Protein kinase C activation increases release of secreted amyloid precursor protein without decreasing Abeta production in human primary neuron cultures

Overexpression and altered metabolism of amyloid precursor protein (APP) resulting in increased 4 kDa amyloid beta peptide (Abeta) production are believed to play a major role in Alzheimer's disease (AD). Therefore, reducing Abeta production in the brain is a possible therapy for AD. Because AD pathology is fairly restricted to the CNS of humans, we have established human cerebral primary neuron cultures to investigate the metabolism of APP. In many cell lines and rodent primary neuron cultures, phorbol ester activation of protein kinase C (PKC) increases the release of the secreted large N-terminal fragment of amyloid precursor protein (sAPP) and decreases Abeta release (; ; ). In contrast, we find that PKC activation in human primary neurons increases the rate of sAPP release and the production of APP C-terminal fragments and 4 kDa Abeta. Our results indicate species- and cell type-specific regulation of APP metabolism. Therefore, our results curtail the use of PKC activators in controlling human brain Abeta levels.     

Modulation of secreted beta-amyloid precursor protein and amyloid beta-peptide in brain by cholesterol

The effects of dietary cholesterol on brain amyloid precursor protein (APP) processing were examined using an APP gene-targeted mouse, genetically humanized in the amyloid beta-peptide (Abeta) domain and expressing the Swedish familial Alzheimer's disease mutations. These mice express endogenous levels of APP holoprotein and abundant human Abeta. Increased dietary cholesterol led to significant reductions in brain levels of secreted APP derivatives, including sAPPalpha, sAPPbeta, Abeta1-40, and Abeta1-42, while having little to no effect on cell-associated species, including full-length APP and the COOH-terminal APP processing derivatives. The changes in levels of sAPP and Abeta in brain all were negatively correlated with serum cholesterol levels and levels of serum and brain apoE. These results demonstrate that secreted APP processing derivatives and Abeta can be modulated in the brain of an animal by diet and provide evidence that cholesterol plays a role in the modulation of APP processing in vivo. APP gene-targeted mice lacking apoE, also have high serum cholesterol levels but do not show alterations in APP processing, suggesting that effects of cholesterol on APP processing require the presence of apoE.     

Presenilin 1 regulates the processing of beta-amyloid precursor protein C-terminal fragments and the generation of amyloid beta-protein in endoplasmic reticulum and Golgi

Progressive cerebral deposition of the amyloid beta-protein (Abeta) is believed to play a pivotal role in the pathogenesis of Alzheimer's disease (AD). The highly amyloidogenic 42-residue form of Abeta (Abeta42) is the first species to be deposited in both sporadic and familial AD. Mutations in two familial AD-linked genes, presenilins 1 (PS1) and 2 (PS2), selectively increase the production of Abeta42 in cultured cells and the brains of transgenic mice, and gene deletion of PS1 shows that it is required for normal gamma-secretase cleavage of the beta-amyloid precursor protein (APP) to generate Abeta. To establish the subcellular localization of the PS1 regulation of APP processing to Abeta, fibroblasts from PS1 wild-type (wt) or knockout (KO) embryos as well as Chinese hamster ovary (CHO) cells stably transfected with wt or mutant PS1 were subjected to subcellular fractionation on discontinuous Iodixanol gradients. APP C-terminal fragments (CTF) were markedly increased in both endoplasmic reticulum- (ER-) and Golgi-rich fractions of fibroblasts from KO mice; moreover, similar increases were documented directly in KO brain tissue. No change in the subcellular distribution of full-length APP was detectable in fibroblasts lacking PS1. In CHO cells, a small portion of APP, principally the N-glycosylated isoform, formed complexes with PS1 in both ER- and Golgi-rich fractions, as detected by coimmunoprecipitation. When the same fractions were analyzed by enzyme-linked immunosorbent assays for Abetatotal and Abeta42, Abeta42 was the major Abeta species in the ER fraction (Abeta42:Abetatotal ratio 0.5-1.0), whereas absolute levels of both Abeta42 and Abeta40 were higher in the Golgi fraction and the Abeta42:Abetatoal ratio was 0.05-0.16 there. Mutant PS1 significantly increased Abeta42 levels in the Golgi fraction. Our results indicate PS1 and APP can interact in the ER and Golgi, where PS1 is required for proper gamma-secretase processing of APP CTFs, and that PS1 mutations augment Abeta42 levels principally in Golgi-like vesicles.     

Increased susceptibility to ischemic brain damage in transgenic mice overexpressing the amyloid precursor protein

We studied the role of the amyloid precursor protein (APP) in ischemic brain damage using transgenic mice overexpressing APP. The middle cerebral artery (MCA) was occluded in FVB/N mice expressing APP695.SWE (Swedish mutation) and in nontransgenic littermates. Infarct volume (cubic millimeters) was assessed 24 hr later in thionin-stained brain sections. The infarct produced by MCA occlusion was enlarged in the transgenics (+32 +/- 6%; n = 12; p < 0. 05; t test). Measurement of APP by ELISA revealed that, although relatively high levels of Abeta were present in the brain of the transgenics (Abeta1-40 = 80 +/- 19 pmol/g; n = 6), there were no differences between ischemic and nonischemic hemispheres (p > 0.05). The reduction in cerebral blood flow produced by MCA occlusion at the periphery of the ischemic territory was more pronounced in APP transgenics (-42 +/- 8%; n = 9) than in controls (-20 +/- 8%; n = 9). Furthermore, the vasodilatation produced by neocortical application of the endothelium-dependent vasodilator acetylcholine (10 microM) was reduced by 82 +/- 5% (n = 8; p < 0.05) in APP transgenics. The data demonstrate that APP overexpression increases the susceptibility of the brain to ischemic injury. The effect is likely to involve the Abeta-induced disturbance in endothelium-dependent vascular reactivity that leads to more severe ischemia in regions at risk for infarction. The cerebral vascular actions of peptides deriving from APP metabolism may play a role in the pathogenic effects of APP.     

The chaperone BiP/GRP78 binds to amyloid precursor protein and decreases Abeta40 and Abeta42 secretion

Recent studies of cellular amyloid precursor protein (APP) metabolism demonstrate a beta-/gamma-secretase pathway resident to the endoplasmic reticulum (ER)/Golgi resulting in intracellular generation of soluble APP (APPsbeta) and Abeta42 peptide. Thus, these intracellular compartments may be key sites of amyloidogenic APP metabolism and Alzheimer's disease pathogenesis. We hypothesized that the ER chaperone immunoglobulin binding protein (BiP/GRP78) binds to and facilitates correct folding of nascent APP. Metabolic labeling and immunoprecipitation of transiently transfected human embryonic kidney 293 cells demonstrated co-precipitation of APP with GRP78, revealing their transient interaction in the ER. Maturation of cellular APP was impaired by this interaction. Furthermore, the levels of APPs, Abeta40, and Abeta42 recovered in conditioned medium were lower compared with cells transfected with APP alone. Co-expression with APP of GRP78 T37G, an ATPase mutant, almost completely blocked cellular APP maturation as well as recovery of APPs, Abeta40, and Abeta42 in conditioned medium. The inhibitory effects of GRP78 and GRP78 T37G on Abeta40 and Abeta42 secretion were magnified by co-expression with the Swedish mutation of APP (K670N/M671L). Collectively, these data suggest a transient and direct interaction of GRP78 with APP in the ER that modulates intracellular APP maturation and processing and may facilitate its correct folding.     

Interaction of a neuron-specific protein containing PDZ domains with Alzheimer's amyloid precursor protein

A novel protein, human X11-like (human X11L), contains a phosphotyrosine interaction (PI) domain and two PDZ domains and displays 55.2% amino acid homology with the human X11 (human X11). The PI domain of human X11L interacts with a sequence containing the NPXY motif found in the cytoplasmic domain of Alzheimer's amyloid precursor protein. A construct lacking the carboxyl-terminal domain, which comprises two PDZ domains (N + PI), enhances PI binding to APP, whereas another construct lacking an amino-terminal domain relative to PI domain (PI + C) suppresses PI binding to APP. Overexpression of full-length human X11L (N + PI + C) in cells that express APP695 stably decreased the secretion of Abeta40 but not that of Abeta42. However, overexpression of the PI domain alone and the N + PI construct in cells did not affect the secretion of Abeta despite their ability to bind to the cytoplasmic domain of Alzheimer's amyloid precursor protein. These observations suggest that the amino-terminal domain regulates PI binding to APP and that the carboxyl-terminal domain containing PDZ motifs is essential to modulate APP processing. Because expression of the human X11L gene is specific to brain, the present observations should contribute to shedding light on the molecular mechanism of APP processing in Alzheimer's disease.     

Amyloid beta peptide of Alzheimer's disease downregulates Bcl-2 and upregulates bax expression in human neurons

Neuronal apoptosis is a suspected cause of neurodegeneration in Alzheimer's disease (AD). Increased levels of amyloid beta peptide (Abeta) induce neuronal apoptosis in vitro and in vivo. The underlying molecular mechanism of Abeta neurotoxicity is not clear. The normal concentration of Abeta in cerebrospinal fluid is 4 nM. We treated human neuron primary cultures with 100 nM amyloid beta peptides Abeta(1-40) and Abeta(1-42) and the control reverse peptide Abeta(40-1). We find that although little neuronal apoptosis is induced by either peptide after 3 d of treatment, Abeta(1-42) provokes a rapid and sustained downregulation of a key anti-apoptotic protein, bcl-2, whereas it increases levels of bax, a protein known to promote cell death. In contrast, the Abeta(1-40) downregulation of bcl-2 is gradual, although the levels are equivalent to those of Abeta(1-42)-treated neurons by 72 hr of treatment. Abeta(1-40) does not upregulate bax levels. The control, reverse peptide Abeta(40-1), does not affect either bcl-2 or bax protein levels. In addition, we found that the Abeta(1-40)- and Abeta(1-42)- but not Abeta(40-1)-treated neurons had increased vulnerability to low levels of oxidative stress. Therefore, we propose that although high physiological amounts of Abeta are not sufficient to induce apoptosis, Abeta depletes the neurons of one of its anti-apoptotic mechanisms. We hypothesize that increased Abeta in individuals renders the neurons vulnerable to age-dependent stress and neurodegeneration.     

Amyloid precursor protein processing in sterol regulatory element-binding protein site 2 protease-deficient Chinese hamster ovary cells

Amyloid peptides of 39-43 amino acids (Abeta) are the major constituents of amyloid plaques present in the brains of Alzheimer's (AD) patients. Proteolytic processing of the amyloid precursor protein (APP) by the yet unidentified beta- and gamma-secretases leads to the generation of the amyloidogenic Abeta peptides. Recent data suggest that all of the known mutations leading to early onset familial AD alter the processing of APP such that increased amounts of the 42-amino acid form of Abeta are generated by a gamma-secretase activity. Identification of the beta- and/or gamma-secretases is a major goal of current AD research, as they are prime targets for therapeutic intervention in AD. It has been suggested that the sterol regulatory element-binding protein site 2 protease (S2P) may be identical to the long sought gamma-secretase. We have directly tested this hypothesis using over-expression of the S2P cDNA in cells expressing APP and by characterizing APP processing in mutant Chinese hamster ovary cells that are deficient in S2P activity and expression. The data demonstrate that S2P does not play an essential role in the generation or secretion of Abeta peptides from cells, thus it is unlikely to be a gamma-secretase.     

Cleavage of Alzheimer's amyloid precursor protein (APP) by secretases occurs after O-glycosylation of APP in the protein secretory pathway. Identification of intracellular compartments in which APP cleavage occurs without using toxic agents that interfere with protein metabolism

beta-Amyloid peptide (Abeta) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. Abeta is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N- and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and Abeta generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defective O-glycosylation, we demonstrated that the majority of APP cleavage by alpha-, beta-, and gamma-secretases occurs after O-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (alphaAPPCOOH), a product of alpha-secretase, and both Abeta40 and Abeta42 in medium, a product of beta- and gamma-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by alpha-, beta-, and gamma-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway.     

Human infection by Rhizopus azygosporus and the occurrence of azygospores in Zygomycetes

We characterized the secretion of amyloid precursor protein (APP) in rat primary neurons and conditionally immortalized central nervous system (CNS) derived progenitor cell lines, to evaluate their suitability as models for studies on APP metabolism. We observed that primary cells dissociated from different regions of the embryonic brain secreted progressively more APP during in vitro maturation. The increase in basal secretion resulted in an offset of the response to protein kinase C (PKC) activated secretion and was correlated with an increased PKC alpha expression. The same type of analysis was performed on CNS derived conditionally immortalized cells whose growth potential becomes restricted upon shifting of the culture conditions to a non-permissive temperature (39 degrees C), an event that coincides with their progression toward differentiation. With a pattern consistent with that observed in primary neurons, conditionally immortalized cells exposed to 39 degrees C showed an offset of the secretory response to activation by PdBu and also an increased expression of PKC alpha. These data suggest that APP secretion and its regulation in CNS derived cells is influenced by the proliferative status of the cells.     

Disturbances of the blood-brain barrier without expression of amyloid precursor protein- containing neuritic clusters or neuronal loss during late stages of thiamine deficiency in guinea pigs

Generalized oxidative deficits associated with experimental thiamine deficiency (TD) lead to selective neurodegeneration. In mouse brain, TD produces region-specific breach of the blood-brain barrier (BBB), neuronal loss and an accumulation of amyloid precursor protein (APP) in abnormal neurites. The APP-laden abnormal neurites within the damaged areas of mouse brain aggregate into neuritic clusters which strikingly resemble the neuritic component of Alzheimer amyloid plaques. However, amyloid beta-peptide (Abeta) immunoreactivity has not been demonstrated in these neuritic clusters, possibly because the Abeta region of APP in mice contains three amino acid substitutions as compared with the amino acid sequence of human Abeta. In contrast, the guinea pig nucleic acid sequence is more related to the human sequence and the Abeta region is identical in sequence to that of human APP. Thus, the current studies tested whether the presence of an authentic Abeta fragment of APP (i.e., identical to that of man) might make guinea pigs more vulnerable to the development of Abeta-containing neuritic clusters following TD. During late stages of TD, BBB abnormalities, manifested by immunoglobulin G (IgG) extravasation and increased NADPH diaphorase reactivity in microvessels, occurred in brain areas known to be damaged by TD in mice. However, despite the prolonged thiamine deprivation and the advanced neurological symptoms of guinea pigs, no significant neuronal loss or altered APP/Abeta immunostaining occurred in any brain region. Microglial activation, another early marker of damage in mice, was not evident in thiamine-deficient guinea pig brain. Ferritin immunoreactivity and iron deposition in oligodendrocytes within areas of BBB abnormalities were either slightly enhanced or unchanged as compared to controls. This is the first report of brain abnormalities in the guinea pig model of dietary and pyrithiamine-induced TD. The results demonstrate species differences in the response to TD-induced damage, and further support the role of BBB and nitric oxide in the initial events in TD pathology.     

Evidence that tumor necrosis factor alpha converting enzyme is involved in regulated alpha-secretase cleavage of the Alzheimer amyloid protein precursor

The amyloid protein, Abeta, which accumulates in the brains of Alzheimer patients, is derived by proteolysis of the amyloid protein precursor (APP). APP can undergo endoproteolytic processing at three sites, one at the amino terminus of the Abeta domain (beta-cleavage), one within the Abeta domain (alpha-cleavage), and one at the carboxyl terminus of the Abeta domain (gamma-cleavage). The enzymes responsible for these activities have not been unambiguously identified. By the use of gene disruption (knockout), we now demonstrate that TACE (tumor necrosis factor alpha converting enzyme), a member of the ADAM family (a disintegrin and metalloprotease-family) of proteases, plays a central role in regulated alpha-cleavage of APP. Our data suggest that TACE may be the alpha-secretase responsible for the majority of regulated alpha-cleavage in cultured cells. Furthermore, we show that inhibiting this enzyme affects both APP secretion and Abeta formation in cultured cells.     

Amyloid beta-protein reduces acetylcholine synthesis in a cell line derived from cholinergic neurons of the basal forebrain

The characteristic features of a brain with Alzheimer disease (AD) include the presence of neuritic plaques composed of amyloid beta-protein (Abeta) and reductions in the levels of cholinergic markers. Neurotoxic responses to Abeta have been reported in vivo and in vitro, suggesting that the cholinergic deficit in AD brain may be secondary to the degeneration of cholinergic neurons caused by Abeta. However, it remains to be determined if Abeta contributes to the cholinergic deficit in AD brain by nontoxic effects. We examined the effects of synthetic Abeta peptides on the cholinergic properties of a mouse cell line, SN56, derived from basal forebrain cholinergic neurons. Abeta 1-42 and Abeta 1-28 reduced the acetylcholine (AcCho) content of the cells in a concentration-dependent fashion, whereas Abeta 1-16 was inactive. Maximal reductions of 43% and 33% were observed after a 48-h treatment with 100 nM of Abeta 1-42 and 50 pM of Abeta 1-28, respectively. Neither Abeta 1-28 nor Abeta 1-42 at a concentration of 100 nM and a treatment period of 2 weeks was toxic to the cells. Treatment of the cells with Abeta 25-28 (48 h; 100 nM) significantly decreased AcCho levels, suggesting that the sequence GSNK (aa 25-28) is responsible for the AcCho-reducing effect of Abeta. The reductions in AcCho levels caused by Abeta 1-42 and Abeta 1-28 were accompanied by proportional decreases in choline acetyltransferase activity. In contrast, acetylcholinesterase activity was unaltered, indicating that Abeta specifically reduces the synthesis of AcCho in SN56 cells. The reductions in AcCho content caused by Abeta 1-42 could be prevented by a cotreatment with all-trans-retinoic acid (10 nM), a compound previously shown to increase choline acetyltransferase mRNA expression in SN56 cells. These results demonstrate a nontoxic, suppressive effect of Abeta on AcCho synthesis, an action that may contribute to the cholinergic deficit in AD brain.     

Zyme, a novel and potentially amyloidogenic enzyme cDNA isolated from Alzheimer's disease brain

The deposition of the beta amyloid peptide in neuritic plaques and cerebral blood vessels is a hallmark of Alzheimer's disease (AD) pathology. The major component of the amyloid deposit is a 4.2-kDa polypeptide termed amyloid beta-protein of 39-43 residues, which is derived from processing of a larger amyloid precursor protein (APP). It is hypothesized that a chymotrypsin-like enzyme is involved in the processing of APP. We have discovered a new serine protease from the AD brain by polymerase chain reaction amplification of DNA sequences representing active site homologous regions of chymotrypsin-like enzymes. A cDNA clone was identified as one out of one million that encodes Zyme, a serine protease. Messenger RNA encoding Zyme can be detected in some mammalian species but not in mice, rats, or hamster. Zyme is expressed predominantly in brain, kidney, and salivary gland. Zyme mRNA cannot be detected in fetal brain but is seen in adult brain. The Zyme gene maps to chromosome 19q13.3, a region which shows genetic linkage with late onset familial Alzheimer's disease. When Zyme cDNA is co-expressed with the APP cDNA in 293 (human embryonic kidney) cells, amyloidogenic fragments are detected using C-terminal antibody to APP. These co-transfected cells release an abundance of truncated amyloid beta-protein peptide and shows a reduction of residues 17-42 of Abeta (P3) peptide. Zyme is immunolocalized to perivascular cells in monkey cortex and the AD brain. In addition, Zyme is localized to microglial cells in our AD brain sample. The amyloidogenic potential and localization in brain may indicate a role for this protease in amyloid precursor processing and AD.     

Increased amyloidogenic secretion in cerebellar granule cells undergoing apoptosis

Some clues suggest that neuronal damage induces a secondary change of amyloid beta protein (Abeta) metabolism. We investigated this possibility by analyzing the secretion of Abeta and processing of its precursor protein (amyloid precursor protein, APP) in an in vitro model of neuronal apoptosis. Primary cultures of rat cerebellar granule neurons were metabolically labeled with [35S]methionine. Apoptosis was induced by shifting extracellular KCl concentration from 25 mM to 5 mM for 6 h. Control and apoptotic neurons were then subjected to depolarization-stimulated secretion. Constitutive and stimulated secretion media and cell lysates were immunoprecipitated with antibodies recognizing regions of Abeta, full-length APP, alpha- and beta-APP secreted forms. Immunoprecipitated proteins were separated by SDS/PAGE and quantitated with a PhosphorImager densitometer. Although intracellular full-length APP was not significantly changed after apoptosis, the monomeric and oligomeric forms of 4-kDa Abeta were 3-fold higher in depolarization-stimulated secretion compared with control neurons. Such increments were paralleled by a corresponding increase of the beta-APPs/alpha-APPs ratio in apoptotic secretion. Immunofluorescence studies performed with an antibody recognizing an epitope located in the Abeta sequence showed that the Abeta signal observed in the cytoplasm and in the Golgi apparatus of control neurons is uniformly redistributed in the condensed cytoplasm of apoptotic cells. These studies indicate that neuronal apoptosis is associated with a significant increase of metabolic products derived from beta-secretase cleavage and suggest that an overproduction of Abeta may be the consequence of neuronal damage from various causes.     

Cholesterol depletion inhibits the generation of beta-amyloid in hippocampal neurons

The amyloid precursor protein (APP) plays a crucial role in the pathogenesis of Alzheimer's disease. During intracellular transport APP undergoes a series of proteolytic cleavages that lead to the release either of an amyloidogenic fragment called beta-amyloid (Abeta) or of a nonamyloidogenic secreted form consisting of the ectodomain of APP (APPsec). It is Abeta that accumulates in the brain lesions that are thought to cause the disease. By reducing the cellular cholesterol level of living hippocampal neurons by 70% with lovastatin and methyl-beta-cyclodextrin, we show that the formation of Abeta is completely inhibited while the generation of APPsec is unperturbed. This inhibition of Abeta formation is accompanied by increased solubility in the detergent Triton X-100 and is fully reversible by the readdition of cholesterol to previously depleted cells. Our results show that cholesterol is required for Abeta formation to occur and imply a link between cholesterol, Abeta, and Alzheimer's disease.     

Inhibition of Alzheimer beta-fibrillogenesis by melatonin

It is generally postulated that the amyloid beta protein (Abeta) plays a central role in the progressive neurodegeneration observed in Alzheimer's disease. Important pathologic properties of this protein, such as neurotoxicity and resistance to proteolytic degradation, depend on the ability of Abeta to form beta-sheet structures or amyloid fibrils. We report that melatonin, a hormone recently found to protect neurons against Abeta toxicity, interacts with Abeta1-40 and Abeta1-42 and inhibits the progressive formation of beta-sheets and amyloid fibrils. These interactions between melatonin and the amyloid peptides were demonstrated by circular dichroism and electron microscopy for Abeta1-40 and Abeta1-42 and by nuclear magnetic resonance spectroscopy for Abeta1-40. Inhibition of beta-sheets and fibrils could not be accomplished in control experiments when a free radical scavenger or a melatonin analog were substituted for melatonin under otherwise identical conditions. In sharp contrast with conventional anti-oxidants and available anti-amyloidogenic compounds, melatonin crosses the blood-brain barrier, is relatively devoid of toxicity, and constitutes a potential new therapeutic agent in Alzheimer's disease.     

Alzheimer amyloid protein precursor in the rat hippocampus: transport and processing through the perforant path

Amyloid deposition is a neuropathological hallmark of Alzheimer's disease. The principal component of amyloid deposits is beta amyloid peptide (Abeta), a peptide derived by proteolytic processing of the amyloid precursor protein (APP). APP is axonally transported by the fast anterograde component. Several studies have indicated that Abeta deposits occur in proximity to neuritic and synaptic profiles. Taken together, these latter observations have suggested that APP, axonally transported to nerve terminals, may be processed to Abeta at those sites. To examine the fate of APP in the CNS, we injected [35S]methionine into the rat entorhinal cortex and examined the trafficking and processing of de novo synthesized APP in the perforant pathway and at presynaptic sites in the hippocampal formation. We report that both full-length and processed APP accumulate at presynaptic terminals of entorhinal neurons. Finally, we demonstrate that at these synaptic sites, C-terminal fragments of APP containing the entire Abeta domain accumulate, suggesting that these species may represent the penultimate precursors of synaptic Abeta.     

Atomic force microscopic imaging of seeded fibril formation and fibril branching by the Alzheimer's disease amyloid-beta protein

BACKGROUND: Amyloid plaques composed of the fibrillar form of the amyloid-beta protein (Abeta) are the defining neuropathological feature of Alzheimer's disease (AD). A detailed understanding of the time course of amyloid formation could define steps in disease progression and provide targets for therapeutic intervention. Amyloid fibrils, indistinguishable from those derived from an AD brain, can be produced in vitro using a seeded polymerization mechanism. In its simplest form, this mechanism involves a cooperative transition from monomeric Abeta to the amyloid fibril without the buildup of intermediates. Recently, however, a transient species, the Abeta amyloid protofibril, has been identified. Here, we report studies of Abeta amyloid protofibril and its seeded transition into amyloid fibrils using atomic force microscopy. RESULTS: Seeding of the protofibril-to-fibril transition was observed. Preformed fibrils, but not protofibrils, effectively seeded this transition. The assembly state of Abeta influenced the rate of seeded growth, indicating that protofibrils are fibril assembly precursors. The handedness of the helical surface morphology of fibrils depended on the chirality of Abeta. Finally, branched and partially wound fibrils were observed. CONCLUSIONS: The temporal evolution of morphologies suggests that the protofibril-to-fibril transition is nucleation-dependent and that protofibril winding is involved in that transition. Fibril unwinding and branching may be essential for the post-nucleation growth process. The protofibrillar assembly intermediate is a potential target for AD therapeutics aimed at inhibiting amyloid formation and AD diagnostics aimed at detecting presymptomatic disease.