Yvonne Kress

Mitochondrial Abnormalities in Alzheimer's Disease

Mitochondria from persons with Alzheimers disease (AD) differ from those of age-matched control subjects. Differences in mitochondrial morphology and function are well documented, and are not brain-limited. Some of these differences are present during all stages of AD, and are even seen in individuals who are without AD symptoms and signs but who have an increased risk of developing AD. This chapter considers the status of mitochondria in AD subjects, the potential basis for AD subject mitochondrial perturbations, and the implications of these perturbations. Data from multiple lines of investigation, including epidemiologic, biochemical, molecular, and cytoplasmic hybrid studies, are reviewed. The possibility that mitochondria could potentially constitute a reasonable AD therapeutic target is discussed, as are several potential mitochondrial medicine treatment strategies.

Hyperphosphorylation and aggregation of tau in mice expressing normal human tau isoforms

Neurofibrillary tangles are composed of insoluble aggregates of the microtubule‐associated protein tau. In Alzheimers disease the accumulation of neurofibrillary tangles occurs in the absence of tau mutations. Here we present mice that develop pathology from non‐mutant human tau, in the absence of other exogenous factors, including β‐amyloid. The pathology in these mice is Alzheimer‐like, with hyperphosphorylated tau accumulating as aggregated paired helical filaments. This pathologic tau accumulates in the cell bodies and dendrites of neurons in a spatiotemporally relevant distribution.

Cell-Cycle Reentry and Cell Death in Transgenic Mice Expressing Nonmutant Human Tau Isoforms

Mutations in the microtubule-associated protein tau gene have been linked to neurofibrillary tangle (NFT) formation in several neurodegenerative diseases known as tauopathies; however, no tau mutations occur in Alzheimers disease, although this disease is also characterized by NFT formation and cell death. Importantly, the mechanism of tau-mediated neuronal death remains elusive. Aged mice expressing nonmutant human tau in the absence of mouse tau (htau mice) developed NFTs and extensive cell death. The mechanism of neuron death was investigated in htau mice, and surprisingly, the presence of tau filaments did not correlate directly with death within individual cells, suggesting that cell death can occur independently of NFT formation. Our observations show that the mechanism of neurodegeneration involved reexpression of cell-cycle proteins and DNA synthesis, indicating that nonmutant tau pathology and neurodegeneration may be linked via abnormal, incomplete cell-cycle reentry.

Cryptococcus neoformans is a facultative intracellular pathogen in murine pulmonary infection.

ABSTRACT To produce chronic infection, microbial pathogens must escape host immune defenses. Infection with the human pathogenic fungusCryptococcus neoformans is typically chronic. To understand the mechanism by which C. neoformans survives in tissue after the infection of immunocompetent hosts, we systematically studied the course of pulmonary infection in mice by electron microscopy. The macrophage was the primary phagocytic cell at all times of infection, but neutrophils also ingested yeast. Alveolar macrophages rapidly internalized yeast cells after intratracheal infection, and intracellular yeast cells were noted at all times of infection from 2 h through 28 days. However, the proportion of yeast cells in the intracellular and extracellular spaces varied with the time of infection. Early in infection, yeast cells were found predominantly in the intracellular compartment. A shift toward extracellular predominance occurred by 24 h that was accompanied by macrophage cytotoxicity and disruption. Later in infection, intracellular persistence in vivo was associated with replication, residence in a membrane-bound phagosome, polysaccharide accumulation inside cells, and cytotoxicity to macrophages, despite phagolysosomal fusion. Many phagocytic vacuoles with intracellular yeast had discontinuous membranes. Macrophage infection resulted in cells with a distinctive appearance characterized by large numbers of vacuoles filled with polysaccharide antigen. Similar results were observed in vitro using a macrophage-like cell line. Our results show that C. neoformans is a facultative intracellular pathogen in vivo. Furthermore, our observations suggest that C. neoformansoccupies a unique niche among the intracellular pathogens whereby survival in phagocytic cells is accompanied by intracellular polysaccharide production.

Conformational change as one of the earliest alterations of tau in Alzheimer's disease

Paired helical filaments (PHFs) found in Alzheimers disease (AD) are mainly comprised of an abnormal form of tau (PHF-tau) that has undergone several post-translational modifications. Previous studies have shown that the monoclonal antibody MCI identifies a distinct conformation of tau in AD. We have assessed the temporal and spatial occurrence of the tau conformation recognized by MC1, and found its appearance in hippocampal neurons vulnerable to neurofibrillary tangle (NFT) formation in Braak Stage I and II cases. Electron microscopy has clearly demonstrated that this conformation precedes the formation of PHF. MC1 immunoaffinity chromatography also has identified a nonfilamentous, soluble pool of this abnormal tau. ELISA and immunoblotting have shown that this material is indistinguishable from that found in NFTs. This soluble component has the ability to self-assemble into PHFs in a concentration-dependent manner. Because the conformational change recognized by MCI appears before the assembly of and is found in PHF, but is not present in the normal brain, we suggest that the formation of the MCI epitope is one of the earliest pathological alterations of tau in AD.

Hippocampal degeneration differentiates diffuse Lewy body disease (DLBD) from Alzheimer's disease Light and electron microscopic immunocytochemistry of CA2–3 neurites specific to DLBD

Immunocytochemistry with antibodies to ubiquitin is currently the most sensitive method for detecting cortical Lewy bodies, which are a sine qua non for the diagnosis of diffuse Lewy body disease (DLBD), an increasingly recognized form of primary degenerative dementia. In the systematic application of ubiquitin immunocytochemistry to sections of hippocampus from control subjects and patients with a wide spectrum of neurodegenerative diseases, we noted the frequent occurrence of ubiquitin-immunoreactive neurites in the CA2–3 region in DLBD. The nature of these neurites was investigated with immunocytochemistry in DLBD, Alzheimers disease (AD), normal elderly subjects, and Parkinsons disease (PD). Although the number of neurites varied from case to case, they were virtually always detected in DLBD but not in normal, AD, or PD brains. Double immunolabeling studies with anti-ubiquitin demonstrated a small fraction of double-stained neurites with antibodies to neurofilament or Alz-50, but no double staining with an antibody to Alzheimer neurofibrillary tangles. These results are different from those for neurites in AD, which are rarely seen in CA2–3 and which are immunoreactive with all these antibodies. Neuritic degeneration in the CA2–3 region of the hippocampus appears to be a specific histopathologic feature of DLBD.

Diffuse lewy body disease: light and electron microscopic immunocytochemistry of senile plaques

SummaryThe nature of senile plaques (SP) in 27 cases of diffuse Lewy body disease (LBD) was investigated using immunocytochemistry and antibodies to beta amyloid protein synthetic peptides (BetaSP), ubiquitin (UBQ), paired helical filaments (PHF; Ab39) and a 68-kDa protein in Alzheimer brains (Alz50). Lewy bodies were present in widespread areas of the neocortex of all cases and were more easily detected with ubiquitin immunocytochemistry than with conventional stains. All cases had neocortical SP, but only six cases had neocortical neurofibrillary tangles (NFT). SP were very numerous in most cases and were usually “pale”, “diffuse” or “very primitive” plaques with thioflavin S fluorescent microscopy. SP in diffuse LBD were immunostained with BetaSP. Several cases had extensive amyloid angiopathy that was also immunoreactive with BetaSP. SP in diffuse LBD were characterized by amyloid deposits with few or no neuritic elements that could be detected with thioflavin S, Bielschowskys stain or double staining with BetaSP and Bodians silver stain. They differed from plaques in Alzheimers disease by lack of PHF-type neurites that could be stained with Ab39. In diffuse LBD, SP contained PHF-type neurites only in areas coexistent with NFT. Some SP had round, granular neurites that were immunoreactive with UBQ, but weakly argyrophilic with Bodians stain and nonfluorescent with thioflavin S. Diffuse LBD lacked significant neuritic change in the neuropil that could be detected with UBQ, Ab39 and Alz50. The latter finding is a characteristic feature that distinguishes Alzheimers disease from diffuse LBD.

Mitotic phosphoepitopes precede paired helical filaments in Alzheimer's disease

We have shown previously that the TG-3 and MPM-2 antibodies recognize phosphoepitopes common to mitosis and degenerating neurons of Alzheimers disease(AD) brain. Here, we have evaluated their occurrence in human brain biopsy tissue, and confirm that they are absent in mature neurons of adult brain, but reappear during neurodegeneration in AD. The TG-3 epitope appears ahead of the MPM-2 epitope and is distributed throughout the neuronal soma. Tau is the major TG-3 antigen in AD brain. The initial localization of MPM-2 immunoreactivity in primary dendrites, its robust occurrence in granulovacuolar bodies, and the increased immunoreactivity with 300-350-kDa proteins, suggest MAPI B as a candidate MPM-2 antigen in AD. Production of mitotic phosphepitopes in more than one type of human neurodegenerative lesion implicates mitotic kinases as common mediators of neuronal death. Because mitotic phosphoepitopes appear before paired helical filaments, it is suggested that mitotic kinase activation triggers neurofibrillary tangle formation. Future studies will need to focus on factors influencing mitotic kinase activity, a point with potential for early diagnosis and disease abrogation.

Dynamic changes in the morphology of Cryptococcus neoformans during murine pulmonary infection

The pathogenesis of Cryptococcus neoformans infection has been studied extensively with respect to inflammatory and pathological changes, but very little information is available regarding the morphology of yeast cells during the course of infection. Electron microscopy of Cryptococcus neoformans in murine pulmonary infection revealed increased cell wall thickness with time, but this difference was only partially accounted for by increases in cell diameter. Cell walls of melanized cells were thicker than those of nonmelanized cells 2 h after infection, and the cell wall of yeast became blacker with time, suggesting that melanization contributes to the increased cell wall thickness. Heterogeneous cell populations emerged, with the appearance of giant forms. While for C. neoformans ATCC strain 24067 (serotype D) the full spectrum of cell sizes were observed, for strains H99 (serotype A) and 3501 (serotype D) cells were divisible into two populations, giant and micro forms. In contrast to cellular heterogeneity, the epitope recognized by a protective mAb on the capsular glucuronoxylomannan (GXM) was found at all times of infection. Immunoelectron microscopy using mAbs to GXM demonstrated reactivity with intracellular structures, suggesting that synthesis of capsular polysaccharide occurs, at least in part, in the cytoplasm. In summary, the results indicate that: (i) the infection is dynamic with respect to yeast cell morphology; (ii) giant cell forms arise in tissue during the course of infection; (iii) cell walls blacken and thicken during the course of infection, consistent with melanin synthesis during infection; and (iv) GXM epitopes are found in the capsule, cell wall and cytoplasm, consistent with intracellular polysaccharide synthesis. The results indicate that the population of C. neoformans cells in tissue is in a highly dynamic state, implying that the immune system must confront cells with varying characteristics during the course of infection.

cAMP-Dependent Protein Kinase Phosphorylations on Tau in Alzheimer’s Disease

To elucidate the role cAMP-dependent protein kinase (PKA) phosphorylations on tau play in Alzheimer’s disease, we have generated highly specific monoclonal antibodies, CP-3 and PG-5, which recognize the PKA-dependent phosphorylations of ser214 and ser409 in tau respectively. The present study demonstrates by immunohistochemical analysis, CP-3 and PG-5 immunoreactivity with neurofibrillary pathology in both early and advanced Alzheimer’s disease, but not in normal brain tissue and demonstrates that cAMP-dependent protein kinase phosphorylations on tau precede or are coincident with the initial appearance of filamentous aggregates of tau. Studies using heat-stable preparations demonstrate that neither site appears to be phosphorylated to any appreciable extent in normal rodent or human brain. Further analysis demonstrates that the β catalytic subunit of PKA (Cβ), the β II regulatory subunit of PKA (RIIβ), and the 79 kDa A-kinase-anchoring-protein (AKAP79), are tightly associated with the neurofibrillary pathology, positioning cAMP-dependent protein kinase to participate directly in the pathological hyperphosphorylation of tau seen in Alzheimer’s disease.