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Intraneuronal accumulation of ubiquitin conjugates in inclusion bodies and neurofibrillary tangles is a pathological feature of neurodegenerative disorders such as Alzheimer's disease and Down's syndrome and of normal aging of the brain. Amyloid beta-protein (A beta) and its precursor are found in neurofibrillary tangle-containing neurons. A beta is the major component of extracellular plaques. We showed that A beta acts as an inhibitor of the ubiquitin-dependent protein degradation in vitro. We examined the effect of A beta on the steps of this proteolytic pathway that contribute to the level of ubiquitin conjugates in the cell. Neither conjugate formation nor conjugate deubiquitination was affected by the presence of A beta. However, A beta significantly reduced the rate of conjugate degradation. Our results indicate that A beta interacts with the proteolytic step of the ubiquitin degradative pathway. Since this step is performed by the 26 S proteasome, the effect of A beta on the catalytic core of this proteolytic complex, the 20 S proteasome, was determined. We found that A beta selectively inhibits the chymotrypsin-like activity of the 20 S proteasome. Under pathological conditions in the affected neuron, A beta could interfere with ubiquitin-dependent degradation by inhibiting the 26 S proteasome activity. This finding may explain the origin of the accumulation of ubiquitin conjugates.

Apolipoprotein E, type 4 allele (APOE 4), is associated with late-onset familial Alzheimer's disease (AD). There is high avidity and specific binding of amyloid β-peptide with the protein ApoE. To test the hypothesis that late-onset familial AD may represent the clustering of sporadic AD in families large enough to be studied, we extended the analyses of APOE alleles to several series of sporadic AD patients. APOE 4 is significantly associated with a series of probable sporadic AD patients (0.36 ± 0.042, AD, versus 0.16 ± 0.027, controls [allele frequency estimate ± standard error], p0.00031). Spouse controls did not differ from CEPH grandparent controls from the Centre d'Etude du Polymorphisme Humain (CEPH) or from literature controls. A large combined series of autopsy-documented sporadic AD patients also demonstrated highly significant association with the APOE 4 allele (0.40 ± 0.026, p≤ 0.00001). These data support the involvement of ApoE 4 in the pathogenesis of late-onset familial and sporadic AD. ApoE isoforms may play an important role in the metabolism of β-peptide, and APOE 4 may operate as a susceptibility gene (risk factor) for the clinical expression of AD.

We have analyzed the modulation of amyloid beta-protein precursor (APP) gene expression in human umbilical vein endothelial cells (HUVEC). The level of the APP mRNA transcripts increased as HUVEC reached confluency. In confluent culture the half-life of the APP mRNA was 4 hr. Treatment of the cells with human-recombinant interleukin 1 (IL-1), phorbol 12-myristate 13-acetate, or heparin-binding growth factor 1 enhanced the expression of APP gene in these cells, but calcium ionophore A23187 and dexamethasone did not. The protein kinase C inhibitor 1-(isoquinolinsulfonyl)-2-methylpiperazine (H7) inhibited IL-1-mediated increase of the level of APP transcripts. To map IL-1-responsive elements of the APP promoter, truncated portions of the APP promoter were fused to the human growth hormone reporter gene. The recombinant plasmids were transfected into mouse neuroblastoma cells, and the cell medium was assayed for the human growth hormone. A 180-base-pair region of the APP promoter located between position -485 and -305 upstream from the transcription start site was necessary for IL-1-mediated induction of the reporter gene. This region contains the upstream transcription factor AP-1 binding site. These results suggest that IL-1 upregulates APP gene expression in HUVEC through a pathway mediated by protein kinase C, utilizing the upstream AP-1 binding site of the APP promoter.

Neurodegenerative disorders of aging are characterized by the intraneuronal accumulation of ubiquitin conjugates into tangles and inclusions. Ubiquitin conjugates are degraded by cellular particles known as proteasomes. We have previously shown that amyloid beta protein (Abeta) inhibits proteasomal activity and thereby blocks ubiquitin conjugate degradation. In the present studies, we found that Abeta binds the 20 S proteasome and forms a proteasome-Abeta complex. The complex was detected by Western blot with anti-Abeta antibodies. Using a 1.4 nm Nanogold-labeled Abeta, we visualized proteasome-Abeta complexes by scanning transmission electron microscopy (STEM). Analysis of the side-on oriented proteasome-Abeta complexes revealed a single gold particle, corresponding to one gold-labeled Abeta, in the middle portion of the proteasome. On end-on views of proteasome-Abeta complexes, gold was detected within the area delimited by the proteasome circular projection. Both STEM views are consistent with Abeta localization inside the proteasome along the peptide channel. Direct interaction of Abeta with the inner catalytic compartment of the proteasome may explain the generation of ubiquitin-containing lesions in Alzheimer's disease and other neurodegenerative disorders. In addition, detection of Nanogold-labeled peptide inside the 20 S eukaryotic proteasome suggests that conformational constraints for protein degradation in eukaryotic proteasomes are different from those in archaebacteria proteasomes.

Neurofibrillary tangles (NFT) and neuritic plaques (NP), two neuropathological markers of Alzheimer disease, may both contain peptide fragments derived from the human amyloid beta protein. However, the nature of the relationship between NFT and NP and the source of the amyloid beta proteins found in each have remained unclear. We used in situ hybridization techniques to map the anatomical distribution of precursor amyloid-beta-protein mRNA in the neocortex of brains from three subjects with no known neurologic disease and from five patients with Alzheimer disease. In brains from control subjects, positively hybridizing neurons were present in cortical regions and layers that contain a high density of neuropathological markers in Alzheimer disease, as well as in those loci that contain NP but few NFT. Quantitative analyses of in situ hybridization patterns within layers III and V of the superior frontal cortex revealed that the presence of high numbers of NFT in Alzheimer-diseased brains was associated with a decrease in the number of positively hybridizing neurons compared to controls and Alzheimer-diseased brains with few NFT. In contrast, no correlation was found between the densities of NP and neurons containing precursor amyloid-beta-protein mRNA transcripts. These findings suggest that the expression of precursor amyloid-beta-protein mRNA may be a necessary but is clearly not a sufficient prerequisite for NFT formation. In addition, these results may indicate that the amyloid beta protein, present in NP in a given region or layer of cortex, is not derived from the resident neuronal cell bodies that express the mRNA for the precursor protein.

We have mapped the neuroanatomical distribution of amyloid-beta-protein mRNA within neuronal subpopulations of the hippocampal formation in the cynomolgus monkey (Macaca fascicularis), normal aged human, and patients with Alzheimer disease. Amyloid-beta-protein mRNA appears to be expressed in all hippocampal neurons, but at different levels of abundance. In the central nervous system of monkey and normal aged human, image analysis shows that neurons of the dentate gyrus and cornu Ammonis fields contain a 2.5-times-greater hybridization signal than is present in neurons of the subiculum and entorhinal cortex. In contrast, in the Alzheimer disease hippocampal formation, the levels of amyloid-beta-protein mRNA in the cornu Ammonis field 3 and parasubiculum are equivalent. These findings suggest that within certain neuronal subpopulations cell type-specific regulation of amyloid-beta-protein gene expression may be altered in Alzheimer disease.

Neurofilaments (NFs) are the intermediate filaments specific to nervous tissue. They are probably essential to the tensile strength of the neuron, as well as to transport of molecules and organelles within the axon. Three peptides with apparent molecular masses of approximately 68 (NF-L), 145 (NF-M), and 200 (NF-H) kDa appear to be the major components of NF. The expression of these peptides is specific to nervous tissue and is developmentally regulated. Recently, complete cDNAs encoding NF-L and NF-M, and partial cDNAs encoding NF-H, have been described. To better understand the normal and pathophysiology of NFs we chose to clone the cDNA encoding the rat NF-H peptide. Using monoclonal antibodies that recognized NF-H, we screened a rat brain lambda gt11 library and identified a clone that contained a 2100-nucleotide cDNA insert representing the carboxyl-terminal portion of the NF-H protein. Anti-fusion protein antibodies recognized the NF-H peptide on immunoblots and stained fibrillar structures only in neurons. The cDNA recognized a 4500-nucleotide polyadenylated mRNA that was present only in nervous tissue and a 3500-nucleotide mRNA in adrenal. Brain NF-H mRNA levels were tightly developmentally regulated and paralleled the levels of NF-H peptide on immunoblots. Nuclear runoff studies showed that the 20-fold developmental increase in the NF-H message was due only in part to a 4-fold increase in its transcription rate. Levels of NF-H mRNA varied 20-fold among brain regions, with highest levels in pons/medulla, spinal cord, and cerebellum, and lowest levels in olfactory bulb and hypothalamus. Transcription studies revealed only a 2-fold difference in the transcription rates among these brain regions. Based on these results, we infer that half of the developmental increase and most of the interregional variation in the levels of the NF-H mRNA are mediated through message stabilization. Sequence information revealed that the carboxyl-terminal region of the NF-H peptide contained a unique serine-, proline-, alanine-, glutamic acid-, and lysine-rich repeat. The serine residues are likely sites of phosphorylation in the mature peptide. Genomic blots revealed a single copy of the gene in the rat genome and two copies in the human genome. In situ hybridizations performed on human chromosomes mapped the NF-H gene to chromosomes 1 and 22. Whether one copy is a pseudogene remains to be determined.