Abstract Pathways traversed by peripherally administered protein tracers for entry to the mammalian brain were investigated by light and electron microscopy. Native horseradish peroxidase (HRP) and wheat germ agglutinin (WGA) conjugated to peroxidase were administered intranasally, intravenously, or intraventricularly to mice; native HRP was delivered intranasally or intravenously to rats and squirrel monkeys. Unlike WGA‐HRP, native HRP administered intranasally passed freely through intercellular junctions of the olfactory epithelia to reach the olfactory bulbs of the CNS extracelluarly within 45–90 minutes in all species. The olfactory epithelium labeled with intravenously delivered HRP, which readily escaped vasculature supplying this epithelium. Blood‐borne peroxidase also exited fenestrated vessels of the dura mater and circumventricular organs. This HRP in the mouse, but not in the other species, passed from the dura mater through patent intercellular junctions within the arachnoid mater; in time, peroxidase reaction product in the mouse brain was associated with the pial surface, the Virchow‐Robin spaces of vessels penetrating the pial surface, perivascular clefts, and with phagocytic pericytes located on the abluminal surface of superficial and deep cerebral microvasculature. Blood‐borne HRP was endocytosed avidly at the luminal face of the cerebral endothelium in all species. WGA‐HRP and native HRP delivered intraventricularly to the mouse were not endocytosed appreciably at the abluminal surface of the endothelium; hence, the endocytosis of protein and internalization of cell surface membrane within the cerebral endothelium are vectorial. The low to non‐existent endocytic activity and internalization of membrane from the abluminal endothelial surface suggests that vesicular transport through the cerebral endothelium from blood to brain and from brain to blood does not occur. The extracellular pathways through which probe molecules enter the mammalian brain offer potential routes of passage for blood‐borne and airborne toxic, carcinogenic, infectious, and neurotoxic agents and addictive drugs, and for the delivery of chemotherapeutic agents to combat CNS infections and deficiency states. Methodological considerations are discussed for the interpretation of data derived from application of peroxidase to study the blood brain barrier.
We draw attention to a historic case of a boy who suffered from scarlet fever (typically caused by the bacterium Streptococcus pyogenes) at age 7 years and went on to develop the symptoms of Alzheimer's disease (AD). His physicians believed that the subsequent dementia was related to the infection. After death at 24 years of age, postmortem brain examination revealed abundant AD-type senile plaques and fibrils, formally confirming AD. Other potential causes of early-onset dementia are discussed, but these are distinct from patient E.H. This case is pertinent regarding the current debate about the potential role of infection in AD.
Abstract Background Chlamydia pneumoniae is an obligate intracellular respiratory pathogen for humans. Infection by C. pneumoniae may be linked etiologically to extra-respiratory diseases of aging, especially atherosclerosis. We have previously shown that age promotes C. pneumoniae respiratory infection and extra-respiratory spread in BALB/c mice. Findings Aged C57BL/6 mice had a greater propensity to develop chronic and/or progressive respiratory infections following experimental intranasal infection by Chlamydia pneumoniae when compared to young counterparts. A heptavalent CTL epitope minigene (CpnCTL7) vaccine conferred equal protection in the lungs of both aged and young mice. This vaccine was partially effective in protecting against C. pneumoniae spread to the cardiovascular system of young mice, but failed to provide cardiovascular protection in aged animals. Conclusions Our findings suggest that vaccine strategies that target the generation of a C. pneumoniae -specific CTL response can protect the respiratory system of both young and aged animals, but may not be adequate to prevent dissemination of C. pneumoniae to the cardiovascular system or control replication in those tissues in aged animals.
Studies have suggested that apoptosis may contribute to the neuronal cell loss observed in Alzheimer's disease (AD). Aβ 1–42 has been shown to induce apoptosis in neurons and may be an initiating factor in AD. Caspase is an effector in neuronal apoptosis that could also play a role in AD. However, the extent to which apoptosis contributes to cell death in AD has yet to be delineated. In an earlier study, we identified and isolated Chlamydia pneumoniae from brains of patients that had been diagnosed with sporadic AD. These in vitro studies suggested that neurons infected with C. pneumoniae are resistant to apoptosis, and that the processing or production of APP into Aβ1–42 was increased by the infection. In addition, we have developed a novel murine model in which non–transgenic mice infected with C. pneumoniae formed deposits of amyloid in areas of the brain most affected in Alzheimer's disease. Interestingly, some of the neurons in these areas showed a high level of Aβ 1–42 immunoreactivity, and these neurons did not appear to be undergoing apoptosis. The focus of the current studies was to delineate whether caspase is activated following a C. pneumoniae infection in neuronal cells. Apoptosis was experimentally induced by staurosporine in uninfected SK–N–MC cells and cells infected with C. pneumoniae. Caspase activity was analyzed using the Apo–ONE caspase 3/7 assay (Promega). We found that staurosporine induced an increase in caspase 3/7 activity in both infected and uninfected cells, however the staurosporine–treated infected cells had lower activity than even the basal activity of uninfected cells. These data were consistent with immunocytochemistry, which showed decreased labeling by antibodies that recognize cleaved (active) caspase 3 in the infected cells. These results suggest that inhibition of apoptosis by suppression of caspase 3/7 activity, and/or by decreasing levels of active caspase 3, may be mechanisms by which C. pneumoniae can sustain a persistent infection in the host and optimize its intracellular environment. In this way, C. pneumoniae may participate in the pathogenesis characteristic of Alzheimer's disease.
Guam Amyotrophic Lateral Sclerosis/Parkinsonism-Dementia Complex (Guam ALS/PDC) is a progressive neurodegenerative disorder characterized by abundant neurofibrillary tangles (NFTs) composed of aggregated paired helical filaments (PHFs). These abnormal filaments resemble the PHFs in neurofibrillary lesions of classic Alzheimer's disease (AD), and recent studies demonstrated that tau in Guam ALS/PDC is aberrantly phosphorylated and biochemically similar to the abnormal tau proteins (PHFtau) in classic AD. However, unlike PHFtau in AD, there is little information on the specific sites of phosphorylation in PHFtau from Guam ALS/PDC. Thus, to address this important issue, we examined tangle-rich Guam ALS/PDC and AD brains by Western blot, immunoelectron microscopy and immunohistochemistry using 13 antibodies to defined phosphate-dependent or -independent epitopes distributed throughout AD PHFtau. These studies identified 7 previously unknown sites of phosphorylation in PHFtau from Guam ALS/PDC (i.e. Thr181, Thr231, Ser262, Ser396, Ser404, Ser422, and the site defined by monoclonal antibody AT10), all of which also are found in AD PHFtau. Indeed, the Western blot, light and immunoelectron microscopic data suggest that NFTs, PHFs and PHFtau in Guam ALS/PDC are very similar to their counterparts in classic AD. Thus, insights into mechanisms leading to the accumulation of neurofibrillary lesions in Guam ALS/PDC may advance understanding of the pathogenesis and biological consequences of these lesions in classic AD.
Several studies have suggested an infectious etiology as a trigger in the cascade of events leading to Alzheimer's disease (AD). Previously, our laboratory identified and isolated Chlamydophila (Chlamydia) pneumoniae (Cpn) from autopsied sporadic late-onset AD brains, as well as developed a BALB/c mouse model that demonstrated that intranasal infection with Cpn induced amyloid plaques similar to those found in the AD brain. An additional pathogen, herpes simplex virus type 1 (HSV1), also may be a factor in the pathogenesis of AD. In this regard, HSV1, in addition to Cpn, may be triggering abnormal cleavage of the beta amyloid precursor protein (βAPP) into Aβ1–42, thereby contributing to amyloid plaque formation. Our current study examines amyloid processing following infection of murine astrocytes (ATCC- C8DIA) and human neuroblastoma cells (ATCC -SKNMC) with Cpn and HSV1. Astrocytes and neuroblastoma cells were infected with each organism singularly or in tandem. Infected cells were analyzed by immunocytochemistry using antibodies targeting Aβ 1–42 amyloid to determine how an infection by these two pathogens would affect amyloid processing. Cpn infection resulted in an increase in cytoplasmic labeling of Aβ 1–42 relative to uninfected cells, while increased nuclear labeling of Aβ 1–42 was observed following HSV1 infection. Co-infections with Cpn and HSV1 resulted in amyloid labeling resembling that of HSV1 infection alone, although cells solely infected with Cpn of the co-infected monolayers demonstrated reduced Aβ1–42 labeling. These data suggest that infection of astrocytes and neuronal cells by Herpes Simplex Virus 1 and Chlamydophila (Chlamydia) pneumoniae alters the processing of βAPP, whereby Aβ1–42 is processed in an intracellular manner. Therefore, these studies, in addition to the previous research reported by our laboratory, support an emerging linkage of the infectious process to the neuropathology characteristic of Alzheimer's disease.
Purified bovine neurofilament (NF) subunit proteins were reassembled in vitro to form either homopolymeric or heteropolymeric intermediate-sized filaments using single or paired combinations of NF triplet proteins. Using conditions established for the reassembly of bovine NF triplet proteins2, we demonstrated that the low Mr NF subunit (NF-L) alone and in combination with the middle Mr NF subunit (NF-M) reassembled very efficiently, i.e. 95% of these proteins formed filaments within 90 min from the start of reassembly. In contra-distinction, the high Mr NF subunit (NF-H) alone and in combination with NF-M or NF-L underwent reassembly to a lesser extent, i.e. 62–88% of these proteins reassembled within 90 min. Immunolabeling of the reassembled NF polymers revealed striking differences in the organization of rod domain determinants. Specifically, antibodies specific for epitopes in the rod domains of NF-H, NF-M and NF-L failed to bind heteropolymeric filaments but recognized rod domains in the homopolymers. In contrast, antibodies specific to head and tail domains of all NF proteins labeled the reassembled hetero- and homopolymeric NFs. Double-labeling of heteropolymers demonstrated that pairs of different NF subunits coassembled into intermediate-sized filaments. Our results also showed that only copolymeric filaments of NF-L and NF-M, but not NF-L/NF-H and NF-M/NF-H were able to form long and stable 10 nm wide filaments. These observations provide new insights into the requirements for stable filament formation from NF subunits. In particular, they support the notion that only NF-L/NF-M, but not NF-L/NF-H or NF-M/NF-H might assemble into a stable filamentous network in vivo.
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. SKNMC neuronal cells obtained from ATCC were infected with the AR39 strain of Chlamydia pneumoniae at an MOI = 1 for 24, 48, and 72 hrs and were analyzed using Real-time PCR arrays from SABiosciences specific for autophagy and apoptosis genetic markers. Some major genes associated with apoptosis such as BID, DAPK1, TP53, TP73 were down regulated by 72 hrs 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 72 hrs post-infection. With regards to genes involved with co-regulation of autophagy and apoptosis, BNIP3 was significantly up-regulated within 48-72 hrs post-infection. Of the genes linking autophagosomes to lysosomes, FAM176A was up-regulated throughout 24-72 hrs post-infection. Modulation of autophagy and apoptosis genes occurs in neuronal cells at 24, 48, and 72 hrs 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.
