In Alzheimer's disease brains, more than 90% of pyramidal neurons in lamina V and 70% in lamina III displayed 2- to 5-fold elevated levels of cathepsin D (Cat D) mRNA by in situ hybridization compared with neurologically normal controls. Most of these cells appeared histologically normal. The less vulnerable nonpyramidal neuron population in lamina IV had relatively normal message levels. Neuronal populations expressing more Cat D mRNA also displayed quantitatively increased Cat D immunoreactive protein. Cat D mRNA expression was only moderately increased in astrocytes. Degenerating neurons exhibited intense immunoreactivity but lowered Cat D mRNA levels. The upregulation of Cat D synthesis and accumulation of hydrolase-laden lysosomes indicate an early activation of the endosomal-lysosomal system in vulnerable neuronal populations, possibly reflecting early regenerative or repair processes. These abnormalities also represent a basis for altered regulation of amyloid precursor protein processing.

We developed a new approach to study single- and double-stranded DNA breaks during chronic, moderate excitotoxicity resulting from the inhibition of the glutamate transporter in cerebellar granule cell primary cultures. A 24 hr treatment of 2-week-old cultures with L-alpha-amino adipate (LAA), an inhibitor of the cerebellar glutamate uptake transporter, caused a gradual extracellular accumulation of endogenous glutamate that induced reversible morphological change of granule neurons but no neuronal cell death despite sustained, but moderate, elevations of the free intracellular calcium concentrations. Nick translation experiments on isolated nuclei or cells from cerebellar cultures chronically exposed to LAA revealed increased radioactive nucleotide incorporation indicative of DNA nicking. This LAA effect was dose-dependent and suppressed by NMDA receptor antagonists. Cultures treated for 24 hr with LAA and subjected to in situ nick translation showed an intense nuclear labeling of neurons but not glia, which could be abolished by MK801. A similar labeling was also observed in altered nuclei of granule neurons acutely exposed to high glutamate concentrations or undergoing an apoptotic cell death. Although the TUNEL labeling method detected no DNA double-strand breaks in LAA-treated cerebellar cultures, it displayed clear evidence of DNA damage during acute glutamate excitotoxicity or during apoptosis. However, Southern blot analysis of nuclear DNA revealed a DNA laddering only in apoptotic cell death. Our results demonstrate that DNA damage, characterized by DNA single-strand breaks, is an early event in chronic, moderate excitotoxicity. This type of DNA degradation, which appears before any nuclear morphological changes, is distinct from the massive DNA single- and/or double-strand damages observed during acute glutamate excitotoxicity or apoptosis.

Programmed cell death, or apoptosis, has been implicated in Alzheimer's disease (AD). DNA damage was assessed in primary cortical neurons infected with herpes simplex virus (HSV) vectors expressing the familial Alzheimer's disease (FAD) gene presenilin-1 (PS-1) or an FAD mutant of this gene, A246E. After infection, immunoreactivity for PS-1 was shown to be enhanced in infected cells. The infected cells exhibited no cytotoxicity, as evaluated by trypan blue exclusion and mitochondrial function assays. Quantitative analysis of cells that were immunohistochemically labeled using a Klenow DNA fragmentation assay or the TUNEL method revealed no enhancement of apoptosis in PS-1-infected cells. This result was confirmed using assays for chromatin condensation and for DNA fragmentation. Expression of PS-1 protected against induction of apoptosis in the cortical neurons by etoposide or staurosporine. The specificity of this phenotype was demonstrated by the fact that cortical cultures infected with recombinant HSV vectors expressing the amyloid precursor protein (APP-695) showed, in contrast, a significant increase in the number of apoptotic cells and an increase in DNA fragmentation for all parameters tested. Our results indicate that overexpression of wild-type or A246E mutant PS-1 does not enhance apoptosis in postmitotic cortical cells and suggest that the previously reported enhancement of apoptosis by presenilins may be dependent on cell type.

In a previous study of murine delayed-type hypersensitivity (DTH) 1 we suggested that the release of serotonin (5-hydroxytryptamine) (5-HT) by local tissue mast cells was required for the elicitation of these responses (1). We noted that DTH was preferentially elicited in those cutaneous sites that were especially rich in 5-HTcontaining mast cells, and that administration of the 5-HT-depleting agent, reserpine, abolished the ability to elicit DTH. Because the effect of reserpine could be prevented by administration of a monoamine oxidase (MAO) inhibitor, the attribution of the action of reserpine to depletion of a monoamine (such as 5-HT) was confirmed (1). Subsequent studies, which have employed two additional and independent pharmacological maneuvers (5-HT tachyphylaxis of the vasculature [2] and the use of 5-HT antagonists2), have been consistent with our hypothesis that in murine DTH, specifically sensitized T cells interact with tissue mast ceils leading to the release of 5-HT, which acts on the local endothelium allowing the emigration of bone marrow-derived leukocytes from the intravascular to the extravascular space (3). The current study was undertaken to test our hypothesis by determining if we could obtain direct evidence for activation of mast cells to release 5-HT during murine DTH. Immunization and Skin Testing (Challenge). BDFI mice (The Jackson Laboratory, Bar Harbor, Maine) received an optimal immunization for the eticitation of DTH (0.2 ml of 0.01% sheep erythrocytes [SRBC]; [Colorado Serum Labs, Boulder, Colo.] diluted in sterile saline) (4). 4 d later, the animals were tested for DTH by injecting 0.03 ml of 20% SRBC into the ventral surface of a rear footpad. The thickness of the footpad was measured with a micrometer before and after challenge with antigen, and the percent change calculated (5). Immunized controls were challenged by footpad injection of 0.03 ml saline. Nonimmunized controls received an intravenous injection of saline on day 0 and were tested by footpad injection of 20% SRBC on day 4. In some experiments, contact hypersensitivity reactions were elicited in the skin of the * Materials and Methods