N-terminally truncated A beta 4-x proteoforms and their relevance for Alzheimer's pathophysiology

BACKGROUND: The molecular heterogeneity of Alzheimer's amyloid-β (Aβ) deposits extends well beyond the classic Aβ1-40/Aβ1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain Aβ peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. Aβ4-x peptides have been reported as major components of plaque cores and the limited studies available indicate their relative abundance in Alzheimer's disease (AD). METHODS: Immunohistochemistry was used to assess the topographic distribution of Aβ4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6-specific for Aβ4-x species and blind for full-length Aβ1-40/Aβ1-42-in conjunction with thioflavin-S and antibodies recognizing Aβx-40 and Aβx-42 proteoforms. Circular dichroism, thioflavin-T binding, and electron microscopy evaluated the biophysical and aggregation/oligomerization properties of full-length and truncated synthetic homologues, whereas stereotaxic intracerebral injections of monomeric and oligomeric radiolabeled homologues in wild-type mice were used to evaluate their brain clearance characteristics. RESULTS: All types of amyloid deposits contained the probed Aβ epitopes, albeit expressed in different proportions. Aβ4-x species showed preferential localization within thioflavin-S-positive cerebral amyloid angiopathy and cored plaques, strongly suggesting poor clearance characteristics and consistent with the reduced solubility and enhanced oligomerization of their synthetic homologues. In vivo clearance studies demonstrated a fast brain efflux of N-terminally truncated and full-length monomeric forms whereas their oligomeric counterparts-particularly of Aβ4-40 and Aβ4-42-consistently exhibited enhanced brain retention. CONCLUSIONS: The persistence of aggregation-prone Aβ4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways

Mitochondrial dysfunction induced by a post-translationally modified amyloid linked to a familial mutation in an alternative model of neurodegeneration

AbstractFamilial British dementia (FBD) is an early-onset non-amyloid-β (Aβ) cerebral amyloidosis that presents with severe cognitive decline and strikingly similar neuropathological features to those present in Alzheimer's disease (AD). FBD is associated with a T to A single nucleotide transition in the stop codon of a gene encoding BRI2, leading to the production of an elongated precursor protein. Furin-like proteolytic processing at its C-terminus releases a longer-than-normal 34 amino acid peptide, ABri, exhibiting amyloidogenic properties not seen in its 23 amino acid physiologic counterpart Bri1–23. Deposited ABri exhibits abundant post-translational pyroglutamate (pE) formation at the N-terminus, a feature seen in truncated forms of Aβ found in AD deposits, and co-exists with neurofibrillary tangles almost identical to those found in AD. We tested the impact of the FBD mutation alone and in conjunction with the pE post-translational modification on the structural properties and associated neurotoxicity of the ABri peptide. The presence of pE conferred to the ABri molecule enhanced hydrophobicity and accelerated aggregation/fibrillization properties. ABri pE was capable of triggering oxidative stress, loss of mitochondrial membrane potential and activation of caspase-mediated apoptotic mechanisms in neuronal cells, whereas homologous peptides lacking the elongated C-terminus and/or the N-terminal pE were unable to induce similar detrimental cellular pathways. The data indicate that the presence of N-terminal pE is not in itself sufficient to induce pathogenic changes in the physiologic Bri1–23 peptides but that its combination with the ABri mutation is critical for the molecular pathogenesis of FBD

Brain Neprilysin Activity and Susceptibility to Transgene-Induced Alzheimer Amyloids

Neprilysin (NEP) is a zinc metalloproteinase that degrades enkephalins, endothelins, and the Alzheimer’s disease amyloid ß (Aß) peptides. NEP-deficient mice possess increased levels of brain Aß1-40 and Aß1-42. The objective of this study was to determine whether tissue NEP specific activity differs according to age and/or across mouse strains, especially those strains predisposed toward formation of Aß-amyloid plaques following overexpression of the human Alzheimer amyloid precursor protein (APP). The C57Bl/6J mouse strain appears to be relatively susceptible to cerebral amyloidosis, whereas the Swiss Webster (SW) strain appears more resistant. We investigated whether NEP specific activity in brain and kidney homogenates from SW and C57 mice of 6, 40, and 80 weeks old varied according to mouse strain, age, and gender. Among the variables tested, NEP specific activity varied most dramatically across mouse strain, with the kidney and brain of SW mice displaying the highest activities. Aging was associated with a reduction in brain NEP specific activity in both trains. Gender-specific differences were identified in kidney but not in brain. We conclude that aging- and strain-dependent ifferences in NEP specific activity may play a role in the differential susceptibility of some mouse strains for developing cerebral amyloidosis following human APP overexpression

Association of clusterin with the BRI2-derived amyloid molecules ABri and ADan

Familial British and Danish dementias (FBD and FDD) share striking neuropathological similarities with Alzheimer's disease (AD), including intraneuronal neurofibrillary tangles as well as parenchymal and vascular amyloid deposits. Multiple amyloid associated proteins with still controversial role in amyloidogenesis colocalize with the structurally different amyloid peptides ABri in FBD, ADan in FDD, and Aβ in AD. Genetic variants and plasma levels of one of these associated proteins, clusterin, have been identified as risk factors for AD. Clusterin is known to bind soluble Aβ in biological fluids, facilitate its brain clearance, and prevent its aggregation. The current work identifies clusterin as the major ABri- and ADan-binding protein and provides insight into the biochemical mechanisms leading to the association of clusterin with ABri and ADan deposits. Mirroring findings in AD, the studies corroborate clusterin co-localization with cerebral parenchymal and vascular amyloid deposits in both disorders. Ligand affinity chromatography with downstream Western blot and amino acid sequence analyses unequivocally identified clusterin as the major ABri- and ADan-binding plasma protein. ELISA highlighted a specific saturable binding of clusterin to ABri and ADan with low nanomolar Kd values within the same range as those previously demonstrated for the clusterin-Aβ interaction. Consistent with its chaperone activity, thioflavin T binding assays clearly showed a modulatory effect of clusterin on ABri and ADan aggregation/fibrillization properties. Our findings, together with the known multifunctional activity of clusterin and its modulatory activity on the complex cellular pathways leading to oxidative stress, mitochondrial dysfunction, and the induction of cell death mechanisms - all known pathogenic features of these protein folding disorders - suggests the likelihood of a more complex role and a translational potential for the apolipoprotein in the amelioration/prevention of these pathogenic mechanisms.This work was supported by grants from the National Institutes of Health NS051715 (to AR) and AG030539, AG051266, AG059695, and AG065651 (to JG) and from CIBERNED and the Spanish Ministry of Science (SAF2016-78603-R and PID2019-110401RB-I00) and Institutional grants from the Queen Sofia Foundation, CIEN Foundation and the Carlos III Institutes of Health (to MC). TL is supported by an Alzheimer's Research UK senior fellowship. TR is supported by a grant from the Karin & Sten Mortstedt CBD Solutions AB, Stockholm, Sweden and by the National Institute for Health Research (NIHR) Queen Square Biomedical Research Unit in Dementia based at University College London Hospitals (UCLH), University College London (UCL). The views expressed are those of the authors and not necessarily those of the NIH, NHS, the NIHR or the Department of Health.S

Differential Degradation of Amyloid β Genetic Variants Associated with Hereditary Dementia or Stroke by Insulin-degrading Enzyme

Inherited amino acid substitutions at position 21, 22, or 23 of amyloid beta (Abeta) lead to presenile dementia or stroke. Insulin-degrading enzyme (IDE) can hydrolyze Abeta wild type, yet whether IDE is capable of degrading Abeta bearing pathogenic substitutions is not known. We studied the degradation of all of the published Abeta genetic variants by recombinant rat IDE (rIDE). Monomeric Abeta wild type, Flemish (A21G), Italian (E22K), and Iowa (D23N) variants were readily degraded by rIDE with a similar efficiency. However, proteolysis of Abeta Dutch (E22Q) and Arctic (E22G) was significantly lower as compared with Abeta wild type and the rest of the mutant peptides. In the case of Abeta Dutch, inefficient proteolysis was related to a high content of beta structure as assessed by circular dichroism. All of the Abeta variants were cleaved at Glu3-Phe4 and Phe4-Arg5 in addition to the previously described major sites within positions 13-15 and 18-21. SDS-stable Abeta dimers were highly resistant to proteolysis by rIDE regardless of the variant, suggesting that IDE recognizes a conformation that is available for interaction only in monomeric Abeta. These results raise the possibility that upregulation of IDE may promote the clearance of soluble Abeta in hereditary forms of Abeta diseases.Fil: Morelli, Laura. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Llovera, Ramiro Esteban. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; ArgentinaFil: Gonzalez, Silvia Adriana. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Affranchino, Jose Luis. Universidad de Buenos Aires. Facultad de Ciencias Exactas y Naturales. Departamento de Química Biológica. Laboratorio de Virología; ArgentinaFil: Prelli, Frances. University of New York; Estados UnidosFil: Frangione, Blas. University of New York; Estados UnidosFil: Ghiso, Jorge. University of New York; Estados UnidosFil: Castaño, Eduardo Miguel. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Houssay. Instituto de Química y Físico-Química Biológicas "Prof. Alejandro C. Paladini". Universidad de Buenos Aires. Facultad de Farmacia y Bioquímica. Instituto de Química y Físico-Química Biológicas; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; Argentin

Human blood-brain barrier receptors for Alzheimer's amyloid-beta 1- 40. Asymmetrical binding, endocytosis, and transcytosis at the apical side of brain microvascular endothelial cell monolayer.

This is the published version. Copyright 1998 by American Society for Clinical Investigation.A soluble monomeric form of Alzheimer's amyloid-beta (1-40) peptide (sAbeta1-40) is present in the circulation and could contribute to neurotoxicity if it crosses the brain capillary endothelium, which comprises the blood-brain barrier (BBB) in vivo. This study characterizes endothelial binding and transcytosis of a synthetic peptide homologous to human sAbeta1-40 using an in vitro model of human BBB. 125I-sAbeta1-40 binding to the brain microvascular endothelial cell monolayer was time dependent, polarized to the apical side, and saturable with high- and low-affinity dissociation constants of 7.8+/-1.2 and 52.8+/-6.2 nM, respectively. Binding of 125I-sAbeta1-40 was inhibited by anti-RAGE (receptor for advanced glycation end products) antibody (63%) and by acetylated low density lipoproteins (33%). Consistent with these data, transfected cultured cells overexpressing RAGE or macrophage scavenger receptor (SR), type A, displayed binding and internalization of 125I-sAbeta1-40. The internalized peptide remains intact > 94%. Transcytosis of 125I-sAbeta1-40 was time and temperature dependent, asymmetrical from the apical to basolateral side, saturable with a Michaelis constant of 45+/-9 nM, and partially sensitive to RAGE blockade (36%) but not to SR blockade. We conclude that RAGE and SR mediate binding of sAbeta1-40 at the apical side of human BBB, and that RAGE is also involved in sAbeta1-40 transcytosis

Preamyloid Lesions and Cerebrovascular Deposits in the Mechanism of Dementia: Lessons from Non-β-Amyloid Cerebral Amyloidosis

The importance of amyloid plaques in the pathogenesis of dementia is usually centered on β-amyloid (Aβ) and its role in Alzheimer's disease (AD). However, since fibrillar plaques correlate poorly with neurodegeneration, challenging their importance in the mechanism(s) of dementia, investigators turned their focus to the importance of soluble oligomers and the role of preamyloid and cerebrovascular deposits. Two non-Aβ cerebral amyloidoses, familial British and Danish dementias (FBD and FDD), share many aspects of AD, including cognitive impairment and the presence of neurofibrillary tangles in limbic areas. The lack of compact plaques in FDD and in many areas in FBD further questions the importance of these lesions in the mechanism of dementia. The main components of the deposits – ABri and ADan – are structurally unrelated to Aβ and yet they all have a high tendency to oligomerize and assemble into amyloid fibrils in vitro and form ion-like channels in lipid membranes. Thus, different amyloid species have the capability to induce similar neuropathological changes, which are neither exclusive for Aβ nor dependent on the presence of compact plaques. These findings reaffirm the notion that non-Aβ amyloidoses constitute alternative models to study the role of preassembled amyloid subunits in neuronal death

Human chorionic gonadotropin (a Luteinizing Hormone homologue) decreases spatial memory and increases brain amyloid-ß levels in female rats

Numerous studies have suggested that estradiol (E) improves spatial memory as female rats with E perform better than those without E. However there is an inverse relationship between E and luteinizing hormone (LH) levels and LH could play a role. We examined whether treatment with the LH homologue human chorionic gonadotropin (hCG), would impair spatial memory of adult E-treated female rats. In the object location memory task, ovariectomized (ovxed) rats treated with E and either a single high dose (400 IU/kg) or a lower repeated dose of hCG (75 IU/kg hourly for 8 h) showed spatial memory disruption compared to ovxed rats treated with estradiol alone. Impairment was attributed to memory disruption as performance improved with shortened delay between task exposure and testing. Tests on another spatial memory task, the Barnes maze, confirmed that hCG (400 IU/kg) can impair memory: although E + veh treated animals made significantly fewer hole errors across time, E + hCG-treated did not. In humans, high LH levels have been correlated with Alzheimer\u27s disease (AD). Because brain amyloid-beta (Aβ) species have been implicated as a toxic factor thought to cause memory loss in AD, we analyzed whether hCG-treated animals had increased Aβ levels. Levels of Aβ from whole brains or hippocampi were assessed by Western blot. hCG treatment to E-implanted females significantly increased soluble Aβ40 and Aβ42 levels. These results indicate that high levels of LH/hCG can impair spatial memory, and an increase in brain Aβ species may account for the memory impairment in hCG-treated rats