Faculty Bibliography

Previous studies established that the populations of neurons that frequently degenerate in Alzheimer's disease exhibit robust up-regulation of the lysosomal system. In this study, we investigated alterations of the lysosomal system during different forms of experimental injury in rat hippocampal neurons in culture, utilizing a combination of immunocytochemical and biochemical methods. Using triple-label immnocytochemistry for activated caspase-3, fragmentation of DNA and the microtubule-associated protein-2, we characterized treatment paradigms as models of the apoptotic (staurosporine, camptothecin), the oncotic (high-dose menadione, glutamate), and the mixed apoptotic and oncotic (low-dose menadione) pathways of neuronal death. Slowly developing apoptotic or slowly developing mixed apoptotic and oncotic forms of neuronal injury were associated with substantial increases in the number and size of cathepsin D-positive vesicles (late endosomes and mature lysosomes) as determined by immunocytochemistry, and elevated levels of cathepsin D by western blotting. In agreement with our previous findings in Alzheimer's disease, where lysosomal system activation was not restricted to overtly degenerating neurons, up-regulation of this system was also detected quite early during the course of experimental neuronal injury, preceding the development of dystrophic neurites, nuclear segmentation or fragmentation of DNA. These findings implicate lysosomal system activation, both in Alzheimer's disease and in experimental models of neuronal injury, as an important event associated with early stages of neurodegeneration

A pathway to Alzheimer's disease (AD) relevant to sporadic AD pathogenesis is described that involves the early and progressive activation of proteolytic systems including, but not limited to, the calpain-calpastatin and endosomal-lysosomal systems. Activation of these proteolytic systems is initiated by normal brain aging and is propelled by the genetic and environmental factors known to increase AD risk. Recent studies show how cathepsins and calpains, acting directly or indirectly through other proteolytic pathways and cellular signaling cascades, may promote beta-amyloidogenesis, neurofibrillary pathology, as well as mediate neurodegeneration in AD

The goal of this study was to investigate the presence of DNA damage in Alzheimer's disease (AD) utilizing independent assays for three different types of DNA strand breaks. Sections from hippocampi of AD brains, brains with Alzheimer neurofibrillary changes (Ch) from non-demented individuals, and controls (C) were labeled with (1) the TUNEL assay to identify blunt-ended and 3' protruding termini of breaks in double-stranded DNA, (2) the Klenow assay to detect single-stranded and double-stranded breaks with protruding 5' termini, and (3) the Apostain assay which utilizes a monoclonal antibody to single-stranded DNA and is based on the decreased stability of apoptotic DNA to thermal denaturation caused by DNA breaks. The highest incidence of nuclei positive for either molecular form of DNA strand breaks was detected in AD, followed by Ch, and controls (C). In either AD and Ch, the incidence of TUNEL- or Klenow-positive nuclei did not differ significantly, but was higher than the incidence of Apostain-positive nuclei. With all three assays, the highest incidence of positive nuclei was in the molecular layer of CA1. In the majority of nuclei positive for either the Klenow or the Apostain assay, the product of the labeling reaction was localized either to the periphery of the nucleus or to distinct clumps of chromatin (or both). With the TUNEL assay, the majority of positive nuclei were diffusely labeled. In both AD and Ch, the individual positive nuclei were labeled with both the Klenow and the TUNEL assays. The results indicate high incidence of nuclei with either double-stranded or single-stranded DNA breaks in AD, which, for the forms detectable with the Klenow or TUNEL assays, were colocalized

Presenilins 1 (PS1) and 2 (PS2) are multispanning transmembrane proteins associated with familial Alzheimer disease (FAD). They are developmentally regulated, being expressed at highest levels during neuronal differentiation and are sustained at a lower level throughout life. We investigated the distribution and metabolism of endogenous murine PS1 as well as human wild-type (wtPS1) and the familial AD Met146Leu (M146L) mutant presenilins in dissociated cultures of hippocampal neurons derived from control and transgenic mice. We found that the PS1 endoproteolytic fragments and, to a lesser extent, the full-length protein, were expressed as early as day 3 post-plating. Both species increased until the cells were fully differentiated at day 12. Confocal microscopy revealed that presenilin is present in the Golgi and endoplasmic reticulum and, as in punctate, vesicle-like structures within developing neurites and growth cones. Using a human-specific PS1 antibody, we were able to independently examine the distribution of the transgenic protein which, although similar to the endogenous, showed some unique qualities. These included (i) some heterogeneity in the proteolytic fragments of human PS1; (ii) significantly reduced levels of full-length human PS1, possibly as a result of preferential processing; and (iii) a more discrete intracellular distribution of human PS1. Colocalization with organelle-specific proteins revealed that PS1 was located in a diffuse staining pattern in the MAP2-positive dendrites and in a punctate manner in GAP43-positive axons. PS1 showed considerable overlap with GAP43, particularly at the growth cones. Similar patterns of PS1 distribution were detected in cultures derived from transgenic animals expressing human wild-type or mutant presenilins. The studies demonstrate that mutant presenilins are not grossly different in their processing or distribution within cultured neurons, which may represent more physiological models as compared to transfection systems. Our data also suggest that the molecular pathology associated with PS1 mutations results from subtle alterations in presenilin function, which can be further investigated using these transgenic neuronal cell culture models

BACKGROUND: Alzheimer disease (AD) neuropathology is present in Down syndrome (DS) after age 35, but dementia onset varies from ages 40 to 70 years. Because of small sample sizes and nonuniform determination of dementia, previous studies produced differing results on the influence of APOE subtypes on AD in DS. OBJECTIVE: To determine the influence of the APOE genotype and gender on development of AD in adults with DS to ascertain similarities with AD in the general population. METHODS: A total of 100 adults with DS (ages 35 to 79 years), almost all of whom were longitudinally assessed by neurologists, underwent APOE genotyping. Dementia onset was determined using criteria applied from the Tenth International Classification of Mental and Behavioral Disorders. This cohort contains the largest number of DS subjects with dementia (n = 57) in a single study, thus increasing reliability of the results. RESULTS: The epsilon2 allele frequency was 4% in those with dementia versus 13% in those without dementia (p = 0.03); epsilon4 allele frequency was 18% in those with dementia versus 13% in those without dementia (p = 0.45). Using APOE-epsilon3/3 as the reference group, the risk ratio for the development of AD at any given time was 0.34 for the APOE-epsilon2/3 group (p = 0.04) and 1.44 for the APOE-epsilon(3/4,4/4) group (p = 0.25). Women were 1.77 times as likely to dement as men at any given point in time (p = 0.04). CONCLUSIONS: The epsilon2 allele confers a protective effect, and women with DS have an increased risk for AD, as in the general population. In this sample, epsilon4 does not confer a significantly increased risk for AD in DS

High concentrations of glutamate, the major excitatory neurotransmitter in the mammalian brain, lead to intracellular calcium overload resulting in excitotoxic damage and death of neurons. Since protein kinase C (PKC) is involved in neuronal degeneration resulting from cerebral ischemia and from glutamate excitotoxicity, we investigated the effect of glutamate on changes in the cellular distribution of various PKC isoforms in cultured hippocampal neurons in comparison with the effects elicited by the PKC activator phorbol ester. Out of the expressed PKC isoforms alpha, gamma,varepsilon,zeta and lambda only the conventional isoforms PKC alpha and gamma responded to glutamate. Using subcellular fractionation and Western blotting with isoform-specific antibodies and immunocytochemical localization with confocal laser scanning microscopy, we observed that phorbol ester and glutamate have different effects on PKC isoform redistribution: Whereas phorbol ester resulted in translocation of PKC alpha and PKC gamma toward a membrane fraction, the glutamate-mediated rise in intracellular calcium concentration induced a translocation mainly toward a detergent-insoluble, cytoskeletal fraction. Immunocytochemical analysis revealed an isoform-specific translocation following glutamate treatment: PKC gamma was translocated mainly to cytoplasmic, organelle-like structures, whereas PKC alpha redistributed to the plasma membrane and into the cell nucleus. The latter result is of special interest, as it indicates that nuclear PKC may play a role in processes of excitotoxic cell damage

We have shown previously that phosphate groups on the amino-terminal head domain region of the middle molecular mass subunit of neurofilament proteins (NF-M) are added by second messenger-dependent protein kinases. Here, we have identified Ser23 as a specific protein kinase A phosphorylation site on the native NF-M subunit and on two synthetic peptides, S1 (14RRVPTETRSSF24) and S2 (21RSSFSRVSGSPSSGFRSQSWS41), localized within the amino-terminal head domain region. Ser23 was identified as a phosphorylation site on the 32P-labeled alpha-chymotryptic peptide that carried >80% of the 32P-phosphates incorporated into the NF-M subunit by protein kinase A. The synthetic peptides S1 and S2 were phosphorylated 18 and two times more efficiently by protein kinase A than protein kinase C, respectively. Neither of the peptides was phosphorylated by casein kinase II. The sequence analyses of the chemically modified phosphorylated serine residues showed that Ser23 was the major site of phosphorylation for protein kinase A on both S1 and S2 peptides. Low levels of incorporation of 32P-phosphates into Ser22, Ser28, and Ser32 by protein kinase A were also observed. Protein kinase C incorporated 32P-phosphates into Ser22, Ser23, Ser25, Ser28, Ser32, and a threonine residue, but none of these sites could be assigned as a major site of phosphorylation. Analyses of the phosphorylated synthetic peptides by liquid chromatography-tandem mass spectrometry also showed that protein kinase A phosphorylated only one site on peptide S1 and that ions with up to four phosphates were detected on peptide S2. Analysis of the data from the tandem ion trap mass spectrometry by using the computer program PEPSEARCH did not unequivocally identify the specific sites of phosphorylation on these serine-rich peptides. Our data suggest that Ser23 is a major protein kinase A-specific phosphorylation site on the amino-terminal head region of the NF-M subunit. Phosphorylation of Ser23 on the NF-M subunit by protein kinase A may play a regulatory role in neurofilament assembly and/or the organization of neurofilaments in the axon