Cerebrospinal fluid biomarkers of ß-amyloid metabolism in multiple sclerosis.

BACKGROUND: Amyloid precursor protein (APP) and amyloid ß (Aß) peptides are intensely studied in neuroscience and their cerebrospinal fluid (CSF) measurements may be used to track the metabolic pathways of APP in vivo. Reduced CSF levels of Aß and soluble APP (sAPP) fragments are reported in inflammatory diseases, including multiple sclerosis (MS); but in MS, the precise pathway of APP metabolism and whether it can be affected by disease-modifying treatments remains unclear. OBJECTIVE: To characterize the CSF biomarkers of APP degradation in MS, including the effects of disease-modifying therapy. METHODS: CSF samples from 87 MS patients (54 relapsing-remitting (RR) MS; 33 secondary progressive (SP) MS and 28 controls were analyzed for sAPP and Aß peptides by immunoassays, plus a subset of samples was analyzed by immunoprecipitation and mass spectrometry (IP-MS). Patients treated with natalizumab or mitoxantrone were examined at baseline, and after 1-2 years of treatment. RESULTS: CSF sAPP and Aß peptide levels were reduced in MS patients; but they increased again towards normal, after natalizumab treatment. A multivariate model of IP-MS-measured Aß species separated the SPMS patients from controls, with RRMS patients having intermediate levels. CONCLUSIONS: We confirmed and extended our previous observations of altered CSF sAPP and Aß peptide levels in MS patients. We found that natalizumab therapy may be able to counteract the altered APP metabolism in MS. The CSF Aß isoform distribution was found to be distinct in SPMS patients, as compared to the controls.

Amyloid-ß metabolism in Niemann-Pick C disease models and patients.

Niemann-Pick type C (NPC) is a progressive neurodegenerative lysosomal disease with altered cellular lipid trafficking. The metabolism of amyloid-ß (Aß) - previously mainly studied in Alzheimer's disease - has been suggested to be altered in NPC. Here we aimed to perform a detailed characterization of metabolic products from the amyloid precursor protein (APP) in NPC models and patients. We used multiple analytical technologies, including immunoassays and immunoprecipitation followed by mass spectrometry (IP-MS) to characterize Aß peptides and soluble APP fragments (sAPP-α/ß) in cell media from pharmacologically (U18666A) and genetically (NPC1 ( -/- ) ) induced NPC cell models, and cerebrospinal fluid (CSF) from NPC cats and human patients. The pattern of Aß peptides and sAPP-α/ß fragments in cell media was differently affected by NPC-phenotype induced by U18666A treatment and by NPC1 ( -/- ) genotype. U18666A treatment increased the secreted media levels of sAPP-α, AßX-40 and AßX-42 and reduced the levels of sAPP-ß, Aß1-40 and Aß1-42, while IP-MS showed increased relative levels of Aß5-38 and Aß5-40 in response to treatment. NPC1 ( -/- ) cells had reduced media levels of sAPP-α and Aß1-16, and increased levels of sAPP-ß. NPC cats had altered CSF distribution of Aß peptides compared with normal cats. Cats treated with the potential disease-modifying compound 2-hydroxypropyl-ß-cyclodextrin had increased relative levels of short Aß peptides including Aß1-16 compared with untreated cats. NPC patients receiving ß-cyclodextrin had reduced levels over time of CSF Aß1-42, AßX-38, AßX-40, AßX-42 and sAPP-ß, as well as reduced levels of the axonal damage markers tau and phosphorylated tau. We conclude that NPC models have altered Aß metabolism, but with differences across experimental systems, suggesting that NPC1-loss of function, such as in NPC1 ( -/- ) cells, or NPC1-dysfunction, seen in NPC patients and cats as well as in U18666A-treated cells, may cause subtle but different effects on APP degradation pathways. The preliminary findings from NPC cats suggest that treatment with cyclodextrin may have an impact on APP processing pathways. CSF Aß, sAPP and tau biomarkers were dynamically altered over time in human NPC patients.

An online nano-LC-ESI-FTICR-MS method for comprehensive characterization of endogenous fragments from amyloid ß and amyloid precursor protein in human and cat cerebrospinal fluid.

Amyloid precursor protein (APP) is the precursor protein to amyloid ß (Aß), the main constituent of senile plaques in Alzheimer's disease (AD). Endogenous Aß peptides reflect the APP processing, and greater knowledge of different APP degradation pathways is important to understand the mechanism underlying AD pathology. When one analyzes longer Aß peptides by low-energy collision-induced dissociation tandem mass spectrometry (MS/MS), mainly long b-fragments are observed, limiting the possibility to determine variations such as amino acid variants or post-translational modifications (PTMs) within the N-terminal half of the peptide. However, by using electron capture dissociation (ECD), we obtained a more comprehensive sequence coverage for several APP/Aß peptide species, thus enabling a deeper characterization of possible variants and PTMs. Abnormal APP/Aß processing has also been described in the lysosomal storage disease Niemann-Pick type C and the major large animal used for studying this disease is cat. By ECD MS/MS, a substitution of Asp7 → Glu in cat Aß was identified. Further, sialylated core 1 like O-glycans at Tyr10, recently discovered in human Aß (a previously unknown glycosylation type), were identified also in cat cerebrospinal fluid (CSF). It is therefore likely that this unusual type of glycosylation is common for (at least) species belonging to the magnorder Boreoeutheria. We here describe a detailed characterization of endogenous APP/Aß peptide species in CSF by using an online top-down MS-based method.

Evaluation of the performance of novel Aß isoforms as theragnostic markers in Alzheimer's disease: from the cell to the patient.

BACKGROUND: Alzheimer's disease (AD) is the most common neurodegenerative disorder in the aging population and is characterized by extracellular plaques in the brain. The last decades have witnessed an explosion in studies of the role of amyloid-ß (Aß) metabolism and aggregation in the pathogenesis of AD which has been translated into novel promising therapies with putative disease-modifying effects. OBJECTIVE: The aim is to investigate the performance of truncated Aß isoforms as theragnostic markers in clinical trials. METHODS: Aß isoforms were immunoprecipitated from human, mouse and dog cerebrospinal fluid (CSF) or cell media and analyzed using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. RESULTS: Aß1-14, Aß1-15, and Aß1-16 are elevated in cell media and in CSF in response to γ-secretase inhibitor treatment. In a clinical trial including AD patients, Aß1-14, Aß1-15, and Aß1-16 increased dose-dependently in response to treatment with the γ-secretase inhibitor LY450139. In dogs, Aß1-37 was significantly increased in response to treatment with the γ-secretase modulator E2012. CONCLUSIONS: The results presented add to the current knowledge on APP processing and that Aß isoforms can be used as novel biomarkers to monitor anti-Aß treatments in clinical trials and may be valuable for making a go/no go decision for large and expensive phase 2 or 3 clinical trials.

The amyloid-ß isoform pattern in cerebrospinal fluid in familial PSEN1 M139T- and L286P-associated Alzheimer's disease.

There are several familial forms of Alzheimer's disease (AD) most of which are caused by mutations in the genes that encode the presenilin enzymes involved in the production of amyloid-ß (Aß) from the amyloid precursor protein (APP). In AD, Aß forms fibrils that are deposited in the brain as plaques. Much of the fibrillar Aß found in the plaques consists of the 42 amino acid form of Aß (Aß1-42) and it is now widely accepted that Aß is related to the pathogenesis of AD and that Aß may both impair memory and be neurotoxic. In human cerebrospinal fluid (CSF) several C- and N-terminally truncated Aß isoforms have been detected and their relative abundance pattern is thought to reflect the production and clearance of Aß. By using immunoprecipitation and mass spectrometry, we have previously demonstrated that carriers of the familial AD (FAD)-associated PSEN1 A431E mutation have low CSF levels of C-terminally truncated Aß isoforms shorter than Aß1-40. Here we replicate this finding in symptomatic carriers of the FAD-causing PSEN1 L286P mutation. Furthermore, we show that preclinical carriers of the PSEN1 M139T mutation may overexpress Aß1-42 suggesting that this particular mutation may cause AD by stimulating γ-secretase-mediated cleavage at amino acid 42 in the Aß sequence.

Pyroglutamate amyloid ß (Aß) aggravates behavioral deficits in transgenic amyloid mouse model for Alzheimer disease.

Pyroglutamate-modified Aß peptides at amino acid position three (Aß(pE3-42)) are gaining considerable attention as potential key players in the pathogenesis of Alzheimer disease (AD). Aß(pE3-42) is abundant in AD brain and has a high aggregation propensity, stability and cellular toxicity. The aim of the present work was to study the direct effect of elevated Aß(pE3-42) levels on ongoing AD pathology using transgenic mouse models. To this end, we generated a novel mouse model (TBA42) that produces Aß(pE3-42). TBA42 mice showed age-dependent behavioral deficits and Aß(pE3-42) accumulation. The Aß profile of an established AD mouse model, 5XFAD, was characterized using immunoprecipitation followed by mass spectrometry. Brains from 5XFAD mice demonstrated a heterogeneous mixture of full-length, N-terminal truncated, and modified Aß peptides: Aß(1-42), Aß(1-40), Aß(pE3-40), Aß(pE3-42), Aß(3-42), Aß(4-42), and Aß(5-42). 5XFAD and TBA42 mice were then crossed to generate transgenic FAD42 mice. At 6 months of age, FAD42 mice showed an aggravated behavioral phenotype compared with single transgenic 5XFAD or TBA42 mice. ELISA and plaque load measurements revealed that Aß(pE3) levels were elevated in FAD42 mice. No change in Aß(x)(-42) or other Aß isoforms was discovered by ELISA and mass spectrometry. These observations argue for a seeding effect of Aß(pE-42) in FAD42 mice.

SILAC zebrafish for quantitative analysis of protein turnover and tissue regeneration.

Defective tissue regeneration is thought to contribute to several human diseases, including neurodegenerative disorders, heart failure and various lung diseases. Boosting the regenerative capacity has been suggested a possible therapeutic approach. Methods to metabolically label newly synthesized proteins in vivo with stable isotopic forms of amino acids holds promise for the study of protein turnover and tissue regeneration that are fundamental to the sustained life of all organisms. Here, we used the "stable isotope labeling with amino acids in cell culture" (SILAC) approach to explore normal protein turnover and tissue regeneration in adult zebrafish. The ratio of labeled and unlabeled proteins/peptides in specific organs of zebrafish fed a SILAC diet containing (13)C(6)-labeled lysine was determined by liquid chromatography and tandem mass spectrometry. Labeling was highest in tissues with high regenerative capacity, including intestine, liver, and fin, whereas brain and heart displayed the lowest labeling. Proteins with high degree of labeling were mainly involved in catalytic or transport activity pathways. The technique also verified increased protein synthesis during regeneration of the caudal fin following amputation. This newly developed SILAC zebrafish model constitutes a novel tool to analyze tissue regeneration in an animal model amenable to genetic and pharmacologic manipulation.

Characterization of the brain ß-amyloid isoform pattern at different ages of Tg2576 mice.

BACKGROUND: Although genetic and biochemical studies have suggested a cardinal role for ß-amyloid (Aß) in Alzheimer's disease, the underlying mechanism(s) of how Aß induces neurodegeneration is still unclear. Our objective was to investigate the consequences of Aß, especially on tau phosphorylation at specific epitopes important for Alzheimer's disease. METHODS: We used cortices from Tg2576 mice at 7 days to 15 months of age. RESULTS: MALDI-TOF MS revealed an age-dependent shift in the Aß isoform pattern. Young animals displayed high cortical levels of the shorter Aß isoforms (Aß1-16 and Aß1-17) compared to 15-month-old Tg2576 mice which mainly expressed Aß1-40 and Aß1-42. The Aß1-42 showed an age-dependent increase, whereas total Aß1-40 levels remained constant. The highest levels of TBS-soluble Aß oligomers were found at 90 days of age. Brain Aß build-up did not affect the phosphorylation of tau at the epitopes investigated. CONCLUSIONS: This study provides new information about age-dependent Aß isoforms and oligomers as well as their effect on site-specific tau phosphorylation in this transgenic mouse model. Our observations suggest that the different human Aß isoforms do not directly cause increased tau phosphorylation and that the cognitive deficits seen in this mouse model are only related to the Aß overexpression.

Acute effect on the Aß isoform pattern in CSF in response to γ-secretase modulator and inhibitor treatment in dogs.

Alzheimer's disease (AD) is associated with deposition of amyloid-ß (Aß) in the brain, which is reflected by low concentration of the Aß(1-42) peptide in the cerebrospinal fluid (CSF). The γ-secretase inhibitor LY450139 (semagacestat) lowers plasma Aß(1-40) and Aß(1-42) in a dose-dependent manner but has no clear effect on the CSF level of these isoforms. Less is known about the potent γ-secretase modulator E2012. Using targeted proteomics techniques, we recently identified several shorter Aß isoforms in CSF, such as Aß(1-16), which is produced by a novel pathway. In a Phase II clinical trial on AD patients, Aß(1-14), Aß(1-15) and Aß(1-16) increased several-fold during γ-secretase inhibitor treatment. In the present study, 9 dogs were treated with a single dose of the γ-secretase modulator E2012, the γ-secretase inhibitor LY450139, or vehicle with a dosing interval of 1 week. The CSF Aß isoform pattern was analyzed by immunoprecipitation combined with MALDI-TOF mass spectrometry. We show here that Aß(1-15) and Aß(1-16) increase while Aß(1-34) decreases in response to treatment with the γ-secretase inhibitor LY450139, which is in agreement with previous studies. The isoform Aß(1-37) was significantly increased in a dose-dependent manner in response to treatment with E2012, while Aß(1-39), Aß(1-40) and A(1-42) decreased. The data presented suggests that the γ-secretase modulator E-2012 alters the cleavage site preference of γ-secretase. The increase in Aß(1-37) may inhibit Aß(1-42) oligomerization and toxicity.

Mass spectrometric characterization of brain amyloid beta isoform signatures in familial and sporadic Alzheimer's disease.

A proposed key event in the pathogenesis of Alzheimer's disease (AD) is the formation of neurotoxic amyloid beta (Abeta) oligomers and amyloid plaques in specific brain regions that are affected by the disease. The main plaque component is the 42 amino acid isoform of Alphabeta (Abeta1-42), which is thought to initiate plaque formation and AD pathogenesis. Numerous isoforms of Abeta, e.g., Abeta1-42, Abeta1-40 and the 3-pyroglutamate derivate of Abeta3-42 (pGluAbeta3-42), have been detected in the brains of sporadic AD (SAD) and familial AD (FAD) subjects. However, the relative importance of these isoforms in the pathogenesis of AD is not fully understood. Here, we report a detailed study using immunoprecipitation in combination with mass spectrometric analysis to determine the Abeta isoform pattern in the cerebellum, cortex and hippocampus in AD, including subjects with a mutation in the presenilin (M146V) or amyloid precursor protein (KM670/671NL) genes, SAD subjects and non-demented controls. We show that the dominating Abeta isoforms in the three different brain regions analyzed from control, SAD, and FAD are Abeta1-42, pGluAbeta3-42, Abeta4-42 and Abeta1-40 of which Abeta1-42 and Abeta4-42 are the dominant isoforms in the hippocampus and the cortex in all groups analyzed, controls included. No prominent differences in Abeta isoform patterns between FAD and SAD patients were seen, underscoring the similarity in the amyloid pathology of these two disease entities.

A novel Abeta isoform pattern in CSF reflects gamma-secretase inhibition in Alzheimer disease.

INTRODUCTION: LY450139 (semagacestat) inhibits gamma-secretase, a key enzyme for generation of amyloid beta (Abeta), the peptide deposited in plaques in Alzheimer disease (AD). Previous data have shown that LY450139 lowers plasma Abeta, but has no clear effect on Abeta1-40 or Abeta1-42 levels in cerebrospinal fluid (CSF). By using targeted proteomics techniques, we recently identified several shorter Abeta isoforms, such as Abeta1-16, that in experimental settings increase during gamma-secretase inhibitor treatment, and thus may serve as sensitive biochemical indices of the treatment effect. Here, we test the hypothesis that these shorter Abeta isoforms may be biomarkers of gamma-secretase inhibitor treatment in clinical trials. METHODS: In a phase II clinical trial, 35 individuals with mild to moderate AD were randomized to placebo (n = 10) or LY450139 (100 mg (n = 15) or 140 mg (n = 10)) and underwent lumbar puncture at baseline and after 14 weeks of treatment. The CSF Abeta isoform pattern was analyzed with immunoprecipitation combined with MALDI-TOF mass spectrometry. RESULTS: The CSF levels of Abeta1-14, Abeta1-15, and Abeta1-16 showed a dose-dependent increase by 57% and 74%, 21% and 35%, and 30% and 67%, respectively in the 100-mg and 140-mg treatment groups. Abeta1-40 and Abeta1-42 were unaffected by treatment. CONCLUSIONS: CSF Abeta1-14, Abeta1-15, and Abeta1-16 increase during gamma-secretase inhibitor treatment in AD, even at doses that do not affect Abeta1-42 or Abeta1-40, probably because of increased substrate availability of the C99 APP stub (APP beta-CTF) induced by gamma-secretase inhibition. These Abeta isoforms may be novel sensitive biomarkers to monitor the biochemical effect in clinical trials. TRIAL REGISTRATION: Clinical Trials.gov NCT00244322.

Distinct cerebrospinal fluid amyloid beta peptide signatures in sporadic and PSEN1 A431E-associated familial Alzheimer's disease.

BACKGROUND: Alzheimer's disease (AD) is associated with deposition of amyloid beta (Abeta) in the brain, which is reflected by low concentration of the Abeta1-42 peptide in the cerebrospinal fluid (CSF). There are at least 15 additional Abeta peptides in human CSF and their relative abundance pattern is thought to reflect the production and degradation of Abeta. Here, we test the hypothesis that AD is characterized by a specific CSF Abeta isoform pattern that is distinct when comparing sporadic AD (SAD) and familial AD (FAD) due to different mechanisms underlying brain amyloid pathology in the two disease groups. RESULTS: We measured Abeta isoform concentrations in CSF from 18 patients with SAD, 7 carriers of the FAD-associated presenilin 1 (PSEN1) A431E mutation, 17 healthy controls and 6 patients with depression using immunoprecipitation-mass spectrometry. Low CSF levels of Abeta1-42 and high levels of Abeta1-16 distinguished SAD patients and FAD mutation carriers from healthy controls and depressed patients. SAD and FAD were characterized by similar changes in Abeta1-42 and Abeta1-16, but FAD mutation carriers exhibited very low levels of Abeta1-37, Abeta1-38 and Abeta1-39. CONCLUSION: SAD patients and PSEN1 A431E mutation carriers are characterized by aberrant CSF Abeta isoform patterns that hold clinically relevant diagnostic information. PSEN1 A431E mutation carriers exhibit low levels of Abeta1-37, Abeta1-38 and Abeta1-39; fragments that are normally produced by gamma-secretase, suggesting that the PSEN1 A431E mutation modulates gamma-secretase cleavage site preference in a disease-promoting manner.

Effects of gamma-secretase inhibition on the amyloid beta isoform pattern in a mouse model of Alzheimer's disease.

BACKGROUND: Accumulation of amyloid beta (Abeta) in the brain is believed to represent one of the earliest events in the Alzheimer disease process. Abeta is generated from amyloid precursor protein after sequential cleavage by beta- and gamma-secretase. Alternatively, alpha-secretase cleaves within the Abeta sequence, thus, precluding the formation of Abeta. A lot of research has focused on Abeta production, while less is known about the non-amyloidogenic pathway. We have previously shown that Abeta is present in human cerebrospinal fluid (CSF) as several shorter C-terminal truncated isoforms (e.g. Abeta1-15 and Abeta1-16), and that the levels of these shorter isoforms are elevated in media from cells that have been treated with gamma-secretase inhibitors. OBJECTIVE: To explore the effect of N-[N-(3,5-difluorophenacetyl-L-alanyl)]-S-phenylglycine t-butyl ester (DAPT), a gamma-secretase-inhibitor, treatment on the Abeta isoform pattern in brain tissue and CSF from Tg2576 mice. METHODS: Immunoprecipitation using the anti-Abeta antibodies 6E10 and 4G8 was combined with either matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or nanoflow liquid chromatography and tandem mass spectrometry. RESULTS: All fragments longer than and including Abeta1-17 displayed a tendency towards decreased levels upon gamma-secretase inhibition, whereas Abeta1-15 and Abeta1-16 indicated slightly elevated levels during treatment. CONCLUSION: These data suggest that Abeta1-15 and Abeta1-16 may be generated through a third metabolic pathway independent of gamma-secretase, and that these Abeta isoforms may serve as biomarkers for secretase inhibitor treatment.