Astrocytes Infected with Chlamydia pneumonia Alter Amyloid Processing Implicated in Alzheimer’s Disease

Alzheimer’s Disease (AD) is a chronic, progressive neurodegenerative disease whose pathogenesis centers around the abnormal processing of amyloid precursor protein (APP) by proteases, resulting in the formation of neuritic plaques composed of toxic, insoluble fragments of amyloid protein (Aβ), including Aβ1-40 and Aβ1-42. Previously, our laboratory identified Chlamydia pneumoniae (Cpn) in autopsied sporadic AD brains. Additionally, an infection based animal model was developed using BALB/c mice that were intranasally inoculated with Cpn, in which the deposition of amyloid was consistent with that observed in the human AD brain. These studies have led to the pathogen hypothesis of AD that implicates Cpn as a trigger for the cleavage of APP into Aβ1-40 and Aβ1-42. Objective: Several studies have demonstrated the presence of astrocytes surrounding neuritic plaques within the AD brain; therefore, we speculate that astrocytes may be specifically involved in the pathological processes leading to Aβ deposition. This investigation addresses if an in vitro Cpn infection of human astrocytes affects processing of the ß amyloid precursor protein (ßAPP) and the enzyme ß APP cleaving enzyme-1 (BACE1), a type 1 transmembrane aspartyl protease directly involved in the processing of APP to Aβ and implicated in numerous neurodegenerative diseases, such as traumatic brain injury. Methods: Human astrocytes (CCF-STTG1) were infected in vitro with the respiratory strain AR39 Cpn (MOI=1). Analysis of protein levels for Aβ and the enzyme BACE1 post-infection was detected by immunocytochemistry and captured with the Olympus Confocal FV1000 microscope. Results: Amyloid processing in infected astrocytes was altered relative to that of uninfected astrocytes. BACE1 immunolabeling appeared more diffuse in the infected astrocytes as compared to membrane-localized BACE1 in the uninfected astrocytes. Conclusions: Neurons have been presumed to be the primary source of beta-amyloid peptides in AD brains; however, when astrocytes are activated, as occurs during infection with Cpn, astrocytic beta-amyloid generation may contribute to amyloid plaque formation. These data imply that infection of human astrocytes with Cpn affects the processing of ßAPP through altering the localization of BACE1 protein from the membrane to the cytoplasm. These data suggest an activation of BACE1 in the processing of amyloid by astrocytes as a major contributor to the neurotoxic amyloid deposition linked to pathology observed in AD

Chlamydia pneumoniae Infection of Monocytes in vitro Stimulates Innate and Adaptive Immune Responses Relevant to those in Alzheimer\u27s Disease.

Background: Alzheimer\u27s disease (AD) is a progressive neurodegenerative disorder in which infection with Chlamydia pneumoniae (Cpn) has been associated. Cpn is an obligate intracellular respiratory pathogen that may enter the central nervous system (CNS) following infection and trafficking of monocytes through the blood-brain barrier. Following this entry, these cells may secrete pro-inflammatory cytokines and chemokines that have been identified in the AD brain, which have been thought to contribute to AD neurodegeneration. The objectives of this work were: (i) to determine if Cpn infection influences monocyte gene transcript expression at 48 hours post-infection and (ii) to analyze whether pro-inflammatory cytokines are produced and secreted from these cells over 24 to 120 hours post-infection. Methods: Gene transcription was analyzed by RT-PCR using an innate and adaptive immunity microarray with 84 genes organized into 5 functional categories: inflammatory response, host defense against bacteria, antibacterial humoral response, septic shock, and cytokines, chemokines and their receptors. Statistical analysis of the results was performed using the Student\u27s t-test. P-values ≤ 0.05 were considered to be significant. ELISA was performed on supernatants from uninfected and Cpn-infected THP1 monocytes followed by statistical analysis with ANOVA. Results: When Cpn-infected THP1 human monocytes were compared to control uninfected monocytes at 48 hours post-infection, 17 genes were found to have a significant 4-fold or greater expression, and no gene expression was found to be down-regulated. Furthermore, cytokine secretion (IL-1ß, IL-6, IL-8) appears to be maintained for an extended period of infection. Conclusions: Utilizing RT-PCR and ELISA techniques, our data demonstrate that Cpn infection of THP1 human monocytes promotes an innate immune response and suggests a potential role in the initiation of inflammation in sporadic/late-onset Alzheimer\u27s disease

Herpes simplex virus type 1 and Chlamydia pneumoniae infection of astrocytes: the effects of co-infection on pathogen replication

Background: Chlamydia pneumoniae (Cpn) and Herpes simplex virus type 1 (HSV-1) have been studied as pathogens contributing to neurodegenerative diseases. Cpn and HSV-1 are both ubiquitous, thus many individuals presumably are exposed to both pathogens during their life time. Since Cpn can establish persistence under adverse environmental conditions, we speculate that productive HSV infection might induce Cpn persistence in cells infected with both pathogens. Intermittent reactivation of either or both pathogens might contribute to progressive pathology associated with neurodegenerative diseases. Objectives: Determine whether Cpn and HSV-1 can co-infect cells and whether the presence of one pathogen alters replication of the other. Methods: The ability of Cpn and HSV-1 to co-infect an astrocyte cell line (STTG-1) was analyzed by immunofluorescence (IF) labeling using antibodies specific for HSV and Cpn, and RT-PCR using primers specific for each pathogen. Cells were infected with HSV or Cpn alone, or co infected with both pathogens for 24 or 48 hours. Results: IF revealed that cells could be simultaneously infected with both pathogens. Gene expression data support the observation that HSV replication is somewhat diminished in the presence of Cpn; similarly, the developmental cycle of Cpn appears to be disrupted by HSV. Conclusions: IF and gene expression data suggest both HSV-1 and Cpn inhibit, but do not prevent, infection by the second pathogen, possibly by competing for the same cellular receptors. Moreover, disruption of host cell transcription by HSV-1 may modify normal Cpn developmen

Astrocytes Infected with Chlamydia Pneumoniae Demonstrate Altered Expression and Activity of Secretases Involved in the Generation of Β-amyloid Found in Alzheimer Disease

BACKGROUND: Epidemiologic studies strongly suggest that the pathophysiology of late-onset Alzheimer disease (AD) versus early-onset AD has environmental rather than genetic causes, thus revealing potentially novel therapeutic targets to limit disease progression. Several studies supporting the pathogen hypothesis of AD demonstrate a strong association between pathogens and the production of β-amyloid, the pathologic hallmark of AD. Although the mechanism of pathogen-induced neurodegeneration of AD remains unclear, astrocytes, a key player of the CNS innate immune response and producer/metabolizer of β-amyloid, have been implicated. We hypothesized that Chlamydia pneumoniae infection of human astrocytes alters the expression of the amyloid precursor protein (APP)-processing secretases, ADAM10, BACE1, and PSEN1, to promote β-amyloid formation. Utilizing immunofluorescent microscopy, molecular, and biochemical approaches, these studies explore the role of an intracellular respiratory pathogen, Chlamydia pneumoniae, as an environmental trigger for AD pathology. Human astrocytoma cells in vitro were infected with Chlamydia pneumoniae over the course of 6-72 h. The gene and protein expression, as well as the enzymatic activity of non-amyloidogenic (ADAM10), and pro-amyloidogenic (BACE1 and PSEN1) secretases were qualitatively and quantitatively assessed. In addition, the formation of toxic amyloid products as an outcome of pro-amyloidogenic APP processing was evaluated through various modalities. RESULTS: Chlamydia pneumoniae infection of human astrocytoma cells promoted the transcriptional upregulation of numerous genes implicated in host neuroinflammation, lipid homeostasis, microtubule function, and APP processing. Relative to that of uninfected astrocytes, BACE1 and PSEN1 protein levels were enhanced by nearly twofold at 48-72 h post-Chlamydia pneumoniae infection. The processing of APP in Chlamydia pneumoniae-infected astrocytes favors the pro-amyloidogenic pathway, as demonstrated by an increase in enzymatic activity of BACE1, while that of ADAM10 was decreased. Fluorescence intensity of β-amyloid and ELISA-quantified levels of soluble-APP by products revealed temporally similar increases, confirming a BACE1/PSEN1-mediated processing of APP. CONCLUSIONS: Our findings suggest that Chlamydia pneumoniae infection of human astrocytes promotes the pro-amyloidogenic pathway of APP processing through the upregulation of expression and activity of β-secretase, upregulated expression of γ-secretase, and decreased activity of α-secretase. These effects of astrocyte infection provide evidence for a direct link between Chlamydia pneumoniae and AD pathology

Mechanisms of Mouse Hepatitis Virus Entry into Cells

Background: Viruses can enter cells through several mechanisms, two common ones being clathrin-mediated and caveolin-mediated endocytosis. The clathrin pathway delivers viral particles to endosomes, with subsequent acidification of the endosome and endosome/lysosome fusion often a prerequisite for release of the viral genome into the cytoplasm. The caveolin-mediated pathway delivers virus initially into vesicles called caveosomes, which have a neutral pH, before viral uncoating occurs. Viral entry pathways can be examined by using various drugs to inhibit the different endocytosis pathways, as well as by siRNA technology to down-regulate expression of clathrin or caveolin proteins on the surface of host cells

Chlamydia pneumoniae infection of neuronal cells induces changes in calcium-associated gene expression consistent with Alzheimer’s disease

Background and Significance: Previous studies have shown that cells infected with Chlamydia pneumoniae (Cpn) exhibit altered gene expression consistent with that observed in Alzheimer’s disease (AD). Furthermore, AD neurodegeneration has been linked to dysregulation of intracellular calcium and calcium-related processes. Therefore, we hypothesize that one mechanism by which pathogenesis evolves in AD is through infection-induced changes in expression of calcium-related genes. Objectives: To determine if infection of neuronal cells with Cpn alters expression of calciumrelated genes associated with neurodegeneration. Methods: SK-N-MC neuronal cells were infected with Cpn (AR39 strain; MOI=1) for 3 to 72 hours, then calcium-related genes were screened with real-time PCR microarrays (SABiosciences PAHS-066). Results: Following infection, approximately 29 genes displayed regulation changes of 2-fold or greater, including genes pertaining to neurotransmitters, cell cycle and immune regulators, and other calcium-responsive elements. Genes involved in synaptic function and memory such as AREG, ATF3, EGR2 and GEM were initially up regulated, then fell to baseline or below by 72 hours. Many of the affected genes have been implicated in AD pathogenesis. Conclusions: Our data suggest that Cpn alters calcium-related gene expression in host neurons consistent with calcium dysfunction previously documented in AD. This study may elucidate how, in its effort to establish a favorable environment, Cpn could affect cellular processes that contribute to AD pathogenesis.https://digitalcommons.pcom.edu/posters/1006/thumbnail.jp

Infection of neuronal cells by Chlamydia pneumoniae and Herpes simplex virus type 1 alters expression of genes associated with Alzheimer’s disease

Several studies have suggested an infectious etiology for Alzheimer’s disease (AD). We have been investigating a potential role for both Chlamydia pneumoniae and Herpes simplex virus type 1 (HSV1) in the initiation of sporadic late-onset AD. Our current study focuses on investigation of gene expression using Alzheimer-specific Real-Time PCR microarrays on RNA derived from SKNMC human neuronal cells infected with C. pneumoniae and/or HSV1. There are distinct differences in the patterns of gene regulation by the two pathogens. For example, C. pneumoniae induces expression of genes involved in amyloid production and processing, such as β-amyloid precursor protein (APP), β-site APP-cleaving enzyme 1 (BACE1), a γ-secretase complex protein (nicastrin [NCSTN]), NEDD8 activating enzyme E1 (NAE1), as well as a mitochondria-associated protein (hydroxysteroid (17-β) dehydrogenase 10 [HSD17B10]), α-2-macroglobulin (A2M) and the metallopeptidase ADAM9. Conversely, HSV1 tends to down-regulate expression of many genes, including those encoding a component of the γ-secretase complex (anterior pharynx defective 1 homolog A [APH1A]), low density lipoprotein related proteins (LRP1, LRP6, and LRP8), β-synuclein (SNCB) and ubiquinols (UQCRC1, UQCRC2). Co-infection with C. pneumoniae and HSV-1 produced a greater down-regulation of gene expression than that seen with HSV1 alone for several genes, including APP-like proteins (APLP1, APLP2) and kinases (cell division cycle 2 protein [CDC2], cyclin-dependent kinase [CDK5] and CDC2-related kinase [CDKL1]). Our data indicate that both C. pneumoniae and HSV1 can modulate expression of genes associated with AD, and thus could contribute to AD pathology, however these two pathogens likely act via different pathways. Furthermore, for several genes, co-infection with both C. pneumoniae and HSV1 appears to exacerbate the changes in gene expression seen with HSV1 alone.https://digitalcommons.pcom.edu/posters/1007/thumbnail.jp

Autophagy and apoptotic genes implicated in Alzheimer’s disease are modulated following infection of neuronal cells with Chlamydia pneumoniae

Background: The focus of the current studies was to determine the relationship between the molecular mechanisms interconnecting autophagy and apoptosis following Chlamydia pneumoniae infection in neuronal cells. Dysfunctions in apoptosis and autophagy have been implicated in the neurodegeneration associated with Alzheimer’s disease (AD). Autophagy in AD pathogenesis has been shown to play a role in amyloid processing through the endosomal-lysosomal system. Apoptosis may contribute to the neuronal cell loss observed in AD; however, there is limited evidence of the apoptotic process proceeding to terminal completion. Although Aβ1-42 has been shown to induce apoptosis in neurons and may be an early factor in AD, our previous investigations demonstrated that neurons infected with Chlamydia pneumoniae are resistant to apoptosis, and that Aβ1-42 is induced following this infection. Thus, these studies address infection as an initiator/trigger or inhibitor for the processes of autophagy and apoptosis observed in Alzheimer’s disease. Methods: SKNMC neuronal cells obtained from ATCC were infected with the AR39 strain of Chlamydia pneumoniae at an MOI=1 for 24, 48, and 72hrs and were analyzed using Real-time PCR arrays from SABiosciences specific for autophagy and apoptosis genetic markers. Results: Some major genes associated with apoptosis such as BID, DAPK1, TP53, TP73 were down regulated by 72hrs post-infection. Genes associated with the regulation of autophagic vacuole formation such as ATG3, ATG4B, ATG4C, ATG9A, ATG9B, ATG12, IRGM, and BECN1 were up-regulated within 72hrs post-infection. With regards to genes involved with co-regulation of autophagy and apoptosis, BNIP3 was significantly up-regulated within 48-72hrs post-infection. Of the genes linking autophagosomes to lysosomes, FAM176A was up-regulated throughout 24-72hrs post-infection. Conclusions: Modulation of autophagy and apoptosis genes occurs in neuronal cells at 24, 48, and 72hrs post- infection with Chlamydia pneumoniae. These genetic changes lead to dysfunction in these basic cellular processes; dysfunction in these processes has been shown to contribute to the neuropathology of late-onset Alzheimer’s disease. This work will allow future studies to further focus on the apoptotic and autophagic pathways to better understand how a pathogen such as Chlamydia pneumoniae plays a role in the development of late-onset Alzheimer’s disease.https://digitalcommons.pcom.edu/posters/1009/thumbnail.jp

Changes in Expression of Genes Associated with Autophagy and Apoptosis in Neuronal Cells Infected with HSV-1may Suggest Infection-induced Mechanisms of Neurodegeneration

Background:This study investigates the potential role of herpes simplex virus type 1 (HSV-1) in the pathogenesis of neurodegenerative disorders, such as Alzheimer’s disease (AD), by exploring changes in gene expression related to antiviral immunity and the autophagic pathway. Autophagy is a process that recycles organelles and proteins to create more energy for the cell. This pathway has been linked to neurodegeneration, as malfunctions in the completion of this process lead to a decline in overall cellular health and function. Interestingly, HSV-1 has been shown to block the completion of autophagy, which would potentially contribute to the cytopathic changes observed in AD