Astrocytes infected with Chlamydia pneumoniae demonstrate altered expression and activity of secretases involved in the generation of ß-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 ß-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 ß-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 ß-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 ß-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 ß-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.

Chlamydia pneumoniae: An Etiologic Agent for Late-Onset Dementia.

The disease known as late-onset Alzheimer's disease is a neurodegenerative condition recognized as the single most commonform of senile dementia. The condition is sporadic and has been attributed to neuronal damage and loss, both of which have been linked to the accumulation of protein deposits in the brain. Significant progress has been made over the past two decades regarding our overall understanding of the apparently pathogenic entities that arise in the affected brain, both for early-onset disease, which constitutes approximately 5% of all cases, as well as late-onset disease, which constitutes the remainder of cases. Observable neuropathology includes: neurofibrillary tangles, neuropil threads, neuritic senile plaques and often deposits of amyloid around the cerebrovasculature. Although many studies have provided a relatively detailed knowledge of these putatively pathogenic entities, understanding of the events that initiate and support the biological processes generating them and the subsequent observable neuropathology and neurodegeneration remain limited. This is especially true in the case of late-onset disease. Although early-onset Alzheimer's disease has been shown conclusively to have genetic roots, the detailed etiologic initiation of late-onset disease without such genetic origins has remained elusive. Over the last 15 years, current and ongoing work has implicated infection in the etiology and pathogenesis of late-onset dementia. Infectious agents reported to be associated with disease initiation are various, including several viruses and pathogenic bacterial species. We have reported extensively regarding an association between late-onset disease and infection with the intracellular bacterial pathogen Chlamydia pneumoniae. In this article, we review previously published data and recent results that support involvement of this unusual respiratory pathogen in disease induction and development. We further suggest several areas for future research that should elucidate details relating to those processes, and we argue for a change in the designation of the disease based on increased understanding of its clinical attributes.

Age-dependent increase of blood-brain barrier permeability and neuron-binding autoantibodies in S100B knockout mice.

S100B is a calcium-sensor protein that impacts multiple signal transduction pathways. It is widely considered to be an important biomarker for several neuronal diseases as well as blood-brain barrier (BBB) breakdown. In this report, we demonstrate a BBB deficiency in mice that lack S100B through detection of leaked Immunoglobulin G (IgG) in the brain parenchyma. IgG leaks and IgG-binding to selected neurons were observed in S100B knockout (S100BKO) mice at 6 months of age but not at 3 months. By 9 months, IgG leaks persisted and the density of IgG-bound neurons increased significantly. These results reveal a chronic increase in BBB permeability upon aging in S100BKO mice for the first time. Moreover, coincident with the increase in IgG-bound neurons, autoantibodies targeting brain proteins were detected in the serum via western blots. These events were concurrent with compromise of neurons, increase of activated microglia and lack of astrocytic activation as evidenced by decreased expression of microtubule-associated protein type 2 (MAP2), elevated number of CD68 positive cells and unaltered expression of glial fibrillary acidic protein (GFAP) respectively. Results suggest a key role for S100B in maintaining BBB functional integrity and, further, propose the S100BKO mouse as a valuable model system to explore the link between chronic functional compromise of the BBB, generation of brain-reactive autoantibodies and neuronal dysfunctions.

Sevoflurane and Isoflurane induce structural changes in brain vascular endothelial cells and increase blood-brain barrier permeability: Possible link to postoperative delirium and cognitive decline.

A large percentage of patients subjected to general anesthesia at 65 years and older exhibit postoperative delirium (POD). Here, we test the hypothesis that inhaled anesthetics (IAs), such as Sevoflurane and Isoflurane, act directly on brain vascular endothelial cells (BVECs) to increase blood-brain barrier (BBB) permeability, thereby contributing to POD. Rats of young (3-5 months), middle (10-12 months) and old (17-19 months) ages were anesthetized with Sevoflurane or Isoflurane for 3h. After exposure, some were euthanized immediately; others were allowed to recover for 24h before sacrifice. Immunohistochemistry was employed to monitor the extent of BBB breach, and scanning electron microscopy (SEM) was used to examine changes in the luminal surfaces of BVECs. Quantitative immunohistochemistry revealed increased BBB permeability in older animals treated with Sevoflurane, but not Isoflurane. Extravasated immunoglobulin G showed selective affinity for pyramidal neurons. SEM demonstrated marked flattening of the luminal surfaces of BVECs in anesthetic-treated rats. Results suggest an aging-linked BBB compromise resulting from exposure to Sevoflurane. Changes in the luminal surface topology of BVECs indicate a direct effect on the plasma membrane, which may weaken or disrupt their BBB-associated tight junctions. Disruption of brain homeostasis due to plasma influx into the brain parenchyma and binding of plasma components (e.g., immunoglobulins) to neurons may contribute to POD. We propose that, in the elderly, exposure to some IAs can cause BBB compromise that disrupts brain homeostasis, perturbs neuronal function and thereby contributes to POD. If unresolved, this may progress to postoperative cognitive decline and later dementia.

Detection of bacterial antigens and Alzheimer's disease-like pathology in the central nervous system of BALB/c mice following intranasal infection with a laboratory isolate of Chlamydia pneumoniae.

Pathology consistent with that observed in Alzheimer's disease (AD) has previously been documented following intranasal infection of normal wild-type mice with Chlamydia pneumoniae (Cpn) isolated from an AD brain (96-41). In the current study, BALB/c mice were intranasally infected with a laboratory strain of Cpn, AR-39, and brain and olfactory bulbs were obtained at 1-4 months post-infection (pi). Immunohistochemistry for amyloid beta or Cpn antigens was performed on sections from brains of infected or mock-infected mice. Chlamydia-specific immunolabeling was identified in olfactory bulb tissues and in cerebrum of AR-39 infected mice. The Cpn specific labeling was most prominent at 1 month pi and the greatest burden of amyloid deposition was noted at 2 months pi, whereas both decreased at 3 and 4 months. Viable Cpn was recovered from olfactory bulbs of 3 of 3 experimentally infected mice at 1 and 3 months pi, and in 2 of 3 mice at 4 months pi. In contrast, in cortical tissues of infected mice at 1 and 4 months pi no viable organism was obtained. At 3 months pi, only 1 of 3 mice had a measurable burden of viable Cpn from the cortical tissues. Mock-infected mice (0 of 3) had no detectable Cpn in either olfactory bulbs or cortical tissues. These data indicate that the AR-39 isolate of Cpn establishes a limited infection predominantly in the olfactory bulbs of BALB/c mice. Although infection with the laboratory strain of Cpn promotes deposition of amyloid beta, this appears to resolve following reduction of the Cpn antigen burden over time. Our data suggest that infection with the AR-39 laboratory isolate of Cpn results in a different course of amyloid beta deposition and ultimate resolution than that observed following infection with the human AD-brain Cpn isolate, 96-41. These data further support that there may be differences, possibly in virulence factors, between Cpn isolates in the generation of sustainable AD pathology.

NLRP3 inflammasome is a target for development of broad-spectrum anti-infective drugs.

We describe the molecular mode of action and pharmacodynamics of a new molecular entity (NME) that induces the NLRP3 inflammasome-mediated innate immune response. This innate response reduces the pathogen load in an experimentally induced methicillin-resistant Staphylococcos aureus infection, enhances survival in an experimentally induced Gram-negative bacteremia, and overrides the escape mechanism of an obligate intracellular pathogen, viz. Chlamydia pneumoniae. Furthermore, the NME is more effective than standard-of-care antibiotic therapy in a clinically established multifactorial bacterial infection. Analysis of transcriptional regulation of inflammasome signaling genes and innate/adaptive immune genes revealed consistent and significant host changes responsible for the improved outcomes in these infections. These studies pave the way for the development of first-in-class drugs that enhance inflammasome-mediated pathogen clearance and identify the NLRP3 inflammasome as a drug target to address the global problem of emerging new infectious diseases and the reemergence of old diseases in an antibiotic-resistant form.

Modern care for patients with Alzheimer disease: rationale for early intervention.

More than 5 million people in the United States are afflicted with Alzheimer disease, a condition that is the seventh leading cause of death in the nation. Lacking definitive disease-modifying treatments, modern care for individuals with Alzheimer disease is necessarily multimodal, combining the use of approved pharmaceutic agents (ie, acetylcholinesterase inhibitors, N-methyl-D-aspartate receptor antagonists, antipsychotics), lifestyle and behavioral interventions, and components of palliative care. Some promising experimental treatments are undergoing clinical trials, including immunotherapy to prevent the deposition of ß-amyloid, a protein implicated as an etiologic factor in the disease. The authors briefly examine the rationale and methods for screening patients for early indications of the onset of Alzheimer disease. They also describe current and potential treatments for patients with this disease.

Immunohistological detection of Chlamydia pneumoniae in the Alzheimer's disease brain.

BACKGROUND: Sporadic late-onset Alzheimer's disease (AD) appears to evolve from an interplay between genetic and environmental factors. One environmental factor that continues to be of great interest is that of Chlamydia pneumoniae infection and its association with late-onset disease. Detection of this organism in clinical and autopsy samples has proved challenging using a variety of molecular and histological techniques. Our current investigation utilized immunohistochemistry with a battery of commercially available anti-C. pneumoniae antibodies to determine whether C. pneumoniae was present in areas typically associated with AD neuropathology from 5 AD and 5 non-AD control brains. RESULTS: Immunoreactivity for C. pneumoniae antigens was observed both intracellularly in neurons, neuroglia, endothelial cells, and peri-endothelial cells, and extracellularly in the frontal and temporal cortices of the AD brain with multiple C. pneumoniae-specific antibodies. This immunoreactivity was seen in regions of amyloid deposition as revealed by immunolabeling with two different anti-beta amyloid antibodies. Thioflavin S staining, overlaid with C. pneumoniae immunolabeling, demonstrated no direct co-localization of the organism and amyloid plaques. Further, the specificity of C. pneumoniae labeling of AD brain sections was demonstrated using C. pneumoniae antibodies pre-absorbed against amyloid ß 1-40 and 1-42 peptides. CONCLUSIONS: Anti-C. pneumoniae antibodies, obtained commercially, identified both typical intracellular and atypical extracellular C. pneumoniae antigens in frontal and temporal cortices of the AD brain. C. pneumoniae, amyloid deposits, and neurofibrillary tangles were present in the same regions of the brain in apposition to one another. Although additional studies are required to conclusively characterize the nature of Chlamydial immunoreactivity in the AD brain, these results further implicate C. pneumoniae infection with the pathogenesis of Alzheimer's disease.

Chlamydophila pneumoniae and the etiology of late-onset Alzheimer's disease.

Sporadic, late-onset Alzheimer's disease (LOAD) is a non-familial, progressive neurodegenerative disease that is now the most common and severe form of dementia in the elderly. That dementia is a direct result of neuronal damage and loss associated with accumulations of abnormal protein deposits in the brain. Great strides have been made in the past 20 years with regard to understanding the pathological entities that arise in the AD brain, both for familial AD ( approximately 5% of all cases) and LOAD ( approximately 95% of all cases). The neuropathology observed includes: neuritic senile plaques (NSPs), neurofibrillary tangles (NFTs), neuropil threads (NPs), and often deposits of cerebrovascular amyloid. Genetic, biochemical, and immunological analyses have provided a relatively detailed knowledge of these entities, but our understanding of the "trigger" events leading to the many cascades resulting in this pathology and neurodegeneration is still quite limited. For this reason, the etiology of AD, in particular LOAD, has remained elusive. However, a number of recent and ongoing studies have implicated infection in the etiology and pathogenesis of LOAD. This review focuses specifically on infection with Chlamydophila (Chlamydia) pneumoniae in LOAD and how this infection may function as a "trigger or initiator" in the pathogenesis of this disease.

Inhibition of apoptosis in neuronal cells infected with Chlamydophila (Chlamydia) pneumoniae.

BACKGROUND: Chlamydophila (Chlamydia) pneumoniae is an intracellular bacterium that has been identified within cells in areas of neuropathology found in Alzheimer disease (AD), including endothelia, glia, and neurons. Depending on the cell type of the host, infection by C. pneumoniae has been shown to influence apoptotic pathways in both pro- and anti-apoptotic fashions. We have hypothesized that persistent chlamydial infection of neurons may be an important mediator of the characteristic neuropathology observed in AD brains. Chronic and/or persistent infection of neuronal cells with C. pneumoniae in the AD brain may affect apoptosis in cells containing chlamydial inclusions. RESULTS: SK-N-MC neuroblastoma cells were infected with the respiratory strain of C. pneumoniae, AR39 at an MOI of 1. Following infection, the cells were either untreated or treated with staurosporine and then examined for apoptosis by labeling for nuclear fragmentation, caspase activity, and membrane inversion as indicated by annexin V staining. C. pneumoniae infection was maintained through 10 days post-infection. At 3 and 10 days post-infection, the infected cell cultures appeared to inhibit or were resistant to the apoptotic process when induced by staurosporine. This inhibition was demonstrated quantitatively by nuclear profile counts and caspase 3/7 activity measurements. CONCLUSION: These data suggest that C. pneumoniae can sustain a chronic infection in neuronal cells by interfering with apoptosis, which may contribute to chronic inflammation in the AD brain.

Differential expression of E-cadherin, N-cadherin and beta-catenin in proximal and distal segments of the rat nephron.

BACKGROUND: The classical cadherins such as E- and N-cadherin are Ca2+-dependent cell adhesion molecules that play important roles in the development and maintenance of renal epithelial polarity. Recent studies have shown that a variety of cadherins are present in the kidney and are differentially expressed in various segments of the nephron. However, the interpretation of these findings has been complicated by the fact that the various studies focused on different panels of cadherins and utilized different species. Moreover, since only a few of the previous studies focused on the rat, information regarding the expression and localization of renal cadherins in this important species is lacking. In the present study, we have employed dual immunofluorescent labeling procedures that utilized specific antibodies against either E- or N-cadherin, along with antibodies that target markers for specific nephron segments, to characterize the patterns of cadherin expression in frozen sections of adult rat kidney. RESULTS: The results showed that N-cadherin is the predominant cadherin in the proximal tubule, but is essentially absent in other nephron segments. By contrast, E-cadherin is abundant in the distal tubule, collecting duct and most medullary segments, but is present only at very low levels in the proximal tubule. Additional results revealed different patterns of N-cadherin labeling along various segments of the proximal tubule. The S1 and S2 segments exhibit a fine threadlike pattern of labeling at the apical cell surface, whereas the S3 segment show intense labeling at the lateral cell-cell contacts. CONCLUSIONS: These results indicate that E- and N-cadherin are differentially expressed in the proximal and distal tubules of rat kidney and they raise the possibility that differences in cadherin expression and localization may contribute to the differences in the susceptibility of various nephron segments to renal pathology or nephrotoxic injury.

Chlamydia pneumoniae induces Alzheimer-like amyloid plaques in brains of BALB/c mice.

Amyloid deposits resembling plaques found in Alzheimer's disease (AD) brains were formed in the brains of non-transgenic BALB/c mice following intranasal infection with Chlamydia pneumoniae. The mice were infected at 3 months of age with C. pneumoniae isolated from an AD brain. Infection was confirmed by light and electron microscopy in olfactory tissues of the mice. C. pneumoniae was still evident in these tissues 3 months after the initial infection indicating that a persistent infection had been established. Amyloid beta (Abeta) 1-42 immunoreactive deposits were identified in the brains of infected BALB/c mice up to 3 months post-infection with the density, size, and number of deposits increasing as the infection progressed. A subset of deposits exhibited thioflavin-s labeling. Intracellular Abeta1-42 labeling was observed in neuronal cells. Experimental induction of amyloid deposition in brains of non-transgenic BALB/c mice following infection with C. pneumoniae may be a useful model for furthering our understanding of mechanisms, linked to infection, involved in the initiation of the pathogenesis of sporadic AD.

Chlamydia pneumoniae infection alters the junctional complex proteins of human brain microvascular endothelial cells.

Chlamydia pneumoniae has been identified and associated with multiple sclerosis (MS) and Alzheimer's disease (AD) pathogenesis, although the relationship of this organism in these diseases remains controversial. We have hypothesized that one potential avenue of infection is through the junctional complexes between the blood-brain barrier (BBB) endothelia. C. pneumoniae is characteristically a respiratory pathogen, but has been implicated in atherosclerosis, coronary artery disease, and neuroinflammatory conditions. C. pneumoniae infection may lead to endothelial damage, junctional alterations, and BBB breakdown. Therefore, in this study, C. pneumoniae infection of human brain microvascular endothelial cells (HBMECs) resulted in increased expression of the zonula adherens proteins beta-catenin, N-cadherin, and VE-cadherin, and decreased expression of the tight junctional protein occludin, as determined by immunocytochemistry and Western blot analyses. These events may underlie a mechanism for the regulation of paracellular permeability while maintaining barrier integrity during C. pneumoniae infection associated with neuropathologies such as MS and AD.

Ultrastructural Analysis of Chlamydia Pneumoniae in the Alzheimer's Brain.

We recently reported identification of the bacterium Chlamydia pneumoniae in affected brain regions of patients with Alzheimer's disease (AD) (Balin et al., 1998). In this report, we extend those initial observations to demonstrate that, in addition to the frequently described, standard morphological forms of the organism, pleiomorphic forms are also present in the AD brain. All AD and control brain tissues examined were verified to be PCR-positive and negative, respectively, for the organism. DNA sequence determination of PCR products so derived from total DNA of infected AD brains, as well as from total DNA of cell lines infected with the organism following isolation from these same patient samples, confirmed the presence of organism in relevant samples. Various morphologic forms of C. pneumoniae were identified in PCR-positive tissues and these were characterized based on membrane structure, core density, size, and immunolabeling profiles. Structures identified include the typical pear-shaped elementary body, as well as larger, spherical and oblong reticulate bodies. Intact C. pneumoniae were found both intracellularly and extracellularly in the sampled autopsy brains. Intracellular organisms were located principally within microglia, astroglia, and presumptive pericytes. These results suggest that C. pneumoniae found in cells indigenous to the AD brain do not conform universally to the classical morphology observed in other infected cell types. This pleiomorphism may reflect an adaptive response and/or persistent state of infection for these organisms in Alzheimer's Disease.