N-terminally truncated AÎ²4-x proteoforms and their relevance for Alzheimer's pathophysiology
BACKGROUND:The molecular heterogeneity of Alzheimer's amyloid-Î² (AÎ²) deposits extends well beyond the classic AÎ²1-40/AÎ²1-42 dichotomy, substantially expanded by multiple post-translational modifications that increase the proteome diversity. Numerous truncated fragments consistently populate the brain AÎ² peptidome, and their homeostatic regulation and potential contribution to disease pathogenesis are largely unknown. AÎ²4-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 AÎ²4-x species in well-characterized AD cases using custom-generated monoclonal antibody 18H6-specific for AÎ²4-x species and blind for full-length AÎ²1-40/AÎ²1-42-in conjunction with thioflavin-S and antibodies recognizing AÎ²x-40 and AÎ²x-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 AÎ² epitopes, albeit expressed in different proportions. AÎ²4-x species showed preferential localization within thioflavin-S-positiveÂ 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 AÎ²4-40 and AÎ²4-42-consistently exhibited enhanced brain retention. CONCLUSIONS:The persistence of aggregation-prone AÎ²4-x proteoforms likely contributes to the process of amyloid formation, self-perpetuating the amyloidogenic loop and exacerbating amyloid-mediated pathogenic pathways.
Identification of Clusterin as a Major ABri- and ADan-Binding Protein Using Affinity Chromatography
Affinity chromatography has, for many years, been at the research forefront as one of the simplest although highly versatile techniques capable of identifying biologically relevant protein-protein interactions. In the field of amyloid disorders, the use of ligands immobilized to a variety of affinity matrices was the method of choice to individualize proteins with affinity for soluble circulating forms of amyloid subunits. The methodology has also played an important role in the identification of proteins that interact with different amyloidogenic peptides and, as a result, are capable of modulating their physiological and pathological functions by altering solubility, aggregation propensity, and fibril formation proclivity. Along this line, classical studies conducted in the field of Alzheimer's disease (AD) identified clusterin as a major binding protein to both circulating soluble AÎ² as well as to the brain deposited counterpart. The affinity chromatography-based approach employed herein, individualized clusterin as the major protein capable of binding the amyloid subunits associated with familial British and Danish dementias, two non-AÎ² neurodegenerative conditions also exhibiting cerebral amyloid deposition and sharing striking similarities to AD. The data demonstrate that clusterin binding ability to amyloid molecules is not restricted to AÎ², suggesting a modulating effect on the aggregation/fibrillization propensity of the amyloidogenic peptides that is consistent with its known chaperone activity.
Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain
A population of more than six million people worldwide at high risk of Alzheimer's disease (AD) are those with Down Syndrome (DS, caused by trisomy 21 (T21)), 70% of whom develop dementia during lifetime, caused by an extra copy of Î²-amyloid-(AÎ²)-precursor-protein gene. We report AD-like pathology in cerebral organoids grown in vitro from non-invasively sampled strands of hair from 71% of DS donors. The pathology consisted of extracellular diffuse and fibrillar AÎ² deposits, hyperphosphorylated/pathologically conformed Tau, and premature neuronal loss. Presence/absence of AD-like pathology was donor-specific (reproducible between individual organoids/iPSC lines/experiments). Pathology could be triggered in pathology-negative T21 organoids by CRISPR/Cas9-mediated elimination of the third copy of chromosome 21 gene BACE2, but prevented by combined chemical Î² and Î³-secretase inhibition. We found that T21 organoids secrete increased proportions of AÎ²-preventing (AÎ²1-19) and AÎ²-degradation products (AÎ²1-20 and AÎ²1-34). We show these profiles mirror in cerebrospinal fluid of people with DS. We demonstrate that this protective mechanism is mediated by BACE2-trisomy and cross-inhibited by clinically trialled BACE1 inhibitors. Combined, our data prove the physiological role of BACE2 as a dose-sensitive AD-suppressor gene, potentially explaining the dementia delay in ~30% of people with DS. We also show that DS cerebral organoids could be explored as pre-morbid AD-risk population detector and a system for hypothesis-free drug screens as well as identification of natural suppressor genes for neurodegenerative diseases.
Correction: Patient-specific Alzheimer-like pathology in trisomy 21 cerebral organoids reveals BACE2 as a gene dose-sensitive AD suppressor in human brain
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 AÎ² 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 AÎ² 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-AÎ² 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.
N-terminal heterogeneity of parenchymal and vascular amyloid-Î² deposits in Alzheimer's disease
AIMS/OBJECTIVE:The deposition of amyloid-Î² (AÎ²) peptides in the form of extracellular plaques in the brain represents one of the classical hallmarks of Alzheimer's disease (AD). In addition to "full length" AÎ² starting with aspartic acid (Asp-1), considerable amounts of various shorter, N-terminally truncated AÎ² peptides have been identified by mass spectrometry in autopsy samples from individuals with AD. METHODS:Selectivity of several antibodies detecting full-length, total or N-terminally truncated AÎ² species has been characterized with capillary isoelectric focusing assays using a set of synthetic AÎ² peptides comprising different N-termini. We further assessed the N-terminal heterogeneity of extracellular and vascular AÎ² peptide deposits in the human brain by performing immunohistochemical analyses using sporadic AD cases with antibodies targeting different N-terminal residues, including the biosimilar antibodies Bapineuzumab and Crenezumab. RESULTS:showed a much weaker staining of extracellular plaques and tended to accentuate cerebrovascular amyloid deposits, antibodies detecting AÎ² starting with phenylalanine at position 4 of the AÎ² sequence showed abundant amyloid plaque immunoreactivity in the brain parenchyma. The biosimilar antibody Bapineuzumab recognized AÎ² starting at Asp-1 and demonstrated abundant immunoreactivity in AD brains. DISCUSSION/CONCLUSIONS:specific antibodies, Bapineuzumab displayed stronger immunoreactivity on fixed tissue samples than with SDS-denatured samples on Western blots. This suggests conformational preferences of this antibody. The diverse composition of plaques and vascular deposits stresses the importance of understanding the roles of various AÎ² variants during disease development and progression in order to generate appropriate target-developed therapies.
Alzheimer's amyloid Î² heterogeneous species differentially affect brain endothelial cell viability, blood-brain barrier integrity, and angiogenesis
Impaired clearance in the Alzheimer's Disease (AD) brain is key in the formation of AÎ² parenchymal plaques and cerebrovascular deposits known as cerebral amyloid angiopathy (CAA), present in >80% of AD patients and ~50% of non-AD elderly subjects. AÎ² deposits are highly heterogeneous, containing multiple fragments mostly derived from catabolism of AÎ²40/AÎ²42, which exhibit dissimilar aggregation properties. Remarkably, the role of these physiologically relevant AÎ² species in cerebrovascular injury and their impact in vascular pathology is unknown. We sought to understand how heterogeneous AÎ² species affect cerebral endothelial health and assess whether their diverse effects are associated with the peptides aggregation propensities. We analyzed cerebral microvascular endothelial cell (CMEC) viability, blood-brain barrier (BBB) permeability, and angiogenesis, all relevant aspects of brain microvascular dysfunction. We found that AÎ² peptides and fragments exerted differential effects on cerebrovascular pathology. Peptides forming mostly oligomeric structures induced CMEC apoptosis, whereas fibrillar aggregates increased BBB permeability without apoptotic effects. Interestingly, all AÎ² species tested inhibited angiogenesis in vitro. These data link the biological effects of the heterogeneous AÎ² peptides to their primary structure and aggregation, strongly suggesting that the composition of amyloid deposits influences clinical aspects of the AD vascular pathology. As the presence of predominant oligomeric structures in proximity of the vessel walls may lead to CMEC death and induction of microhemorrhages, fibrillar amyloid is likely responsible for increased BBB permeability and associated neurovascular dysfunction. These results have the potential to unveil more specific therapeutic targets and clarify the multifactorial nature of AD.
Ion channel formation by N-terminally truncated AÎ² (4-42): relevance for the pathogenesis of Alzheimer's disease
AÎ² deposition is a pathological hallmark of Alzheimer's disease (AD). Besides the full-length amyloid forming peptides (AÎ²1-40 and AÎ²1-42), biochemical analyses of brain deposits have identified a variety of N- and C-terminally truncated AÎ² variants in sporadic and familial AD patients. However, their relevance for AD pathogenesis remains largely understudied. We demonstrate that AÎ²4-42 exhibits a high tendency to form Î²-sheet structures leading to fast self-aggregation and formation of oligomeric assemblies. Atomic force microscopy and electrophysiological studies reveal that AÎ²4-42 forms highly stable ion channels in lipid membranes. These channels that are blocked by monoclonal antibodies specifically recognizing the N-terminus of AÎ²4-42. An AÎ² variant with a double truncation at phenylalanine-4 and leucine 34, (AÎ²4-34), exhibits unstable channel formation capability. Taken together the results presented herein highlight the potential benefit of C-terminal proteolytic cleavage and further support an important pathogenic role for N-truncated AÎ² species in AD pathophysiology.
Correction to: Nrf2 activation through the PI3K/GSK-3 axisprotects neuronal cells from AÎ²-mediatedoxidative and metabolic damage
After the publication of this article , we became aware that there were errors in Figs. 4 and 31.
Nrf2 activation through the PI3K/GSK-3 axis protects neuronal cells from AÎ²-mediated oxidative and metabolic damage
BACKGROUND:Mounting evidence points to a crucial role of amyloid-Î² (AÎ²) in the pathophysiology of Alzheimer's disease (AD), a disorder in which brain glucose hypometabolism, downregulation of central elements of phosphorylation pathways, reduced ATP levels, and enhanced oxidative damage coexist, and sometimes precede, synaptic alterations and clinical manifestations. Since the brain has limited energy storage capacity, mitochondria play essential roles in maintaining the high levels of energy demand, but, as major consumers of oxygen, these organelles are also the most important generators of reactive oxygen species (ROS). Thus, it is not surprising that mitochondrial dysfunction is tightly linked to synaptic loss and AD pathophysiology. In spite of their relevance, the mechanistic links among ROS homeostasis, metabolic alterations, and cell bioenergetics, particularly in relation to AÎ², still remain elusive. METHODS:We have used classic biochemical and immunocytochemical approaches together with the evaluation of real-time changes in global energy metabolism in a Seahorse Metabolic Analyzer to provide insights into the detrimental role of oligAÎ² in SH-SY5Y and primary neurons testing their pharmacologic protection by small molecules. RESULTS:Our findings indicate that oligomeric AÎ² induces a dramatic increase in ROS production and severely affects neuronal metabolism and bioenergetics. Assessment of global energy metabolism in real time demonstrated AÎ²-mediated reduction in oxygen consumption affecting basal and maximal respiration and causing decreased ATP production. Pharmacologic targeting of AÎ²-challenged neurons with a set of small molecules of known antioxidant and cytoprotective activity prevented the metabolic/bioenergetic changes induced by the peptide, fully restoring mitochondrial function while inducing an antioxidant response that counterbalanced the ROS production. Search for a mechanistic link among the protective small molecules tested identified the transcription factor Nrf2-compromised by age and downregulated in AD and transgenic models-as their main target and the PI3K/GSK-3 axis as the central pathway through which the compounds elicit their AÎ² protective action. CONCLUSIONS:Our study provides insights into the complex molecular mechanisms triggered by oligAÎ² which profoundly affect mitochondrial performance and argues for the inclusion of small molecules targeting the PI3K/GSK-3 axis and Nrf2-mediated pathways as part of the current or future combinatorial therapies.