Correlation of statin-increased platelet APP ratios and reduced blood lipids in AD patients.

Platelets, like neurons, contain 120- to 130- and 110-kd amyloid precursor proteins (APPs). Their ratio is reduced in AD, further reductions correlating with reduced Mini-Mental Status Examination scores [r(11) = 0.69, p < 0.05]. As statins alter APP processing, platelet APPs were analyzed in patients with AD given anticholesterol drugs for 6 weeks. APP ratios increased [t(37) = -3.888, p = 0.0004], proportionally with reduced cholesterol [r(36) = -0.45, p = 0.005]. Longer trials may reveal slowed cognitive loss, validating this index.

Platelet APP isoform ratios in asymptomatic young adults expressing an AD-related presenilin-1 mutation.

The Alzheimer's disease (AD) related amyloid precursor protein (APP) is stored, cleaved and released similarly from neurons and from platelets. We have reported that the proportion of 120-130 to 110 kDa carboxyl-cleaved APP present in the platelets of AD patients is significantly lower than that of platelets of age-matched controls. This reduced APP isoform ratio, not seen in several other disease groups, is further reduced as the severity of AD increases. Since the neuropathology of AD is believed to begin many years before the onset of cognitive loss, we have also compared platelet APP ratios of four pre-symptomatic young adults carrying a presenilin-1 mutation to seven siblings homozygous for the normal PS-1 gene in an effort to determine whether reduced APP ratios are present before apparent cognitive loss in familial AD. Decreased platelet APP ratios were not seen in any of these subjects at this time. We will continue to monitor these subjects as they near the mean age of AD onset in these families. As the magnitude of the APP ratio reduction is proportional to the severity of cognitive loss in sporadic AD, these cognitively normal incipient AD subjects would not be expected to present significant reductions in this AD severity index at this time. Alternatively, the absence of platelet APP ratio reductions may result from a failure of platelets from familial PS-1 AD subjects to manifest altered APPs, as has been reported for PS-2 AD subjects, unlike those of sporadic AD patients. Continued monitoring of cognitive status in our sub-set of controls with AD-like low APP ratios may yet validate the ability of this assay to detect incipient sporadic AD.

Platelet APP isoform ratios correlate with declining cognition in AD.

BACKGROUND: Platelets and neurons both contain large quantities of two carboxyl-truncated 120 to 130 and 110 kDa Alzheimer amyloid precursor proteins (APPs). Platelets taken from patients with AD have been reported to contain a reduced ratio of these APPs. OBJECTIVE: To further study the AD specificity of reduced platelet APP ratios and to determine whether, after 3 years, cognitive losses in AD are accompanied by similarly reduced platelet APP ratios. METHODS: To test the AD specificity of reduced platelet APP ratios, we quantitated these APPs in eight patients with PD and six patients with hemorrhagic stroke (HS). To determine whether further cognitive losses correlate with platelet APP ratio reductions in patients with AD, the authors re-examined platelet APPs and Mini-Mental State Examination (MMSE) scores of 10 patients with AD and 11 controls, who were tested 3 years ago. APP ratios were determined by the average of six assays using Western blotting with m22C11 monoclonal antibody, enhanced chemoluminescence, and digital scanning of autoradiographs. RESULTS: APP ratios were normal in the patients with PD and HS, further supporting the AD specificity of this assay. After 3 years, the MMSE scores and APP ratios of our control subjects changed by <4%. However, the average MMSE scores of our patients with AD declined from 16.4 to 8.3, and their average 120 to 130/110 kDa APP ratios declined from 5.8 to 3.6. The difference between AD and control APP ratios, with no overlap, is significant and the correlation between the 3-year decline in AD MMSE scores and reduced APP ratios (r = 0.69) was significant. CONCLUSIONS: Although the number of subjects analyzed was limited, reduced platelet APP ratios appear to be a specific biological marker of AD and a biological index of the severity of cognitive loss in AD.

Altered apolipoprotein E secretion in cytokine treated human astrocyte cultures.

Apolipoprotein E (ApoE), postulated to be a major lipid carrier protein in brain, is synthesized and secreted primarily by astrocytes and is involved in brain development and repair. We have analyzed its secretion in primary cultures of older (high passage) slowly dividing and younger (lower passage) rapidly dividing fetal human astrocytes exposed to various inflammatory and anti-inflammatory cytokines, alone and in combination. ApoE secretion was reduced in high passage astrocytes when compared to lower passage astrocytes. A further reduction in ApoE secretion in high passage cells was consistently produced by the combination of cytokines interleukin 1 (IL-1) alpha and beta and interferon (IFN-gamma) cytokines or by the basic fibroblast growth factor (basic-FGF) alone. Epidermal growth factor (EGF) increased ApoE secretion. The combination of these cytokine effects in chronically degenerating brain regions of Alzheimer's disease and other neurodegenerative diseases could reduce the amount of ApoE available for neuronal regeneration. EGF, or agents inducing EGF, could ameliorate these ApoE deficiencies.

An AP-2 binding sequence within exon 1 of human and porcine choline acetyltransferase genes enhances transcription in neural cells.

The gene for choline acetyltransferase, synthesizing acetylcholine, is induced by several neurotrophic factors. A role for AP-2 in enhancing this transcription and limiting it to neural cells is strongly suggested. Previous studies demonstrated that base pairs +465-727 within the untranslated exon 1 of the porcine gene enhanced the expression of a reporter gene transfected into PC-12 cells. Deletion and mutation experiments indicate that base pairs +465-472 (CCGCGGGG) in the porcine gene, or +307-314 (CCTCGGGG) in the human sequence, were necessary and sufficient for increased gene expression in cholinergic or adrenergic but not liver cells. Constructs containing active sequences, but not inactive mutated sequences, specifically bind nuclear proteins from neuroblastoma cells, but not liver cells, in gel shift experiments. The human and porcine sequences are in agreement with an AP-2 consensus binding sequence, a nuclear transcription factor expressed only in cells derived from the neural crest. Gel shift experiments using recombinant AP-2 confirm this identification. AP-2 antibody further retarded the mobility of these DNA-nuclear extract or DNA-AP-2 complexes. These results support the importance of this AP-2 binding sequence in enhancing and limiting choline acetyltransferase expression in neural cells.

Altered amyloid protein processing in platelets of patients with Alzheimer disease.

BACKGROUND: beta-Amyloid peptide, the core component of neuritic plaques in brain areas in patients with Alzheimer disease (AD), is 1 cleavage product of the beta-amyloid precursor protein (APP) in neurons and platelets. Alternate cleavage products of intact 140- to 150-kd APPs in platelets include nonamyloidogenic 120- to 130-kd and 110-kd isoforms. The possible differential significance of these 2 isoforms, structurally similar to protease nexin II, is unknown. OBJECTIVE: To determine whether the ratio of the 120- to 130-kd APP isoform to the 110-kd APP isoform as processed in platelets correlates with the presence of AD and/or the apolipoprotein E4 (ApoE4) allele, which is a major risk factor for AD. SETTING: The Alzheimer Disease Center at The University of Texas Southwestern Medical Center at Dallas. METHODS: The APP isoforms were quantitated with the use of 2 different Western blot detection methods in platelets from 15 patients with AD and 19 control subjects in whom genotyping of apolipoprotein E was performed. RESULTS: The mean ratio of the 120- to 130-kd APP isoform to the 110-kd APP isoform in the patients with AD was significantly lower than that of the control subjects (5.98 vs 7.64; P = .03 [method 1] and 5.98 vs 7.92; P = .01 [method 2]) after adjusting for age and the increased incidence of ApoE4 in patients with AD. The lower APP ratios were also associated with increased age and with the presence of an ApoE4 allele. CONCLUSIONS: The APP processing in platelets of patients with AD is different from that of control subjects. This difference, largely caused by factors other than the ApoE4 genotype, may reflect chronic platelet activation in patients with AD. The use of these data to estimate "AD risk," by using the APP isoform ratio, indicates an odds ratio of 1.75, suggesting possible utility as an adjunct in the diagnosis of AD. Moreover, these findings may relate to analogous alterations in APP processing that may occur in brain areas affected by AD.

A cell type-specific silencer in the human choline acetyltransferase gene requiring two distinct and interactive E boxes.

We have previously reported that cholinergic neuron-specific expression of the human choline acetyltransferase gene is mediated by two co-operative silencers. We have now localized the proximal silencer to the region from nucleotide -2195 to -2409, which contains two distinct E boxes (CACCTG and CATGTG). Deletion or mutation of either of these E boxes results in a loss of silencer activity. There are specific nuclear proteins in adrenergic cells which bind to each of the two E boxes. However, nuclear proteins from cholinergic cells only bind the 5' E box not the 3' E box. It is this interaction which appears to be the cause of the inactivity of this silencer in these cells.

Cholinergic neuron-specific expression of the human choline acetyltransferase gene is controlled by silencer elements.

Choline acetyltransferase (ChAT) is specifically expressed in cholinergic neurons. To identify control mechanisms regulating the cell-specific expression of the gene encoding ChAT, transient expression of the luciferase gene driven by human ChAT gene 5'flanking sequences was compared in cholinergic and noncholinergic cell lines. Analysis of the gene indicated the presence of two regulatory elements with selective silencing activity. These elements, located between nucleotides -2043 to -3347 and nucleotides -3347 to -6550, act cooperatively to repress promoter activity > 10-fold in a human adrenergic neuroblastoma cell line, SHSY5Y, and a human osteosarcoma cell line, 143 TK-, while exhibiting less than a two-fold effect in cholinergic cell lines. Deletion of either nucleotides -2043 to -3347 or nucleotides -3348 to -6550 reduced cell-specific repression by approximately half. Such differential repression appears to be responsible for the selective expression of the ChAT component of the cholinergic phenotype.

Morphological differentiation and proteoglycan synthesis regulate Alzheimer amyloid precursor protein processing in PC-12 and human astrocyte cultures.

A morphologically differentiated strain of rat pheochromocytoma (PC-12H) metabolically labeled with [35S]methionine and incubated with a phorbol ester displayed reduced 140-kDa and increased 15 kDa bands relative to cells incubated without phorbol ester after immunoprecipitation with antisera elicited by the C-terminal peptide of the Alzheimer amyloid precursor protein (APP). These bands correspond to glycosylated full length APP and a C-terminal fragment previously reported by Anderson et al. (Neurosci. Lett. 120:126-128, 1991) to result from a cleavage within the amyloidotic A4 region of APP, which releases a 120 kDa extracellular fragment. The 15 kDa fragment, not immunoprecipitated with an antisera elicited by the N-terminal portion of A4 amyloid, is nonamyloidogenic. Incubation of these cells with p-nitrophenylxyloside, known to inhibit proteoglycan formation, also increased this nonamyloidogenic cleavage of APP. In contrast to these results, an undifferentiated low passage PC-12-L strain constitutively displayed rapid nonamyloidogenic APP cleavage. Incubation of PC-12-L with phorbol ester did not affect the relative abundance of 140 or 15 kDa bands. Growth of PC-12-L with 7 S NGF or dibutyryl cAMP resulted in increased morphological differentiation and decreased APP cleavage which was now phorbol-inducible. Similar analyses of dividing and senescent human astrocytes and normal and F-AD fibroblasts indicate 5-fold lower rates of mid-A4 APP cleavage. Phorbol esters decreased the 140 kDa APP band without affecting the intensity of the 15 kDa band in these cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Increased release of an amyloidogenic C-terminal Alzheimer amyloid precursor protein fragment from stressed PC-12 cells.

Amyloid plaques, found in characteristically large numbers in specific brain areas of Alzheimer's disease (AD) and Down's Syndrome (DS) patients, are composed of a 41-43 amino acid peptide, A4, derived from a transmembrane glycoprotein, amyloid precursor protein (APP). In transformed cells APP has been shown to be cleaved within the extracellular portion of the A4 region causing the release of 100-120 kDa soluble N-terminal APP products. If this cleavage occurs in human tissue, neither the soluble product nor the remaining 10-12 kDa transmembrane fragment could be further degraded to yield A4. It has been hypothesized that an alternate APP cleavage product containing the intact A4 region is released in increased amounts in AD and DS brain where subsequent extracellular degradation produces the amyloidogenic A4 peptide. In support of this hypothesis, we have found that PC-12 cells maintained in serum-free media with or without additional injurious agents release a 60 kDa protein which has been detected by immunoprecipitation and immunoblot analyses with 9 antisera elicited by 4 distinct peptides within the carboxyl-terminal half of APP. Six of these antisera, elicited by peptides corresponding to the carboxyl-terminal 20 amino acids of APP, or the A4 peptide itself, do not bind the normally released 120 kDa APP product which is detected by 11 other antisera elicited by peptides with the N-terminal portion of APP. Controls in which two 60 kDa-detecting antisera were preabsorbed with the peptides used to elicit them, produced markedly reduced 60 kd bands on immunoblots.(ABSTRACT TRUNCATED AT 250 WORDS)

Frequent alterations of specific reiterated DNA sequence abundances in human cancer.

An in-gel renaturation method allows the visualization, quantitation, and initial characterization of reiterated DNA restriction fragments (RRFs) without prior possession of their probes. Using this method we analyzed EcoRI restricted DNAs from ten human tumors, paired normal tissues from these patients, ten unpaired tumors, and 26 noncancer patients. Frequent qualitative and quantitative differences in the relative radioactive intensities of all or specific RRFs in the DNA from different individuals have been observed. However, we also report frequent two- to ten-fold alterations in the relative radioactive intensities of several specific RRFs in tumor-control analyses of DNA from the same individual. A 0.65-kb alphoid-like DNA RRF was decreased in six tumors and increased in none. Moreover, a new 1.55-kb RRF was observed in two colon cancer DNAs. Several new RRFs, suggestive of gene amplification, were detected in a neuroblastoma, two of which were also seen in a glioma. These data support frequent qualitative and quantitative alterations of specific reiterated DNA sequences in human cancer when compared with paired controls. This approach will allow the future characterization of specific DNA sequences whose patterns of altered reiteration suggest a role in the emergence of specific malignancies.

Thymidylate synthase gene amplification in fluorodeoxyuridine-resistant mouse cell lines.

We have previously isolated fluorodeoxyuridine-resistant mouse fibroblast (LU3-7) and neuroblastoma (FUdR-R) cell lines that overproduce thymidylate synthase and the mRNA for this enzyme up to 50-fold as compared to the parental cell lines. We have also cloned cDNA corresponding to mouse thymidylate synthase mRNA into pBR322. In the present study, we used this cloned cDNA as a hybridization probe in Southern blot analysis of DNA from the parental and overproducing cell lines. These analyses showed that the thymidylate synthase gene is amplified 50-100 fold in LU3-7 cells and about 30-fold in FUdR-R cells when compared to the respective parental cells. The sizes of the restriction fragments were the same in the parental and overproducing cells of each type, suggesting that extensive rearrangements have not occurred in the vicinity of the thymidylate synthase gene during the amplification process. However, not all of the fragments in the parental cells were amplified in the overproducing cells, suggesting that there may be multiple genes or pseudogenes for the enzyme. Restriction fragment length polymorphisms were detected when analyzing DNA from several different mouse cell lines. When LU3-7 cells were grown in the absence of selective pressure, the level of thymidylate synthase overproduction and the number of copies of the thymidylate synthase gene decreased in parallel.

Joseph disease and Huntington disease: protein patterns in fibroblasts and brain.

Proteins were separated on two-dimensional acrylamide gels obtained from brain samples of patients with Joseph disease, Huntington disease (HD) and multiple sclerosis. Similar protein separations were made from cultured skin fibroblasts of Joseph disease patients. Two major classes of proteins, one with a MW of 50,000 probably representing the glial filamentous acidic protein, or another class with a MW of 40,000 (proteins Jc, Jd, L1 and L2) were increased in the cerebellum of six Joseph disease patients. The same protein species were abnormally increased in HD brains, mainly in the basal ganglia and frontal cortex. These identical classes of protein changes were present in two nosologically separate autosomal dominant neurological disorders, Joseph disease (a spinocerebellar degeneration) and HD (a basal ganglia and cerebral cortical degeneration) and may reflect a biochemical correlation of gliosis and neuronal disease. However, these changes may be evidence that the two diseases are allelic mutations of the same gene. The dominantly inherited spinocerebellar degenerations may result from a primary deficit of glial-neuronal interaction, resulting in neuronal loss but with a compensatory increase in the number of glial cells attempting to provide additional trophic-metabolic support.

Correlation of double-minute chromosomes with unstable multidrug cross-resistance in uptake mutants of neuroblastoma cells.

A series of increasingly drug-resistant cell populations were selected and cloned from C-46 murine neuroblastoma with the chemotherapeutic drugs maytansine, vincristine, adriamycin, or Baker's antifol. All clones demonstrated reciprocal cross-resistance to these structurally and functionally diverse drugs and failed to accumulate radiolabeled vincristine, colchicine, or Baker's antifol despite normal drug binding to cell homogenates. Initial isolates of drug-resistant populations were genetically unstable, rapidly reverting to a drug-sensitive phenotype when grown without drug, at 0.05 reversion per cell division. After prolonged growth in drug, this drug-resistant genotype stabilized. Mean chromosome number increased 300% in an initially isolated 20-fold maytansine-resistant clone, which also displayed numerous double-minute chromosomes. Descendants 240-fold more resistant than the parent, also unstable, possessed the wild-type complement of 80 chromosomes, but 45% of these cells possessed 24 double-minute chromosomes per cell; such chromosomes were absent from the drug-sensitive parental clone. Only 1.0 and 1.2 double-minute chromosomes per cell were seen in a 7-fold stably resistant revertant or 1200-fold stably resistant descendants, respectively. Double-minute chromosomes containing amplified genes for the drug target dihydrofolate reductase (tetrahydrofolate dehydrogenase; 5,6,7,8-tetrahydrofolate:NADP+ oxidoreductase, EC 1.5.1.3) have been reported in an unstable methotrexate-resistant R1-A sarcoma. These extrachromosomal gene copies were absent in stably resistant progeny. The presence of similar particles in unstably drug-resistant uptake mutants of neuroblastoma and their diminution in stably resistant descendants supports and extends their possible role in the rapid onset and instability of epigenetic drug resistance in cancer chemotherapy.

Cyclic adenosine 3':5'-monophosphate-binding protein, a biochemical marker of neuroblastoma differentiation.

Mouse neuroblastoma tumors show reduced amounts of cyclic adenosine 3':5'-monophosphate (cAMP) binding protein. However, the levels of cAMP-binding protein were increased by 2-fold when the tumor cells were established in tissue culture, and these levels were comparable to that found in mouse brain. This binding protein is a free cAMP-binding protein that is not associated with protein kinase. The reduced amounts of free cAMP-binding protein in tumors are not a consequence of a defective gene, but the synthesis of this protein is regulated at the transcriptional and/or translational levels. The free cAMP-binding protein like the neurotransmitter-synthesizing enzymes can be used as a biochemical marker of differentiation, and this protein may play a role in neuronal differentiation.

Phosphorus 31 NMR of neuroblastoma clonal lines. Effect of cell confluency state and dibutyryl cyclic AMP.

Phosphorus nuclear magnetic resonance is used to study changes in the levels of the major phosphate-containing intermediary metabolites concomitant with induced cellular differentiation in the N-18 and C-46 neuroblastoma clonal lines. The study reveals differences between the 31P-NMR profiles of the two clonal lines and also striking differences attendant to dibutyryl cAMP-mediated morphological differentiation in the N-18 clone. Phosphorus-31 NMR would appear to provide a new technique with which to study genetic differentiation.

Joseph disease: protein patterns in fibroblasts and brain.

The separation of brain and fibroblast proteins was analyzed on two-dimensional acrylamide gels. Proteins were examined from skin fibroblast cultures and brain homogenates from the frontal cerebral cortex, putamen, and cerebellum. Protein species from skin fibroblast cultures of controls and patients with Joseph disease or Huntington disease were not significantly different. The proteins from homogenates of the cerebral cortex, putamen, and cerebellum from controls differed from those of one Joseph disease patient. Two major classes of proteins were increased in the patient's putamen and cerebellum. Proteins of 40,000 and 50,000 daltons--including the glial filamentous acidic protein complex (molecular weight 50,000), and two proteins which migrated near actin--were increased in the cerebellum. The glial filamentous acidic protein complex increased 3.7-fold in the putamen of the patient. These protein changes probably represent gliosis, but may also be an expression of the primary genetic mutation.