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.
TGF-β1; glutamate uptake; hippocampus; neuronal calcium; extrasynaptic; astrocytes
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.
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.
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.
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.
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.
Alzheimer’s disease; amyloid β-peptide; oligomers; cerebrospinal fluid; brain extracts; ELISAs
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.
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.
Prospective studies published between 1995 and 2011 indexed in the PubMed, EMBASE, and PsycInfo databases were searched.
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.
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.
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.
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.
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.
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.
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.
DISC1; APP; schizophrenia; Alzheimer’s; development; migration
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.
alpha synuclein; Parkinson’s disease; peroxisome proliferator-activated receptors (PPAR)γ; Retinoic X receptor (RXR); protein oligomerization and aggregation; docosahexaenoic acid
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.
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.
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.
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.
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.
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.
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.
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.
amyloid beta peptide; insulin; epidemiology
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.
Alzheimer disease; amyloid β-protein; β-amyloid precursor protein; angiotensin-converting enzyme; Aβ degradation
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.
Amyloid β-protein; oligomers; memory consolidation; Alzheimer’s disease; synapse ultrastructure
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.
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.
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.
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.
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.
Five point mutations within the amyloid β-protein (Aβ) sequence of the APP gene are associated with hereditary diseases which are similar or identical to Alzheimer’s disease and encode: the A21G (Flemish), E22G (Arctic), E22K (Italian), E22Q (Dutch) and the D23N (Iowa) amino acid substitutions. Although a substantial body of data exists on the effects of these mutations on Aβ production, whether or not intra-Aβ mutations alter degradation and how this relates to their aggregation state remain unclear. Here we report that the E22G, E22Q and the D23N substitutions significantly increase fibril nucleation and extension, whereas the E22K substitution exhibits only an increased rate of extension and the A21G substitution actually causes a decrease in the extension rate.
These substantial differences in aggregation together with our observation that aggregated wild type Aβ(1–40) was much less well degraded than monomeric wild type Aβ(1–40), prompted us to assess whether or not disease-associated intra-Aβ mutations alter proteolysis independent of their effects on aggregation. Neprilysin (NEP), insulin degrading enzyme (IDE) and plasmin play a major role in Aβ catabolism, therefore we compared the ability of these enzymes to degrade wild type and mutant monomeric Aβ peptides. Experiments investigating proteolysis revealed that all monomeric peptides are degraded similarly by IDE and plasmin, but that the Flemish peptide was degraded significantly more slowly by NEP than wild type Aβ or any of the other mutant peptides. This finding suggests that resistance to NEP-mediated proteolysis may underlie the pathogenicity associated with the A21G mutation.
Alzheimer’s disease; amyloid beta-protein; aggregation; degradation; insulin degrading enzyme; plasmin
The molecular pathways leading to Alzheimer-type dementia are not well understood, but the amyloid β-protein is believed to be centrally involved. The quantity of amyloid β-protein containing plaques does not correlate well with clinical status, suggesting that if amyloid β-protein is pathogenic it involves soluble non-plaque material. Using 43 brains from the Newcastle cohort of the population-representative Medical Research Council Cognitive Function and Ageing Study, we examined the relationship between biochemically distinct forms of amyloid β-protein and the presence of Alzheimer-type dementia. Cortical samples were serially extracted with Tris-buffered saline, Tris-buffered saline containing 1% TX-100 and with 88% formic acid and extracts analysed for amyloid β-protein by immunoprecipitation/western blotting. The cohort was divisible into those with dementia at death with (n = 14) or without (n = 10) significant Alzheimer-type pathology, and those who were not demented (n = 19). Amyloid β-protein monomer in extracts produced using Tris-buffered saline and Tris-buffered saline containing 1% TX-100 were strongly associated with Alzheimer type dementia (P < 0.001) and sodium dodecyl sulphate-stable amyloid β-protein dimer was detected specifically and sensitively in Tris-buffered saline, Tris-buffered saline containing 1% TX-100 and formic acid extracts of Alzheimer brain. Amyloid β-protein monomer in the formic acid fraction closely correlated with diffuse and neuritic plaque burden, but was not specific for dementia. These findings support the hypothesis that soluble amyloid β-protein is a major correlate of dementia associated with Alzheimer-type pathology and is likely to be intimately involved in the pathogenesis of cognitive failure.
Alzheimer’s disease pathology; Alzheimer’s disease; amyloid β-protein; biochemistry; cognitive impairment
The amyloid precursor family of proteins are of considerable interest both because of their role in Alzheimer’s disease pathogenesis and because of their normal physiological functions. In mammals, the amyloid precursor protein (APP) has two homologs, amyloid precursor-like protein 1 and amyloid precursor-like protein 2. All 3 proteins undergo ectodomain shedding and regulated intramembrane proteolysis, and important functions have been impunged to the full-length proteins, shed ectodomains, C-terminal fragments and intra-cellular domains (ICDs). One of the proteases known to cleave APP and which is essential for generation of the amyloid β-protein is the β-site APP cleaving enzyme 1 (BACE1). Here we investigated the effects of genetic manipulation of BACE1 on the processing of the APP family of proteins. BACE1 expression regulated the levels and species of full-length APLP1, APP and APLP2, of their shed ectodomains and membrane-bound C-terminal fragments. In particular, APP processing appears to be tightly regulated, with changes in APPsβ being compensated with changes in APPsα. In contrast, the total levels of soluble cleaved APLP1 and APLP2 species were less tightly regulated and fluctuated depending on BACE1 expression. Importantly, the production of ICDs for all three proteins was not decreased by loss of BACE1 activity. These results indicate that BACE1 is involved in regulating ectodomain shedding, maturation and trafficking of the APP family of proteins. Consequently, while inhibition of BACE1 is unlikely to adversely affect potential ICD-mediated signalling it may alter other important facets of APLP/APP biology.
APP; APLP1; APLP2; BACE1; Alzheimer’s disease
Previously we reported 1 μM synthetic human amyloid beta1-42 oligomers induced cofilin dephosphorylation (activation) and formation of cofilin-actin rods within rat hippocampal neurons primarily localized to the dentate gyrus.
Here we demonstrate that a gel filtration fraction of 7PA2 cell-secreted SDS-stable human Aβ dimers and trimers (Aβd/t) induces maximal neuronal rod response at ~250 pM. This is 4,000-fold more active than traditionally prepared human Aβ oligomers, which contain SDS-stable trimers and tetramers, but are devoid of dimers. When incubated under tyrosine oxidizing conditions, synthetic human but not rodent Aβ1-42, the latter lacking tyrosine, acquires a marked increase (620 fold for EC50) in rod-inducing activity. Gel filtration of this preparation yielded two fractions containing SDS-stable dimers, trimers and tetramers. One, eluting at a similar volume to 7PA2 Aβd/t, had maximum activity at ~5 nM, whereas the other, eluting at the void volume (high-n state), lacked rod inducing activity at the same concentration. Fractions from 7PA2 medium containing Aβ monomers are not active, suggesting oxidized SDS-stable Aβ1-42 dimers in a low-n state are the most active rod-inducing species. Aβd/t-induced rods are predominantly localized to the dentate gyrus and mossy fiber tract, reach significance over controls within 2 h of treatment, and are reversible, disappearing by 24 h after Aβd/t washout. Overexpression of cofilin phosphatases increase rod formation when expressed alone and exacerbate rod formation when coupled with Aβd/t, whereas overexpression of a cofilin kinase inhibits Aβd/t-induced rod formation.
Together these data support a mechanism by which Aβd/t alters the actin cytoskeleton via effects on cofilin in neurons critical to learning and memory.
The amyloid β-protein (Aβ) is believed to play a causal role in Alzheimer’s disease, however, the mechanism by which Aβ mediates its’ effect and the assembly form(s) of Aβ responsible remain unclear. Several APP transgenic mice have been shown to accumulate Aβ and to develop cognitive deficits. We have studied one such model, the J20 mouse. Using an immunoprecipitation/Western blotting technique we find an age-dependent increase in Aβ monomer and SDS-stable dimer. But prior to the earliest detection of Aβ dimers, immunohistochemical analysis revealed an increase in oligomer immunoreactivity that was coincident with reduced hippocampal MAP2 and synaptophysin staining. Moreover, biochemical fractionation and ELISA analysis revealed evidence of TBS and triton-insoluble sedimentable Aβ aggregates at the earliest ages studied. These data demonstrate the presence of multiple assembly forms of Aβ throughout the life of J20 mice and highlight the difficulty in attributing synaptotoxicity to a single Aβ species.
Amyloid β-protein; aggregation; oligomers; amyloid precursor protein; J20 mice; synaptophysin; MAP2