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Several studies have shown the protective effects of dietary enrichment of various lipids in several late-onset animal models of Alzheimer Disease (AD); however, none of the studies has determined which structure within a lipid determines its detrimental or beneficial effects on AD. High-sensitivity enzyme-linked immunosorbent assay (ELISA) shows that saturated fatty acids (SFAs), upstream omega-3 FAs, and arachidonic acid (AA) resulted in significantly higher secretion of both Aβ 40 and 42 peptides compared with long chain downstream omega-3 and monounsaturated FAs (MUFA). Their distinct detrimental action is believed to be due to a structural template found in their fatty acyl chains that lack SFAs, upstream omega-3 FAs, and AA. Immunoblotting experiments and use of APP-C99-transfected COS-7 cells suggest that FA-driven altered production of Aβ is mediated through γ-secretase cleavage of APP. An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein (APP); Swedish (K670N/M671L) and Indiana (V717F), corroborated in vitro findings by showing lower levels of Aβ and amyloid plaques in the brain, when they were fed a low fat diet enriched in DHA. Our work contributes to the clarification of aspects of structure-activity relationships.

alpha-Synuclein (alphaSN), also termed the precursor of the non-Abeta component of Alzheimer's disease (AD) amyloid (NACP), is a major component of Lewy bodies and Lewy neurites pathognomonic of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). A fragment of alphaSN termed the non-Abeta component of AD amyloid (NAC) had previously been identified as a constituent of AD amyloid plaques. To clarify the relationship of NAC and alphaSN with Abeta plaques, antibodies were raised to three domains of alphaSN. All antibodies produced punctate labeling of human cortex and strong labeling of Lewy bodies. Using antibodies to alphaSN(75-91) to label cortical and hippocampal sections of pathologically proven AD cases, we found no evidence for NAC in Abeta amyloid plaques. Double labeling of tissue sections in mixed DLB/AD cases revealed alphaSN in dystrophic neuritic processes, some of which were in close association with Abeta plaques restricted to the CA1 hippocampal region. In brain homogenates alphaSN was predominantly recovered in the cytosolic fraction as a 16-kd protein on Western analysis; however, significant amounts of aggregated and alphaSN fragments were also found in urea extracts of SDS-insoluble material from DLB and PD cases. NAC antibodies identified an endogenous fragment of 6 kd in the cytosolic and urea-soluble brain fractions. This fragment may be produced as a consequence of alphaSN aggregation or alternatively may accelerate aggregation of the full-length alphaSN.

A distinguishing feature of Alzheimer's disease (AD) is the deposition of amyloid plaques in brain parenchyma. These plaques arise by the abnormal accumulation of beta A4, a proteolytic fragment of amyloid precursor protein (APP). Despite the fact that neurons are dramatically affected in the course of the disease, little is known about the neuronal processing of APP. To address this question we have expressed in fully mature, synaptically active rat hippocampal neurons, the neuronal form of human APP (APP695), two mutant forms of human APP associated with AD, and the mouse form of APP (a species known not to develop amyloid plaques). Protein expression was achieved via the Semliki Forest Virus system. Expression of wild type human APP695 resulted in the secretion of beta A4-amyloid peptide and the intracellular accumulation of potential amyloidogenic and non-amyloidogenic fragments. The relative amount of amyloid-containing fragments increased dramatically during expression of the clinical mutants, while it decreased strongly when the mouse form of APP was expressed. 'Humanizing' the rodent APP sequence by introducing three mutations in the beta A4-region also led to increased production of amyloid peptide to levels similar to those obtained with human APP. The single Gly601 to Arg substitution alone was sufficient to triple the ratio of beta A4-peptide to non-amyloidogenic p3-peptide. Due to the capacity of these cells to secrete and accumulate intracellular amyloid fragments, we hypothesize that in the pathogenesis of AD there is a positive feed-back loop where neurons are both producers and victims of amyloid, leading to neuronal degeneration and dementia.(ABSTRACT TRUNCATED AT 250 WORDS)

The proteolytic fragments derived from the amyloid precursor protein (APP) in primary cultures of rat hippocampal neurons were analyzed by two-dimensional gel electrophoresis. The Semliki Forest Virus expression vector was used to express human APP695 and a mutant form associated with familial Alzheimer's disease (APP-FAD670/671). Hippocampal neurons expressing wtAPP695 or APP-FAD670/671 secrete at least six APP fragments of 100-110 kDa with isoelectric focusing points ranging from 4.5 to 4.0. The heterogeneity of the secreted APP forms is shown to be in part due to differences in glycosylation. In contrast to wtAPP695, neurons producing the APP-FAD670/671 variant did not secrete detectable amounts of secretory APP derived from cleavage within the amyloid beta A4 domain. This result suggests that there is little alpha-secretase cleavage in neurons expressing the APP-FAD670/671 mutant.

Alzheimer's disease is one of the prevalent forms of human dementia. Its pathology is distinguished by proteinaceous deposits ("amyloid") in the brain. They contain a peptide (beta A4) that is proteolytically derived from a larger transmembrane protein. To follow the different metabolic pathways of this Amyloid Precursor Protein (APP) may thus lead to the elucidation of the molecular basis of Alzheimer's disease. Specific antibodies are necessary tools for this task. Using synthetic peptides, we have characterized the epitope of the APP-specific monoclonal antibody 22C11; it is localized between residues 66 and 81 of APP. Some of the peptides flanking this site exhibited properties generally associated with amyloid, i.e. low solubility, filament formation, and birefringence after Congo Red staining. Exploiting differences in the peptides' aggregational properties, we present evidence that the two dyes Eosin and Direct Red 254, in conjunction with classical amyloid staining by Congo Red, can be used to characterize aggregating, amyloid-like peptides in vitro.

The evolution of senile plaques and the relationships among neuritic elements, extracellular deposits of the beta-amyloid protein (beta/A4), and vascular beta/A4 are poorly understood. Immunocytochemical methods were used to examine fixed-frozen prefrontal cortices of 14 rhesus monkeys (Macaca mulatta) (14 to 37 years of age) for the presence of abnormal fibers/neurites, alpha 1-antichymotrypsin (alpha-ACT), and beta/A4. Age-associated alterations included abnormal fibers/neurites, presence of beta/A4, and association of alpha-ACT with beta/A4 in plaques and blood vessels. Vascular amyloid was present only in the oldest monkeys. The topographic distribution of abnormal fibers/neurites was mapped with acetylcholinesterase (AChE) histochemistry, and deposits of amyloid were visualized with immunocytochemistry for beta/A4. beta/A4 often was associated with neurites, but many neurites lacked demonstrable beta/A4. Thus in aged monkeys, abnormal neurites may provide one type of focus for the accumulation of the amyloid precursor, which is subsequently abnormally processed to form beta/A4. Our data in rhesus monkeys suggest that fiber and neuritic abnormalities increase with age and that they may precede the majority of beta/A4 deposits; the initial stages of neurite formation and parenchymal amyloid deposits may be independent of the appearance of vascular amyloid; and these processes may be synergistic with advanced age.

Retinoic acid (RA) induced differentiation of SH-SY5Y neuroblastoma cells is associated with more than a tenfold induction of total Alzheimer's disease beta A4 amyloid protein precursor (APP) mRNA as analyzed by Northern blot hybridisation. S1 nuclease protection experiments reveal that the splicing pattern of these differentiated cells is altered in favor of APP695 mRNA, coding for the shortest amyloidogenic beta A4 amyloid precursor protein. Induction of differentiation of SH-SY5Y cells with NGF leads to a fivefold increase of total APP mRNA without change in the splicing pattern. This suggests that RA but not NGF induces factor(s) which are responsible for an APP hnRNA splicing favoring APP695 mRNA.

The beta-amyloid protein (beta/A4), derived from a larger amyloid precursor protein (APP), is the principal component of senile plaques in Alzheimer's disease. APP is an integral membrane glycoprotein and is secreted as a carboxyl-terminal truncated molecule. APP cleavage, which is a membrane-associated event, occurred at a site located within the beta/A4 region. This suggests that an intact amyloidogenic beta/A4 fragment is not generated during normal APP catabolism. Therefore, an early event in amyloid formation may involve altered APP processing that results in the release and subsequent deposition of intact beta/A4.

In the brains of aged humans and cases of Alzheimer disease, deposits of amyloid in senile plaques are located in proximity to nerve processes. The principal component of this extracellular amyloid is beta/A4, a peptide derived from a larger amyloid precursor protein (APP), which is actively expressed in brain and systemic organs. Mechanisms that result in the proteolysis of APP to form beta/A4, previously termed beta-amyloid protein, and the subsequent deposition of the peptide in brain are unknown. If beta/A4 in senile plaques is derived from neuronally synthesized APP and deposited at locations remote from sites of synthesis, then APP must be transported from neuronal cell bodies to distal nerve processes in proximity to deposits of amyloid. In this study, using several immunodetection methods, we demonstrate that APP is transported axonally in neurons of the rat peripheral nervous system. Moreover, our investigations show that APP is transported by means of the fast anterograde component. These findings are consistent with the hypothesis of a neuronal origin of beta/A4, in which amyloid is deposited in the brain parenchyma of aged individuals and cases of Alzheimer disease. In this setting, we suggest that APP is synthesized in neurons and delivered to dystrophic nerve endings, where subsequent alterations of local processing of APP result in deposits of brain amyloid.

To the triad of neuronal loss, gliosis and spongiform change as characteristic morphological changes associated with infection of the central nervous system, one can now add the presence of scrapie-associated filaments (SAF)/PrP rods. While the host's immune response is conspicuous by its absence, the vigorous astrocytic response is presumptive evidence of the host's ability to recognize and respond to the primary neuronal insult. We assume that the spongiform change and vacuolation of neurons are of fundamental importance in the pathogenesis of the disease, realizing that neither is specific or essential for the replication of the infectious agent. The topographical distribution of lesions is partly explained by the portal of entry and retrograde spread of the virus. The temporal progression of the lesions is more clearly determined by the host genes, best illustrated by studies of the incubation period. The molecular basis of the spongiform change is unknown but it is presumed to involve some disturbance of membrane metabolism. The recognition of PrP as a membrane glycoprotein invites proposals for its role in the development of these spongiform lesions. Extracellular amyloid occurs as plaques or congophilic angiopathy in some instances, and provides the best evidence that Alzheimer's disease (AD) is in some way related to the unconventional virus diseases. However, the protein subunit (A4) of the amyloid fibril in AD and its precursor are quite distinct from the PrP subunit which constitutes the amyloid fibril in these infectious diseases. It is still unclear whether the PrP subunit in the SAF has exactly the same composition as in the extracellular amyloid fibril. Our results suggest that only a fragment of the PrP molecule is the major constituent of the extracellular fibril. Since both PrP and A4 are derived from membrane glycoproteins, the elucidation of their normal function is likely to lead to a better understanding of the spongiform and amyloidogenic lesions in these diseases.

The histologic diagnosis of Alzheimer's disease (AD) might be aided if a more sensitive marker of aberrant A4 amyloid protein deposition were available. We screened a sample of aged brains, using immunocytochemical methods to detect the A4 protein deposition, and found that, in comparison with conventional histologic techniques (silver impregnation and Congo red), immunocytochemistry is more sensitive and allows an easier demarcation between "normal" and "abnormal." If A4 protein deposition is accepted as a definitive marker for AD, then the age-related prevalence of AD increases dramatically. To what degree these prevalence rates are reflected in clinically detectable impairment of higher cortical function remains to be determined.

The protein component of Alzheimer's disease amyloid (neurofibrillary tangles, amyloid plaque cores and congophilic angiopathy) is an aggregated polypeptide with a subunit mass of approximately 4 kDa (the A4 monomer). The aggregational properties of this monomer may explain the amyloidogenic nature of the protein: the native monomer forms dimers, tetramers and higher oligomeric species which are dependent on pH, ionic strength and concentration; the synthetic peptide corresponding to residues 1-28 spontaneously forms fibrils in vitro. Based on the degree of N-terminal heterogeneity, the A4 monomer aggregates first in neurons and later in the extracellular space. Using antisera raised against synthetic peptides, we can demonstrate that the N-terminus contains an epitope for neurofibrillary tangles, and the inner region of the molecule contains an epitope for the extracellular amyloid fibrils. There is a non-protein component of the amyloid (inorganic residues of aluminium silicate) which may be important in the deposition of the amyloid fibrils. There are several intriguing similarities between the amyloid fibrils and proteins of Alzheimer's disease when compared to the scrapie-associated filaments and proteins of the unconventional virus diseases (scrapie, kuru, Creutzfeldt-Jakob disease). Although there is no sequence homology between the proteins, we suspect they are formed as a result of similar biochemical processes. If the scrapie proteins and filaments are an integral part of the infectious agent, it follows that Alzheimer's disease is also an infectious process similar to scrapie. As they are host-encoded proteins, it is still feasible that both types are pathological by-products of independent diseases.

Neurofibrillary tangles and neuritic plaques are the neuropathological hallmarks of Alzheimer's disease. The latter consist of a core of A4 amyloid protein. We now report that some neurofibrillary tangles ('tombstone tangles') are also A4 immunoreactive. This observation is consistent with the hypothesis that A4 amyloid accumulation is a component of both neurofibrillary tangles and neuritic plaques.

The protein component of Alzheimer's disease amyloid [neurofibrillary tangles (NFT), amyloid plaque core and congophilic angiopathy] is an aggregated polypeptide with a subunit mass of 4 kd (the A4 monomer). Based on the degree of N-terminal heterogeneity, the amyloid is first deposited in the neuron, and later in the extracellular space. Using antisera raised against synthetic peptides, we show that the N terminus of A4 (residues 1-11) contains an epitope for neurofibrillary tangles, and the inner region of the molecule (residues 11-23) contains an epitope for plaque cores and vascular amyloid. The non-protein component of the amyloid (aluminum silicate) may form the basis for the deposition or amplification (possible self-replication) of the aggregated amyloid protein. The amyloid of Alzheimer's disease is similar in subunit size, composition but not sequence to the scrapie-associated fibril and its constituent polypeptides. The sequence and composition of NFT are not homologous to those of any of the known components of normal neurofilaments.

The A4 amyloid precursor gene of Alzheimer's Disease (PreA4 gene, App gene, PAD gene) gives rise to three different transcripts, which are generated by alternative splicing. The three transcripts are PreA4695, PreA4751 and PreA4770, according to the number of amino-acids in the primary translation product. Previous expression studies did not discriminate between PreA4751 and PreA4770. We have analyzed the distribution of PreA4 transcripts in four cortical brain areas in a way that allows us the selective identification of each transcript. We used a sensitive S1 nuclease protection assay with a probe derived from PreA4770 cDNA. This radiolabeled probe gives rise to three specifically protected DNA fragments, each corresponding to one of the three PreA4 transcripts. These fragments could readily be resolved in a single lane of a denaturing polyacrylamide gel. PreA4 transcripts were quantified by scanning autoradiograms. In younger individuals PreA4695 mRNA is the dominant transcript with 55% (mean values). PreA4770 mRNA is the minor PreA4 transcript with less than 5% and PreA4751 ranges at 40%. The exception is observed in occipital cortex, where PreA4751 (50%) is higher than PreA695 (45%). A different situation was observed in older individuals. In the latter PreA4(695) transcripts are significantly reduced, with the exception of frontal cortex, whereas PreA4770 transcripts show an increase. PreA4751 has a constant level of 45% and becomes the dominant transcript.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4. We suggest that besides proteolytic cleavage, other events, such as membrane damage are primary events that precede the release of the small, aggregating amyloid A4 subunit.

The prevalence of deposition of the Alzheimer's disease A4 amyloid protein in representative areas of the cerebral cortex was compared using a sensitive immunohistochemical technique in a group of 26 cases of idiopathic Parkinson's disease (PD) and in a control group of 82 subjects of comparable age who had died unexpectedly from non-cerebral causes. Cortical A4 protein deposition was found in 54% of PD subjects and 48% of the control group, while deposition of A4 protein in meningeal or cortical blood vessels was found in 38% of PD subjects and 25% of controls. When allowance was made for age and sex, the differences between the two groups were not found to be statistically significant. Heavy cortical A4 protein deposition was found in a number of PD cases, including two of four cases with dementia, and was absent in the other two cases. The present findings indicate that a large proportion of cases of idiopathic PD have associated Alzheimer's disease changes. In some cases these lesions are of sufficient severity to account for dementia. In other cases the changes are less severe and are probably subclinical but may be a contributory factor to the development of cognitive impairment and dementia. The absence of Alzheimer changes in some demented PD cases indicates that the dementia of PD is heterogeneous.

Alzheimer's disease is characterized by a widespread functional disturbance of the human brain. Fibrillar amyloid proteins are deposited inside neurons as neurofibrillary tangles and extracellularly as amyloid plaque cores and in blood vessels. The major protein subunit (A4) of the amyloid fibril of tangles, plaques and blood vessel deposits is an insoluble, highly aggregating small polypeptide of relative molecular mass 4,500. The same polypeptide is also deposited in the brains of aged individuals with trisomy 21 (Down's syndrome). We have argued previously that the A4 protein is of neuronal origin and is the cleavage product of a larger precursor protein. To identify this precursor, we have now isolated and sequenced an apparently full-length complementary DNA clone coding for the A4 polypeptide. The predicted precursor consists of 695 residues and contains features characteristic of glycosylated cell-surface receptors. This sequence, together with the localization of its gene on chromosome 21, suggests that the cerebral amyloid deposited in Alzheimer's disease and aged Down's syndrome is caused by aberrant catabolism of a cell-surface receptor.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.

The cloned cDNA encoding the rat cognate of the human A4 amyloid precursor protein was isolated from a rat brain library. The predicted primary structure of the 695-amino acid-long protein displays 97% identity to its human homologue shown previously to resemble an integral membrane protein. The protein was detected in rodent brain and muscle by Western blot analysis. Using in situ hybridization and immunocytochemistry on rat brain sections, we discovered that rat amyloidogenic glycoprotein (rAG) and its mRNA are ubiquitously and abundantly expressed in neurons indicating a neuronal original for the amyloid deposits observed in humans with Alzheimer's disease (AD). The protein appears in patches on or near the plasma membranes of neurons suggesting a role for this protein in cell contact. Highest expression was seen in rat brain regions where amyloid is deposited in AD but also in areas which do not contain deposits in AD. Since amyloid deposits are rarely observed in rat brain, we conclude that high expression of AG is not the sole cause of amyloidosis.

The presence of abundant intraneuronal amyloid in the form of neurofibrillary tangles (NFT) in the brains of Guamanian parkinsonism-dementia patients and the absence of extraneuronal amyloid in the form of vascular amyloid deposits or senile plaques permit the purification of NFT without contamination with extraneuronal amyloid. Thus, we have isolated and determined the amino acid sequence of the polypeptide subunit of the amyloid fibrils of these NFT and describe their ultrastructure. The NFT, which consist of single and paired helical filaments, similar to those of Alzheimer disease, and occasionally triple helical filaments, are composed of multimeric aggregates of a polypeptide of 42 amino acids (A4 protein). The relative molecular mass of the subunit protein, 4.0-4.5 kDa, is the same as the molecular mass of the amyloid of NFT, of the amyloid plaque cores, and of vascular amyloid deposits in Alzheimer disease and Down syndrome; the sequence of 15 amino acid residues at the N-terminus of the amyloid fibrils in the NFT of Guamanian parkinsonism-dementia is identical to that of the amyloid of NFT, amyloid plaque cores, and cerebrovascular deposits in Alzheimer disease and Down syndrome. Furthermore, the heterogeneity, or variation in polypeptide length, of the N-terminus of the amyloid of Guamanian parkinsonism-dementia is the same as in Alzheimer disease and Down syndrome. Our observations indicate that the brain amyloids of these diseases have a common subunit protein, which would also indicate a common pathogenesis.

The promoter of the gene for the human precursor of Alzheimer's disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. It lacks a typical TATA box and shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in an in vivo assay. Transcription initiates at multiple sites. Sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein were found upstream of the RNA start sites. Six copies of a 9-bp-long GC-rich element are located between positions -200 and -100. A protein--DNA interaction could be mapped to this element. The 3.8 kb of the 5' region of the PAD gene include two Alu-type repetitive sequences. These findings suggest that four mechanisms may participate in the regulation of the PAD gene and could be of relevance for the progression of amyloid deposition in Alzheimer's disease.

In patients with Alzheimer's disease, amyloid fibrils that are aggregates of A4 protein subunits are deposited in the brain. A similar process occurs at an earlier age in persons with Down's syndrome. To investigate the deposition of amyloid in these diseases, we used a radioimmunoassay to measure levels of the amyloid precursor (PreA4) in the serum of 17 patients with Down's syndrome, 15 patients with Alzheimer's disease, and 33 normal elderly controls. The mean (+/- SD) concentration of serum PreA4 was increased 1.5-fold in patients with Down's syndrome (2.49 +/- 1.13 nmol per liter) as compared with that in controls (1.68 +/- 0.49 nmol per liter; P less than 0.007); the levels in patients with Alzheimer's disease were similar to those in controls (1.83 +/- 0.78; P less than 0.98). We also found that the concentration of PreA4 in the brain tissue of two adults with Down's syndrome (100 and 190 pmol per gram) was higher than that in the brain tissue of either 26 patients with Alzheimer's disease (64.4 +/- 17.3 pmol per gram) or 17 elderly controls with neurologic disease (68.5 +/- 26.3 pmol per gram). Immunocytochemical studies of brain tissue from 26 patients with Down's syndrome showed that the deposition of A4 protein amyloid began in these patients approximately 50 years earlier than it began in 127 normal aging subjects studied previously, although the rate of deposition was the same. We conclude that, since the gene for PreA4 is on the long arm of chromosome 21, which is present in triplicate in Down's syndrome, overexpression of this gene may lead to increased levels of PreA4 and amyloid deposition in Down's syndrome. However, since increased levels of PreA4 are not present in Alzheimer's disease, additional factors must account for the amyloid deposition in that disorder.

The promoter of the gene for the precursor of Alzheimer's Disease A4 amyloid protein (PAD gene) resembles promoters of housekeeping genes. A typical TATA box is missing, and transcription initiates at multiple sites. It shows a high GC content of 72% in a DNA region that confers promoter activity to a reporter gene in vivo. Upstream of the RNA start sites we found sequences homologous to the consensus binding sites of transcription factor AP-1 and the heat shock control element binding protein. Six copies of a 9bp long GC-rich element are located between positions -100 and -200 of the sequence. A protein-DNA interaction could be mapped to this element. The ratio of the dinucleotide CpG, the target for DNA methylation, versus GpC is about 1:1 around the RNA start site, in contrast to the normal ratio of 1:5 in eucaryotic DNA. These findings suggest that four mechanisms may participate in the regulation of the PAD gene: the stress-related heat shock; the AP-1/Fos binding; the GC-rich element, and the possible methylation of the CpG region. PAD gene regulation could be of relevance for the progression of amyloid deposition in Alzheimer's Disease.

The A4 amyloid protein is the major subunit present in the amyloid of Alzheimer's disease. It is derived by proteolytic cleavage from a larger precursor (PreA4) which is a neuronal membrane glycoprotein. Whereas in Down's syndrome, over-expression of the gene coding for PreA4 is likely to be responsible for the premature development of cerebral amyloidosis, a similar mechanism is yet to be demonstrated in Alzheimer's disease.

To study the putative precursor proteins (PreA4(695), PreA4(751), and PreA4(770] of Alzheimer's disease A4 amyloid protein, polyclonal and monoclonal antibodies were raised against a recombinant bacterial PreA4(695) fusion protein. These antibodies were used to identify the precursors in different cell lines as well as in human brain homogenates and cerebrospinal fluid (CSF). The precursors are tyrosine-sulfated, O- and N-glycosylated membrane proteins and have half-lives of 20-30 min in cells. Cells express the polypeptides at their surface but also secrete C-terminal truncated proteins into the medium. These proteins are also found in CSF of both Alzheimer's disease patients and normal individuals. The proteins are derived from their cognate membrane-associated forms by proteolysis and have apparently lost the cytoplasmic and the transmembrane domains. Since the latter contributes to the A4 amyloid sequence, it seems possible that this proteolytic cleavage represents the first step in the formation of A4 amyloid deposits.

We present a 22-year follow-up of a large and unusual kindred previously reported as familial Alzheimer's disease (FAD). However, detailed clinical and neuropathologic evaluation of family members and brain autopsy on another affected individual now make the diagnosis of FAD unlikely. Our patient, as well as members of this family, had numerous amyloid plaques and rare neurofibrillary tangles. These plaques were quite atypical for Alzheimer's disease (AD); many were quite large (up to 500 microns in diameter) and contained several amyloid cores, some with neuritic components. The plaques were present throughout the cerebral cortex and striatum, but not in the cerebellum. By electron microscopy, they had radiating star-shaped amyloid cores containing 8- to 10-nm fibrils, and a few dystrophic neurites. They were strongly immunoreactive with antiserum to prion protein but did not react with the antiserum to the amyloid A4 protein of AD. Although the cerebellum was uninvolved, this family appears to represent another clinical and neuropathologic variant of Gerstmann-Sträussler syndrome.

Azzarelli et al reported an Indiana kindred affected by a hereditary disorder, characterized clinically by ataxia, parkinsonism, and dementia. Recently, we studied neuropathologically the 3rd and 4th cases that came to autopsy among the patients of this family. As in 2 patients examined previously, amyloid plaques were widespread throughout the cerebrum and the cerebellum, whereas neurofibrillary tangles were numerous in the cerebral cortex, the hippocampus, and the substantia innominata. Amyloid plaques were not recognized by polyclonal antibodies against the Alzheimer's disease amyloid A4 protein, but did contain epitopes recognized by antibodies against a prion protein. Spongiform changes were occasionally observed and were mild. Our findings indicate that this familial disorder is a form of or is related to Gerstmann-Sträussler-Scheinker disease. The consistent presence of numerous neurofibrillary tangles may be important in differentiating a distinct subgroup of patients with familial Gerstmann-Sträussler-Scheinker disease, and indicates that a disturbance of the cytoskeleton might be part of the neuronal pathology of Gerstmann-Sträussler-Scheinker disease.

This study was designed to investigate the role of serum proteins, microglia, glial fibrillary acidic protein (GFAP) positive cells and dystrophic neurites in the genesis of cerebral amyloid. Using A4 protein antisera, we found an amorphous non-congophilic, form of plaque, which was not seen in Bielschowsky silver staining or Bodian impregnations. GFAP-positive glial cells, cells immunolabelled for some macrophage markers and dystrophic neurites were detected in congophilic plaques with crystalline amyloid, but not in the amorphous, non-congophilic plaques. The presence of alpha l-antichymotrypsin, complement factors and P component, but not of common serum proteins in both the amorphous and congophilic plaques, indicates that these three proteins may have a pathogenetic role in amyloid formation. Amorphous plaques may be the earlier forms of plaque and consequently, the presence of reactive cells and dystrophic neurites may be secondary phenomena.

We have purified and characterized the cerebral amyloid protein that forms the plaque core in Alzheimer disease and in aged individuals with Down syndrome. The protein consists of multimeric aggregates of a polypeptide of about 40 residues (4 kDa). The amino acid composition, molecular mass, and NH2-terminal sequence of this amyloid protein are almost identical to those described for the amyloid deposited in the congophilic angiopathy of Alzheimer disease and Down syndrome, but the plaque core proteins have ragged NH2 termini. The shared 4-kDa subunit indicates a common origin for the amyloids of the plaque core and of the congophilic angiopathy. There are superficial resemblances between the solubility characteristics of the plaque core and some of the properties of scrapie infectivity, but there are no similarities in amino acid sequences between the plaque core and scrapie polypeptides.

Plaques containing amyloid β-peptides (Aβ) are a major feature in Alzheimer's disease (AD), and GM1 ganglioside is an important component of cellular plasma membranes and especially enriched in lipid raft. GM1-bound Aβ (GM1/Aβ), found in brains exhibiting early pathological changes of AD including diffuse plaques, has been suggested to be involved in the initiation of amyloid fibril formation in vivo by acting as a seed. However, the role of GM1 in amyloid β- protein precursor (APP) processing is not yet defined. In this study, we report that exogenous GM1 ganglioside promotes Aβ biogenesis and decreases sAPPα secretion in SH-SY5Y and COS7 cells stably transfected with human APP695 cDNA without affecting full-length APP and the sAPPβ levels. We also observe that GM1 increases extracellular levels of Aβ in primary cultures of mixed rat cortical neurons transiently transfected with human APP695 cDNA. These findings suggest a regulatory role for GM1 in APP processing pathways. [ABSTRACT FROM AUTHOR]

Previous studies have shown that a minor glycoform of acetylcholinesterase (AChE) is increased in Alzheimer's disease brain and cerebrospinal fluid. This glycoform can be distinguished from other AChE species by its lack of binding to concanavalin A (Con A). In this study, the temporal relationship between AChE glycosylation and Aβ deposition was examined in Tg2576 mice. There was a significant (p < 0.05) difference in AChE glycosylation in Tg2576 mice compared with age-matched background strain control mice at 4 months of age. This difference in glycosylation was also observed in 8- and 12-month-old Tg2576 mice. In contrast, Aβ plaques were only seen in the Tg2576 mice at 12 months of age, and were not detected at 4 and 8 months of age. Soluble human-sequence Aβ was detected as early as 4 months of age in the transgenic mice. The altered AChE glycosylation was due to an increase in a minor AChE isoform, which did not bind Con A, similar to that previously observed to be increased in Alzheimer's disease brain and cerebrospinal fluid. The results demonstrate that in transgenic mice altered AChE glycosylation is associated with very early events in the development of AD-like pathology. The study supports the possibility that glycosylation may also be a useful biomarker of AD. [ABSTRACT FROM AUTHOR]

The accumulation of amyloid plaques and amyloid congophilic angiopathy (ACA) in the brains of affected individuals is one of the main pathological features of Alzheimer's disease. Within these deposits, the βA4 (Aß) polypeptide represents a major component with the C-terminal 39–43 amino acid variants being most abundant. Using a mouse IgG1 MoAb produced by hybridoma βA4[35–43]-95.2 3B9, which reacts with the epitope is defined by the amino acid residues βA438[GVV]40, this study has identified a unique conformation within the carboxyl terminus of human βA4[1–42]. Although the βA438[GVV]40 sequence is present within the C-termini of human βA4[1–40] and βA4[1–43] and the βA4-containing region of human APP, the βA4[35–43]-95.2 3B9 MoAb (designated MoAb 3B9) does not bind these polypeptides, demonstrating a high degree of specificity for the βA438[GVV]40 epitope as presented within the βA4[1–42] sequence. The βA4[1–42] epitope bound by MoAb 3B9 is sensitive to heating (100°C for 5 min) and is denatured by SDS but not by oxidative radio-iodination of βA4 or by adsorption to plastic surfaces or nitrocellulose. The recognition of βA4 plaque deposits and ACA by MoAb 3B9 within formalin-fixed sections of human AD brain demonstrates the potential of these antibodies for investigating the role of the unique βA4[1–42] conformation in the development of Alzheimer's disease. [ABSTRACT FROM AUTHOR]

Peptide aldehyde inhibitors of the chymotrypsin-like activity of the proteasome (CLIP) such as N-acetyl-Leu-Leu-Nle-H (or ALLN) have been shown previously to inhibit the secretion of β-amyloid peptide (Aβ) from cells. To evaluate more fully the role of the proteasome in this process, we have tested the effects on Aβ formation of a much wider range of peptide-based inhibitors of CLIP than published previously. The inhibitors tested included several peptide boronates, some of which proved to be the most potent peptide-based inhibitors of β-amyloid production reported so far. We found that the ability of the peptide aldehyde and boronate inhibitors to suppress Aβ formation from cells correlated extremely well with their potency as CLIP inhibitors. Thus, we conclude that the proteasome may be involved either directly or indirectly in Aβ formation. [ABSTRACT FROM AUTHOR]