Faculty Bibliography

A central pathological feature of Alzheimer's disease is the profuse deposition of amyloid-beta protein (Abeta) in the brain parenchyma and vessel walls. Abeta also forms deposits in the brains of a variety of mammals, including all aged non-human primates studied to date. The sequence of Abeta in these animals is identical to that in humans. No Abeta deposits have been found in the brains of wild-type rats and mice, suggesting that the three amino acid differences between their Abeta and that of amyloid-bearing mammals impedes the fibrillogenicity of Abeta. Analysis of the primary sequence of the beta-amyloid precursor protein in tree shrews revealed a 98% similarity and 97% identity with the human protein. Furthermore, the predicted amino acid sequence of Abeta in tree shrews is identical to that in humans. However, immunohistochemical analysis failed to reveal beta-amyloid deposits in the neural parenchyma or vasculature of eight aged (7-8 years) tree shrews (Tupaia belangeri). The lack of correlation between the Abeta sequence and amyloid formation suggests that other factors contribute to cerebral amyloid deposition in aged animals

The protein interaction domain of the neuronal protein X11 binds to the YENPTY motif within the cytoplasmic domain of beta-amyloid precursor protein (betaAPP). Amyloid-beta protein (Abeta), the major constituent of the amyloid deposited in brain of Alzheimer's disease patients, is generated by proteolytic processing of betaAPP, which occurs in part following betaAPP internalization. Because the YENPTY motif has a role in the internalization of betaAPP, the effect of X11 binding on betaAPP processing was studied in mouse neuroblastoma N2a, human embryonic kidney 293, monkey kidney COS-1, and human glial U251 cell lines transfected with wild type or mutated betaAPP cDNAs. Secretion of soluble betaAPP via alpha-secretase activity increased significantly in cells transfected with betaAPP variants containing mutations that impair interaction with X11 when compared with cells transfected with wild type cDNA. Cotransfection of betaAPP and X11 caused retention of cellular betaAPP, decreased secretion of sbetaAPPalpha, and decreased Abeta secretion. Thus, betaAPP interaction with the protein interaction domain of X11 stabilizes cellular betaAPP and thereby participates in the regulation of betaAPP processing pathways

Most of the cases of early-onset familial Alzheimer's disease (FAD) are related to missense mutations in the presenilin 1 (PS-1) gene on chromosome 14. Although PS-1 mutations are distributed throughout the entire open reading frame, most mutations are found in transmembrane region II and hydrophilic loop VI encoded by exons 5 and 8, respectively. These two groups of substitutions are associated with an age of onset of 40-43 years for exon 5 and 45-55 years for exon 8, respectively. We have previously described a South American pedigree from Argentina with early-onset FAD (mean age of onset 38.9 +/- 3.9 years) with no mutations in exons 16 and 17 of the beta-protein precursor gene (betaPP770 transcript). Here we report the identification of an A --> T transversion at the first position of codon 146 of PS-1 in these patients. This missense mutation results in a Met --> Leu substitution, as reported for the Italian pedigrees Tor1.1 and FAD4. The significant differences in ages of onset and death among members of generations II-III and IV suggest that other genetic and/or environmental factors may influence disease phenotype in this pedigree

A cystatin C variant with L68Q substitution and a truncation of 10 NH2-terminal residues is the major constituent of the amyloid deposited in the cerebral vasculature of patients with the Icelandic form of hereditary cerebral hemorrhage with amyloidosis (HCHWA-I). Variant and wild type cystatin C production, processing, secretion, and clearance were studied in human cell lines stably overexpressing the cystatin C genes. Immunoblot and mass spectrometry analyses demonstrated monomeric cystatin C in cell homogenates and culture media. While cystatin C formed concentration-dependent dimers, the HCHWA-I variant dimerized at lower concentrations than the wild type protein. Amino-terminal sequence analysis revealed that the variant and normal proteins produced and secreted are the full-length cystatin C. Pulse-chase experiments demonstrated similar levels of normal and variant cystatin C production and secretion. However, the secreted variant cystatin C exhibited an increased susceptibility to a serine protease in conditioned media and in human cerebrospinal fluid, explaining its depletion from the cerebrospinal fluid of HCHWA-I patients. Thus, the amino acid substitution may induce unstable cystatin C with intact inhibitory activity and predisposition to self-aggregation and amyloid fibril formation

Alzheimer's disease is a neurodegenerative disorder characterized by protein depositions in intracellular and extracellular spaces in the brain. The intraneuronal deposits are formed by neurofibrillary tangles composed mainly of abnormally phosphorylated tau, a microtubule-associated protein, whereas the major constituent of the amyloid deposited extracellularly in the brain parenchyma and vessel walls is amyloid beta-protein (A beta). The proteolytic processing of the beta-amyloid precursor protein (beta PP) results in the generation of a complex set of carboxyl-terminal peptides that contain A beta. In this study, we have used fusion proteins containing carboxyl-terminal fragments of beta PP to investigate the association of beta PP with cellular components. We demonstrate that specific domains within the carboxyl end of beta PP contain binding sites for cytoskeletal components; one, within residues 1 to 28 of A beta, binds directly to tubulin, and the second one, within sequences carboxyl-terminal to A beta, binds tau and tubulin. We propose that the two neuropathological hallmarks of Alzheimer's disease, A beta deposition and neurofibrillary tangles, represent the residual of a disrupted beta PP-tubulin-tau complex

BACKGROUND AND PURPOSE: Cerebral amyloid angiopathy (CAA) occurs as a sporadic disorder in aged humans, as a frequent component of Alzheimer's disease, or in hereditary cerebral hemorrhage with amyloidosis (HCHWA). The primary histological locus of cerebral amyloid deposition varies in aged humans and in different species of nonhuman primates. In aged rhesus monkeys, amyloid deposition occurs most frequently in senile plaques, whereas in aged squirrel monkeys CAA is more common. We hypothesized that the preponderance of CAA in squirrel monkeys is related to a species-specific amino acid change in cystatin C, a cysteine protease inhibitor, similar to the Leu68Gln substitution found in the amyloid protein of Icelandic patients with HCHWA-I, also termed hereditary cystatin C amyloid angiopathy. METHODS: We performed immunohistochemical analyses of brain sections of aged squirrel and rhesus monkeys with anti-amyloid-beta and anti-cystatin C antibodies and sequenced the cystatin C cDNA of these monkeys. RESULTS: Cerebral amyloid in aged squirrel and rhesus monkeys, previously shown to be immunoreactive with anti-amyloid-beta anti-bodies, reacts also with antibodies to cystatin C. While the predicted amino acid sequence in rhesus monkeys differs from the human sequence by four residues, that of the squirrel monkeys has seven additional amino acid substitutions, one of which is Leu68Met. CONCLUSIONS: The presence of a mutation in squirrel monkeys similar to the one found in HCHWA-I suggests that alterations in cystatin C may influence the likelihood that amyloid will be deposited in the walls of cerebral blood vessels. These observations support the utilization of the monkeys as models to study CAA

The phosphotyrosine interaction (PI) domains (also known as the PTB, or phosphotyrosine binding, domains) of Shc and IRS-1 are recently described domains that bind peptides phosphorylated on tyrosine residues. The PI/PTB domains differ from Src homology 2 (SH2) domains in that their binding specificity is determined by residues that lie amino terminal and not carboxy terminal to the phosphotyrosine. Recently, it has been appreciated that other cytoplasmic proteins also contain PI domains. We now show that the PI domain of X11 and one of the PI domains of FE65, two neuronal proteins, bind to the cytoplasmic domain of the amyloid precursor protein ((beta)APP). (beta)APP is an integral transmembrane glycoprotein whose cellular function is unknown. One of the processing pathways of (beta)APP leads to the secretion of A(beta), the major constituent of the amyloid deposited in the brain parenchyma and vessel walls of Alzheimer's disease patients. We have found that the X11 PI domain binds a YENPTY motif in the intracellular domain of (beta)APP that is strikingly similar to the NPXY motifs that bind the Shc and IRS-1 PI/PTB domains. However, unlike the case for binding of the Shc PI/PTB domain, tyrosine phosphorylation of the YENPTY motif is not required for the binding of (beta)APP to X11 or FE65. The binding site of the FE65 PI domain appears to be different from that of X11, as mutations within the YENPTY motif differentially affect the binding of X11 and FE65. Using site-directed mutagenesis, we have identified a crucial residue within the PI domain involved in X11 and FE65 binding to (beta)APP. The binding of X11 or FE65 PI domains to residues of the YENPTY motif of (beta)APP identifies PI domains as general protein interaction domains and may have important implications for the processing of (beta)APP

Familial amyloidosis, British type, is an autosomal dominant disease characterized by progressive dementia, spastic paralysis and ataxia. The identity of the accumulating amyloid is not known, thus preventing the definitive classification of the disease. Biochemical methods were used to characterize the nature of the amyloid deposits from the brain tissue of one individual who died with this disease. The purified tissue material was subjected to trypsin digestion and subsequent N-terminal sequence analysis. Major tryptic fragments yielded the sequences VGINYQPPTVVPGGDLAK, FDLMYAK, GLTVPEL and GYLTVAAVFR, which are all tryptic fragments of the C-termini of human tubulin subunits alpha and beta. Synthetic peptides based on the sequences of these fragments formed amyloid fibrils in vitro fitting the characteristic definition of amyloid. These findings suggest that the C-terminal fragments of both alpha- and beta-tubulin are closely associated to the amyloid deposits of familial amyloidosis, British type