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1.  Systematic evaluation of candidate ligands regulating ectodomain shedding of Amyloid Precursor Protein 
Biochemistry  2013;52(19):10.1021/bi400165f.
Despite intense interest in the proteolysis of the ß-Amyloid Precursor Protein (APP) in Alzheimer's disease (AD), how the normal processing of this type I receptor-like glycoprotein is physiologically regulated remains ill-defined. In recent years, several candidate protein ligands for APP, including F-spondin, Reelin, β1 Integrin, Contactins, Lingo-1 and Pancortin, have been reported. However, a cognate ligand for APP that regulates its processing by α- or β-secretase has yet to be widely confirmed in multiple laboratories. Here, we developed new assays in an effort to confirm a role for one or more of these candidate ligands in regulating APP ectodomain shedding in a biologically relevant context. A comprehensive quantification of APPsα and APPsβ, the immediate products of secretase processing, in both non-neuronal cell lines and primary neuronal cultures expressing endogenous APP yielded no evidence that any of these published candidate ligands stimulate ectodomain shedding. Rather, Reelin, Lingo-1 and Pancortin-1 emerged as the most consistent ligands for significantly inhibiting ectodomain shedding. These findings led us to conduct further detailed analyses of the interactions of Reelin and Lingo-1 with APP.
doi:10.1021/bi400165f
PMCID: PMC3809327  PMID: 23597280
Regulated intramembrane proteolysis; Amyloid Precursor Protein; ectodomain shedding; Lingo-1/Reelin; Alzheimer's disease
2.  Alzheimer Disease in 2020 
Remarkable advances in unraveling the biological underpinnings of Alzheimer disease (AD) have occurred during the last 25 years. Despite this, we have made only the smallest of dents in the development of truly disease-modifying treatments. What will change over the next 10 years? While the answer is not clear, we make several predictions on the state of the field in 2020, based on the rich knowledge described in the other contributions in this collection. As such, our predictions represent some of the principal unresolved questions that we believe deserve special investigative attention in the coming decade.
By 2020, thanks to a significantly advanced understanding of Alzheimer disease pathogenesis, we may be on the verge of treatments that delay the onset of the disease in many millions worldwide.
doi:10.1101/cshperspect.a011585
PMCID: PMC3543098  PMID: 23125202
3.  N-Alpha-Acetylation of α-Synuclein Increases Its Helical Folding Propensity, GM1 Binding Specificity and Resistance to Aggregation 
PLoS ONE  2014;9(7):e103727.
A 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.
doi:10.1371/journal.pone.0103727
PMCID: PMC4116227  PMID: 25075858
4.  Secreted Amyloid β-Proteins in a Cell Culture Model Include N-Terminally Extended Peptides That Impair Synaptic Plasticity 
Biochemistry  2014;53(24):3908-3921.
Evidence 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.
doi:10.1021/bi5003053
PMCID: PMC4070750  PMID: 24840308
5.  Dynamic analysis of amyloid β-protein in behaving mice reveals opposing changes in ISF vs. parenchymal Aβ during age-related plaque formation 
Growing evidence supports the hypothesis that soluble, diffusible forms of the amyloid β-peptide (Aβ) are pathogenically important in Alzheimer’s disease (AD) and thus have both diagnostic and therapeutic salience. To learn more about the dynamics of soluble Aβ economy in vivo, we sampled by microdialysis the brain interstitial fluid (ISF), which contains the most soluble Aβ species in brain at steady state, in >40 wake, behaving APP transgenic mice before and during the process of Aβ plaque formation (age 3–28 months). Diffusible forms of Aβ, especially Aβ42, declined significantly in ISF as mice underwent progressive parenchymal deposition of Aβ. Moreover, radiolabeled Aβ administered at physiological concentrations into ISF revealed a striking difference in the fate of soluble Aβ in plaque-rich (vs. -free) mice: it clears more rapidly from the ISF and becomes more associated with the TBS-extractable pool, suggesting that cerebral amyloid deposits can rapidly sequester soluble Aβ from the ISF. Likewise, acute γ-secretase inhibition in plaque-free mice showed a marked decline of Aβ38, Aβ40 and Aβ42, whereas in plaque- rich mice, Aβ42 declined significantly less. These results suggest that most of the Aβ42 that populates the ISF in plaque-rich mice is derived not from new Aβ biosynthesis but rather from the large reservoir of less soluble Aβ42 in brain parenchyma. Together, these and other findings herein illuminate the in vivo dynamics of soluble Aβ during the development of AD-type neuropathology and after γ-secretase inhibition and help explain the apparent paradox that cerebrospinal fluid Aβ42 levels fall as humans develop AD.
doi:10.1523/JNEUROSCI.3272-11.2011
PMCID: PMC3227224  PMID: 22049429
6.  α-Synuclein occurs physiologically as a helically folded tetramer that resists aggregation 
Nature  2011;477(7362):107-110.
Parkinson disease (PD) is the second most common neurodegenerative disorder1,2. Growing evidence suggests a causative role of misfolded forms of the protein, α-synuclein (αSyn), in the pathogenesis of PD3,4. Intraneuronal aggregates of αSyn occur in Lewy bodies and Lewy neurites5, the cytopathological hallmarks of PD and the related disorders called synucleinopathies. αSyn has long been defined as a “natively unfolded” monomer of ∼14 kDa6 that is believed to acquire α-helical secondary structure only upon binding to lipid vesicles7. This concept derives from the widespread use of recombinant bacterial expression protocols for in vitro studies, and of overexpression, sample heating and/or denaturing gels for cell culture and tissue studies. In contrast, we report that endogenous αSyn isolated and analyzed under non-denaturing conditions from neuronal and non-neuronal cell lines, brain tissue and living human cells occurs in large part as a folded tetramer of ∼58 kDa. Multiple methods, including analytical ultracentrifugation, scanning transmission electron microscopy and in vivo cell crosslinking, confirmed the occurrence of the tetramer. Native, cell-derived αSyn showed α-helical structure without lipid addition and had much greater lipid binding capacity than the recombinant αSyn studied heretofore. Whereas recombinantly expressed monomers readily aggregated into amyloid-like fibrils in vitro, native human tetramers underwent little or no amyloid-like aggregation. Based on these findings, we propose that destabilization of the helically folded tetramer precedes αSyn misfolding and aggregation in PD and other human synucleinopathies and that small molecules which stabilize the physiological tetramer could reduce αSyn pathogenicity.
doi:10.1038/nature10324
PMCID: PMC3166366  PMID: 21841800
7.  Impaired glutamate recycling and GluN2B-mediated neuronal calcium overload in mice lacking TGF-β1 in the CNS 
Glia  2013;61(6):985-1002.
Transforming growth factor β1 (TGF-β1) is a pleiotropic cytokine expressed throughout the CNS. Previous studies demonstrated that TGF-β1 contributes to maintain neuronal survival, but mechanistically this effect is not well understood. We generated a CNS-specific TGF-β1-deficient mouse model to investigate the functional consequences of TGF-β1-deficiency in the adult mouse brain. We found that depletion of TGF-β1 in the CNS resulted in a loss of the astrocyte glutamate transporter (GluTs) proteins GLT-1 (EAAT2) and GLAST (EAAT1) and decreased glutamate uptake in the mouse hippocampus. Treatment with TGF-β1 induced the expression of GLAST and GLT-1 in cultured astrocytes and enhanced astroglial glutamate uptake. Similar to GLT-1-deficient mice, CNS-TGF-β1-deficient mice had reduced brain weight and neuronal loss in the CA1 hippocampal region. CNS-TGF-β1-deficient mice showed GluN2B-dependent aberrant synaptic plasticity in the CA1 area of the hippocampus similar to the glutamate transport inhibitor DL-TBOA and these mice were highly sensitive to excitotoxic injury. In addition, hippocampal neurons from TGF-β1-deficient mice had elevated GluN2B-mediated calcium signals in response to extrasynaptic glutamate receptor stimulation, whereas cells treated with TGF-β1 exhibited reduced GluN2B-mediated calcium signals. In summary, our study demonstrates a previously unrecognized function of TGF-β1 in the CNS to control extracellular glutamate homeostasis and GluN2B-mediated calcium responses in the mouse hippocampus.
doi:10.1002/glia.22490
PMCID: PMC3981075  PMID: 23536313
TGF-β1; glutamate uptake; hippocampus; neuronal calcium; extrasynaptic; astrocytes
8.  Biochemical and functional interaction of DISC1 and APP regulates neuronal migration during mammalian cortical development 
Although clinically distinct, schizophrenia and Alzheimer’s disease are common and devastating disorders that profoundly impair cognitive function. For Alzheimer’s disease, key mechanistic insights have emerged from genetic studies that identified causative mutations in Amyloid Precursor Protein (APP) and Presenilin. Several genes have been associated with schizophrenia and other major psychoses, and understanding their normal functions will help elucidate the underlying causes of these disorders. One such gene is Disrupted in Schizophrenia-1 (DISC1). DISC1 and APP have been implicated separately in cortical development, with each having roles in both neuronal migration and neurite outgrowth. Here, we report a previously unrecognized biochemical and functional interaction between DISC1 and APP. Using in utero electroporation in the living rat brain, we show that DISC1 acts downstream of APP and Disabled-1 to regulate cortical precursor cell migration. Specifically, overexpression of DISC1 rescues the migration defect caused by a loss of APP expression. Moreover, knock-down of APP in cultured embryonic neurons results in altered subcellular localization of DISC1. Using transfected cells and normal brain tissue, we show that APP and DISC1 co-immunoprecipitate and that the intracellular domain of APP interacts with the N-terminal domain of DISC1. Based on these findings, we hypothesize that the APP cytoplasmic region transiently interacts with DISC1 to help regulate the translocation of DISC1 to the centrosome, where it plays a key role in controlling neuronal migration during cortical development.
doi:10.1523/JNEUROSCI.1445-10.2010
PMCID: PMC3018837  PMID: 20685985
DISC1; APP; schizophrenia; Alzheimer’s; development; migration
9.  Environmental Novelty Activates β2-Adrenergic Signaling to Prevent the Impairment of Hippocampal LTP by Aβ Oligomers 
Neuron  2013;77(5):929-941.
Summary
A central question about human brain aging is whether cognitive enrichment slows the development of Alzheimer changes. Here we show that prolonged exposure to an enriched environment (EE) facilitated signaling in the hippocampus of wild-type mice that promoted long-term potentiation. A key feature of the EE effect was activation of β2-adrenergic receptors and downstream cAMP/PKA signaling. This EE pathway prevented LTP inhibition by soluble oligomers of amyloid β-protein (Aβ) isolated from AD cortex. Protection by EE occurred in both young and middle-aged wild-type mice. Exposure to novelty afforded greater protection than did aerobic exercise. Mice chronically fed a β-adrenergic agonist without EE were protected from hippocampal impairment by Aβ oligomers. Thus, EE enhances hippocampal synaptic plasticity by activating β-adrenoceptor signaling and mitigating synaptotoxicity of human Aβ oligomers. These mechanistic insights support using prolonged exposure to cognitive novelty and/or oral β-adrenergic agonists to lessen the effects of Aβ accumulation during aging.
doi:10.1016/j.neuron.2012.12.040
PMCID: PMC3596823  PMID: 23473322
10.  New ELISAs with high specificity for soluble oligomers of amyloid β-protein detect natural Aβ oligomers in human brain but not CSF 
Background
Soluble oligomers of amyloid β-protein (Aβ) have been increasingly linked to synaptic dysfunction, tau alteration and neuritic dystrophy in Alzheimer’s disease (AD) and mouse models. There is a great need for assays that quantify Aβ oligomers with high specificity and sensitivity.
Methods
We designed and validated two oligomer-specific (o-) ELISAs using either an Aβ aggregate-selective monoclonal for capture and a monoclonal to the free N-terminus for detection or the latter antibody for both capture and detection.
Results
The o-ELISAs specifically quantified pure oligomers of synthetic Aβ with sizes from dimers up to much larger assemblies and over a wide dynamic range of concentrations, whereas Aβ monomers were undetectable. Natural Aβ oligomers of similarly wide size and concentration ranges were measured in extracts of AD and control brains, revealing >1,000-fold higher concentrations of Aβ oligomers than monomers in the soluble fraction of AD cortex. The assays quantified the age-related rise in oligomers in hAPP transgenic mice. Unexpectedly, none of 90 human CSF samples gave a specific signal in either o-ELISA.
Conclusions
These new o-ELISAs with rigorously confirmed specificity can quantify oligomer burden in human and mouse brains for diagnostic and mechanistic studies and for AD biomarker development. However, our data raise the likelihood that the hydrophobicity of Aβ oligomers makes them very low or absent in aqueous CSF.
doi:10.1016/j.jalz.2012.11.005
PMCID: PMC3604133  PMID: 23375565
Alzheimer’s disease; amyloid β-peptide; oligomers; cerebrospinal fluid; brain extracts; ELISAs
11.  Pink1 forms a multi-protein complex with Miro and Milton, linking Pink1 function to mitochondrial trafficking‡ 
Biochemistry  2009;48(9):2045-2052.
Recessive mutations in Pink1 lead to a selective degeneration of dopaminergic neurons in the substantia nigra that is characteristic of Parkinson disease. Pink1 is a kinase that is targeted in part to mitochondria, and loss of Pink1 function can alter mitochondrial morphology and dynamics, thus supporting a link between mitochondrial dysfunction and Parkinson disease etiology. Here, we report the unbiased identification and confirmation of a mitochondrial multi-protein complex that contains Pink1, the atypical GTPase Miro, and the adaptor protein Milton. Our screen also identified an interaction between Pink1 and Mitofilin. Based on previously established functions for Miro and Milton in the trafficking of mitochondria along microtubules, we postulate here a role for Pink1 in mitochondrial trafficking. Using subcellular fractionation, we show that the overexpression of Miro and Milton, both of which are known to reside at the outer mitochondrial membrane, increases the mitochondrial Pink1 pool, suggesting a function of Pink1 at the outer membrane. Further, we document that Pink1 expressed without a mitochondrial targeting sequence can still be targeted to a mitochondria-enriched subcellular fraction via Miro and Milton. The latter finding is important for the interpretation of a previously reported protective effect of Pink1 expressed without a mitochondrial targeting sequence. Finally, we find that Miro and Milton expression suppresses altered mitochondrial morphology induced by loss of Pink1 function in cell culture. Our findings suggest that Pink1 functions in the trafficking of mitochondria in cells.
doi:10.1021/bi8019178
PMCID: PMC2693257  PMID: 19152501
12.  Effects of Prolonged Angiotensin-converting Enzyme Inhibitor Treatment on Amyloid β-Protein Metabolism in Mouse Models of Alzheimer Disease 
Neurobiology of disease  2007;26(1):273-281.
Genetic and pathologic studies have associated angiotensin-converting enzyme (ACE) with Alzheimer disease. Previously, we and others have reported that ACE degrades in vitro the amyloid β-protein (Aβ), a putative upstream initiator of Alzheimer disease. These studies support the hypothesis that deficiency in ACE-mediated Aβ proteolysis could increase Alzheimer disease risk, and raise the question of whether ACE inhibitors, a commonly prescribed class of anti-hypertensive medications, can elevate Aβ levels in vivo. To test this hypothesis, we administered the ACE inhibitor captopril to two lines of APP transgenic mice harboring either low levels of Aβ or high levels of Aβ with associated plaque deposition. In both models, we show that captopril does not affect cerebral Aβ levels in either soluble or insoluble pools. Further, we find no change in plaque deposition or in peripheral Aβ levels. Data from these Alzheimer models suggest that captopril and similar ACE inhibitors do not cause Aβ accumulation in vivo.
doi:10.1016/j.nbd.2007.01.004
PMCID: PMC2377010  PMID: 17321748
Alzheimer disease; amyloid β-protein; β-amyloid precursor protein; angiotensin-converting enzyme; Aβ degradation
13.  Plasma Amyloid β as a Predictor of Dementia and Cognitive Decline: A Systematic Review and Meta-analysis 
Archives of neurology  2012;69(7):824-831.
Context
Preclinical prediction of Alzheimer’s disease is important, critical to effective intervention. Plasma levels of amyloid β-peptides have been a principal focus of the growing literature on blood-based biomarkers, but studies to date have varied in design, assay methods and sample size, making it difficult to readily interpret the overall data.
Objective
To conduct a systematic review and meta-analysis of relevant prospective studies in order to determine if plasma amyloid β levels may predict development of dementia, Alzheimer’s disease, and cognitive decline.
Data Sources
Prospective studies published between 1995 and 2011 indexed in the PubMed, EMBASE, and PsycInfo databases were searched.
Study Selection
Selected studies included those measuring at least one relevant plasma amyloid β species (Aβ40, Aβ42, Aβ42:Aβ40 ratio) and reporting an effect estimate for dementia, Alzheimer’s disease, or cognitive change.
Data Extraction
Using a standardized extraction form, appropriate study parameters on subject information, exposure, and outcome were extracted. Random effects models were utilized to generate summary risk ratios and 95% confidence intervals, comparing the bottom versus top quantile for each plasma measure.
Results
Thirteen studies with a total of 10,303 subjects met inclusion criteria for meta-analysis. Lower Aβ42:Aβ40 ratios were significantly associated with development of Alzheimer’s disease (summary RR=1.60, 95% CI=1.04,2.46; p=0.03) and dementia (RR=1.67 95% CI=1.02,2.75; p=0.04). Significant heterogeneity was found for both summary estimates, which could not be explained by participants’ age, sex distribution, the study’s follow-up time, or year of publication. Plasma levels of Aβ40 and Aβ42 alone were not significantly associated with either outcome.
Conclusions
Overall, the literature indicates that plasma Aβ42:Aβ40 ratios predict development of Alzheimer’s disease and dementia. However, significant heterogeneity in the meta-analysis underlines the need for substantial further investigation of plasma amyloid β levels as a preclinical biomarker.
doi:10.1001/archneurol.2011.1841
PMCID: PMC3772635  PMID: 22451159
14.  Alzheimer’s Disease 
Over the last three decades, advances in biochemical pathology and human genetics have illuminated one of the most enigmatic subjects in biomedicine—neurodegeneration. Eponymic diseases of the nervous system such as Alzheimer's, Parkinson's, and Huntington's diseases that were long characterized by mechanistic ignorance have yielded striking progress in our understanding of their molecular underpinnings. A central theme in these and related disorders is the concept that certain normally soluble neuronal proteins can misfold and aggregate into oligomers and amyloid fibrils which can confer profound cytotoxicity. Perhaps the foremost example, both in terms of its societal impact and how far knowledge has moved toward the clinic, is that of Alzheimer's disease (AD). Here, we will review the classical protein lesions of the disorder that have provided a road map to etiology and pathogenesis. We will discuss how elucidating the genotype-to-phenotype relationships of familial forms of Alzheimer's disease has highlighted the importance of the misfolding and altered proteostasis of two otherwise soluble proteins, amyloid β-protein and tau, suggesting mechanism-based therapeutic targets that have led to clinical trials.
Misfolded amyloid β and tau proteins cause the neurodegeneration seen in Alzheimer's. Small compounds that target amyloid β production and antibodies that prevent aggregate formation could be effective therapies.
doi:10.1101/cshperspect.a004457
PMCID: PMC3119915  PMID: 21576255
15.  α-Synuclein neuropathology is controlled by nuclear hormone receptors and enhanced by docosahexanoic acid in a mouse model for Parkinson’s disease 
α-synuclein (α-Syn) is a neuronal protein that accumulates progressively in Parkinson’s disease and related synucleinopathies. Attempting to identify cellular factors that affect α-Syn neuropathology, we previously reported that polyunsaturated fatty acids (PUFAs) promote α-Syn oligomerization and aggregation in cultured cells. We now report that docosahexaenoic acid (DHA) a 22:6 PUFA affects α-Syn oligomerization by activating retinoic X receptor (RXR) and peroxisome proliferator-activated receptor γ2 (PPARγ2). In addition, we show that dietary changes in brain DHA levels affect α-Syn cytopathology in mice transgenic for the Parkinson’s disease-causing A53T mutation in human α-Syn. A diet enriched in docosahexaenoic acid, an activating ligand of RXR, increased the accumulation of soluble and insoluble neuronal α-Syn, neuritic injury and astrocytosis. Conversely, abnormal accumulations of α-Syn and its deleterious effects were significantly attenuated by low dietary docosahexaenoic acid levels. Our results suggest a role for activated RXR/PPARγ 2, obtained by elevated brain polyunsaturated fatty acids levels, in α-Syn neuropathology.
doi:10.1111/j.1750-3639.2011.00530.x
PMCID: PMC3253141  PMID: 21929559
alpha synuclein; Parkinson’s disease; peroxisome proliferator-activated receptors (PPAR)γ; Retinoic X receptor (RXR); protein oligomerization and aggregation; docosahexaenoic acid
16.  The effects of membrane lipids on the activity and processivity of purified γ-secretase 
Biochemistry  2012;51(17):3565-3575.
The 19-transmembrane multi-subunit γ-secretase complex generates the amyloid β-peptide (Aβ) of Alzheimer’s disease (AD) by intramembrane proteolysis of the β-amyloid precursor protein (APP). Despite substantial advances in elucidating how this protein complex functions, the effect of the local membrane lipid microenvironment on γ-secretase cleavage of substrates is still poorly understood. Using detergent-free proteoliposomes to reconstitute purified human γ-secretase, we examined the effects of fatty acyl (FA) chain length, saturation and double-bond isomerisation, and membrane lipid polar head groups on γ-secretase function. We analyzed γ-secretase activity and processivity (i.e., sequential cleavages of the APP transmembrane domain that convert longer Aβ species (e.g., Aβ46) into shorter ones (e.g, Aβ40)) by quantifying the APP intracellular domain (AICD) and various Aβ peptides, including via a bicine/urea gel system that detects multiple Aβ lengths. These assays revealed several trends: (1) switching from a cis to a trans isomer of a monounsaturated FA chain in phosphatidylcholine (PC) increased γ-activity, did not affect Aβ42/40 ratios, but decreased the ratio of long (≥42) vs. short (≤41) Aβ peptides; (2) increasing FA carbon chain length (14<16<18<20) increased γ-activity, reduced longer Aβ species and reduced Aβ42/40; (3) shifting the position of the double bond in 18:1(Δ9-cis) PC to the Δ6 position substantially reduced activity; (4) gangliosides increased γ-activity but decreased processivity, thus elevating Aβ42/40; (5) phosphatidylserine decreased γ-activity but increased processivity; and (6) phosphatidylinositol strongly inhibited γ-activity. Overall, our results show that subtle changes in membrane lipid composition can greatly influence γ-secretase activity and processivity, suggesting that relatively small changes in lipid membrane composition may affect the risk of AD at least as much as do presenilin or APP mutations.
doi:10.1021/bi300303g
PMCID: PMC3347702  PMID: 22489600
18.  A Specific ELISA for Measuring Amyloid β-Protein Oligomers in Human Plasma and the Brains of Alzheimer Patients 
Archives of neurology  2009;66(2):190-199.
1. Objective
A new ELISA specific for oligomeric assemblies of amyloid β protein (oAβ) was developed to examine in vivo levels of oAβ vs. monomeric Aβ in sporadic and familial Alzheimer disease (AD) plasma and brain tissue.
2. Design
To establish the oAβ ELISA, the same N-terminal Aβ antibody was used for antigen capture and detection. Plasmas and postmortem brains from AD and control subjects were systematically analyzed by conventional monomeric Aβ and new oAβ ELISAs.
3. Subjects
We measured oAβ species in plasma samples from 36 clinically well-characterized AD patients and 10 controls. In addition, postmortem samples were obtained from brain autopsies of 9 verified AD and 7 control subjects.
4. Results
The specificity of oAβ ELISA was validated with a disulfide crossed-linked, synthetic Aβ1–40Ser26Cys dimer that was specifically detected before but not after the dissociation of the dimers in β-mercaptoethanol. Plasma assays showed that relative oAβ levels were closely associated with relative Aβ42 monomer levels across all subjects. Analysis of sequential plasma samples from a subset of the AD patients, including a patient with AD caused by a presenilin mutation, revealed decreases in both oAβ and Aβ42 monomer levels over a 1–2 year period. In brain tissue from 9 AD and 7 control subjects, both oAβ and monomeric Aβ42 were consistently higher in the AD cases.
6. Conclusions
An oAβ-specific ELISA reveals a tight link between oAβ and Aβ42 monomer levels in plasma and brain, and both forms can decline over time in plasma, presumably reflecting their increasing insolubility in the brain.
doi:10.1001/archneurol.2008.565
PMCID: PMC3618974  PMID: 19204155
19.  The relation between insulin, insulin-related factors, and plasma amyloid beta peptide levels at mid-life in a population-based study 
Little is known regarding factors associated with soluble amyloid beta peptide (Aβ) concentrations in humans at late midlife, when Aβ is likely most critical to Alzheimer disease pathogenesis. We examined the association between insulin, insulin-related factors, and plasma Aβ at late midlife. Plasma Aβ42, Aβ40, fasting insulin, and c-peptide were measured in 468 women without diabetes, aged 59–69 years (median 63 years). Prior to blood draw, participants reported body mass index, waist circumference, physical activity, alcohol intake, hypertension, and diabetes family history. Linear regression was used to calculate age-adjusted mean differences in Aβ42 to Aβ40 ratio, and Aβ42 levels, by insulin and insulin-related factors. The ratio of Aβ42 to Aβ40 was statistically significantly lower in women with diabetes family history, and Aβ42 was significantly lower with less physical activity, greater waist circumference, hypertension, and diabetes family history (p<0.05 for all). Aβ42 to Aβ40 ratio, and Aβ42 levels, appeared lower with higher c-peptide levels (p-trend=0.07 and 0.06, respectively), although these were not statistically significant. In summary, insulin-related factors appear associated with lower plasma Aβ42 to Aβ40 ratio, and Aβ42, at late mid-life, consistent with increased brain sequestration of Aβ42 (relative to Aβ40), suggesting insulin merits focus in strategies to prevent dementia.
doi:10.1097/WAD.0b013e31821764ce
PMCID: PMC3140548  PMID: 21502851
amyloid beta peptide; insulin; epidemiology
20.  Aβ oligomers inhibit synapse remodelling necessary for memory consolidation 
Neurobiology of aging  2010;32(12):2211-2218.
Extensive research has implicated the amyloid-β protein (Aβ) in the aetiology of Alzheimer’s disease (AD). This protein has been shown to produce memory deficits when injected into rodent brain and in mouse models of AD Aβ production is associated with impaired learning and/or recall. Here we examined the effects of cell-derived SDS-stable 7PA2-derived soluble Aβ oligomers on consolidation of avoidance learning. At 0, 3, 6, 9 or 12 h after training, animals received an intracerebroventricular injection of Aβ-containing or control media and recall was tested at 24 and 48 h. Immediately after 48 h recall animals were transcardially perfused and the brain removed for sectioning and EM analysis. Rats receiving injections of Aβ at 6 or 9 h post-training showed a significant impairment in memory consolidation at 48 h. Importantly, impaired animals injected at 9 h had significantly fewer synapses in the dentate gyrus. These data suggest that Aβ low-n oligomers target specific temporal facets of consolidation-associated synaptic remodelling whereby loss of functional synapses results in impaired consolidation.
doi:10.1016/j.neurobiolaging.2010.01.001
PMCID: PMC2891223  PMID: 20097446
Amyloid β-protein; oligomers; memory consolidation; Alzheimer’s disease; synapse ultrastructure
21.  Cholesterol and Statins in Alzheimer’s Disease 
Archives of neurology  2011;68(11):1385-1392.
Substantial evidence has accumulated in support of the hypothesis that elevated cholesterol levels increase the risk of developing Alzheimer’s disease (AD). As a result, much work has been done investigating the potential use of lipid-lowering agents (LLAs), particularly statins, as preventive or therapeutic agents for AD. While epidemiology and preclinical statin research (described in Part 1 of this review) have generally supported an adverse role of high cholesterol regarding AD, human studies of statins (reviewed here) show highly variable outcomes, making it difficult to draw firm conclusions. We identify several confounding factors among the human studies, including differing blood-brain barrier permeabilities among statins, the stage in AD at which statins were administered, and the drugs’ pleiotropic metabolic effects, all of which contribute to the substantial variability observed to date. We recommend that future human studies of this important therapeutic topic 1) take the blood-brain barrier permeabilities of statins into account when analyzing results, 2) include specific analyses of effects on low-density and high-density lipoprotein cholesterol, and most importantly, 3) conduct statin treatment trials solely in mild AD patients, who have the best chance for disease modification.
doi:10.1001/archneurol.2011.242
PMCID: PMC3248784  PMID: 22084122
22.  Cholesterol and Statins in Alzheimer’s Disease 
Archives of neurology  2011;68(10):1239-1244.
Over the past twenty years, evidence has accumulated that high cholesterol levels may increase the risk of developing Alzheimer’s disease (AD). With the global use of statins to treat hypercholesterolemia, this finding has led to the hope that statins could prove useful in treating or preventing AD. However, the results of work on this topic are inconsistent: some studies find beneficial effects, others do not. In this first segment of a two-part review, we examine the complex preclinical and clinical literature on cholesterol and AD. First, we review epidemiological research on cholesterol levels and the risk of AD and discuss the relevance of discrepancies among studies as regards participants’ age and clinical status. Next, we assess studies correlating cholesterol with AD-type neuropathology. The potential molecular mechanisms for cholesterol’s apparent adverse effect on the development of AD are then discussed. Finally, we review preclinical studies of statins and AD. Thus, this first portion of our review provides the background and rationale for investigating statins as potential therapeutic agents in AD patients, the subject of the second part.
doi:10.1001/archneurol.2011.203
PMCID: PMC3211071  PMID: 21987540
23.  Association of SNCA with Parkinson: replication in the Harvard NeuroDiscovery Center Biomarker Study 
Movement Disorders  2011;26(12):2283-2286.
Background
Mutations in the α-synuclein gene (SNCA) cause autosomal dominant forms of Parkinson’s disease, but the substantial risk conferred by this locus to the common sporadic disease has only recently emerged from genome-wide association studies.
Methods
Here we genotyped a prioritized non-coding variant in SNCA intron-4 in 344 patients with Parkinson’s and 275 controls from the longitudinal Harvard NeuroDiscovery Center Biomarker Study.
Results
The common minor allele of rs2736990 was associated with elevated disease susceptibility (odds ratio = 1.40, P value = 0.0032).
Conclusions
This result increases confidence in the notion that in many clinically well-characterized patients genetic variation in SNCA contributes to “sporadic” disease.
doi:10.1002/mds.23934
PMCID: PMC3337217  PMID: 21953863
Parkinson’s disease; α-synuclein; GATA transcription factors; biomarker; genome-wide association study
24.  Neurotoxicity of Amyloid β-Protein: Synaptic and Network Dysfunction 
Evidence for an ever-expanding variety of molecular mediators of amyloid β-protein neurotoxicity (membrane lipids, receptor proteins, channel proteins, second messengers and related signaling cascades, cytoskeletal proteins, inflammatory mediators, etc.) has led to the notion that the binding of hydrophobic Aβ assemblies to cellular membranes triggers multiple effects affecting diverse pathways. It appears unlikely that there are only one or two cognate receptors for neurotoxic forms of Aβ and also that there are just one or two assembly forms of the peptide that induce neuronal dysfunction. Rather, various soluble (diffusible) oligomers of Aβ that may be in dynamic equilibrium with insoluble, fibrillar deposits (amyloid plaques) and that can bind to different components of neuronal and non-neuronal plasma membranes appear to induce complex patterns of synaptic dysfunction and network disorganization that underlie the intermittent but gradually progressive cognitive manifestations of the clinical disorder. Modern analyses of this problem utilize electrophysiology coupled with synaptic biochemistry and behavioral phenotyping of animal models to elucidate the affected circuits and assess the effects of potential therapeutic interventions.
Multiple forms of neurotoxic amyloid-beta protein bind to different plasma membrane components. This induces complex patterns of synaptic dysfunction and neural network disorganization associated with Alzheimer disease.
doi:10.1101/cshperspect.a006338
PMCID: PMC3385944  PMID: 22762015
25.  Biochemistry of Amyloid β-Protein and Amyloid Deposits in Alzheimer Disease 
Progressive cerebral deposition of the amyloid β-protein (Aβ) in brain regions serving memory and cognition is an invariant and defining feature of Alzheimer disease. A highly similar but less robust process accompanies brain aging in many nondemented humans, lower primates, and some other mammals. The discovery of Aβ as the subunit of the amyloid fibrils in meningocerebral blood vessels and parenchymal plaques has led to innumerable studies of its biochemistry and potential cytotoxic properties. Here we will review the discovery of Aβ, numerous aspects of its complex biochemistry, and current attempts to understand how a range of Aβ assemblies, including soluble oligomers and insoluble fibrils, may precipitate and promote neuronal and glial alterations that underlie the development of dementia. Although the role of Aβ as a key molecular factor in the etiology of Alzheimer disease remains controversial, clinical trials of amyloid-lowering agents, reviewed elsewhere in this book, are poised to resolve the question of its pathogenic primacy.
Amyloid β-proteins (Aβ) are biochemically heterogeneous, with different lengths, amino and carboxyl termini, and propensities for aggregation. A range of Aβ assemblies may promote neurodegeneration in Alzheimer disease.
doi:10.1101/cshperspect.a006262
PMCID: PMC3367542  PMID: 22675658

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