Joseph H. Su

Immunohistochemical evidence for apoptosis in Alzheimer's disease

Recently, in vitro studies conducted in our laboratory and others have suggested that apoptosis may have a role in the neuronal cell death associated with Alzheimers disease (AD). To evaluate this hypothesis, the hippocampi and entorhinal cortices of AD, aged control, and surgical biopsy tissue were examined using the ApopTag system for the detection of DNA fragmentation and DNA strains to reveal nuclear morphology. Numerous neuronal nuclei displaying distinct morphological characteristics of apoptosis were present within tangle-bearing neurons as well as non-tangle-bearing neurons in AD brain, whereas few or no such nuclei were detected in control brain. Our in vivo results support the hypothesis that apoptosis may be one mechanism leading neuronal cell death in AD.

Mechanisms of Neuronal Death in Alzheimer's Disease

Recent data in cell culture has shown that brain neurons are particularly vulnerable to degeneration by apoptosis. Further the inducers that activate the program (e.g. β‐amyloid, oxidatative damage, low energy metabolism) correspond to conditions present in the Alzheimers disease (AD) brain. This suggests the possibility that apoptosis may be one of the mechanisms contributing to neuronal loss in this disease. Indeed, some neurons in vulnerable regions of the AD brain show evidence of DNA damage, nuclear apoptotic bodies, chromatin condensation, and the induction of select genes characteristic of apoptosis in cell culture and animal models. This suggests the existence of apoptosis in the AD brain, a hypothesis also consistent with evolving research in one of the regulatory functions of the presenilin genes. On the other hand, DNA damage is present in the majority of neurons in vulnerable regions in early and mild cases. In most tissues, cells in fully activated apoptosis degenerate and are removed within hours to days and thus it seems all DNA damage is unlikely to signify terminal apoptosis. The presence of extensive DNA damage suggests an acceleration of damage, faulty repair process, loss of protective mechanisms, or an activation and arrest of aspects of the apoptotic program. DNA damage is unlikely to be an artifact of postmortem delay or agonal state. The existence of protective mechanisms for neurons may exist as these cells are nondividing and essential. In this context it is interesting that Bcl‐2 is upregulated in most neurons with DNA damage. Further, at least one DNA repair enzyme is also upregulated. Thus it appears as if neurons are in a struggle between degeneration and repair. As research advances it is critical to reduce the stimuli that cause the neuronal damage and discover the key intervention points to assist neurons in the repair processes.

β-amyloid accumulation in aged canine brain : a model of early plaque formation in Alzheimer's disease

Abstract We characterized eight aged beagles (maintained from birth in a laboratory colony) and one black Labrador using Bielschowskys, thioflavine S, and Congo red staining, and antibodies to the β-amyloid peptide, dystrophic neurites, and other plaque components. All plaques within these canine brains were of the diffuse subtype and were neither thioflavine S- nor Congo red-positive. The majority of plaques in the entorhinal cortex contained numerous neurons within them while plaques in the dentate gyrus did not. β-Amyloid immunoreactivity was also present within select neurons and neuronal processes and was detected as a diffuse linear zone corresponding to the terminal fields of the perforant path. There was no significant correlation between extent of β-amyloid accumulation and neuron number in entorhinal cortex. Neither tau-1, PHF-1, nor SMI-31-immunostaining revealed dystrophic fibers, confirming the classification of these plaques as diffuse. Canine plaques did not appear to contain bFGF- or HS-positive immunostaining. This may explain why neuritic involvement was not detected within these canine plaques. It is possible that the β-amyloid within the canine brain has a unique primary structure or may not be in an assembly state that adversely affects neurons.

Activated caspase-3 expression in Alzheimer’s and aged control brain: correlation with Alzheimer pathology

Several studies have suggested that activated caspase-3 has properties of a cell death executioner protease. In this study, we examined the expression of activated caspase-3 in AD and aged control brains. Activated caspase-3 immunoreactivity was seen in neurons, astrocytes, and blood vessels, was elevated in AD, and exhibited a high degree of colocalization with neurofibrillary tangles and senile plaques. These data suggest that activated caspase-3 may be a factor in functional decline and may have an important role in neuronal cell death and plaque formation in AD brain.

Neuronal DNA damage precedes tangle formation and is associated with up-regulation of nitrotyrosine in Alzheimer's disease brain.

The relationship of neuronal DNA damage to tangle-bearing neurons and nitrotyrosine (NT) expression, a neurochemical marker of oxidative damage mediated by peroxynitrite, was examined in visual cortex of AD patients. Many terminal deoxynucleotidyl transferase (TdT)-positive neurons were detected and the majority (93%) of these TdT-labeled neurons lacked evidence of tangle formation. NT expression was elevated in AD cases and most TdT-labeled nuclei also showed strong NT immunoreactivity. These data suggest the hypothesis that the neurons with DNA damage in the absence of tangle formation may degenerate by tangle-independent mechanisms and that oxidative damage may contribute to such mechanisms in AD.

Correlation between Caspase Activation and Neurofibrillary Tangle Formation in Alzheimer’s Disease

Although evidence suggests that neurofibrillary tangles (NFTs) and neuronal cell loss are prominent features of Alzheimers disease (AD), the relationship between the two remains unknown. In the present study, the relationship between the activation of apoptotic mechanisms and NFT formation in AD was investigated using a caspase-cleavage site-directed antibody to fodrin, an abundant neuronal cytoskeleton protein. This antibody recognized cleavage products of fodrin after digestion by caspase-3, but did not recognize full-length fodrin. In vitro analysis of this fodrin caspase-cleavage product (CCP) antibody demonstrates that it is a specific probe for the detection of apoptotic but not necrotic pathways in cultured neurons. To determine whether caspases cleave fodrin in vivo, tissue sections from controls and AD were immunostained for fodrin (CCPs). Although no staining was observed in control cases, labeling of neurons was observed in the hippocampus of all AD cases, which increased as a function of disease progression. To determine a possible relationship between caspase activation and NFT formation, double-labeling experiments with fodrin CCP and PHF-1 were performed. Co-localization of these markers was observed in many neurons, and quantitative analysis showed that as the extent of NFT formation increased, there was a significant corresponding increase in fodrin CCP immunolabeling (r = 0.84). Taken together, these results provide evidence for the activation of apoptotic mechanisms in neurons in the AD brain and suggest that there is an association between NFT formation and the activation of apoptotic pathways in AD.

Localization of heparan sulfate glycosaminoglycan and proteoglycan core protein in aged brain and Alzheimer's disease.

Two monoclonal antibodies, one which recognizes a glycosaminoglycan epitope present in heparan sulfate glycosaminoglycan and another which recognizes the core protein of a basement membrane heparan sulfate proteoglycan, were used to study the distribution and localization of these components in Alzheimers disease and control brain. The cytoplasm of neurons, and occasional neurofibrillary tangles, senile plaques and astrocytes were immunopositive for the heparan sulfate glycosaminoglycan antibody in control brains. In Alzheimers tissue, however, the number and intensity of these elements was more extensive than in control brains. In addition, within the Alzheimers brains studied, the nuclei of select neurons and a small number of microglia were also immunopositive for heparan sulfate glycosaminoglycan in contrast to controls, where nuclei and neuroglia were immuno-negative. Some senile plaques in Alzheimers tissue also contained strong heparan sulfate glycosaminoglycan-positive neurites which were not seen in controls. In Alzheimers tissue, double labeling for heparan sulfate glycosaminoglycans and the beta-amyloid protein in adjacent sections revealed that, in general, heparan sulfate glycosaminoglycan- and beta-amyloid protein-immunopositive plaques were co-localized. Occasionally, however, beta-amyloid-positive plaques were seen without heparan sulfate glycosaminoglycan immunoreactivity and vice versa. Heparan sulfate glycosaminoglycan immunoreactivity and Tau immunoreactivity co-localized in many neurofibrillary tangles; however a small number of heparan sulfate glycosaminoglycan-positive neurofibrillary tangles did not co-localize with Tau-positive neurofibrillary tangles. In contrast, the heparan sulfate proteoglycan antibody immunostained only the walls of blood vessels and a few senile plaques in Alzheimers brains and primarily blood vessels in control brains. Heparan sulfate glycosaminoglycan immunostaining was present within neurons, glia, neurofibrillary tangles and senile plaques in Alzheimers tissue. These results suggest that heparan sulfate-like molecules play an important role in the pathogenesis of the characteristic lesions of Alzheimers disease and could serve as a marker reflecting early pathological changes.

DNA damage and activated caspase-3 expression in neurons and astrocytes: evidence for apoptosis in frontotemporal dementia.

Frontotemporal dementia (FTD) is a neurodegenerative disease which affects mainly the frontal and anterior temporal cortex. It is associated with neuronal loss, gliosis, and microvacuolation of lamina I to III in these brain regions. In previous studies we have described neurons with DNA damage in the absence of tangle formation and suggested this may result in tangle-independent mechanisms of neurodegeneration in the AD brain. In the present study, we sought to examine DNA fragmentation and activated caspase-3 expression in FTD brain where tangle formation is largely absent. The results demonstrate that numerous nuclei were TdT positive in all FTD brains examined. Activated caspase-3 immunoreactivity was detected in both neurons and astrocytes and was elevated in FTD cases as compared to control cases. A subset of activated caspase-3-positive cells were also TdT positive. In addition, the cell bodies of a subset of astrocytes showed enlarged, irregular shapes, and vacuolation and their processes appeared fragmented. These degenerating astrocytes were positive for activated caspase-3 and colocalized with robust TdT-labeled nuclei. These findings suggest that a subset of astrocytes exhibit degeneration and that DNA damage and activated caspase-3 may contribute to neuronal cell death and astrocyte degeneration in the FTD brain. Our results suggest that apoptosis may be a mechanism of neuronal cell death in FTD as well as in AD (228).

Up-regulation of Bcl-2 is associated with neuronal DNA damage in Alzheimer's disease

Cell death and neurofibrillary tangle formation are prominent features of Alzheimers disease (AD). It has been suggested that DNA damage may reflect neuronal vulnerability. In this context, the Ced homologue Bcl-2 is able to repress a number of cell death programs. Recently we found both numerous nuclei exhibiting DNA damage within neurons in the AD brain and increases in Bcl-2 immunoreactivity. In this study, we examined the relationship between Bcl-2 expression and nuclear DNA damage or tangle formation. Nuclei exhibiting DNA damage were associated with an up-regulation of Bcl-2 expression, whereas tangle-bearing neurons were associated with a down-regulation of Bcl-2 expression.

The Induction of the TNFα Death Domain Signaling Pathway in Alzheimer's Disease Brain

The tumor necrosis factor-α death domain pathway contributes to cellular degeneration in a variety of conditions. This study investigates the hypothesis that this death domain pathway is progressively induced in the brain during the progression of Alzheimers disease (AD). AD cases had increased levels of proapoptotic markers including tumor necrosis factor-α (TNFα), TNF receptor type 1 (TNF-R1), TNF receptor–associated death domain (TRADD), and caspase-3, 2- to 10-fold higher (P < .01) than age-matched controls and 1 to 3 times higher than transitional cases. In striking contrast, potentially neuroprotective TNF receptor type 2 (TNF-R2), and Fas-associated death domain-like interleukin-1β–converting enzyme (FLICE) inhibitor protein (FLIP) were decreased in AD as compared with age-matched control cases (P < .01). Overall, there was an elevation in proapoptotic elements, including a 5-fold increase in TNF-R1 and a 12-fold decrease in FLIP in AD brains. These changes may translate to increased degenerative potential because the downstream effector caspase-3 and product of the TNF pathway was also increased in parallel with enhanced TNF proapoptotic conditions. Our findings suggest that the TNF death receptor pathway and caspases are activated in the early stages of neuronal degeneration in AD.