Monoclonal antibodies selective for α-synuclein oligomers/protofibrils recognize brain pathology in Lewy body disorders and α-synuclein transgenic mice with the disease-causing A30P mutation.

Inclusions of intraneuronal alpha-synuclein (α-synuclein) can be detected in brains of patients with Parkinson's disease and dementia with Lewy bodies. The aggregation of α-synuclein is a central feature of the disease pathogenesis. Among the different α-synuclein species, large oligomers/protofibrils have particular neurotoxic properties and should therefore be suitable as both therapeutic and diagnostic targets. Two monoclonal antibodies, mAb38F and mAb38E2, with high affinity and strong selectivity for large α-synuclein oligomers were generated. These antibodies, which do not bind amyloid-beta or tau, recognize Lewy body pathology in brains from patients with Parkinson's disease and dementia with Lewy bodies and detect pathology earlier in α-synuclein transgenic mice than linear epitope antibodies. An oligomer-selective sandwich ELISA, based on mAb38F, was set up to analyze brain extracts of the transgenic mice. The overall levels of α-synuclein oligomers/protofibrils were found to increase with age in these mice, although the levels displayed a large interindividual variation. Upon subcellular fractionation, higher levels of α-synuclein oligomers/protofibrils could be detected in the endoplasmic reticulum around the age when behavioral disturbances develop. In summary, our novel oligomer-selective α-synuclein antibodies recognize relevant pathology and should be important tools to further explore the pathogenic mechanisms in Lewy body disorders. Moreover, they could be potential candidates both for immunotherapy and as reagents in an assay to assess a potential disease biomarker.

γ-Secretase complexes containing caspase-cleaved presenilin-1 increase intracellular Aß(42) /Aß(40) ratio.

Markers for caspase activation and apoptosis have been shown in brains of Alzheimer's disease (AD) patients and AD-mouse models. In neurons, caspase activation is associated with elevated amyloid ß-peptide (Aß) production. Caspases cleave numerous substrates including presenilin-1 (PS1). The cleavage takes place in the large cytosolic loop of PS1-C-terminal fragment (PS1CTF), generating a truncated PS1CTF lacking half of the loop domain (caspCTF). The loop has been shown to possess important regulatory functions with regard to Aß(40) and Aß(42) production. Previously, we have demonstrated that γ-secretase complexes are active during apoptosis regardless of caspase cleavage in the PS1CTF-loop. Here, a PS1/PS2-knockout mouse blastocyst-derived cell line was used to establish stable or transient cell lines expressing either caspCTF or full-length CTF (wtCTF). We show that caspCTF restores γ-secretase activity and forms active γ-secretase complexes together with Nicastrin, Pen-2, Aph-1 and PS1-N-terminal fragment. Further, caspCTF containing γ-secretase complexes have a sustained capacity to cleave amyloid precursor protein (APP) and Notch, generating APP and Notch intracellular domain, respectively. However, when compared to wtCTF cells, caspCTF cells exhibit increased intracellular production of Aß(42) accompanied by increased intracellular Aß(42) /Aß(40) ratio without changing the Aß secretion pattern. Similarly, induction of apoptosis in wtCTF cells generate a similar shift in intracellular Aß pattern with increased Aß(42) /Aß(40) ratio. In summary, we show that caspase cleavage of PS1 generates a γ-secretase complex that increases the intracellular Aß(42) /Aß(40) ratio. This can have implications for AD pathogenesis and suggests caspase inhibitors as potential therapeutic agents.

Gelsolin co-occurs with Lewy bodies in vivo and accelerates α-synuclein aggregation in vitro.

Deposition of fibrillar α-synuclein as Lewy bodies is the neuropathological hallmark of Parkinson's disease (PD) and dementia with Lewy bodies (DLB). Apart from α-synuclein, these intraneuronal inclusions contain over 250 different proteins. The actin binding protein gelsolin, has previously been suggested to be part of the Lewy body, but its potential role in α-synuclein aggregation remains unknown. Here, we studied the association between gelsolin and α-synuclein in brain tissue from PD and DLB patients as well as in a cell model for α-synuclein aggregation. Moreover, the potential effect of gelsolin on α-synuclein fibrillization was also investigated. Our data demonstrate that gelsolin co-occured with α-synuclein in Lewy bodies from affected human brain as well as with Lewy body-like inclusions in α-synuclein over expressing cells. Furthermore, in the presence of calcium chloride, gelsolin was found to enhance the aggregation rate of α-synuclein in vitro. Moreover, no apparent structural differences could be observed between fibrils formed in the presence or absence of gelsolin. Further studies on gelsolin and other Lewy body associated proteins are warranted to learn more about their potential role in the α-synuclein aggregation process.

Affinity pulldown of γ-secretase and associated proteins from human and rat brain.

γ-Secretase is a transmembrane protease complex responsible for the processing of a multitude of type 1 transmembrane proteins, including amyloid precursor protein (APP) and Notch. A functional complex is dependent on the assembly of four proteins: presenilin (PS), nicastrin, Aph-1 and Pen-2. Little is known about how the substrates are selected by γ-secretase, but it has been suggested that γ-secretase associated proteins (GSAPs) could be of importance. For instance, it was recently reported from studies in cell lines that TMP21, a transmembrane protein involved in trafficking, binds to γ-secretase and regulates the processing of APP-derived substrates without affecting Notch cleavage. Here, we present an efficient and selective method for purification and analysis of γ-secretase and GSAPs. Microsomal membranes were prepared from rat or human brain and incubated with a γ-secretase inhibitor coupled to biotin via a long linker and a S-S bridge. After pulldown using streptavidin beads, bound proteins were eluted under reducing conditions and digested by trypsin. The tryptic peptides were subjected to LC-MS/MS analysis, and proteins were identified by sequence data from MS/MS spectra. All of the known γ-secretase components were identified. Interestingly, TMP21 and the PS associated protein syntaxin1 were associated to γ-secretase in rat brain. We suggest that the present method can be used for further studies on the composition of the γ-secretase complex.

Abeta43 is more frequent than Abeta40 in amyloid plaque cores from Alzheimer disease brains.

One hallmark of Alzheimer disease (AD) is the extracellular deposition of the amyloid beta-peptide (Abeta) in senile plaques. Two major forms of Abeta are produced, 40 (Abeta40) and 42 (Abeta42) residues long. The most abundant form of Abeta is Abeta40, while Abeta42 is more hydrophobic and more prone to form toxic oligomers and the species of particular importance in early plaque formation. Thus, the length of the hydrophobic C-terminal seems to be very important for the oligomerization and neurotoxicity of the Abeta peptide. Here we investigated which Abeta species are deposited in AD brain. We analyzed plaque cores, prepared from occipital and frontal cortex, from sporadic and familial AD cases and performed a quantitative study using Abeta standard peptides. Cyanogen bromide was used to generate C-terminal Abeta fragments, which were analyzed by HPLC coupled to an electrospray ionisation ion trap mass spectrometer. We found a longer peptide, Abeta43, to be more frequent than Abeta40. No variants longer than Abeta43 could be observed in any of the brains. Immunohistochemistry was performed and was found to be in line with our findings. Abeta1-43 polymerizes rapidly and we suggest that this variant may be of importance for AD.

Active gamma-secretase is localized to detergent-resistant membranes in human brain.

Several lines of evidence suggest that polymerization of the amyloid beta-peptide (Abeta) into amyloid plaques is a pathogenic event in Alzheimer's disease (AD). Abeta is produced from the amyloid precursor protein as the result of sequential proteolytic cleavages by beta-secretase and gamma-secretase, and it has been suggested that these enzymes could be targets for treatment of AD. gamma-Secretase is an aspartyl protease complex, containing at least four transmembrane proteins. Studies in cell lines have shown that gamma-secretase is partially localized to lipid rafts, which are detergent-resistant membrane microdomains enriched in cholesterol and sphingolipids. Here, we studied gamma-secretase in detergent-resistant membranes (DRMs) prepared from human brain. DRMs prepared in the mild detergent CHAPSO and isolated by sucrose gradient centrifugation were enriched in gamma-secretase components and activity. The DRM fraction was subjected to size-exclusion chromatography in CHAPSO, and all of the gamma-secretase components and a lipid raft marker were found in the void volume (> 2000 kDa). Co-immunoprecipitation studies further supported the notion that the gamma-secretase components are associated even at high concentrations of CHAPSO. Preparations from rat brain gave similar results and showed a postmortem time-dependent decline in gamma-secretase activity, suggesting that DRMs from fresh rat brain may be useful for gamma-secretase activity studies. Finally, confocal microscopy showed co-localization of gamma-secretase components and a lipid raft marker in thin sections of human brain. We conclude that the active gamma-secretase complex is localized to lipid rafts in human brain.

Low molar excess of 4-oxo-2-nonenal and 4-hydroxy-2-nonenal promote oligomerization of alpha-synuclein through different pathways.

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal inclusions found in brains with Parkinson's disease and dementia with Lewy bodies. A body of evidence implicates oxidative stress in the pathogenesis of these diseases. For example, a large excess (30:1, aldehyde:protein) of the lipid peroxidation end products 4-oxo-2-nonenal (ONE) or 4-hydroxy-2-nonenal (HNE) can induce alpha-synuclein oligomer formation. The objective of the study was to investigate the effect of these reactive aldehydes on alpha-synuclein at a lower molar excess (3:1) at both physiological (7.4) and acidic (5.4) pH. As observed by size-exclusion chromatography, ONE rapidly induced the formation of alpha-synuclein oligomers at both pH values, but the effect was less pronounced under the acidic condition. In contrast, only a small proportion of alpha-synuclein oligomers were formed with low excess HNE-treatment at physiological pH and no oligomers at all under the acidic condition. With prolonged incubation times (up to 96h), more alpha-synuclein was oligomerized at physiological pH for both ONE and HNE. As determined by Western blot, ONE-oligomers were more SDS-stable and to a higher-degree cross-linked as compared to the HNE-induced oligomers. However, as shown by their greater sensitivity to proteinase K treatment, ONE-oligomers, exhibited a less compact structure than HNE-oligomers. As indicated by mass spectrometry, ONE modified most Lys residues, whereas HNE primarily modified the His50 residue and fewer Lys residues, albeit to a higher degree than ONE. Taken together, our data show that the aldehydes ONE and HNE can modify alpha-synuclein and induce oligomerization, even at low molar excess, but to a higher degree at physiological pH and seemingly through different pathways.

The pathogenic aß43 is enriched in familial and sporadic Alzheimer disease.

The amyloid-cascade hypothesis posits that the role of amyloid ß-peptide (Aß) in Alzheimer disease (AD) involves polymerization into structures that eventually are deposited as amyloid plaques. During this process, neurotoxic oligomers are formed that induce synaptic loss and neuronal death. Several different isoforms of Aß are produced, of which the 40 and 42 residue variants (Aß40 and Aß42) are the most common. Aß42 has a strong tendency to form neurotoxic aggregates and is involved in AD pathogenesis. Longer Aß isoforms, like the less studied Aß43, are gaining attention for their higher propensity to aggregate into neurotoxic oligomers. To further investigate Aß43 in AD, we conducted a quantitative study on Aß43 levels in human brain. We homogenized human brain tissue and prepared fractions of various solubility; tris buffered saline (TBS), sodium dodecyl sulfate (SDS) and formic acid (FA). Levels of Aß43, as well as Aß40 and Aß42, were quantified using ELISA. We compared quantitative data showing Aß levels in occipital and frontal cortex from sporadic (SAD) and familial (FAD) AD cases, as well as non-demented (ND) controls. Results showed Aß43 present in each fraction from the SAD and FAD cases, while its level was lower than the detection limit in the majority of the ND-cases. Aß42 and Aß43 were enriched in the less soluble fractions (SDS and FA) of SAD and FAD cases in both occipital and frontal cortex. Thus, although the total levels of Aß43 in human brain are low compared to Aß40 and Aß42, we suggest that Aß43 could initiate the formation of oligomers and amyloid plaques and thereby be crucial to AD pathogenesis.

The PSEN1 I143T mutation in a Swedish family with Alzheimer's disease: clinical report and quantification of Aß in different brain regions.

Early-onset dominantly inherited forms of Alzheimer's disease (AD) are rare, but studies of such cases have revealed important information about the disease mechanisms. Importantly, mutations in amyloid precursor protein (APP), presenilin 1 (PSEN1) and PSEN2, alter the APP processing and lead to an increased amyloid ß-peptide (Aß) 42/40 ratio. This, together with other studies on pathogenic mechanisms, show that Aß42 is a major player in the etiology of AD. Here, we present a clinical and neuropathological description of a Swedish family with an I143T mutation in the PSEN1 gene, which gives rise to a severe form of AD. We also performed an extensive investigation on the concentration and distribution of Aß species of different lengths in six brain regions from two mutation carriers. Our study showed that Aß42 and a longer peptide, Aß43, were present both in plaque cores and in total amyloid preparations, and were each clearly more frequent than Aß40 in all examined regions, as shown by both mass spectrometry and immunohistochemistry.

Rat brain gamma-secretase activity is highly influenced by detergents.

Gamma-secretase is important for the development of Alzheimer's disease, since it is a crucial enzyme for the generation of the pathogenic amyloid beta-peptide (Abeta). Most data on gamma-secretase is derived from studies in cell lines overexpressing gamma-secretase components or amyloid precursor protein (APP), and since gamma-secretase is a transmembrane protein complex, detergents have been frequently used to facilitate the studies. However, no extensive comparison of the influence of different detergents at different concentrations on gamma-secretase activity in preparations from brain has been made. Here, we establish the optimal conditions for gamma-secretase activity in rat brain, using an activity assay detecting endogenous production of the APP intracellular domain, which is generated when gamma-secretase cleaves the APP C-terminal fragments. We performed a subcellular fractionation and noted the highest gamma-secretase activity in the 100000g pellet and that the optimal pH was around 7. We found that gamma-secretase was active for at least 16 h at 37 degrees C and that the endogenous substrate levels were sufficient for activity measurements. The highest activity was obtained in 0.4% CHAPSO, which is slightly below the critical micelle concentration (0.5%) for this detergent, but the complex was not solubilized efficiently at this concentration. On the other hand, 1% CHAPSO solubilized a substantial amount of the gamma-secretase components, but the activity was low. The activity was fully restored by diluting the sample to 0.4% CHAPSO. Therefore, using 1% CHAPSO for solubilization and subsequently diluting the sample to 0.4% is an appropriate procedure for obtaining a soluble, highly active gamma-secretase from rat brain.