Natsuko Goto Yamamoto

Identification of Novel γ-Secretase-associated Proteins in Detergent-resistant Membranes from Brain

Background: In AD, APP can be processed in lipid rafts, and γ-secretase-associated proteins (GSAPs) can affect Aβ production. Results: We identify novel GSAPs in detergent-resistant membranes (DRMs). Conclusion: VDAC1 and CNTNAP1 associate with γ-secretase in DRMs and affect APP processing with less effect on Notch processing. Significance: Novel GSAPs that regulate Aβ production can be used as AD therapeutic targets. In Alzheimer disease, oligomeric amyloid β-peptide (Aβ) species lead to synapse loss and neuronal death. γ-Secretase, the transmembrane protease complex that mediates the final catalytic step that liberates Aβ from its precursor protein (APP), has a multitude of substrates, and therapeutics aimed at reducing Aβ production should ideally be specific for APP cleavage. It has been shown that APP can be processed in lipid rafts, and γ-secretase-associated proteins can affect Aβ production. Here, we use a biotinylated inhibitor for affinity purification of γ-secretase and associated proteins and mass spectrometry for identification of the purified proteins, and we identify novel γ-secretase-associated proteins in detergent-resistant membranes from brain. Furthermore, we show by small interfering RNA-mediated knockdown of gene expression that a subset of the γ-secretase-associated proteins, in particular voltage-dependent anion channel 1 (VDAC1) and contactin-associated protein 1 (CNTNAP1), reduced Aβ production (Aβ40 and Aβ42) by around 70%, whereas knockdown of presenilin 1, one of the essential γ-secretase complex components, reduced Aβ production by 50%. Importantly, these proteins had a less pronounced effect on Notch processing. We conclude that VDAC1 and CNTNAP1 associate with γ-secretase in detergent-resistant membranes and affect APP processing and suggest that molecules that interfere with this interaction could be of therapeutic use for Alzheimer disease.

Visualizing active enzyme complexes using a photoreactive inhibitor for proximity ligation--application on γ-secretase.

Here, we present a highly sensitive method to study protein-protein interactions and subcellular location selectively for active multicomponent enzymes. We apply the method on γ-secretase, the enzyme complex that catalyzes the cleavage of the amyloid precursor protein (APP) to generate amyloid β-peptide (Aβ), the major causative agent in Alzheimer disease (AD). The novel assay is based on proximity ligation, which can be used to study protein interactions in situ with very high sensitivity. In traditional proximity ligation assay (PLA), primary antibody recognition is typically accompanied by oligonucleotide-conjugated secondary antibodies as detection probes. Here, we first performed PLA experiments using antibodies against the γ-secretase components presenilin 1 (PS1), containing the catalytic site residues, and nicastrin, suggested to be involved in substrate recognition. To selectively study the interactions of active γ-secretase, we replaced one of the primary antibodies with a photoreactive γ-secretase inhibitor containing a PEG linker and a biotin group (GTB), and used oligonucleotide-conjugated streptavidin as a probe. Interestingly, significantly fewer interactions were detected with the latter, novel, assay, which is a reasonable finding considering that a substantial portion of PS1 is inactive. In addition, the PLA signals were located more peripherally when GTB was used instead of a PS1 antibody, suggesting that γ-secretase matures distal from the perinuclear ER region. This novel technique thus enables highly sensitive protein interaction studies, determines the subcellular location of the interactions, and differentiates between active and inactive γ-secretase in intact cells. We suggest that similar PLA assays using enzyme inhibitors could be useful also for other enzyme interaction studies.

Identification of two novel synaptic γ-secretase associated proteins that affect amyloid β-peptide levels without altering Notch processing

Synaptic degeneration is one of the earliest hallmarks of Alzheimer disease (AD) and results in loss of cognitive function. One of the causative agents for the synaptic degeneration is the amyloid β-peptide (Aβ), which is formed from its precursor protein by two sequential cleavages mediated by β- and γ-secretase. We have earlier shown that γ-secretase activity is enriched in synaptic compartments, suggesting that the synaptotoxic Aβ is produced locally. Proteins that interact with γ-secretase at the synapse and regulate the production of Aβ can therefore be potential therapeutic targets. We used a recently developed affinity purification approach to identify γ-secretase associated proteins (GSAPs) in synaptic membranes and synaptic vesicles prepared from rat brain. Liquid chromatography-tandem mass spectrometry analysis of the affinity purified samples revealed the known γ-secretase components presenilin-1, nicastrin and Aph-1b along with a number of novel potential GSAPs. To investigate the effect of these GSAPs on APP processing, we performed siRNA experiments to knock down the expression of the GSAPs and measured the Aβ levels. Silencing of NADH dehydrogenase [ubiquinone] iron-sulfur protein 7 (NDUFS7) resulted in a decrease in Aβ levels whereas silencing of tubulin polymerization promoting protein (TPPP) resulted in an increase in Aβ levels. Treatment with γ-secretase inhibitors often results in Notch-related side effects and therefore we also studied the effect of the siRNAs on Notch processing. Interestingly, silencing of TPPP or NDUFS7 did not affect cleavage of Notch. We also studied the expression of TPPP and NDUFS7 in control and AD brain and found NDUFS7 to be highly expressed in vulnerable neurons such as pyramidal neurons in the hippocampus, whereas TPPP was found to accumulate in intraneuronal granules and fibrous structures in hippocampus from AD cases. In summary, we here report on two proteins, TPPP and NDUFS7, which interact with γ-secretase and alter the Aβ levels without affecting Notch cleavage.

Abnormal platelet amyloid-β protein precursor (AβPP) metabolism in Alzheimer's disease: identification and characterization of a new AβPP isoform as potential biomarker.

Previous findings demonstrated an altered pattern of amyloid-β protein precursor (AβPP) expression in platelets of Alzheimers disease (AD) patients compared with either healthy control subjects or patients with non-Alzheimer-type dementia. In an attempt to explore the diagnostic potential of platelet AβPP metabolism, we have generated monoclonal antibodies directed to the N-terminal part of AβPP. We have observed two different antibody recognition patterns of AβPP: one resembling previously described 130 kDa and 105 kDa species and a novel AβPP 115 kDa form. This form was significantly increased in platelets of the mild cognitive impairment and AD group as compared to control subjects. The abundance of AβPP 115 kDa species correlated with the previously described AβPP 130/105 kDa ratio as well as with Mini-Mental State Examination score. Despite the inability of these particular monoclonal antibodies to recognize native forms of AβPP, identification of a new AβPP isoform in platelets as a potential AD biomarker can provide an additional tool for the development of a reliable diagnostic test to detect preclinical stages of AD.

Proton myo‐inositol cotransporter is a novel γ‐secretase associated protein that regulates Aβ production without affecting Notch cleavage

γ‐Secretase is a transmembrane protease complex that is responsible for the processing of a multitude of type 1 transmembrane proteins, including the amyloid precursor protein and Notch. γ‐Secretase processing of amyloid precursor protein results in the release of the amyloid β‐peptide (Aβ), which is involved in the pathogenesis in Alzheimers disease. Processing of Notch leads to the release of its intracellular domain, which is important for cell development. γ‐Secretase associated proteins (GSAPs) could be of importance for substrate selection, and we have previously shown that affinity purification of γ‐secretase in combination with mass spectrometry can be used for finding such proteins. In the present study, we used this methodology to screen for novel GSAPs from human brain, and studied their effect on Aβ production in a comprehensive gene knockdown approach. Silencing of probable phospholipid‐transporting ATPase IIA, brain‐derived neurotrophic factor/neurotrophin‐3 growth factor receptor precursor and proton myo‐inositol cotransporter (SLC2A13) showed a clear reduction of Aβ and these proteins were selected for further studies on Aβ production and Notch cleavage using small interfering RNA‐mediated gene silencing, as well as an overexpression approach. Silencing of these reduced Aβ secretion in a small interfering RNA dose‐dependent manner. Interestingly, SLC2A13 had a lower effect on Notch processing. Furthermore, overexpression of SLC2A13 increased Aβ40 generation. Finally, the interaction between γ‐secretase and SLC2A13 was confirmed using immunoprecipitation and a proximity ligation assay. In summary, SLC2A13 was identified as a novel GSAP that regulates Aβ production without affecting Notch cleavage. We suggest that SLC2A13 could be a target for Aβ lowering therapy aimed at treating Alzheimers disease.

Monoamine oxidase B is elevated in Alzheimer disease neurons, is associated with γ-secretase and regulates neuronal amyloid β-peptide levels

BackgroundIncreased levels of the pathogenic amyloid β-peptide (Aβ), released from its precursor by the transmembrane protease γ-secretase, are found in Alzheimer disease (AD) brains. Interestingly, monoamine oxidase B (MAO-B) activity is also increased in AD brain, but its role in AD pathogenesis is not known. Recent neuroimaging studies have shown that the increased MAO-B expression in AD brain starts several years before the onset of the disease. Here, we show a potential connection between MAO-B, γ-secretase and Aβ in neurons.MethodsMAO-B immunohistochemistry was performed on postmortem human brain. Affinity purification of γ-secretase followed by mass spectrometry was used for unbiased identification of γ-secretase-associated proteins. The association of MAO-B with γ-secretase was studied by coimmunoprecipitation from brain homogenate, and by in-situ proximity ligation assay (PLA) in neurons as well as mouse and human brain sections. The effect of MAO-B on Aβ production and Notch processing in cell cultures was analyzed by siRNA silencing or overexpression experiments followed by ELISA, western blot or FRET analysis. Methodology for measuring relative intraneuronal MAO-B and Aβ42 levels in single cells was developed by combining immunocytochemistry and confocal microscopy with quantitative image analysis.ResultsImmunohistochemistry revealed MAO-B staining in neurons in the frontal cortex, hippocampus CA1 and entorhinal cortex in postmortem human brain. Interestingly, the neuronal staining intensity was higher in AD brain than in control brain in these regions. Mass spectrometric data from affinity purified γ-secretase suggested that MAO-B is a γ-secretase-associated protein, which was confirmed by immunoprecipitation and PLA, and a neuronal location of the interaction was shown. Strikingly, intraneuronal Aβ42 levels correlated with MAO-B levels, and siRNA silencing of MAO-B resulted in significantly reduced levels of intraneuronal Aβ42. Furthermore, overexpression of MAO-B enhanced Aβ production.ConclusionsThis study shows that MAO-B levels are increased not only in astrocytes but also in pyramidal neurons in AD brain. The study also suggests that MAO-B regulates Aβ production in neurons via γ-secretase and thereby provides a key to understanding the relationship between MAO-B and AD pathogenesis. Potentially, the γ-secretase/MAO-B association may be a target for reducing Aβ levels using protein–protein interaction breakers.

Human brain proteins showing neuron‐specific interactions with γ‐secretase

The transmembrane protease complex γ‐secretase is a key enzyme in Alzheimer disease pathogenesis as it liberates the neurotoxic amyloid β‐peptide (Aβ); however, the mechanism of regulation of its activity in various cell types and subcellular compartments is largely unknown. Several γ‐secretase inhibitors have been developed, but none have been released due to side‐effects that appear to arise from reduced processing of Notch, one of many γ‐secretase substrates. Hence, it is desirable to specifically inhibit Aβ production. In our previous studies, we have identified several γ‐secretase‐associated proteins (GSAPs) from brain, which affect Aβ production without having any major effects on Notch processing. In the present study using detergent‐resistant membranes prepared from brain, we have identified four GSAPs that affect Aβ production to a greater extent than Notch processing. We evaluated the interaction between GSAPs and γ‐secretase in various cell types and their mRNA expression in various human organs. Using an in situ proximity ligation assay, we demonstrated that many GSAPs showed considerably greater interaction with γ‐secretase in neurons than in human embryonic kidney cells stably over‐expressing APP, and showed that several GSAPs are highly expressed in human brain. This study underscores the importance of studying protein‐protein interactions in relevant cell types, and suggests that reducing Aβ production by interfering with brain‐ or neuron‐specific γ‐secretase/GSAP interactions may reduce the risk of unwanted side‐effects associated with treatment of Alzheimer disease.

Maturation and processing of the amyloid precursor protein is regulated by the potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2)

The toxic amyloid β-peptide (Aβ) is a key player in Alzheimer Disease (AD) pathogenesis and selective inhibition of the production of this peptide is sought for. Aβ is produced by the sequential cleavage of the Aβ precursor protein (APP) by β-secretase (to yield APP-C-terminal fragment β (APP-CTFβ) and soluble APPβ (sAPPβ)) and γ-secretase (to yield Aβ). We reasoned that proteins that associate with γ-secretase are likely to regulate Aβ production and to be targets of pharmaceutical interventions and therefore performed a pull-down assay to screen for such proteins in rat brain. Interestingly, one of the purified proteins was potassium/sodium hyperpolarization-activated cyclic nucleotide-gated ion channel 2 (HCN2), which has been shown to be involved in epilepsy. We found that silencing of HCN2 resulted in decreased secreted Aβ levels. To further investigate the mechanism behind this reduction, we also determined the levels of full-length APP, sAPP and APP-CTF species after silencing of HCN2. A marked reduction in sAPP and APP-CTF, as well as glycosylated APP levels was detected. Decreased Aβ, sAPP and APP-CTF levels were also detected after treatment with the HCN2 inhibitor ZD7288. These results indicate that the effect on Aβ levels after HCN2 silencing or inhibition is due to altered APP maturation or processing by β-secretase rather than a direct effect on γ-secretase. However, HCN2 and γ-secretase were found to be in close proximity, as evident by proximity ligation assay and immunoprecipitation. In summary, our results indicate that silencing or inhibition of HCN2 affects APP processing and thereby could serve as a potential treatment strategy.

NEURON-SELECTIVE GAMMA-SECRETASE INTERACTIONS WITH PROTEINS EXPRESSED IN HUMAN BRAIN

Background:Alzheimer’s disease (AD) is characterized by the presence of amyloid beta (Ab) plaques and neurofibrillary tangles (NFT). SPPL2b is a novel enzyme that can process TNFa and BRI2, substrates involved in Ab plaque and NFT formation. Since changes in BRI2 and TNFa were previously identified in early stages of AD, we have now extensively characterized SPPL2b in human brain tissue from controls and AD patients at different stages of the disease. We also investigated the relation of SPPL2b with important pathological AD hallmarks including amyloid plaques, NFTs, ER stress and ubiquitin. Methods: SPPL2b expression levels were quantified in post-mortem human homogenates from AD patients (n1⁄414) and controls (n1⁄413) by western blot. SPPL2b immunostaining was evaluated on paraffin sections from AD patients (n 1⁄4 12) and age-matched controls (n1⁄415). Results: SPPL2b levels were 10-fold increased (p < 0.0001) in AD hippocampus compared to non-demented controls. SPPL2b was strongly correlated (r 1⁄4 0.785, p < 0.0001) to Braak stages showing a significant increased from Braak III to IV, stages in which cognitive impairment starts. Strong SPPL2b immunoreactivity in the hippocampus was observed in early AD stages (p <0 .0001), which was associated with both NFT and Ab plaques. Preincubation and comparison of staining patterns of SPPL2b family members proved that the observations were SPPL2b-specific. Conclusions: We found a dramatically increase of SPPL2b in early stages of AD. SPPL2b processes BRI2 and TNF a, proteins involved not only in Ab homeostasis but also in chronic inflammation and NFT formation. These data reveal a novel potential AD etiological factor that links Ab plaques and NFT and thus forms a new and promising target for disease modifying therapies.