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Cleavage of the amyloid precursor protein (APP) is a crucial event in Alzheimer disease pathogenesis that creates the amyloid-beta peptide (Abeta) and liberates the carboxy-terminal APP intracellular domain (AICD) into the cytosol. The interaction of the APP C terminus with the adaptor protein Fe65 mediates APP trafficking and signalling, and is thought to regulate APP processing and Abeta generation. We determined the crystal structure of the AICD in complex with the C-terminal phosphotyrosine-binding (PTB) domain of Fe65. The unique interface involves the NPxY PTB-binding motif and two alpha helices. The amino-terminal helix of the AICD is capped by threonine T(668), an Alzheimer disease-relevant phosphorylation site involved in Fe65-binding regulation. The structure together with mutational studies, isothermal titration calorimetry and nuclear magnetic resonance experiments sets the stage for understanding T(668) phosphorylation-dependent complex regulation at a molecular level. A molecular switch model is proposed.

The neuronal adaptor protein Fe65 is involved in brain development, Alzheimer disease amyloid precursor protein (APP) signaling, and proteolytic processing of APP. It contains three protein-protein interaction domains, one WW domain, and a unique tandem array of phosphotyrosine-binding (PTB) domains. The N-terminal PTB domain (Fe65-PTB1) was shown to interact with a variety of proteins, including the low density lipoprotein receptor-related protein (LRP-1), the ApoEr2 receptor, and the histone acetyltransferase Tip60. We have determined the crystal structures of human Fe65-PTB1 in its apo- and in a phosphate-bound form at 2.2 and 2.7A resolution, respectively. The overall fold shows a PTB-typical pleckstrin homology domain superfold. Although Fe65-PTB1 has been classified on an evolutionary basis as a Dab-like PTB domain, it contains attributes of other PTB domain subfamilies. The phosphotyrosine-binding pocket resembles IRS-like PTB domains, and the bound phosphate occupies the binding site of the phosphotyrosine (Tyr(P)) within the canonical NPXpY recognition motif. In addition Fe65-PTB1 contains a loop insertion between helix alpha2 and strand beta2(alpha2/beta2 loop) similar to members of the Shc-like PTB domain subfamily. The structural comparison with the Dab1-PTB domain reveals a putative phospholipid-binding site opposite the peptide binding pocket. We suggest Fe65-PTB1 to interact with its target proteins involved in translocation and signaling of APP in a phosphorylation-dependent manner.

Alzheimer's disease is associated with typical brain deposits (senile plaques) that mainly contain the neurotoxic amyloid beta peptide. This peptide results from proteolytic processing of the type I transmembrane protein amyloid precursor protein (APP). During this proteolytic pathway the APP intracellular domain (AICD) is released into the cytosol, where it associates with various adaptor proteins. The interaction of the AICD with the C-terminal phosphotyrosine-binding domain of Fe65 (Fe65-PTB2) regulates APP translocation, signalling and processing. Human AICD and Fe65-PTB2 have been cloned, overproduced and purified in large amounts in Escherichia coli. A complex of Fe65-PTB2 with the C-terminal 32 amino acids of the AICD gave well diffracting hexagonal crystals and data have been collected to 2.1 A resolution. Initial phases obtained by the molecular-replacement method are of good quality and revealed well defined electron density for the substrate peptide.

Fe65 is a three-domain neuronal adaptor protein involved in brain development and amyloid precursor protein (APP) signalling. The phosphotyrosine-binding domain 1 (PTB1) of human Fe65 has been cloned, overexpressed, purified and crystallized using the hanging-drop vapour-diffusion method. Native crystals belong to the space group R3 and diffract to 2.6 A resolution. This crystal form suffered from high thermal B factors and pseudo-symmetry, resulting in a bisection of the c axis. Co-crystallization with a mercury compound under similar conditions induced an orthorhombic crystal form in the space group P2(1)2(1)2(1) diffracting to 2.2 A resolution. SAD phases have been computed to the diffraction limit at the wavelength of maximum absorption (L(III) edge).

Understanding the intracellular transport of the beta-amyloid precursor protein (APP) is a major key to elucidate the regulation of APP processing and thus beta-amyloid peptide generation in Alzheimer disease pathogenesis. APP and its two paralogues, APLP1 and APLP2 (APLPs), are processed in a very similar manner by the same protease activities. A putative candidate involved in APP transport is protein interacting with APP tail 1 (PAT1), which was reported to interact with the APP intracellular domain. We show that PAT1a, which is 99.0% identical to PAT1, binds to APP, APLP1, and APLP2 in vivo and describe their co-localization in trans-Golgi network vesicles or endosomes in primary neurons. We further demonstrate a direct interaction of PAT1a with the basolateral sorting signal of APP/APLPs. Moreover, we provide evidence for a direct role of PAT1a in APP/APLP transport as overexpression or RNA interference-mediated knockdown of PAT1a modulates APP/APLPs levels at the cell surface. Finally, we show that PAT1a promotes APP/APLPs processing, resulting in increased secretion of beta-amyloid peptide. Taken together, our data establish PAT1a as a functional link between APP/APLPs transport and their processing.

The amyloid precursor protein (APP) plays a central role in Alzheimer's disease, but its physiological function and that of its mammalian paralogs, the amyloid precursor-like proteins 1 and 2 (APLPs), is still poorly understood. APP has been proposed to form dimers, a process that could promote cell adhesion via trans-dimerization. We investigated the dimerization and cell adhesion properties of APP/APLPs and provide evidence that all three paralogs are capable of forming homo- and heterocomplexes. Moreover, we show that trans-interaction of APP family proteins promotes cell-cell adhesion in a homo- and heterotypic fashion and that endogenous APLP2 is required for cell-cell adhesion in mouse embryonic fibroblasts. We further demonstrate interaction of all the three APP family members in mouse brain, genetic interdependence, and molecular interaction of APP and APLPs in synaptically enriched membrane compartments. Together, our results provide evidence that homo- and heterocomplexes of APP/APLPs promote trans-cellular adhesion in vivo.

The view that only the production and deposition of Abeta plays a decisive role in Alzheimer's disease has been challenged by recent evidence from different model systems, which attribute numerous functions to the amyloid precursor protein (APP). To investigate the potential cellular functions of APP and its paralogs, we use transgenic Drosophila as a model. Upon overexpression of the APP-family members, transformations of cell fates during the development of the peripheral nervous system were observed. Genetic analysis showed that APP, APLP1 and APLP2 induce Notch gain-of-function phenotypes, identified Numb as a potential target and provided evidence for a direct involvement of Disabled and Neurotactin in the induction of the phenotypes. The severity of the induced phenotypes not only depended on the dosage and the particular APP-family member but also on particular domains of the molecules. Studies with Drosophila APPL confirmed the results obtained with human proteins and the analysis of flies mutant for the appl gene further supports an involvement of APP-family members in neuronal development and a crosstalk between the APP family and Notch.

Amyloid precursor protein (APP) processing is of major interest in Alzheimer's disease research, since sequential cleavages by beta- and gamma-secretase lead to the formation of the 4-kDa amyloid Abeta protein peptide that accumulates in Alzheimer's disease brain. The processing of APP involves proteolytic conversion by different secretases leading to alpha-, beta-, gamma-, delta-, and epsilon-cleavages. Since modulation of these cleavages represents a rational therapeutic approach to control amyloid formation, its interference with the processing of the members of the APP gene family is of considerable importance. By using C-terminally tagged constructs of APLP-1 and APLP-2 and the untagged proteins, we have characterized their proteolytic C-terminal fragments produced in stably transfected SH-SY5Y cells. Pharmacological manipulation with specific protease inhibitors revealed that both homologues are processed by alpha- and gamma-secretase-like cleavages, and that their intracellular domains can be released by cleavage at epsilon-sites. APLP-2 processing appears to be the most elaborate and to involve alternative cleavage sites. We show that APLP-1 is the only member of the APP gene family for which processing can be influenced by N-glycosylation. Additionally, we were able to detect p3-like fragments of APLP-1 and p3-like and Abeta-like fragments of APLP-2 in the media of stably transfected SH-SY5Y cells.

The Alzheimer's disease Abeta peptide can increase the levels of cell-associated amyloid precursor protein (APP) in vitro. To determine the specificity of this response for Abeta and whether it is related to cytotoxicity, we tested a diverse range of fibrillar peptides including amyloid-beta (Abeta), the fibrillar prion peptides PrP106-126 and PrP178-193 and human islet-cell amylin. All these peptides increased the levels of APP and amyloid precursor-like protein 2 (APLP2) in primary cultures of astrocytes and neurons. Specificity was shown by a lack of change to amyloid precursor-like protein 1, tau-1 and cellular prion protein (PrP(c)) levels. APP and APLP2 levels were elevated only in cultures exposed to fibrillar peptides as assessed by electron microscopy and not in cultures treated with non-fibrillogenic peptide variants or aggregated lipoprotein. We found that PrP106-126 and the non-toxic but fibril-forming PrP178-193 increased APP levels in cultures derived from both wild-type and PrP(c)-deficient mice indicating that fibrillar peptides up-regulate APP through a non-cytotoxic mechanism and irrespective of parental protein expression. Fibrillar PrP106-126 and Abeta peptides bound recombinant APP and APLP2 suggesting the accumulation of these proteins was mediated by direct binding to the fibrillated peptide. This was supported by decreased APP accumulation following extensive washing of the cultures to remove fibrillar aggregates. Pre-incubation of fibrillar peptide with recombinant APP18-146, the putative fibril binding site, also abrogated the accumulation of APP. These findings show that diverse fibrillogenic peptides can induce accumulation of APP and APLP2 and this mechanism could contribute to pathogenesis in neurodegenerative disorders.

Alpha-synuclein (alphaSN) has been implicated in Parkinson's Disease (PD) and alphaSN is a major component of Lewy bodies (LBs). This study explored platelets as a model system for study of alphaSN metabolism and platelet alphaSN as a diagnostic marker for PD. We used Western blot analysis to characterize and compare platelet and brain alpha-, beta- and gammaSN; and to quantitate alphaSN levels in platelets from PD and age-matched controls. We found that platelets contain full-length alphaSN and 6 and 12 kDa fragments, and gammaSN-like protein. alphaSN and gammaSN were not secreted by thrombin-activated platelets. Furthermore, we also found that the alphaSN and gammaSN levels in sporadic PD patients and age-matched normal controls were not significantly different. This indicates that platelet alphaSN or gammaSN is not a suitable peripheral diagnostic marker for PD. Platelets may be used for study of alphaSN and gammaSN metabolism, and may give some broad insight into the normal functions of alphaSN and gammaSN.

Intracellular inclusions containing alpha-synuclein (alpha SN) are pathognomonic features of several neurodegenerative disorders. Inclusions occur in oligodendrocytes in multiple system atrophy (MSA) and in neurons in dementia with Lewy bodies (DLB) and Parkinson's disease (PD). In order to identify disease-associated changes of alpha SN, this study compared the levels, solubility and molecular weight species of alpha SN in brain homogenates from MSA, DLB, PD and normal aged controls. In DLB and PD, substantial amounts of detergent-soluble and detergent-insoluble alpha SN were detected compared with controls in grey matter homogenate. Compared with controls, MSA cases had significantly higher levels of alpha SN in the detergent-soluble fraction of brain samples from pons and white matter but detergent-insoluble alpha SN was not detected. There was an inverse correlation between buffered saline-soluble and detergent-soluble levels of alpha SN in individual MSA cases suggesting a transition towards insolubility in disease. The differences in solubility of alpha SN between grey and white matter in disease may result from different processing of alpha SN in neurons compared with oligodendrocytes. Highly insoluble alpha SN is not involved in the pathogenesis of MSA. It is therefore possible that buffered saline-soluble or detergent-soluble forms of alpha SN are involved in the pathogenesis of other alpha SN-related diseases.

The contribution of alpha-synuclein accumulation in Alzheimer's disease (AD) plaques is currently a matter of scientific debate. In the present study antisera against the N- and C-terminus, the full-length protein and the central so-called non-amyloid component (NAC) domain of the alpha-synuclein protein were used to address this question in brains of cases with typical AD and of cases with the Lewy body (LB) variant of AD. In typical AD cases, none of the antisera revealed evidence for co-accumulation of alpha-synuclein with extracellular A beta peptides in plaques or in dystrophic neurites decorating the plaque core. Interestingly, cases with mixed pathology of the LB variant of AD revealed accumulation of alpha-synuclein in LBs and in dystrophic neurites of A beta plaques.

The alpha-synuclein (alpha SN) protein is thought to play a central role in the pathogenesis of neurodegenerative diseases where it aggregates to form intracellular inclusions. We have used Western blotting to examine the expression levels and solubility of alpha SN in brain homogenates from dementia with Lewy bodies (DLB), Parkinson's disease (PD), Alzheimer's disease (AD), and normal controls using samples from the parahippocampus/transentorhinal cortex. Compared to controls, DLB brains accumulate significantly greater amounts of sodium dodecyl sulfate (SDS)-soluble and SDS-insoluble alpha SN but levels of TBS-soluble alpha SN did not change. Levels of synaptophysin, a marker of synaptic integrity, were significantly lower in DLB cases than in normal aged controls regardless of whether concurrent changes of AD were present. This limbic synaptic dysfunction may contribute to cognitive impairment in DLB. Whether aggregated alpha SN is a cause or effect of the disease process in DLB and PD remains to be determined, but the presence of aggregated alpha SN is consistent with a pathogenesis similar to that associated with aggregates of Abeta amyloid in AD., (Copyright 2000 Academic Press.)

The amyloid precursor protein (APP) of Alzheimer's disease can reduce copper (II) to copper (I) in a cell-free system potentially leading to increased oxidative stress in neurons. We used neuronal cultures derived from APP knock-out (APP(-/-)) and wild-type (WT) mice to examine the role of APP in copper neurotoxicity. WT cortical, cerebellar, and hippocampal neurons were significantly more susceptible than their respective APP(-/-) neurons to toxicity induced by physiological concentrations of copper but not by zinc or iron. There was no difference in copper toxicity between APLP2(-/-) and WT neurons, demonstrating specificity for APP-associated copper toxicity. Copper uptake was the same in WT and APP(-/-) neurons, suggesting APP may interact with copper to induce a localized increase in oxidative stress through copper (I) production. This was supported by significantly higher levels of copper-induced lipid peroxidation in WT neurons. Treatment of neuronal cultures with a peptide corresponding to the human APP copper-binding domain (APP142-166) potentiated copper but not iron or zinc toxicity. Incubation of APP142-166 with low-density lipoprotein (LDL) and copper resulted in significantly increased lipid peroxidation compared to copper and LDL alone. Substitution of the copper coordinating histidine residues with asparagines (APP142-166(H147N, H149N, H151N)) abrogated the toxic effects. A peptide corresponding to the zinc-binding domain (APP181-208) failed to induce copper or zinc toxicity in neuronal cultures. These data support a role for the APP copper-binding domain in APP-mediated copper (I) generation and toxicity in primary neurons, a process that has important implications for Alzheimer's disease and other neurodegenerative disorders.

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.

The pathological process in Alzheimer's disease (AD) involves amyloid beta (Abeta) deposition and neuronal cell degeneration. The neurotoxic Abeta peptide is derived from the amyloid precursor protein (APP), a member of a larger gene family including the amyloid precursor-like proteins, APLP1 and APLP2. The APP and APLP2 molecules contain metal binding sites for copper and zinc. The zinc binding domain (ZnBD) is believed to have a structural rather than a catalytic role. The activity of the copper binding domain (CuBD) is unknown, however, APP reduces copper (II) to copper (I) and this activity could promote copper-mediated neurotoxicity. The expression of APP and APLP2 in the brain suggests they could have an important direct or indirect role in neuronal metal homeostasis. To examine this, we measured copper, zinc and iron levels in the cerebral cortex, cerebellum and selected non-neuronal tissues from APP (APP(-/-)) and APLP2 (APLP2(-/-)) knockout mice using atomic absorption spectrophotometry. Compared with matched wild-type (WT) mice, copper levels were significantly elevated in both APP(-/-) and APLP2(-/-) cerebral cortex (40% and 16%, respectively) and liver (80% and 36%, respectively). Copper levels were not significantly different between knockout and WT cerebellum, spleen or serum samples. There were no significant differences observed between APP(-/-), APLP2(-/-) and WT mice zinc or iron levels in any tissue examined. These findings indicate APP and APLP2 expression specifically modulates copper homeostasis in the liver and cerebral cortex, the latter being a region of the brain particularly involved in AD. Perturbations to APP metabolism and in particular, its secretion or release from neurons may alter copper homeostasis resulting in increased Abeta accumulation and free radical generation. These data support a novel mechanism in the APP/Abeta pathway which leads to AD.

Alpha-synuclein is a predominantly neuronal presynaptic protein that may play an important role during synaptogenesis and CNS development. In order to elucidate the human developmental expression profile, we used a polyclonal antiserum against the NAC domain of alpha-synuclein. In normal fetal cortex neuroectodermal precursor cells elicited staining in the soma, whereas, in adult cortex, we observed a staining pattern compatible with synaptic function. The same developmental intraneuronal redistribution was found in neurodegenerative disorders, i.e. somatic staining in neuroectodermal precursors in fetal (trisomy 21) and a synaptic pattern in adult (Down's syndrome, Alzheimer's disease) brains. RT-PCR and Western blot analysis revealed expression at all time points studied (4-7.5 months) during human brain development.

A growing body of evidence suggests that the non-Abeta component of Alzheimer's disease amyloid precursor protein (NACP) or alpha-synuclein contributes to the neurodegenerative processes in Alzheimer's disease (AD), Parkinson's disease (PD) and dementia with Lewy bodies (DLB). In the present study antisera to the N terminus and the NAC domain of the alpha-synuclein protein were employed to elucidate the expression pattern in brains of patients with AD, PD, DLB and control specimen. Alpha-synuclein exhibited an overall punctuate expression profile compatible with a synaptic function. Interestingly, while Lewy bodies were strongly immunoreactive, none of the alpha-synuclein antisera revealed staining in mature beta-amyloid plaques in AD. These observations suggest that alpha-synuclein does not contribute to late neurodegenerative processes in AD brains.

The human amyloid precursor-like protein 2 (APLP2) is a member of the Alzheimer's disease amyloid precursor protein (APP) gene family. The human APLP2 ectodomain (sAPLP2) was expressed in the yeast Pichia pastoris and the recombinant sAPLP2 was purified from the culture medium in a single step by metal-chelating Sepharose chromatography. The neuritotrophic activity of APLP2 was compared to the APP isoforms sAPP695 and sAPP751 on chick sympathetic neurones. APLP2 had neurite outgrowth-promoting activity similar to that of the APP isoforms. This suggests that APP and APLP2 have a similar or related role and supports the idea of a redundancy in function between the APP-gene family proteins.

Mutations in the presenilin-1 gene are linked to the majority of early-onset familial Alzheimer's disease cases. We have previously shown that the expression of transforming growth factor-beta is altered in Alzheimer's patients, compared to controls. Here we examine presenilin- expression in human post-mitotic neurons (hNT cells), normal human astrocytes, and human brain tumor cell lines following treatment with three isoforms of transforming growth factor-beta, or glial cell line-derived neurotrophic factor, a member of the transforming growth factor-beta superfamily. As the NT2/D1 teratocarcinoma cell line is treated with retinoic acid to induce differentiation to hNT cells, presenilin-1 messenger RNA expression is dramatically increased. Furthermore, there is a 2-3-fold increase in presenilin-1 messenger RNA expression following treatment of hNT cells with growth factors and similar results are found by Western blotting and with immunohistochemical staining for presenilin-1 protein. However, treatment of normal human astrocytes with cytokines results in minimal changes in presenilin-1 messenger RNA and protein. Interestingly, the expression of presenilin-1 in human U87 MG astrocytoma and human SK-N-SH neuroblastoma cells is only increased when cells are treated with glial cell line-derived neurotrophic factor or transforming growth factor-beta3. These findings suggest that endogenous presenilin-1 gene expression in human neurons can be induced by growth factors present in normal and diseased brain tissue. Cytokines may play a major role in regulating expression of presenilin-1 which may affect its biological actions in physiological and pathological conditions.

The Alzheimer's beta A4-amyloid protein precursor (APP) and the APP-like proteins (APLPs) are transmembrane glycoproteins with a similar modular domain structure. Alternatively spliced exons found in both genes comprise a Kunitz protease inhibitor domain encoding exon, and another exon within the divergent regions adjacent to the transmembrane domain, i.e. exon 15 of the APP gene and an exon encoding 12 residues in APLP2. Omission of the latter exons in L-APP and L-APLP2 isoforms, respectively, generates a functional recognition sequence for xylosyltransferase-mediated addition of glycosaminoglycans and proteoglycan formation. In this paper, we summarize our analyses of the regulated expression of these alternatively spliced exons in APP and APLP2 in primary cultured rat brain cells, rat brain development and aging. In conjunction with additional data for the human brain, these data provide important clues for understanding the functional significance of alternative splicing and glycosylation in APP biology. On the basis of recent results showing a higher amyloidogenicity of exon 15 encoding APP than L-APP isoforms, we further discuss the potential significance of the low levels of L-APP in neurons for the susceptibility of the brain towards Alzheimer's disease.

The formation of beta A4 amyloid protein in neuritic plaques in Alzheimer's disease (AD) and advanced age is a complex process that involves a number of both cellular and molecular mechanisms, the interrelations of which are not yet completely understood. We have examined quantitatively, in AD and aged controls an extended spectrum of amyloid plaque-related cellular and molecular factors and the cortical synaptophysin immunoreactivity (synaptic density) in order to check for interrelations between them by multifactorial analysis. In 3 cases of senile dementia of the Alzheimer type (SDAT) aged 72, 80 and 82 years, and 9 controls aged 43-88 (mean age 65) years, the cortical synaptophysin immunoreactivity was assessed, together with the numbers of neurons, astrocytes and microglial cells, senile plaques, of tangle-bearing neurons, and the amount of beta A4 amyloid precursor protein (APP) with and without the Kunitz type serine protease inhibitor (KPI) domain. The main results were: APP including the KPI domain (KPI-APP) correlated with the number of neuritic plaques, regardless of whether they occurred in SDAT or non-demented controls. There was no significant difference in the amount of KPI-APP between SDAT and controls. Conversely, APP695 (without KPI) was significantly reduced in SDAT. KPI-APP did not correlate with the synaptophysin immunoreactivity (RGVA), while APP695 showed a significant correlation with the latter in all evaluations. It also correlated with the neuron counts, which was not true for KPI-APP. These results support previous findings indicating that KPI-APP is an important local factor for amyloid deposition in the neuritic plaques, both in AD and in non-demented aged people. On the contrary, KPI-APP does not seem to be significantly involved in the mechanisms of synaptic change outside of the plaques.

We present the cDNA sequence of the rat amyloid precursor-like protein 2 (APLP2) comprising the complete coding sequence of 765 amino acid residues. By alternative splicing of two exons, transcripts encoding for 753, 709 and 697 amino acids are also generated. The derived amino acid sequence displays a sequence identity to human APLP2 of approx. 92% and to murine CDEI binding protein of approx. 95%, but differs from both by a deletion of 2 amino acids and an insertion of 4 amino acids within the acidic domain.

Neurofibrillary tangles (NFT), one of the neurodegenerative features of Alzheimer's disease, Down's syndrome and normal aging, is a constant, widespread neuropathological finding in Guamanian amyotrophic lateral sclerosis (ALS), parkinsonism-dementia (PD) and in neurologically normal Guamanians, dying of causes other than ALS and PD. NFT in brain tissue sections of patients with Guamanian ALS and PD were immunoreactive to antibodies directed against a 43-amino acid synthetic peptide homologous to amyloid beta/A4-protein (anti-SP43) associated with Alzheimer's disease. NFT extracted from frozen brain tissues of Guamanian patients with ALS and PD and from tissues of neurologically normal Guamanians were congophilic and birefringent. By negative-stain electron microscopy, NFT preparations contained bundles and/or isolated single, straight, unpaired filaments in Guamanian ALS and occasionally pairing of filaments in neurologically normal Guamanians, measuring 5-20 nm in diameter. Formic acid digestion of NFT preparations, followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and size-exclusion high-pressure liquid chromatography, showed a protein with an apparent molecular mass of 4- to 4.5-kDa, which by Western blot analysis was immunoreactive to anti-SP43. Immunoabsorption of purified NFT or SP43 with anti-SP43 abolished immunostaining. Our study corroborate previous data that amyloid beta/A4-protein is present in NFT in Guamanian PD. Furthermore, our data indicate that amyloid beta/A4-protein is present in NFT in brain tissues of patients with Guamanian ALS and in neurologically normal Guamanians, suggesting a common mechanism of amyloidogenesis with NFT formation in Alzheimer's disease and normal brain aging.

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.

Sheep are the natural hosts of the pathogens that cause scrapie, an infectious degenerative disease of the central nervous system. Scrapie-associated fibrils [and their major protein, prion protein (PrP)] accumulate in the brains of all species affected by scrapie and related diseases. PrP is encoded by a single gene that is linked to (and may be) the major gene controlling the incubation period of the various strains of scrapie pathogens. To investigate the role of PrP in natural scrapie, we have determined its gene structure and expression in the natural host. We have isolated two sheep genomic DNA clones that encode proteins of 256 amino acids with high homology to the PrPs of other species. Sheep PrPs have an arginine/glutamine polymorphism at position 171 that may be related to the alleles of the scrapie incubation-control gene in this species.

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.

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.

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.

During the past two years, more than 1,000 cases of a neurological disorder of cattle, bovine spongiform encephalopathy (BSE), have been confirmed from farms throughout Great Britain. The neurological signs and brain pathology of BSE resemble those produced in other species by the pathogens of scrapie and related disorders. The discovery of fibrils similar to scrapie-associated fibrils in detergent extracts o BSE-affected brain supported the clinical and pathological diagnosis of the disease, but has been controversial. Scrapie-associated fibrils are found in brain extracts of all species affected by scrapie and diseases caused by related pathogens. They are pathological aggregates of a neuronal membrane protein termed PrP and a protease-resistant form of PrP is a molecular marker of scrapie-associated fibrils. In this report, we show the major protein of BSE fibrils is the bovine homologue of PrP as judged by its size, protease resistance, immunoreactivity, lectin binding and partial N-terminal protein sequence. This confirms that BSE is a scrapie-like disease.

Scrapie-associated fibril protein (SAF-protein) extracted from infectious scrapie-associated fibrils (SAF) isolated from scrapie hamster brains is not infectious. SAF-protein is composed of various mol. wt. species of glycoproteins differing in carbohydrate content rather than amino acid composition. The N-linked carbohydrate chains represent approximately 40-60% of the mol. wt. of SAF-protein. The deglycosylated SAF-protein has a surprisingly low mol. wt. of approximately 7 kd, representing approximately 55 amino acid residues. This size and chemical analyses indicate that SAF-protein is an amyloid-type of protein. The simplest explanation for the available data is that SAF-polypeptide is very likely not to be part of the scrapie agent but that it is, like other amyloid proteins, derived from host-encoded proteins and not infectious. It is suggested that the infectivity of fractions rich in SAF is due to co-purification of scrapie virus and SAF caused by the high carbohydrate content of SAF-protein.

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.

Scrapie-associated fibrils (SAF) are unique structures characteristic of the group of unconventional slow infections which includes scrapie and Creutzfeldt-Jakob disease. A major component of hamster fibrils has been described as a protease-resistant glycoprotein with an apparent mol. wt of 27,000-30,000 (PrP27-30). However, we report here that if fibrils are prepared by procedures designed to minimise proteolysis the PrP proteins co-purifying with hamster SAF have mol. wts of 33,000-35,000 (PrP33-35) and 26,000-29,000 (PrP26-29). We find a Lys-Lys-Arg-Pro-Lys sequence at the amino terminus of these SAF proteins, that is absent from PrP27-30, and which has recently been predicted to be the N-terminal sequence of the native PrP protein of uninfected brain. The major SAF protein (PrP33-35) and its normal brain homologue are shown to have the same apparent mol. wt and ionic charge distribution by two-dimensional gel analysis, silver staining and immunoblotting. These results support our view that PrP33-35 and the normal brain PrP protein may have the same covalent structure, and that the PrP protein is recruited into these amyloid-like SAF or into association with a non-protein component of SAF by an irreversible event initiated directly or indirectly by scrapie infection.

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.

Scrapie-associated fibrils (SAF) are disease-specific structures found in extracts of the brains of animals affected with scrapie. These structures are pathological aggregates of a normal host protein (PrP). Abnormal post-translational modification of PrP has been suggested to explain its aberrant properties in scrapie-affected brains and although there is a form of PrP in SAF indistinguishable in size from the protein in uninfected brain, lower-molecular-mass variants of PrP are also found in SAF fractions. We report the characterisation of the multiple forms of PrP found in SAF fractions purified from mouse brain affected by the ME7 strain of scrapie. The quantitatively major forms of PrP in SAF prepared without the use of proteinase K have the amino-terminal sequence Lys-Lys-Arg-Pro-Lys-Pro-Gly-Gly-, identical to that predicted for the amino-terminus of normal mouse brain PrP. However N-terminal cleavage of some PrP does occur in vivo within a domain of repetitive sequences at sites similar to but distinct from those cut by proteinase K in vitro. This suggests the conformation of the protein in aggregates in vivo does not differ extensively from that in detergent-treated SAF in vitro. We conclude that the size diversity of PrP in SAF is only partly due to N-terminal proteolysis and is independent of the proteolysis that occurs if proteinase K is used in the purification of SAF. Apart from proteolytic changes in the structure of PrP, we found a novel, as yet unidentified, amino-acid derivative of the arginine residue at position 3 in mouse PrP, which may predispose PrP to form SAF.