Ann Westman-Brinkmalm

Proteome analysis of cerebrospinal fluid proteins in Alzheimer patients

&NA; By comparing the CSF proteome between Alzheimer disease (AD) patients and controls it may be possible to identify proteins that play a role in the disease process and thus to study the pathogenesis of AD. We used mini‐gel technology in a two‐dimensional electrophoresis procedure, sensitive SYPRO Ruby staining and mass spectrometry for clinical screening of disease‐influenced CSF proteins in 15 AD patients and 12 controls. The levels of six proteins and their isoforms, including proapolipoprotein, apolipoprotein E, &bgr;‐2 microglobulin, retinol‐binding protein, transthyretin, and ubiquitin, were significantly altered in CSF of AD patients. The most prominently altered proteins were the apolipoproteins, especially proapolipoprotein.

Site-specific characterization of threonine, serine, and tyrosine glycosylations of amyloid precursor protein/amyloid β-peptides in human cerebrospinal fluid

The proteolytic processing of human amyloid precursor protein (APP) into shorter aggregating amyloid β (Aβ)-peptides, e.g., Aβ1-42, is considered a critical step in the pathogenesis of Alzheimer’s disease (AD). Although APP is a well-known membrane glycoprotein carrying both N- and O-glycans, nothing is known about the occurrence of released APP/Aβ glycopeptides in cerebrospinal fluid (CSF). We used the 6E10 antibody and immunopurified Aβ peptides and glycopeptides from CSF samples and then liquid chromatography—tandem mass spectrometry for structural analysis using collision-induced dissociation and electron capture dissociation. In addition to 33 unglycosylated APP/Aβ peptides, we identified 37 APP/Aβ glycopeptides with sialylated core 1 like O-glycans attached to Thr(−39, −21, −20, and −13), in a series of APP/AβX-15 glycopeptides, where X was −63, −57, −52, and −45, in relation to Asp1 of the Aβ sequence. Unexpectedly, we also identified a series of 27 glycopeptides, the Aβ1-X series, where X was 20 (DAEFRHDSGYEVHHQKLVFF), 19, 18, 17, 16, and 15, which were all uniquely glycosylated on Tyr10. The Tyr10 linked O-glycans were (Neu5Ac)1-2Hex(Neu5Ac)HexNAc-O- structures with the disialylated terminals occasionally O-acetylated or lactonized, indicating a terminal Neu5Acα2,8Neu5Ac linkage. We could not detect any glycosylation of the Aβ1-38/40/42 isoforms. We observed an increase of up to 2.5 times of Tyr10 glycosylated Aβ peptides in CSF in six AD patients compared to seven non-AD patients. APP/Aβ sialylated O-glycans, including that of a Tyr residue, the first in a mammalian protein, may modulate APP processing, inhibiting the amyloidogenic pathway associated with AD.

A novel pathway for amyloid precursor protein processing

Amyloid precursor protein (APP) can be proteolytically processed along two pathways, the amyloidogenic that leads to the formation of the 40-42 amino acid long Alzheimer-associated amyloid β (Aβ) peptide and the non-amyloidogenic in which APP is cut in the middle of the Aβ domain thus precluding Aβ formation. Using immunoprecipitation and mass spectrometry we have shown that Aβ is present in cerebrospinal fluid (CSF) as several shorter isoforms in addition to Aβ1-40 and Aβ1-42. To address the question by which processing pathways these shorter isoforms arise, we have developed a cell model that accurately reflects the Aβ isoform pattern in CSF. Using this model, we determined changes in the Aβ isoform pattern induced by α-, β-, and γ-secretase inhibitor treatment. All isoforms longer than and including Aβ1-17 were γ-secretase dependent whereas shorter isoforms were γ-secretase independent. These shorter isoforms, including Aβ1-14 and Aβ1-15, were reduced by treatment with α- and β-secretase inhibitors, which suggests the existence of a third and previously unknown APP processing pathway involving concerted cleavages of APP by α- and β-secretase.

An Alzheimer's disease-specific β-amyloid fragment signature in cerebrospinal fluid

Pathogenic events in Alzheimers disease (AD) involve an imbalance between the production and clearance of the neurotoxic beta-amyloid peptide (Abeta), especially the 42 amino acid peptide Abeta1-42. While much is known about the production of Abeta1-42, many questions remain about how the peptide is degraded. To investigate the degradation pattern, we developed a method based on immunoprecipitation combined with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry that determines the Abeta degradation fragment pattern in cerebrospinal fluid (CSF). We found in total 18 C-terminally and 2 N-terminally truncated Abeta peptides and preliminary data indicated that there were differences in the detected Abeta relative abundance pattern between AD and healthy controls. Here, we provide direct evidence that an Abeta fragment signature consisting of Abeta1-16, Abeta1-33, Abeta1-39, and Abeta1-42 in CSF distinguishes sporadic AD patients from non-demented controls with an overall accuracy of 86%.

Comparative genome- and proteome analysis of cerebral cortex from MK-801-treated rats

cDNA microarrays and two‐dimensional gel‐electrophoresis in combination with mass spectrometry, were used to screen alterations in mRNA and protein levels, respectively, in cerebral cortex of MK‐801‐treated rats. The rats were divided in two groups; group 1 (short‐term treated) and group 2 (long‐term treated). In group 1, four genes were up‐regulated and five down‐regulated. In group 2, seven genes were up‐regulated and six down‐regulated. In group 1, the levels of one protein was increased and eight proteins reduced. In group 2, the levels of two proteins were increased and four proteins reduced. Several of the altered genes (casein kinase 2, glutamic acid decarboxylase, synaptotagmin, gamma aminobutyric acid [GABA] transporter, creatine kinase, and cytochrome c oxidase) and proteins (superoxide dismutase, hsp 60, hsp 72 and γ‐enolase) have previously been connected to schizophrenia. Alterations of the genes (microglobulin, c‐jun proto‐oncogene, 40S ribosomal protein S19, adenosine diphosphate (ADP)‐ribosylation factors, platelet‐derived growth factor, fructose‐bisphophate aldolase A, and myelin proteolipid) and the proteins (stathmin, H+‐transp. Adenosine triphosphate (ATP) synthase, pyruvate dehydrogenase, β‐actin and α‐enolase), have not, to our knowledge, earlier been implicated in schizophrenia pathology. Overall, these results with a combined approach of genomics and proteomics add to the validity of subchronic N‐methyl‐D‐aspartate (NMDA)‐receptor antagonist treatment as an animal model of schizophrenia.

Studies of the pathophysiological mechanisms in frontotemporal dementia by proteome analysis of CSF proteins

Comparative proteomic analysis of cerebrospinal fluid (CSF) proteins was employed for studies of the pathophysiological mechanisms in frontotemporal dementia (FTD). Two-dimensional gel electrophoresis and mass spectrometry were used for clinical screening of disease-influenced CSF proteins in 15 FTD patients compared to 12 controls. Six proteins were significantly altered in FTD compared to controls, including granin-like neuroendocrine precursor (proSAAS), pigment-epithelium derived factor (PEDF), retinol-binding protein (RBP), apoE, haptoglobin, and albumin. The levels of ProSAAS, PEDF, and RBP have not been shown earlier to be involved in the FTD pathology. Recently, we have also used proteomic analysis for studies of disease-influenced CSF proteins in Alzheimers disease (AD) patients. The most clearly affected CSF proteins were the apolipoproteins in AD, compared to controls and FTD patients. ApoE seems to be influenced to a lesser degree in FTD compared to AD. Our data showed that several proteins involved in FTD pathology are not influenced in the CSF of AD patients, and vice versa, establishing differences in the pathophysiological mechanisms between FTD and AD, two of the most common neurodegenerative disorders.

Identification of Novel APP/Aβ Isoforms in Human Cerebrospinal Fluid

Background: Aggregation of β-amyloid (Aβ) into oligomers and plaques is the central pathogenic mechanism in Alzheimer’s disease (AD). Aβ is produced from the amyloid precursor protein (APP) by β- and γ-secretases, whereas, in the nonamyloidogenic pathway, α-secretase cleaves within the Aβ sequence, and thus precludes Aβ formation. A lot of research has focused on Aβ production and the neurotoxic 42-amino-acid form of Aβ (Aβ1–42), while less is known about the nonamyloidogenic pathway and how Aβ is degraded. Objective: To study the Aβ metabolism in man by searching for novel Aβ peptides in cerebrospinal fluid (CSF). Methods: Immunoprecipitation, using an anti-Aβ antibody, 6E10, was combined with either matrix-assisted laser desorption/ionization time-of-flight mass spectrometry or nanoflow liquid chromatography and tandem mass spectrometry. Results: We identified 12 truncated APP/Aβ peptides in the CSF, all of which end at amino acid 15 in the Aβ sequence, i.e. 1 amino acid before the proposed α-secretase site. Of these 12 APP/Aβ peptides, 11 are novel peptides and start N-terminally of the β-secretase site. The most abundant APP/Aβ peptide starts 25 amino acids before the β-secretase site, APP/Aβ (–25 to 15), and had a concentration of approximately 80 pg/ml. The identity of all the APP/Aβ peptides was verified in a cohort of AD patients and controls. A first pilot study also showed that the intensity of several APP/Aβ peaks in CSF was higher in AD cases than in controls. Conclusion: These data suggest an enzymatic activity that cleaves the precursor protein in a specific manner that may reflect a novel metabolic pathway for APP and Aβ.

Identification of Novel α-Synuclein Isoforms in Human Brain Tissue by using an Online NanoLC-ESI-FTICR-MS Method

Parkinson’s disease (PD) and Dementia with Lewy bodies (DLB) are neurodegenerative diseases that are characterized by intra-neuronal inclusions of Lewy bodies in distinct brain regions. These inclusions consist mainly of aggregated α-synuclein (α-syn) protein. The present study used immunoprecipitation combined with nanoflow liquid chromatography (LC) coupled to high resolution electrospray ionization Fourier transform ion cyclotron resonance tandem mass spectrometry (ESI-FTICR-MS/MS) to determine known and novel isoforms of α-syn in brain tissue homogenates. N-terminally acetylated full-length α-syn (Ac-α-syn1–140) and two N-terminally acetylated C-terminally truncated forms of α-syn (Ac-α-syn1–139 and Ac-α-syn1–103) were found. The different forms of α-syn were further studied by Western blotting in brain tissue homogenates from the temporal cortex Brodmann area 36 (BA36) and the dorsolateral prefrontal cortex BA9 derived from controls, patients with DLB and PD with dementia (PDD). Quantification of α-syn in each brain tissue fraction was performed using a novel enzyme-linked immunosorbent assay (ELISA).

Characterization of Tau in Cerebrospinal Fluid Using Mass Spectrometry

The neurodegenerative disorder Alzheimers disease (AD) is the most common cause of dementia in the elderly. The presence of neurofibrillary tangles, consisting of hyperphosphorylated tau protein, is one of the major neuropathologic characteristics of the disease, making this protein an attractive biomarker for AD and a possible target for therapy. Here, we describe an optimized immunoprecipitation mass spectrometry method that enables, for the first time, detailed characterization of tau in human cerebrospinal fluid. The identities of putative tau fragments were confirmed using nanoflow liquid chromatography and tandem mass spectrometry. Nineteen tryptic fragments of tau were detected, of which 16 are found in all tau isoforms while 3 represented unique tau isoforms. These results pave the way for clinical CSF studies on the tauopathies.

Identification of novel N-terminal fragments of amyloid precursor protein in cerebrospinal fluid.

Alzheimers disease (AD) is a progressive neurodegenerative disorder of the central nervous system. Two pathological hallmarks in the brain of AD patients are neurofibrillary tangles and senile plaques. The plaques consist mainly of beta-amyloid (Abeta) peptides that are produced from the amyloid precursor protein (APP), by sequential cleavage by beta- and gamma-secretase. Most previous studies have been focused on the C-terminal fragments of APP, where the Abeta sequence is localized. The purpose of this study was to search for N-terminal fragments of APP in cerebrospinal fluid (CSF) using mass spectrometry (MS). By using immunoprecipitation (IP) combined with matrix-assisted laser desorption/ionization time-of-flight MS as well as nanoflow liquid chromatography coupled to high resolution tandem MS we were able to detect and identify six novel N-terminal APP fragments [APP((18-119)), APP((18-121)), APP((18-122)), APP((18-123)), APP((18-124)) and APP((18-126))], having molecular masses of approximately 12 kDa. The presence of these APP derivatives in CSF was also verified by Western blot analysis. Two pilot studies using either IP-MS or Western blot analysis indicated slightly elevated levels of N-terminal APP fragments in CSF from AD patients compared with controls, which are in need of replications in independent and larger patient materials.