Disease modifying effects of the amyloid-beta protofibril-selective antibody mAb158 in aged Tg2576 transgenic mice.

Amyloid beta (Aß) peptides, which aggregate to form neocortical plaques in Alzheimer's disease, exist in states that range from soluble monomers and oligomers/protofibrils to insoluble fibrillar amyloid. The present study evaluated the effects of mAb158, a mouse monoclonal antibody version of lecanemab that preferentially binds to soluble Aß protofibrils, in aged transgenic mice (Tg2576) with Aß pathology. Female Tg2576 mice (12 months old) received weekly intraperitoneal mAb158 (35 mg/kg) or vehicle for 4 weeks or for 18 weeks, with or without a subsequent 12-week off-treatment period. Aß protofibril levels were significantly lower in mAb158-treated animals at both 4 and 18 weeks, while longer treatment duration (18 weeks) was required to observe significantly lower Aß42 levels in insoluble brain fractions and lower Aß plaque load. Following the off-treatment period, comparison of the vehicle- and mAb158-treated mice demonstrated that the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load remained significantly lower in mAb158-treated animals, as compared with age-matched controls. However, there was a significant increase of brain accumulation of both the Aß protofibril levels, insoluble Aß42 levels and Aß plaque load after treatment cessation. Thus, repeated mAb158 treatment of aged Tg2576 mice first reduced Aß protofibril levels within 4 weeks of treatment, which then was followed by a reduction of amyloid plaque pathology within 18 weeks of treatment. These effects were maintained 12 weeks after the final dose, indicating that mAb158 had a disease-modifying effect on the Aß pathology in this mouse model. In addition, brain accumulation of both Aß protofibril levels and amyloid pathology progressed after discontinuation of the treatment which supports the importance of continued treatment with mAb158 to maintain the effects on Aß pathology.

Lecanemab demonstrates highly selective binding to Aß protofibrils isolated from Alzheimer's disease brains.

Recent advances in immunotherapeutic approaches to the treatment of Alzheimer's disease (AD) have increased the importance of understanding the exact binding preference of each amyloid-beta (Aß) antibody employed, since this determines both efficacy and risk for potentially serious adverse events known as amyloid-related imaging abnormalities. Lecanemab is a humanized IgG1 antibody that was developed to target the soluble Aß protofibril conformation. The present study prepared extracts of post mortem brain samples from AD patients and non-demented elderly controls, characterized the forms of Aß present, and investigated their interactions with lecanemab. Brain tissue samples were homogenized and extracted using tris-buffered saline. Aß levels and aggregation states in soluble and insoluble extracts, and in fractions prepared using size-exclusion chromatography or density gradient ultracentrifugation, were analyzed using combinations of immunoassay, immunoprecipitation (IP), and mass spectrometry. Lecanemab immunohistochemistry was also conducted in temporal cortex. The majority of temporal cortex Aß (98 %) was in the insoluble extract. Aß42 was the most abundant form present, particularly in AD subjects, and most soluble Aß42 was in soluble aggregated protofibrillar structures. Aß protofibril levels were much higher in AD subjects than in controls. Protofibrils captured by lecanemab-IP contained high levels of Aß42 and lecanemab bound to large, medium, and small Aß42 protofibrils in a concentration-dependent manner. Competitive IP showed that neither Aß40 monomers nor Aß40-enriched fibrils isolated from cerebral amyloid angiopathy reduced lecanemab's binding to Aß42 protofibrils. Immunohistochemistry showed that lecanemab bound readily to Aß plaques (diffuse and compact) and to intraneuronal Aß in AD temporal cortex. Taken together, these findings indicate that while lecanemab binds to Aß plaques, it preferentially targets soluble aggregated Aß protofibrils. These are largely composed of Aß42, and lecanemab binds less readily to the Aß40-enriched fibrils found in the cerebral vasculature. This is a promising binding profile because Aß42 protofibrils represent a key therapeutic target in AD, while a lack of binding to monomeric Aß and cerebral amyloid deposits should reduce peripheral antibody sequestration and minimize risk for adverse events.

Profiling amyloid-ß peptides as biomarkers for cerebral amyloid angiopathy.

Brain amyloid-ß (Aß) deposits are key pathological hallmarks of both cerebral amyloid angiopathy (CAA) and Alzheimer's disease (AD). Microvascular deposits in CAA mainly consist of the Aß40 peptide, whereas Aß42 is the predominant variant in parenchymal plaques in AD. The relevance in pathogenesis and diagnostic accuracy of various other Aß isoforms in CAA remain understudied. We aimed to investigate the biomarker potential of various Aß isoforms in cerebrospinal fluid (CSF) to differentiate CAA from AD pathology. We included 25 patients with probable CAA, 50 subjects with a CSF profile indicative of AD pathology (AD-like), and 23 age- and sex-matched controls. CSF levels of Aß1-34 , Aß1-37 , Aß1-38 , Aß1-39 , Aß1-40 , and Aß1-42 were quantified by liquid chromatography mass spectrometry. Lower CSF levels of all six Aß peptides were observed in CAA patients compared with controls (p = 0.0005-0.03). Except for Aß1-42 (p = 1.0), all peptides were decreased in CAA compared with AD-like subjects (p = 0.007-0.03). Besides Aß1-42 , none of the Aß peptides were decreased in AD-like subjects compared with controls. All Aß peptides combined differentiated CAA from AD-like subjects better (area under the curve [AUC] 0.84) than individual peptide levels (AUC 0.51-0.75). Without Aß1-42 in the model (since decreased Aß1-42 served as AD-like selection criterion), the AUC was 0.78 for distinguishing CAA from AD-like subjects. CAA patients and AD-like subjects showed distinct disease-specific CSF Aß profiles. Peptides shorter than Aß1-42 were decreased in CAA patients, but not AD-like subjects, which could suggest different pathological mechanisms between vascular and parenchymal Aß accumulation. This study supports the potential use of this panel of CSF Aß peptides to indicate presence of CAA pathology with high accuracy.

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 ß-amyloid-(Aß)-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 Aß 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 ß and γ-secretase inhibition. We found that T21 organoids secrete increased proportions of Aß-preventing (Aß1-19) and Aß-degradation products (Aß1-20 and Aß1-34). 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.

Amyloid pathology and synaptic loss in pathological aging.

Alzheimer's disease (AD) is a neurodegenerative disease characterized by progressive memory dysfunction and cognitive decline. Pathological aging (PA) describes patients who are amyloid-positive but cognitively unimpaired at time of death. Both AD and PA contain amyloid plaques dominated by amyloid ß (Aß) peptides. In this study, we investigated and compared synaptic protein levels, amyloid plaque load, and Aß peptide patterns between AD and PA. Two cohorts of post-mortem brain tissue were investigated. In the first, consisting of controls, PA, AD, and familial AD (FAD) individuals, synaptic proteins extracted with tris(hydroxymethyl)aminomethane-buffered saline (TBS) were analyzed. In the second, consisting of tissue from AD and PA patients from three different regions (occipital lobe, frontal lobe, and cerebellum), a two-step extraction was performed. Five synaptic proteins were extracted using TBS, and from the remaining portion Aß peptides were extracted using formic acid. Subsequently, immunoprecipitation with several antibodies targeting different proteins/peptides was performed for both fractions, which were subsequently analyzed by mass spectrometry. The levels of synaptic proteins were lower in AD (and FAD) compared with PA (and controls), confirming synaptic loss in AD patients. The amyloid plaque load was increased in AD compared with PA, and the relative amount of Aß40 was higher in AD while for Aß42 it was higher in PA. In AD loss of synaptic function was associated with increased plaque load and increased amounts of Aß40 compared with PA cases, suggesting that synaptic function is preserved in PA cases even in the presence of Aß.

Familial Alzheimer's disease patient-derived neurons reveal distinct mutation-specific effects on amyloid beta.

Familial Alzheimer's disease (fAD) mutations alter amyloid precursor protein (APP) cleavage by γ-secretase, increasing the proportion of longer amyloidogenic amyloid-ß (Aß) peptides. Using five control induced pluripotent stem cell (iPSC) lines and seven iPSC lines generated from fAD patients, we investigated the effects of mutations on the Aß secretome in human neurons generated in 2D and 3D. We also analysed matched CSF, post-mortem brain tissue, and iPSCs from the same participant with the APP V717I mutation. All fAD mutation lines demonstrated an increased Aß42:40 ratio relative to controls, yet displayed varied signatures for Aß43, Aß38, and short Aß fragments. We propose four qualitatively distinct mechanisms behind raised Aß42:40. (1) APP V717I mutations alter γ-secretase cleavage site preference. Whereas, distinct presenilin 1 (PSEN1) mutations lead to either (2) reduced γ-secretase activity, (3) altered protein stability or (4) reduced PSEN1 maturation, all culminating in reduced γ-secretase carboxypeptidase-like activity. These data support Aß mechanistic tenets in a human physiological model and substantiate iPSC-neurons for modelling fAD.

Low-dose γ-secretase inhibition increases secretion of Aß peptides and intracellular oligomeric Aß.

γ-Secretase inhibitors have been considered promising drug candidates against Alzheimer's disease (AD) due to their ability to reduce amyloid-ß (Aß) production. However, clinical trials have been halted due to lack of clinical efficacy and/or side effects. Recent in vitro studies suggest that low doses of γ-secretase inhibitors may instead increase Aß production. Using a stem cell-derived human model of cortical neurons and low doses of the γ-secretase inhibitor DAPT, the effects on a variety of Aß peptides were studied using mass spectrometry. One major focus was to develop a novel method for specific detection of oligomeric Aß (oAß), and this was used to study the effects of low-dose γ-secretase inhibitor treatment on intracellular oAß accumulation. Low-dose treatment (2 and 20nM) with DAPT increased the secretion of several Aß peptides, especially Aßx-42. Furthermore, using the novel method for oAß detection, we found that 2nM DAPT treatment of cortical neurons resulted in increased oAß accumulation. Thus, low dose-treatment with DAPT causes both increased production of long, aggregation-prone Aß peptides and accumulation of intracellular Aß oligomers, both believed to contribute to AD pathology.

Amyloid-beta antibody binding to cerebral amyloid angiopathy fibrils and risk for amyloid-related imaging abnormalities.

Therapeutic antibodies have been developed to target amyloid-beta (Aß), and some of these slow the progression of Alzheimer's disease (AD). However, they can also cause adverse events known as amyloid-related imaging abnormalities with edema (ARIA-E). We investigated therapeutic Aß antibody binding to cerebral amyloid angiopathy (CAA) fibrils isolated from human leptomeningeal tissue to study whether this related to the ARIA-E frequencies previously reported by clinical trials. The binding of Aß antibodies to CAA Aß fibrils was evaluated in vitro using immunoprecipitation, surface plasmon resonance, and direct binding assay. Marked differences in Aß antibody binding to CAA fibrils were observed. Solanezumab and crenezumab showed negligible CAA fibril binding and these antibodies have no reported ARIA-E cases. Lecanemab showed a low binding to CAA fibrils, consistent with its relatively low ARIA-E frequency of 12.6%, while aducanumab, bapineuzumab, and gantenerumab all showed higher binding to CAA fibrils and substantially higher ARIA-E frequencies (25-35%). An ARIA-E frequency of 24% was reported for donanemab, and its binding to CAA fibrils correlated with the amount of pyroglutamate-modified Aß present. The findings of this study support the proposal that Aß antibody-CAA interactions may relate to the ARIA-E frequency observed in patients treated with Aß-based immunotherapies.

Specific Mutations in the Cholesterol-Binding Site of APP Alter Its Processing and Favor the Production of Shorter, Less Toxic Aß Peptides.

Excess brain cholesterol is strongly implicated in the pathogenesis of Alzheimer's disease (AD). Here we evaluated how the presence of a cholesterol-binding site (CBS) in the transmembrane and juxtamembrane regions of the amyloid precursor protein (APP) regulates its processing. We generated nine point mutations in the APP gene, changing the charge and/or hydrophobicity of the amino-acids which were previously shown as part of the CBS. Most mutations triggered a reduction of amyloid-ß peptides Aß40 and Aß42 secretion from transiently transfected HEK293T cells. Only the mutations at position 28 of Aß in the APP sequence resulted in a concomitant significant increase in the production of shorter Aß peptides. Mass spectrometry (MS) confirmed the predominance of Aßx-33 and Aßx-34 with the APPK28A mutant. The enzymatic activity of α-, ß-, and γ-secretases remained unchanged in cells expressing all mutants. Similarly, subcellular localization of the mutants in early endosomes did not differ from the APPWT protein. A transient increase of plasma membrane cholesterol enhanced the production of Aß40 and Aß42 by APPWT, an effect absent in APPK28A mutant. Finally, WT but not CBS mutant Aß derived peptides bound to cholesterol-rich exosomes. Collectively, the present data revealed a major role of juxtamembrane amino acids of the APP CBS in modulating the production of toxic Aß species. More generally, they underpin the role of cholesterol in the pathophysiology of AD.

Characterization of monomeric and soluble aggregated Aß in Down's syndrome and Alzheimer's disease brains.

The major characteristics of Alzheimer's disease (AD) are amyloid plaques, consisting of aggregated beta amyloid (Aß) peptides, together with tau pathology (tangles, neuropil treads and dystrophic neurites surrounding the plaques), in the brain. Down's syndrome (DS) individuals are at increased risk to develop AD-type pathology; most DS individuals have developed substantial pathology already at the age of 40. DS individuals have an extra copy of chromosome 21, harbouring the amyloid precursor protein gene (APP). Our aim was to investigate the Aß peptide pattern in DS and AD brains to investigate differences in their amyloid deposition and aggregation, respectively. Cortical tissue from patients with DS (with amyloid pathology), sporadic AD and controls were homogenized and fractionated into TBS (water soluble) and formic acid (water insoluble) fractions. Immunoprecipitation (IP) was performed using a variety of antibodies targeting different Aß species including oligomeric Aß. Mass spectrometry was then used to evaluate the presence of Aß species in the different patient groups. A large number of Aß peptides were identified including Aß1-X, 2-X, 3-X, 4-X, 5-X, 11-X, and Aß peptides extended N terminally of the BACE1 cleavage site and ending at amino 15 in the Aß sequence APP/Aß(-X to 15), as well as peptides post-translationally modified by pyroglutamate formation. Most Aß peptides had higher abundance in AD and DS compared to controls, except the APP/Aß(-X to 15) peptides which were most abundant in DS followed by controls and AD. Furthermore, the abundancies of AßX-40 and AßX-34 were increased in DS compared with AD. Aß1-40, Aß1-42, and Aß4-42 were identified as the main constitutes of protofibrils (IP'd using mAb158) and higher relative Aß1-42 signals were obtained compared with samples IP'd with 6E10 + 4G8, indicating that the protofibrils/oligomers were enriched with peptides ending at amino acid 42. All Aß peptides found in AD were also present in DS indicating similar pathways of Aß peptide production, degradation and accumulation, except for APP/Aß(-X to 15). Likewise, the Aß peptides forming protofibrils/oligomers in both AD and DS were similar, implying the possibility that treatment with clinical benefit in sporadic AD might also be beneficial for subjects with DS.

A distinct brain beta amyloid signature in cerebral amyloid angiopathy compared to Alzheimer's disease.

Cerebral amyloid angiopathy (CAA) is a type of vascular disease present in more than 50% of demented elderly and more than 80% of Alzheimer's disease (AD) patients. Both CAA and AD are characterized by extracellular Aß deposits with the distinction that CAA has vascular deposits while AD has amyloid plaques. In this study, we used immunoprecipitation (IP) in combination with mass spectrometry (MS) to test the hypothesis that the Aß peptide pattern differs between subjects having Aß plaque pathology only and subjects with Aß plaque pathology together with CAA pathology. Occipital lobes from 12 AD brains, ranging from no CAA to severe CAA, were extracted using 70% formic acid followed by IP-MS analysis. The Aß peptide pattern differed greatly between subjects with no CAA compared to subjects with CAA. In cases with CAA, the most abundant Aß peptides ended at amino acid 40 including Aß1-40 (P = .048) and Aß 2-40 (P = .0253) which were significantly increased compared to cases with no CAA. This was in contrast to subjects with no CAA where the most abundant Aß peptides ended at amino acid 42 of which Aß1-42 (P = .0101) and Aß2-42 (P = .0051) as well as the pyroglutamate (pGlu)-modified peptides pGlu Aß3-42 (P = .0177), and pGlu Aß11-42 (P = .0088) were significantly increased compared to CAA subjects. The results are in line with earlier immunohistochemistry data and show that the molecular composition of the Aß deposits found in blood vessels are different to the parenchymal deposits, suggesting they arise from distinct pathogenic pathways. This information may be useful in the development of pathology-specific biomarkers.

Update on biomarkers for amyloid pathology in Alzheimer's disease.

At the center of Alzheimer's disease pathogenesis is the aberrant aggregation of amyloid-ß (Aß) into oligomers, fibrils and plaques. Effective monitoring of Aß deposition directly in patients is essential to assist anti-Aß therapeutics in target engagement and participant selection. In the advent of approved anti-Aß therapeutics, biomarkers will become of fundamental importance in initiating treatments having disease modifying effects at the earliest stage. Two well-established Aß biomarkers are widely utilized: Aß-binding ligands for positron emission tomography and immunoassays to measure Aß42 in cerebrospinal fluid. In this review, we will discuss the current clinical, diagnostic and research state of biomarkers for Aß pathology. Furthermore, we will explore the current application of blood-based markers to assess Aß pathology.

The presubiculum is preserved from neurodegenerative changes in Alzheimer's disease.

In the majority of affected brain regions the pathological hallmarks of Alzheimer's disease (AD) are ß-amyloid (Aß) deposits in the form of diffuse and neuritic plaques, tau pathology in the form of neurofibrillary tangles, neuropil threads and plaque-associated abnormal neurites in combination with an inflammatory response. However, the anatomical area of the presubiculum, is characterised by the presence of a single large evenly distributed 'lake-like' Aß deposit with minimal tau deposition or accumulation of inflammatory markers. Post-mortem brain samples from sporadic AD (SAD) and familial AD (FAD) and two hereditary cerebral amyloid diseases, familial British dementia (FBD) and familial Danish dementia (FDD) were used to compare the morphology of the extracellular proteins deposited in the presubiculum compared to the entorhinal cortex. The level of tau pathology and the extent of microglial activation were quantitated in the two brain regions in SAD and FAD. Frozen tissue was used to investigate the Aß species and proteomic differences between the two regions. Consistent with our previous investigations of FBD and FDD cases we were able to establish that the 'lake-like' pre-amyloid deposits of the presubiculum were not a unique feature of AD but they also found two non-Aß amyloidosis. Comparing the presubiculum to the entorhinal cortex the number of neurofibrillary tangles and tau load were significantly reduced; there was a reduction in microglial activation; there were differences in the Aß profiles and the investigation of the whole proteome showed significant changes in different protein pathways. In summary, understanding why the presubiculum has a different morphological appearance, biochemical and proteomic makeup compared to surrounding brain regions severely affected by neurodegeneration could lead us to understanding protective mechanisms in neurodegenerative diseases.

Ex vivo 18O-labeling mass spectrometry identifies a peripheral amyloid ß clearance pathway.

BACKGROUND: Proteolytic degradation of amyloid ß (Aß) peptides has been intensely studied due to the central role of Aß in Alzheimer's disease (AD) pathogenesis. While several enzymes have been shown to degrade Aß peptides, the main pathway of Aß degradation in vivo is unknown. Cerebrospinal fluid (CSF) Aß42 is reduced in AD, reflecting aggregation and deposition in the brain, but low CSF Aß42 is, for unknown reasons, also found in some inflammatory brain disorders such as bacterial meningitis. METHOD: Using 18O-labeling mass spectrometry and immune-affinity purification, we examined endogenous proteolytic processing of Aß in human CSF. RESULTS: The Aß peptide profile was stable in CSF samples from healthy controls but in CSF samples from patients with bacterial meningitis, showing increased leukocyte cell count, 18O-labeling mass spectrometry identified proteolytic activities degrading Aß into several short fragments, including abundant Aß1-19 and 1-20. After antibiotic treatment, no degradation of Aß was detected. In vitro experiments located the source of the proteolytic activity to blood components, including leukocytes and erythrocytes, with insulin-degrading enzyme as the likely protease. A recombinant version of the mid-domain anti-Aß antibody solanezumab was found to inhibit insulin-degrading enzyme-mediated Aß degradation. CONCLUSION: 18O labeling-mass spectrometry can be used to detect endogenous proteolytic activity in human CSF. Using this technique, we found an enzymatic activity that was identified as insulin-degrading enzyme that cleaves Aß in the mid-domain of the peptide, and could be inhibited by a recombinant version of the mid-domain anti-Aß antibody solanezumab.