹⁰ backward masking,¹¹ visual fields,¹² and other visual performance tasks.^13,14,15,16 In addition to the damage and malfunction in the central visual pathways, retinal abnormalities such as ganglion cell degeneration,¹⁷ decreased thickness of the retinal nerve fiber layer,^18,19 and optic nerve degeneration^20,21 may, in part, account for AD-related visual dysfunction. Although intracellular Aβ deposition has been detected in both ganglion and lens fiber cells of patients with glaucoma, AD, or Down’s syndrome,^22,23,24,25 other typical hallmarks of AD have not yet been demonstrated. Interestingly, thioflavine-S-positive Aβ plaques were recently found in the retinal strata of APPswe/PS1ΔE9 transgenic mice²⁶ but not in the other animal models of AD. The current study used Tg2576 mice that constitutively overexpress APPswe and develop robust Aβ deposits in brain as well as cognitive abnormalities with aging.²⁷ We assessed the pathological changes in the retina of aged mice following different immunization schemes. We immunized Tg2576 with fibrillar Aβ42 and with a prefibrillar oligomer mimetic that gives rise to a prefibrillar oligomer-specific immune response. Both types of immunogens have been shown to be equally effective in reducing plaque deposition and inflammation in Tg2576 mouse brains.²⁸ In this study, we also used another prefibrillar oligomer mimetic antigen that uses the islet amyloid polypeptide (IAPP) instead of Aβ, but which gives rise to the same generic prefibrillar oligomer-specific immune response that also recognizes Aβ prefibrillar oligomers.²⁹ Aβ plaques and microvascular Aβ deposition were observed in the control Tg2576 mouse retinas. In contrast, Aβ and IAPP prefibrillar oligomer vaccinations differentially removed retinal Aβ deposits but exacerbated retinal amyloid angiopathy and inflammation as demonstrated by a significantly enhanced microglial infiltration and astrogliosis.     

Independent Effects of Intra- and Extracellular Aβ on Learning-Related Gene Expression

Alzheimer’s disease is characterized by numerous pathological abnormalities, including amyloid β (Aβ) deposition in the brain parenchyma and vasculature. In addition, intracellular Aβ accumulation may affect neuronal viability and function. In this study, we evaluated the effects of different forms of Aβ on cognitive decline by analyzing the behavioral induction of the learning-related gene Arc/Arg3.1 in three different transgenic mouse models of cerebral amyloidosis (APPPS1, APPDutch, and APP23). Following a controlled spatial exploration paradigm, reductions in both the number of Arc-activated neurons and the levels of Arc mRNA were seen in the neocortices of depositing mice from all transgenic lines (deficits ranging from 14 to 26%), indicating an impairment in neuronal encoding and network activation. Young APPDutch and APP23 mice exhibited intracellular, granular Aβ staining that was most prominent in the large pyramidal cells of cortical layer V; these animals also had reductions in levels of Arc. In the dentate gyrus, striking reductions (up to 58% in aged APPPS1 mice) in the number of Arc-activated cells were found. Single-cell analyses revealed both the proximity to fibrillar amyloid in aged mice, and the transient presence of intracellular granular Aβ in young mice, as independent factors that contribute to reduced Arc levels. These results provide evidence that two independent Aβ pathologies converge in their impact on cognitive function in Alzheimer’s disease.Alzheimer’s disease (AD) is the most common form of dementia in the elderly, with abnormal accumulation of the amyloid β (Aβ) peptide being one of the hallmarks of the disease. The correlation between parenchymal amyloid deposition (plaques) and cognitive decline is, however, controversial. Although the number of plaques does not closely correspond to the degree of dementia,^1,2 some studies point toward a detrimental effect on neuronal connectivity and function.^3,4,5,6 The majority of AD cases show further Aβ deposition in the cerebral vasculature,^7,8,9 referred to as cerebral amyloid angiopathy (CAA). Although CAA is a clear risk factor for hemorrhagic stroke,^10,11,12 indirectly leading to brain damage and cognitive impairments,^9,13,14,15 its direct impact on cognition is more difficult to characterize. A detrimental role of intraneuronal AB accumulation has been indicated prior to the onset of extracellular deposition (see reviews^16,17). Intraneuronal Aβ has been reported in brains of subjects with mild cognitive impairment, AD,^18,19 and in patients with Down syndrome,^20,21 and has also been described for several mouse models of cerebral Aβ amyloidoses.^22,23,24,25 Negative effects have been illustrated by several in vitro studies,^26,27 and coincidence of intraneuronal Aβ appearance with cognitive deficits has been shown in a triple transgenic mouse model of AD.²⁸Here we took advantage of three mouse models replicating distinct aspects of AD pathology to investigate the impact extracellular amyloid (vascular and parenchymal) and factors such as intracellular Aβ accumulation, have on cognitive impairment. APPPS1 mice show predominantly parenchymal amyloid with a high Aβ42 to Aβ40 ratio due to the L166P PS1 mutation in combination with the K670N/M671L “Swedish” double mutation. Both transgenes (as in all models studied) are driven by the neuron-specific Thy-1 minigene promoter.²⁹ In comparison, APPDutch mice exhibit almost exclusively vascular amyloid, due to the overexpression of human amyloid precursor protein (APP) bearing the E693Q “Dutch” mutation. This mutation was identified as the cause of hereditary cerebral hemorrhage with amyloidosis-Dutch type, a rare autosomal dominant disorder, characterized by severe CAA, recurrent strokes, and dementia.³⁰ APPDutch mice develop extensive vascular Aβ deposition initially within the walls of leptomeningeal vessels followed by cortical vessels.³¹ The third analyzed model, APP23 mice, overexpresses human APP with the “Swedish” mutation and exhibits both amyloid plaques and deposition within the vasculature.³²Within these mouse models, we investigated the behavioral induction of activity-regulated cytoskeleton-associated protein (Arc)/Arg3.1, as a marker for the functional status of individual neurons and memory systems. Arc is an effector immediate early gene implicated in synaptic plasticity, and is known to be necessary for memory consolidation and learning.^33,34,35,36 The specificity and characteristic time course of Arc mRNA induction can be used to monitor neural circuit activation following behavior episodes, as initially demonstrated by Guzowski and colleagues³⁷ and expanded in subsequent studies.^38,39,40,41 Recent evidence has demonstrated a role of Arc in alpha-amino-3-hydroxyl-5-methyl-4-isoxazole-propionate (AMPA) receptor trafficking, and thus it’s involvement in the molecular processes of synaptic plasticity.^42,43,44Although a reduction of Arc and other plasticity-related genes has been previously demonstrated in aging rats^45,46 and AD mouse models,^47,48,49 we show for the first time that particular pathological events in AD transiently impact neuronal function.