Person: Selkoe, Dennis
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Selkoe
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Selkoe, Dennis
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Publication A highly sensitive novel immunoassay specifically detects low levels of soluble Aβ oligomers in human cerebrospinal fluid(BioMed Central, 2015) Yang, Ting; O’Malley, Tiernan T; Kanmert, Daniel; Jerecic, Jasna; Zieske, Lynn R; Zetterberg, Henrik; Hyman, Bradley; Walsh, Dominic; Selkoe, DennisIntroduction: Amyloid β-protein oligomers play a key role in Alzheimer’s disease (AD), but well-validated assays that routinely detect them in cerebrospinal fluid (CSF) are just emerging. We sought to confirm and extend a recent study using the Singulex Erenna platform that reported increased mean CSF oligomer levels in AD. Methods: We tested four antibody pairs and chose one pair that was particularly sensitive, using 1C22, our new oligomer-selective monoclonal antibody, for capture. We applied this new assay to extracts of human brain and CSF. Results: A combination of 1C22 for capture and 3D6 for detection yielded an Erenna immunoassay with a lower limit of quantification of approximately 0.15 pg/ml that was highly selective for oligomers over monomers and detected a wide size-range of oligomers. Most CSFs we tested had detectable oligomer levels but with a large overlap between AD and controls and a trend for higher mean levels in mild cognitive impairment (MCI) than controls. Conclusion: Aβ oligomers are detectable in most human CSFs, but AD and controls overlap. MCI CSFs may have a modest elevation in mean value by this assay. Electronic supplementary material The online version of this article (doi:10.1186/s13195-015-0100-y) contains supplementary material, which is available to authorized users.Publication Cell-type Dependent Alzheimer's Disease Phenotypes: Probing the Biology of Selective Neuronal Vulnerability(Elsevier, 2017) Muratore, Christina; Zhou, Constance; Liao, Meichen; Fernandez, Marty; Taylor, Walter M.; Lagomarsino, Valentina N.; Pearse, Richard; Rice, Heather C.; Negri, Joseph; He, Amy; Srikanth, Priya; Callahan, Dana; Shin, Taehwan; Zhou, Monica; Bennett, David A.; Noggle, Scott; Love, J. Christopher; Selkoe, Dennis; Young-Pearse, TracySummary Alzheimer's disease (AD) induces memory and cognitive impairment in the absence of motor and sensory deficits during its early and middle course. A major unresolved question is the basis for this selective neuronal vulnerability. Aβ, which plays a central role in AD pathogenesis, is generated throughout the brain, yet some regions outside of the limbic and cerebral cortices are relatively spared from Aβ plaque deposition and synapse loss. Here, we examine neurons derived from iPSCs of patients harboring an amyloid precursor protein mutation to quantify AD-relevant phenotypes following directed differentiation to rostral fates of the brain (vulnerable) and caudal fates (relatively spared) in AD. We find that both the generation of Aβ and the responsiveness of TAU to Aβ are affected by neuronal cell type, with rostral neurons being more sensitive than caudal neurons. Thus, cell-autonomous factors may in part dictate the pattern of selective regional vulnerability in human neurons in AD.Publication Secreted Amyloid β-Proteins in a Cell Culture Model Include N-Terminally Extended Peptides That Impair Synaptic Plasticity(American Chemical Society, 2014) Welzel, Alfred T.; Maggio, John; Shankar, Ganesh; Walker, Donald E.; Ostaszewski, Beth L.; Li, Shaomin; Klyubin, Igor; Rowan, Michael J.; Seubert, Peter; Walsh, Dominic; Selkoe, DennisEvidence for a central role of amyloid β-protein (Aβ) in the genesis of Alzheimer’s disease (AD) has led to advanced human trials of Aβ-lowering agents. The “amyloid hypothesis” of AD postulates deleterious effects of small, soluble forms of Aβ on synaptic form and function. Because selectively targeting synaptotoxic forms of soluble Aβ could be therapeutically advantageous, it is important to understand the full range of soluble Aβ derivatives. We previously described a Chinese hamster ovary (CHO) cell line (7PA2 cells) that stably expresses mutant human amyloid precursor protein (APP). Here, we extend this work by purifying an sodium dodecyl sulfate (SDS)-stable, ∼8 kDa Aβ species from the 7PA2 medium. Mass spectrometry confirmed its identity as a noncovalently bonded Aβ40 homodimer that impaired hippocampal long-term potentiation (LTP) in vivo. We further report the detection of Aβ-containing fragments of APP in the 7PA2 medium that extend N-terminal from Asp1 of Aβ. These N-terminally extended Aβ-containing monomeric fragments are distinct from soluble Aβ oligomers formed from Aβ1-40/42 monomers and are bioactive synaptotoxins secreted by 7PA2 cells. Importantly, decreasing β-secretase processing of APP elevated these alternative synaptotoxic APP fragments. We conclude that certain synaptotoxic Aβ-containing species can arise from APP processing events N-terminal to the classical β-secretase cleavage site.Publication A New Method for Quantitative Immunoblotting of Endogenous α-Synuclein(Public Library of Science, 2013) Newman, Andrew J.; Selkoe, Dennis; Dettmer, Ulfβ-Sheet-rich aggregates of α-synuclein (αSyn) are the hallmark neuropathology of Parkinson’s disease and related synucleinopathies, whereas the principal native structure of αSyn in healthy cells - unfolded monomer or α-helically folded oligomer - is under debate. Our recent crosslinking analysis of αSyn in intact cells showed that a large portion of endogenous αSyn can be trapped as oligomers, most notably as apparent tetramers. One challenge in such studies is accurately quantifying αSyn Western blot signals among samples, as crosslinked αSyn trends toward increased immunoreactivity. Here, we analyzed this phenomenon in detail and found that treatment with the reducible amine-reactive crosslinker DSP strongly increased αSyn immunoreactivity even after cleavage with the reducing agent β-mercaptoethanol. The effect was observed with all αSyn antibodies tested and in all sample types from human brain homogenates to untransfected neuroblastoma cells, permitting easy detection of endogenous αSyn in the latter, which had long been considered impossible. Coomassie staining of blots before and after several hours of washing revealed complete retention of αSyn after DSP/β-mercaptoethanol treatment, in contrast to a marked loss of αSyn without this treatment. The treatment also enhanced immunodetection of the homologs β- and γ-synuclein and of histones, another group of small, lysine-rich proteins. We conclude that by neutralizing positive charges and increasing protein hydrophobicity, amine crosslinker treatment promotes adhesion of αSyn to blotting membranes. These data help explain the recent report of fixing αSyn blots with paraformaldehyde after transfer, which we find produces similar but weaker effects. DSP/β-mercaptoethanol treatment of Western blots should be particularly useful to quantify low-abundance αSyn forms such as extracellular and post-translationally modified αSyn and splice variants.Publication Purification of α-Synuclein from Human Brain Reveals an Instability of Endogenous Multimers as the Protein Approaches Purity(American Chemical Society, 2014) Luth, Eric S.; Bartels, Tim; Dettmer, Ulf; Kim, Nora C.; Selkoe, DennisDespite two decades of research, the structure–function relationships of endogenous, physiological forms of α-synuclein (αSyn) are not well understood. Most in vitro studies of this Parkinson’s disease-related protein have focused on recombinant αSyn that is unfolded and monomeric, assuming that this represents its state in the normal human brain. Recently, we have provided evidence that αSyn exists in considerable part in neurons, erythrocytes, and other cells as a metastable multimer that principally sizes as a tetramer. In contrast to recombinant αSyn, physiological tetramers purified from human erythrocytes have substantial α-helical content and resist pathological aggregation into β-sheet rich fibers. Here, we report the first method to fully purify soluble αSyn from the most relevant source, human brain. We describe protocols that purify αSyn to homogeneity from nondiseased human cortex using ammonium sulfate precipitation, gel filtration, and ion exchange, hydrophobic interaction, and affinity chromatographies. Cross-linking of the starting material and the partially purified chromatographic fractions revealed abundant αSyn multimers, including apparent tetramers, but these were destabilized in large part to monomers during the final purification step. The method also fully purified the homologue β-synuclein, with a similar outcome. Circular dichroism spectroscopy showed that purified, brain-derived αSyn can display more helical content than the recombinant protein, but this result varied. Collectively, our data suggest that purifying αSyn to homogeneity destabilizes native, α-helix-rich multimers that exist in intact and partially purified brain samples. This finding suggests existence of a stabilizing cofactor (e.g., a small lipid) present inside neurons that is lost during final purification.Publication Parkinson-causing α-synuclein missense mutations shift native tetramers to monomers as a mechanism for disease initiation(Nature Pub. Group, 2015) Dettmer, Ulf; Newman, Andrew J.; Soldner, Frank; Luth, Eric S.; Kim, Nora C.; von Saucken, Victoria E.; Sanderson, John B.; Jaenisch, Rudolf; Bartels, Tim; Selkoe, Dennisβ-Sheet-rich α-synuclein (αS) aggregates characterize Parkinson's disease (PD). αS was long believed to be a natively unfolded monomer, but recent work suggests it also occurs in α-helix-rich tetramers. Crosslinking traps principally tetrameric αS in intact normal neurons, but not after cell lysis, suggesting a dynamic equilibrium. Here we show that freshly biopsied normal human brain contains abundant αS tetramers. The PD-causing mutation A53T decreases tetramers in mouse brain. Neurons derived from an A53T patient have decreased tetramers. Neurons expressing E46K do also, and adding 1-2 E46K-like mutations into the canonical αS repeat motifs (KTKEGV) further reduces tetramers, decreases αS solubility and induces neurotoxicity and round inclusions. The other three fPD missense mutations likewise decrease tetramer:monomer ratios. The destabilization of physiological tetramers by PD-causing missense mutations and the neurotoxicity and inclusions induced by markedly decreasing tetramers suggest that decreased α-helical tetramers and increased unfolded monomers initiate pathogenesis. Tetramer-stabilizing compounds should prevent this.Publication N-Alpha-Acetylation of α-Synuclein Increases Its Helical Folding Propensity, GM1 Binding Specificity and Resistance to Aggregation(Public Library of Science, 2014) Bartels, Tim; Kim, Nora C.; Luth, Eric Sloan; Selkoe, DennisA switch in the conformational properties of α-synuclein (αS) is hypothesized to be a key step in the pathogenic mechanism of Parkinson’s disease (PD). Whereas the beta-sheet-rich state of αS has long been associated with its pathological aggregation in PD, a partially alpha-helical state was found to be related to physiological lipid binding; this suggests a potential role of the alpha-helical state in controlling synaptic vesicle cycling and resistance to β-sheet rich aggregation. N-terminal acetylation is the predominant post-translational modification of mammalian αS. Using circular dichroism, isothermal titration calorimetry, and fluorescence spectroscopy, we have analyzed the effects of N-terminal acetylation on the propensity of recombinant human αS to form the two conformational states in interaction with lipid membranes. Small unilamellar vesicles of negatively charged lipids served as model membranes. Consistent with previous NMR studies using phosphatidylserine, we found that membrane-induced α-helical folding was enhanced by N-terminal acetylation and that greater exothermic heat could be measured upon vesicle binding of the modified protein. Interestingly, the folding and lipid binding enhancements with phosphatidylserine in vitro were weak when compared to that of αS with GM1, a lipid enriched in presynaptic membranes. The resultant increase in helical folding propensity of N-acetylated αS enhanced its resistance to aggregation. Our findings demonstrate the significance of the extreme N-terminus for folding nucleation, for relative GM1 specificity of αS-membrane interaction, and for a protective function of N-terminal-acetylation against αS aggregation mediated by GM1.Publication Physical and functional interaction between the α- and γ-secretases: A new model of regulated intramembrane proteolysis(The Rockefeller University Press, 2015) Chen, Allen C.; Kim, Sumin; Shepardson, Nina; Patel, Sarvagna; Hong, Soyon; Selkoe, DennisMany single-transmembrane proteins are sequentially cleaved by ectodomain-shedding α-secretases and the γ-secretase complex, a process called regulated intramembrane proteolysis (RIP). These cleavages are thought to be spatially and temporally separate. In contrast, we provide evidence for a hitherto unrecognized multiprotease complex containing both α- and γ-secretase. ADAM10 (A10), the principal neuronal α-secretase, interacted and cofractionated with γ-secretase endogenously in cells and mouse brain. A10 immunoprecipitation yielded γ-secretase proteolytic activity and vice versa. In agreement, superresolution microscopy showed that portions of A10 and γ-secretase colocalize. Moreover, multiple γ-secretase inhibitors significantly increased α-secretase processing (r = −0.86) and decreased β-secretase processing of β-amyloid precursor protein. Select members of the tetraspanin web were important both in the association between A10 and γ-secretase and the γ→α feedback mechanism. Portions of endogenous BACE1 coimmunoprecipitated with γ-secretase but not A10, suggesting that β- and α-secretases can form distinct complexes with γ-secretase. Thus, cells possess large multiprotease complexes capable of sequentially and efficiently processing transmembrane substrates through a spatially coordinated RIP mechanism.Publication The amyloid-beta forming tripeptide cleavage mechanism of γ-secretase(eLife Sciences Publications, Ltd, 2016) Bolduc, David M; Montagna, Daniel R; Seghers, Matthew C; Wolfe, Michael; Selkoe, Dennisγ-secretase is responsible for the proteolysis of amyloid precursor protein (APP) into short, aggregation-prone amyloid-beta (Aβ) peptides, which are centrally implicated in the pathogenesis of Alzheimer’s disease (AD). Despite considerable interest in developing γ-secretase targeting therapeutics for the treatment of AD, the precise mechanism by which γ-secretase produces Aβ has remained elusive. Herein, we demonstrate that γ-secretase catalysis is driven by the stabilization of an enzyme-substrate scission complex via three distinct amino-acid-binding pockets in the enzyme’s active site, providing the mechanism by which γ-secretase preferentially cleaves APP in three amino acid increments. Substrate occupancy of these three pockets occurs after initial substrate binding but precedes catalysis, suggesting a conformational change in substrate may be required for cleavage. We uncover and exploit substrate cleavage preferences dictated by these three pockets to investigate the mechanism by which familial Alzheimer’s disease mutations within APP increase the production of pathogenic Aβ species. DOI: http://dx.doi.org/10.7554/eLife.17578.001Publication The amyloid hypothesis of Alzheimer's disease at 25 years(John Wiley and Sons Inc., 2016) Selkoe, Dennis; Hardy, JohnAbstract Despite continuing debate about the amyloid β‐protein (or Aβ hypothesis, new lines of evidence from laboratories and clinics worldwide support the concept that an imbalance between production and clearance of Aβ42 and related Aβ peptides is a very early, often initiating factor in Alzheimer's disease (AD). Confirmation that presenilin is the catalytic site of γ‐secretase has provided a linchpin: all dominant mutations causing early‐onset AD occur either in the substrate (amyloid precursor protein, APP) or the protease (presenilin) of the reaction that generates Aβ. Duplication of the wild‐type APP gene in Down's syndrome leads to Aβ deposits in the teens, followed by microgliosis, astrocytosis, and neurofibrillary tangles typical of AD. Apolipoprotein E4, which predisposes to AD in > 40% of cases, has been found to impair Aβ clearance from the brain. Soluble oligomers of Aβ42 isolated from AD patients' brains can decrease synapse number, inhibit long‐term potentiation, and enhance long‐term synaptic depression in rodent hippocampus, and injecting them into healthy rats impairs memory. The human oligomers also induce hyperphosphorylation of tau at AD‐relevant epitopes and cause neuritic dystrophy in cultured neurons. Crossing human APP with human tau transgenic mice enhances tau‐positive neurotoxicity. In humans, new studies show that low cerebrospinal fluid (CSF) Aβ42 and amyloid‐PET positivity precede other AD manifestations by many years. Most importantly, recent trials of three different Aβ antibodies (solanezumab, crenezumab, and aducanumab) have suggested a slowing of cognitive decline in post hoc analyses of mild AD subjects. Although many factors contribute to AD pathogenesis, Aβ dyshomeostasis has emerged as the most extensively validated and compelling therapeutic target.