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Many neurons in Alzheimer's disease (AD) exhibit terminal deoxynucleotidyl transferase (TdT) labeling for DNA strand breaks with a distribution suggestive of apoptosis. We have shown previously that immunoreactivity for c-Jun is elevated in AD and found in association with neuronal pathology. In addition, cultured neurons undergoing beta-amyloid-mediated apoptosis exhibit a selective and prolonged induction of c-Jun. Consequently, we conducted double-labeling experiments to examine whether c-Jun is associated with DNA strand breaks in AD tissue; we observed a strong colocalization between these markers. As would be predicted based on the distribution of AD pathology, we also found that TdT labeling was prominent in the entorhinal cortex, but absent or at very low levels in cerebellum. Furthermore, we confirmed that postmortem delay (PMD) does not affect TdT labeling within the limits used for tissue used in this study. However, in contrast to previous studies, we report an increase in TdT labeling with more extended PMDs. Finally, gel electrophoresis of genomic DNA isolated from AD and control cases failed to reveal evidence for either an apoptotic or a necrotic mechanism of cell death in AD, possibly because of a low number of cells actually undergoing cell death at any given time. Our findings support the hypothesis that DNA damage labeled using TdT reflects neuronal vulnerability and cell loss associated with AD pathology, and that at least a portion of the cells labeled with this technique is undergoing apoptosis. Furthermore, in agreement with in vitro findings, these results suggest a relationship between the expression of c-Jun and neuronal risk and/or cell death in AD.

Thrombin is a multifunctional serine protease that is rapidly produced from prothrombin at sites of tissue injury and catalyzes the final steps in blood coagulation. Thrombin also regulates gene expression and process outgrowth in neurons and astrocytes and stimulates proliferation of astrocytes. Since thrombin is produced immediately upon breakdown of the blood-brain barrier we examined its effects on astrocytes and neurons cultured under conditions which resemble those found in vivo following cerebrovascular injury. These studies showed that thrombin markedly protected rat primary astrocytes from cell death induced by hypoglycemia or oxidative stress. Thrombin also protected rat primary hippocampal neurons from cell death produced by hypoglycemia or growth supplement deprivation. Synthetic peptides which directly activate the thrombin receptor also protected astrocytes and neurons from these environmental insults, demonstrating that the thrombin effects were mediated through the thrombin receptor. In contrast to these results with stressed cells, high concentrations of thrombin killed both astrocytes and neurons cultured under normal conditions. All of the effects of thrombin on astrocytes and neurons were blocked by the brain thrombin inhibitor, protease nexin-1 (PN-1). This shows that the effects required the proteolytic activity of thrombin and is consistent with the known proteolytic mechanism by which thrombin activates its receptor. These results indicate that thrombin and PN-1 may regulate the viability of both astrocytes and neurons in early moments following trauma to the CNS or other conditions that alter the blood-brain barrier.

Basic fibroblast growth factor (bFGF) is a potent trophic factor for neurons and astrocytes and recently has been implicated in the pathology of Alzheimer's disease. In order to better understand the role of bFGF in normal brain function and during pathology, we have analyzed its anatomical distribution and its response to injury in the CNS. Double-staining immunohistochemistry showed that bFGF immunoreactivity was localized in astrocytes, in select neuronal populations, and occasionally in microglial cells throughout the normal rat brain. Neuronal populations that showed bFGF immunoreactivity included septohippocampal nucleus, cingulate cortex, subfield CA2 of the hippocampus, cerebellar Purkinje cells, cerebellar deep nuclei, facial nerve nucleus, and the motor and spinal subdivisions of the trigeminal nucleus and facial nerve nucleus. The pattern of bFGF immunoreactivity in the hippocampus was examined following entorhinal cortex lesion, or fimbria-fornix transection. After entorhinal cortex lesion, bFGF immunoreactivity increased in the outer molecular layer of the dentate gyrus ipsilateral to the lesion. The lesion effect on bFGF immunoreactivity was expressed as an increase in the number of bFGF astrocytes, as an increase in the intensity of bFGF immunoreactivity within astrocytes, and as an increase of bFGF immunoreactivity in the surrounding extracellular matrix, relative to the contralateral side. The time course and pattern of reorganization paralleled the sprouting of septal cholinergic terminals in response to the same type of lesion, suggesting that bFGF may play an important role in lesion-induced plasticity. After transection of the fimbria-fornix, chronic infusion of bFGF appeared to preserve NGF receptors on neurons within the medial septal complex and, as previously reported, prevent the death of medial septal neurons. Therefore, it appears that bFGF infusion, which has been shown to increase the synthesis of NGF by astrocytes (Yoshida and Gage, 1991), also helps enable neurons to respond to NGF. This suggests that after injury bFGF may participate in a cascade of neurotrophic events, directly and indirectly facilitating neuronal repair and/or promoting neuronal survival.

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