N-terminal heterogeneity of parenchymal and vascular amyloid-ß deposits in Alzheimer's disease.

AIMS: The deposition of amyloid-ß (Aß) peptides in the form of extracellular plaques in the brain represents one of the classical hallmarks of Alzheimer's disease (AD). In addition to 'full-length' Aß starting with aspartic acid (Asp-1), considerable amounts of various shorter, N-terminally truncated Aß peptides have been identified by mass spectrometry in autopsy samples from individuals with AD. METHODS: Selectivity of several antibodies detecting full-length, total or N-terminally truncated Aß species has been characterized with capillary isoelectric focusing assays using a set of synthetic Aß peptides comprising different N-termini. We further assessed the N-terminal heterogeneity of extracellular and vascular Aß peptide deposits in the human brain by performing immunohistochemical analyses using sporadic AD cases with antibodies targeting different N-terminal residues, including the biosimilar antibodies Bapineuzumab and Crenezumab. RESULTS: While antibodies selectively recognizing Aß1-x showed a much weaker staining of extracellular plaques and tended to accentuate cerebrovascular amyloid deposits, antibodies detecting Aß starting with phenylalanine at position 4 of the Aß sequence showed abundant amyloid plaque immunoreactivity in the brain parenchyma. The biosimilar antibody Bapineuzumab recognized Aß starting at Asp-1 and demonstrated abundant immunoreactivity in AD brains. DISCUSSION: In contrast to other studied Aß1-x -specific antibodies, Bapineuzumab displayed stronger immunoreactivity on fixed tissue samples than with sodium dodecyl sulfate-denatured samples on Western blots. This suggests conformational preferences of this antibody. The diverse composition of plaques and vascular deposits stresses the importance of understanding the roles of various Aß variants during disease development and progression in order to generate appropriate target-developed therapies.

Physical activity delays hippocampal neurodegeneration and rescues memory deficits in an Alzheimer disease mouse model.

The evidence for a protective role of physical activity on the risk and progression of Alzheimer's disease (AD) has been growing in the last years. Here we studied the influence of a prolonged physical and cognitive stimulation on neurodegeneration, with special emphasis on hippocampal neuron loss and associated behavioral impairment in the Tg4-42 mouse model of AD. Tg4-42 mice overexpress Aß4-42 without any mutations, and develop an age-dependent hippocampal neuron loss associated with a severe memory decline. We demonstrate that long-term voluntary exercise diminishes CA1 neuron loss and completely rescues spatial memory deficits in different experimental settings. This was accompanied by changes in the gene expression profile of Tg4-42 mice. Deep sequencing analysis revealed an upregulation of chaperones involved in endoplasmatic reticulum protein processing, which might be intimately linked to the beneficial effects seen upon long-term exercise. We believe that we provide evidence for the first time that enhanced physical activity counteracts neuron loss and behavioral deficits in a transgenic AD mouse model. The present findings underscore the relevance of increased physical activity as a potential strategy in the prevention of dementia.

Development of a capillary isoelectric focusing immunoassay to measure DJ-1 isoforms in biological samples.

We report on the development of a novel assay protocol for the separation and detection of charge isoforms of DJ-1 in biological samples by automated capillary isoelectric focusing followed by immunological detection. DJ-1 (PARK7) is considered as a biomarker candidate for Parkinson's disease and may potentially support the differentiation of clinical subtypes of the disease. The new method allows for separation and subsequent relative quantitative comparison of different isoforms of DJ-1 in biological samples. The assay was successfully applied to the analysis of DJ-1 isoform patterns in brains from mice subjected to normal or high-fat diet and revealed statistically significant group differences. Furthermore, in a pooled and concentrated sample of human cerebrospinal fluid that was depleted of albumin and immunoglobulin G, four different charge variants of DJ-1 could be detected. Taken together, the capillary isoelectric focusing immunoassay for DJ-1 represents a promising tool that may ultimately serve in clinical biomarker studies.

Current application of neurochemical biomarkers in the prediction and differential diagnosis of Alzheimer's disease and other neurodegenerative dementias.

In light of the dramatically increasing prevalence of Alzheimer's disease (AD) to be expected in the future, the development of novel therapeutics, improved differential and early diagnostics, and means for the identification of individuals at risk are urgently needed. At present, instruments for a reliable differential diagnosis in clinical dementia, mild cognitive impairment, or prodromal stages have direct practical implications for differentiating secondary dementias from neurodegenerative conditions and for treatment decisions. It may also be reasonable to enforce the incorporation of biomarkers into clinical studies as surrogate outcome parameters and as an attempt to optimize recruitment criteria. Recently, revised research criteria increasingly rely on the interpretation of biomarker patterns, including neuroimaging and CSF-based neurochemical dementia diagnosis (NDD) in supporting the clinical diagnosis. Here, we review the performance of current core CSF biomarkers (Aß(42) peptide, total tau protein and phosphorylated tau species) and try to define objectives for prospective markers, also considering blood-based tests, which would increase the acceptance and wide application of NDD. Moreover, we evaluate the role and the limitations of genotyping in the predictive diagnosis of AD.

Soluble amyloid precursor proteins in the cerebrospinal fluid as novel potential biomarkers of Alzheimer's disease: a multicenter study.

In this report, we present the results of a multicenter study to test analytic and diagnostic performance of soluble forms of amyloid precursor proteins alpha and beta (sAPP alpha and sAPP beta) in the cerebrospinal fluid (CSF) of patients with different forms of dementing conditions. CSF samples were collected from 188 patients with early dementia (mini-mental state examination >or=20 in majority of cases) and mild cognitive impairment (MCI) in 12 gerontopsychiatric centers, and the clinical diagnoses were supported by neurochemical dementia diagnostic (NDD) tools: CSF amyloid beta peptides, Tau and phospho-Tau. sAPP alpha and sAPP beta were measured with multiplexing method based on electrochemiluminescence. sAPP alpha and sAPP beta CSF concentrations correlated with each other with very high correlation ratio (R=0.96, P<0.001). We observed highly significantly increased sAPP alpha and sAPP beta CSF concentrations in patients with NDD characteristic for Alzheimer's disease (AD) compared to those with NDD negative results. sAPP alpha and sAPP beta highly significantly separated patients with AD, whose diagnosis was supported by NDD findings (sAPP alpha: cutoff, 117.4 ng ml(-1), sensitivity, 68%, specificity, 85%, P<0.001; sAPP beta: cutoff, 181.8 ng ml(-1), sensitivity, 75%, specificity, 85%, P<0.001), from the patients clinically assessed as having other dementias and supported by NDD untypical for AD. We conclude sAPP alpha and sAPP beta might be regarded as novel promising biomarkers supporting the clinical diagnosis of AD.

Immune complexes of auto-antibodies against A beta 1-42 peptides patrol cerebrospinal fluid of non-Alzheimer's patients.

The diagnostic potential of large A beta-peptide binding particles (LAPs) in the cerebrospinal fluid (CSF) of Alzheimer's dementia (AD) patients and non-AD controls (nAD) was evaluated. LAPs were detected by confocal spectroscopy in both groups with high inter-individual variation in number. Molecular imaging by confocal microscopy revealed that LAPs are heterogeneous superaggregates that could be subdivided morphologically into four main types (LAP 1-4). LAP-4 type, resembling a 'large chain of pearls', was detected in 42.1% of all nAD controls but it was virtually absent in AD patients. LAP-4 type could be selectively removed by protein A beads, a clear indication that it contained immunoglobulins in addition to beta-amyloid peptides (A beta 1-42). We observed a close correlation between LAPs and immunoglobulin G (IgG) concentration in CSF in controls but not in AD patients. Double labeling of LAPs with anti-A beta and anti-IgG antibodies confirmed that LAP-4 type consisted of A beta and IgG aggregates. Our results assign a central role to the immune system in regulating A beta1-42 homeostasis by clustering this peptide in immunocomplexes.

Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation.

Human lumbar CSF patterns of Abeta peptides were analysed by urea-based beta-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Abeta-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Abeta1-37/38/39 was found in addition to Abeta1-40 and Abeta1-42. Remarkably, Abeta1-38 was present at a higher concentration than Abeta1-42, being the second prominent Abeta peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Abeta peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Abeta peptides relative to their total Abeta peptide concentration (Abeta1-x%, fractional Abeta peptide pattern), which may reflect disease-specific gamma-secretase activities. Remarkably, patients with AD and CID shared elevated Abeta1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE epsilon4 alleles resulted in an overall reduction of CSF Abeta peptides, which was pronounced for Abeta1-42. The severity of dementia was significantly correlated to the fractional Abeta peptide pattern but not to the absolute Abeta peptide concentrations.

Improved electrophoretic separation and immunoblotting of beta-amyloid (A beta) peptides 1-40, 1-42, and 1-43.

Beta-amyloid peptides (A beta peptides) form the main protein component of the amyloid deposits found in the brains of Alzheimer's disease (AD) patients. Soluble A beta peptides, which are proteolytic fragments of the amyloid-precursor protein (APP) are constitutively secreted by cells expressing APP during normal metabolism [1] and are also present in human plasma and cerebrospinal fluid [2]. Missense mutations in Codon 717 of the APP gene are responsible for a small percentage of inherited AD cases (FAD) and increase the amount of A beta peptides containing additional carboxy terminal amino acids (A beta 1-42, A beta 1-43) [3, 4]. Recent findings indicate that FAD mutations in the presenilin 1 and 2 genes also increase the amount of these longer A beta peptides [5]. A beta 1-42 polymerizes more rapidly in vitro [6] than A beta 1-40 and has been identified as the major component of the brain amyloid deposits [7-9]. We recently developed a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) system [10] for the separation of these two peptides. Here we describe a modified version of the original SDS-PAGE procedure, which allows the separation of A beta 1-40, A beta 1-42, and A beta 1-43 for the first time. Detection of the three A beta peptides in the lower ng and pg range is realized by optimized silver staining or immunoblot procedures. These nonradioactive methods may validate results obtained by ELISA procedures used to study the metabolic fate of APP. They may help to define the neurotoxic potential of the longer A beta peptides in relation to their aggregation state.

Amyloid precursor protein truncated at any of the gamma-secretase sites is not cleaved to beta-amyloid.

beta A4 secretion occurs upon processing of amyloid protein precursor (APP) by beta-secretase (N-terminus of beta A4) and gamma-secretase (C-terminus). To determine the sequence of these activities and the processing intermediate of beta A4, we expressed several truncated APP molecules in human HEK-293 cells. Immunofluorescence and biotinylation studies indicated that full-length APP or APP lacking the cytosolic domain both were located intracellularly, associated with the cell surface and secreted. APPs truncated after amino acid 40, 42, or 43 of beta A4 were not inserted into cell membranes, were found intracellularly but not on the cell surface, and were efficiently secreted into the culture medium. The secretion of APP truncated at amino acid 40 of beta A4 occurred without proteolytic processing. Neither beta A4 nor P3 (the product of the alpha-secretase) was secreted from any of the APP molecules truncated at the gamma-secretase sites. In sharp contrast to this, when the C-terminal 100 amino acids of APP were expressed (APP truncated at the N-terminus of beta A4), a robust beta A4 secretion was observed. Thus, the C-terminal fragment of APP produced by beta-secretase activity is likely to be the processing intermediate of beta A4.

The carboxyl termini of beta-amyloid peptides 1-40 and 1-42 are generated by distinct gamma-secretase activities.

We have studied the effects of peptide aldehyde protease inhibitors on the secretion of beta-amyloid peptide 1-40 (Abeta(1-40)) and Abeta(1-42) by HEK 293 and COS-1 cells expressing beta-amyloid precursor protein with the Swedish double mutation. A multiphasic SDS-polyacrylamide gel electrophoresis system was used for the discrimination of Abeta(1-40) and Abeta(1-42). Calpain inhibitor I, carbobenzoxyl-Leu-Leu-leucinal, and calpeptin were found to reduce the amount of Abeta(1-40) released into the medium in a dose-dependent manner. The reduction of Abeta(1-40) after treatment with 50 microM calpain inhibitor I or 5 microM carbobenzoxyl-Leu-Leu-leucinal was accompanied by a slight increase of Abeta(1-42) released into the medium. These observations suggest that the cleavages at residues 40 and 42 are accomplished by different enzyme activities.

Electrophoretic separation of betaA4 peptides (1-40) and (1-42).

Different sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) systems designed for the separation of peptides were compared for their usefulness in separating synthetic beta-amyloid peptides betaA4 (1-40) and betaA4 (1-42). Clear resolution was achieved by addition of 8 M urea to the separation gel and use of a multiphasic buffer system employing bicine and sulfate as trailing and leading ions, respectively (bicine/Tris/urea gels). Under these conditions, the longer peptide migrated faster than the one ending at amino acid 40. The usefulness of this SDS-PAGE system for the analysis of betaA4-related peptides generated during cellular metabolism was demonstrated by immunoprecipitation and electrophoretic separation of radiolabeled peptides secreted by cells transfected with amyloid precursor protein cDNAs.

Calpain inhibitor I decreases beta A4 secretion from human embryonal kidney cells expressing beta-amyloid precursor protein carrying the APP670/671 double mutation.

We have investigated the effects of the cell-penetrating cysteine protease inhibitors calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal) and calpain inhibitor II (N-acetyl-Leu-Leu-methioninal) on the secretion of the beta-amyloid peptide (beta A4) using transiently transfected cells expressing beta-amyloid precursor protein (APP) with the NL670/671 double mutation. Calpain inhibitor I markedly reduced the amounts of immunoprecipitable beta A4 and p3 peptide released into the culture medium. Within the cells C-terminal APP fragments accumulated. Since beta A4 secretion by cells expressing the 100 amino acid long APP C-terminus was also reduced by calpain inhibitor I, we conclude that this substance directly or indirectly interferes with the gamma-secretase activity responsible for generating the beta A4 and p3 C-termini.

Reduction of disulfide bonds in an amyloidogenic Bence Jones protein leads to formation of "amyloid-like" fibrils in vitro.

Motivated by the finding that the amino acid sequence of the Bence Jones protein BJP-DIA was identical to that of the main protein component of the amyloid fibrils obtained from the same patient with AL-amyloidosis, (Klafki, H.-W., Kratzin, H.-D., Pick, A.-I., Eckart, K., Karas, M. & Hilschmann, N. (1992) Biochemistry 31, 3265-3272.), we attempted to create "amyloid-like" fibrils from the Bence Jones protein in vitro, without addition of proteolytic enzymes. Reduction of BJP-DIA, solubilized in PBS, pH 7.4, overnight at 37 degrees C resulted in the formation of a precipitate which had affinity for the dye Congo red. Electron microscopy of negatively stained samples of the reduced protein revealed aggregates of linear unbranched fibrils. SDS-polyacrylamide gel electrophoresis demonstrated that the precipitate consisted almost exclusively of intact light chain molecules. This result makes it possible to deduce a molecular model of these amyloid fibrils generated in vitro.

Synthesis and release of the beta-amyloid precursor protein by bovine chromaffin cells.

Production of the beta-amyloid precursor protein (beta APP) by bovine adrenal chromaffin cells was investigated by polymerase chain reaction (PCR) and by immunoblot. Chromaffin cells were found to synthesize forms of beta APP similar to those found in the rat PC12 pheochromocytoma cell line and to secrete these constitutively into their culture medium. Release of beta APP could be enhanced by stimulation with phorbol ester but not by cholinergic stimulation of secretion. The fact that normal chromaffin cells produce beta APP suggests that beta APP has some (as yet undermined) function in the adrenal medulla in vivo.

Complete amino acid sequence determinations demonstrate identity of the urinary Bence Jones protein (BJP-DIA) and the amyloid fibril protein (AL-DIA) in a case of AL-amyloidosis.

The complete primary structures of both the main amyloid fibril protein component (AL-DIA) and the soluble Bence Jones protein (BJP-DIA) obtained from the same patient with AL-amyloidosis are reported for the first time. The amino acid sequences were determined by automated Edman degradation following proteolytic digestion of the isolated proteins and HPLC separation of the resulting fragments and by amino-terminal sequencing after treatment with pyroglutamate aminopeptidase. Sequencing data were confirmed by amino acid analysis and plasma desorption mass spectrometry (PDMS). Molecular weights of the complete proteins were determined by laser desorption mass spectrometry. The amyloid fibril preparation contained a complete monoclonal lambda immunoglobulin light chain (subgroup 1.2) as well as different-sized fragments thereof which were identified by immunoblotting and amino-terminal sequencing following immobilization of electrophoretically-separated proteins on poly(vinylidene difluoride) (PVDF) membranes. The soluble urinary Bence Jones protein (BJP-DIA) was a dimer of monoclonal L-chains with a primary structure identical to that of the amyloid L-chain (AL-DIA) and thus represented the amyloid precursor protein.