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.

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.

Aß and tau prions feature in the neuropathogenesis of Down syndrome.

Down syndrome (DS) is caused by the triplication of chromosome 21 and is the most common chromosomal disorder in humans. Those individuals with DS who live beyond age 40 y develop a progressive dementia that is similar to Alzheimer's disease (AD). Both DS and AD brains exhibit numerous extracellular amyloid plaques composed of Aß and intracellular neurofibrillary tangles composed of tau. Since AD is a double-prion disorder, we asked if both Aß and tau prions feature in DS. Frozen brains from people with DS, familial AD (fAD), sporadic AD (sAD), and age-matched controls were procured from brain biorepositories. We selectively precipitated Aß and tau prions from DS brain homogenates and measured the number of prions using cellular bioassays. In brain extracts from 28 deceased donors with DS, ranging in age from 19 to 65 y, we found nearly all DS brains had readily measurable levels of Aß and tau prions. In a cross-sectional analysis of DS donor age at death, we found that the levels of Aß and tau prions increased with age. In contrast to DS brains, the levels of Aß and tau prions in the brains of 37 fAD and sAD donors decreased as a function of age at death. Whether DS is an ideal model for assessing the efficacy of putative AD therapeutics remains to be determined.

Mutation analysis of disease causing genes in patients with early onset or familial forms of Alzheimer's disease and frontotemporal dementia.

BACKGROUND: Most dementia disorders have a clear genetic background and a number of disease genes have been identified. Mutations in the tau gene (MAPT) lead to frontotemporal dementia (FTD), whereas mutations in the genes for the amyloid-ß precursor protein (APP) and the presenilins (PSEN1, PSEN2) cause early-onset, dominantly inherited forms of Alzheimer's disease (AD). Even if mutations causing Mendelian forms of these diseases are uncommon, elucidation of the pathogenic effects of such mutations have proven important for understanding the pathogenic processes. Here, we performed a screen to identify novel pathogenic mutations in known disease genes among patients undergoing dementia investigation. RESULTS: Using targeted exome sequencing we have screened all coding exons in eleven known dementia genes (PSEN1, PSEN2, APP, MAPT, APOE, GRN, TARDBP, CHMP2B, TREM2, VCP and FUS) in 102 patients with AD, FTD, other dementia diagnoses or mild cognitive impairment. We found three AD patients with two previously identified pathogenic mutations in PSEN1 (Pro264Leu and Met146Val). In this screen, we also identified the recently reported APP mutation in two siblings with AD. This mutation, named the Uppsala mutation, consists of a six amino acid intra-amyloid ß deletion. In addition, we found several potentially pathogenic mutations in PSEN2, FUS, MAPT, GRN and APOE. Finally, APOE ε4 was prevalent in this patient group with an allele frequency of 54%. CONCLUSIONS: Among the 102 screened patients, we found two disease causing mutations in PSEN1 and one in APP, as well as several potentially pathogenic mutations in other genes related to neurodegenerative disorders. Apart from giving important information to the clinical investigation, the identification of disease mutations can contribute to an increased understanding of disease mechanisms.

The Amyloid-ß Pathway in Alzheimer's Disease.

Breakthroughs in molecular medicine have positioned the amyloid-ß (Aß) pathway at the center of Alzheimer's disease (AD) pathophysiology. While the detailed molecular mechanisms of the pathway and the spatial-temporal dynamics leading to synaptic failure, neurodegeneration, and clinical onset are still under intense investigation, the established biochemical alterations of the Aß cycle remain the core biological hallmark of AD and are promising targets for the development of disease-modifying therapies. Here, we systematically review and update the vast state-of-the-art literature of Aß science with evidence from basic research studies to human genetic and multi-modal biomarker investigations, which supports a crucial role of Aß pathway dyshomeostasis in AD pathophysiological dynamics. We discuss the evidence highlighting a differentiated interaction of distinct Aß species with other AD-related biological mechanisms, such as tau-mediated, neuroimmune and inflammatory changes, as well as a neurochemical imbalance. Through the lens of the latest development of multimodal in vivo biomarkers of AD, this cross-disciplinary review examines the compelling hypothesis- and data-driven rationale for Aß-targeting therapeutic strategies in development for the early treatment of AD.

Elevated soluble amyloid beta protofibrils in Down syndrome and Alzheimer's disease.

Down syndrome (DS) is caused by trisomy of chromosome 21, which leads to a propensity to develop amyloid ß (Aß) brain pathology in early adulthood followed later by cognitive and behavioral deterioration. Characterization of the Aß pathology is important to better understand the clinical deterioration of DS individuals and to identify interventive strategies. Brain samples from people with DS and Alzheimer's disease (AD), as well as non-demented controls (NDC), were analyzed with respect to different Aß species. Immunohistochemical staining using antibodies towards Aß was also performed. Elevated levels of soluble Aß protofibrils and insoluble Aßx-40 and Aßx-42 in formic acid brain extracts, and elevated immunohistochemical staining of Aß deposits were demonstrated with the antibody BAN2401 (lecanemab) in DS and AD compared with NDC. These data and the promising data in a large phase 2 CE clinical trial with lecanemab suggest that lecanemab may have the potential to preserve cognitive capacity in DS. Lecanemab is currently in a phase 3 CE clinical trial.

ABBV-0805, a novel antibody selective for soluble aggregated α-synuclein, prolongs lifespan and prevents buildup of α-synuclein pathology in mouse models of Parkinson's disease.

A growing body of evidence suggests that aggregated α-synuclein, the major constituent of Lewy bodies, plays a key role in the pathogenesis of Parkinson's disease and related α-synucleinopathies. Immunotherapies, both active and passive, against α-synuclein have been developed and are promising novel treatment strategies for such disorders. Here, we report on the humanization and pharmacological characteristics of ABBV-0805, a monoclonal antibody that exhibits a high selectivity for human aggregated α-synuclein and very low affinity for monomers. ABBV-0805 binds to a broad spectrum of soluble aggregated α-synuclein, including small and large aggregates of different conformations. Binding of ABBV-0805 to pathological α-synuclein was demonstrated in Lewy body-positive post mortem brains of Parkinson's disease patients. The functional potency of ABBV-0805 was demonstrated in several cellular assays, including Fcγ-receptor mediated uptake of soluble aggregated α-synuclein in microglia and inhibition of neurotoxicity in primary neurons. In vivo, the murine version of ABBV-0805 (mAb47) displayed significant dose-dependent decrease of α-synuclein aggregates in brain in several mouse models, both in prophylactic and therapeutic settings. In addition, mAb47 treatment of α-synuclein transgenic mice resulted in a significantly prolonged survival. ABBV-0805 selectively targets soluble toxic α-synuclein aggregates with a picomolar affinity and demonstrates excellent in vivo efficacy. Based on the strong preclinical findings described herein, ABBV-0805 has been progressed into clinical development as a potential disease-modifying treatment for Parkinson's disease.

η-Secretase processing of APP inhibits neuronal activity in the hippocampus.

Alzheimer disease (AD) is characterized by the accumulation of amyloid plaques, which are predominantly composed of amyloid-ß peptide. Two principal physiological pathways either prevent or promote amyloid-ß generation from its precursor, ß-amyloid precursor protein (APP), in a competitive manner. Although APP processing has been studied in great detail, unknown proteolytic events seem to hinder stoichiometric analyses of APP metabolism in vivo. Here we describe a new physiological APP processing pathway, which generates proteolytic fragments capable of inhibiting neuronal activity within the hippocampus. We identify higher molecular mass carboxy-terminal fragments (CTFs) of APP, termed CTF-η, in addition to the long-known CTF-α and CTF-ß fragments generated by the α- and ß-secretases ADAM10 (a disintegrin and metalloproteinase 10) and BACE1 (ß-site APP cleaving enzyme 1), respectively. CTF-η generation is mediated in part by membrane-bound matrix metalloproteinases such as MT5-MMP, referred to as η-secretase activity. η-Secretase cleavage occurs primarily at amino acids 504-505 of APP695, releasing a truncated ectodomain. After shedding of this ectodomain, CTF-η is further processed by ADAM10 and BACE1 to release long and short Aη peptides (termed Aη-α and Aη-ß). CTFs produced by η-secretase are enriched in dystrophic neurites in an AD mouse model and in human AD brains. Genetic and pharmacological inhibition of BACE1 activity results in robust accumulation of CTF-η and Aη-α. In mice treated with a potent BACE1 inhibitor, hippocampal long-term potentiation was reduced. Notably, when recombinant or synthetic Aη-α was applied on hippocampal slices ex vivo, long-term potentiation was lowered. Furthermore, in vivo single-cell two-photon calcium imaging showed that hippocampal neuronal activity was attenuated by Aη-α. These findings not only demonstrate a major functionally relevant APP processing pathway, but may also indicate potential translational relevance for therapeutic strategies targeting APP processing.

Structural heterogeneity and intersubject variability of Aß in familial and sporadic Alzheimer's disease.

Point mutations in the amyloid-ß (Aß) coding region produce a combination of mutant and WT Aß isoforms that yield unique clinicopathologies in familial Alzheimer's disease (fAD) and cerebral amyloid angiopathy (fCAA) patients. Here, we report a method to investigate the structural variability of amyloid deposits found in fAD, fCAA, and sporadic AD (sAD). Using this approach, we demonstrate that mutant Aß determines WT Aß conformation through prion template-directed misfolding. Using principal component analysis of multiple structure-sensitive fluorescent amyloid-binding dyes, we assessed the conformational variability of Aß deposits in fAD, fCAA, and sAD patients. Comparing many deposits from a given patient with the overall population, we found that intrapatient variability is much lower than interpatient variability for both disease types. In a given brain, we observed one or two structurally distinct forms. When two forms coexist, they segregate between the parenchyma and cerebrovasculature, particularly in fAD patients. Compared with sAD samples, deposits from fAD patients show less intersubject variability, and little overlap exists between fAD and sAD deposits. Finally, we examined whether E22G (Arctic) or E22Q (Dutch) mutants direct the misfolding of WT Aß, leading to fAD-like plaques in vivo. Intracerebrally injecting mutant Aß40 fibrils into transgenic mice expressing only WT Aß induced the deposition of plaques with many biochemical hallmarks of fAD. Thus, mutant Aß40 prions induce a conformation of WT Aß similar to that found in fAD deposits. These findings indicate that diverse AD phenotypes likely arise from one or more initial Aß prion conformations, which kinetically dominate the spread of prions in the brain.

Mosaic Loss of Chromosome Y in Blood Is Associated with Alzheimer Disease.

Men have a shorter life expectancy compared with women but the underlying factor(s) are not clear. Late-onset, sporadic Alzheimer disease (AD) is a common and lethal neurodegenerative disorder and many germline inherited variants have been found to influence the risk of developing AD. Our previous results show that a fundamentally different genetic variant, i.e., lifetime-acquired loss of chromosome Y (LOY) in blood cells, is associated with all-cause mortality and an increased risk of non-hematological tumors and that LOY could be induced by tobacco smoking. We tested here a hypothesis that men with LOY are more susceptible to AD and show that LOY is associated with AD in three independent studies of different types. In a case-control study, males with AD diagnosis had higher degree of LOY mosaicism (adjusted odds ratio = 2.80, p = 0.0184, AD events = 606). Furthermore, in two prospective studies, men with LOY at blood sampling had greater risk for incident AD diagnosis during follow-up time (hazard ratio [HR] = 6.80, 95% confidence interval [95% CI] = 2.16-21.43, AD events = 140, p = 0.0011). Thus, LOY in blood is associated with risks of both AD and cancer, suggesting a role of LOY in blood cells on disease processes in other tissues, possibly via defective immunosurveillance. As a male-specific risk factor, LOY might explain why males on average live shorter lives than females.

The Aß protofibril selective antibody mAb158 prevents accumulation of Aß in astrocytes and rescues neurons from Aß-induced cell death.

BACKGROUND: Currently, several amyloid beta (Aß) antibodies, including the protofibril selective antibody BAN2401, are in clinical trials. The murine version of BAN2401, mAb158, has previously been shown to lower the levels of pathogenic Aß and prevent Aß deposition in animal models of Alzheimer's disease (AD). However, the cellular mechanisms of the antibody's action remain unknown. We have recently shown that astrocytes effectively engulf Aß42 protofibrils, but store rather than degrade the ingested Aß aggregates. In a co-culture set-up, the incomplete degradation of Aß42 protofibrils by astrocytes results in increased neuronal cell death, due to the release of extracellular vesicles, containing N-truncated, neurotoxic Aß. METHODS: The aim of the present study was to investigate if the accumulation of Aß in astrocytes can be affected by the Aß protofibril selective antibody mAb158. Co-cultures of astrocytes, neurons, and oligodendrocytes, derived from embryonic mouse cortex, were exposed to Aß42 protofibrils in the presence or absence of mAb158. RESULTS: Our results demonstrate that the presence of mAb158 almost abolished Aß accumulation in astrocytes. Consequently, mAb158 treatment rescued neurons from Aß-induced cell death. CONCLUSION: Based on these findings, we conclude that astrocytes may play a central mechanistic role in anti-Aß immunotherapy.

Alzheimer's disease pathology propagation by exosomes containing toxic amyloid-beta oligomers.

The gradual deterioration of cognitive functions in Alzheimer's disease is paralleled by a hierarchical progression of amyloid-beta and tau brain pathology. Recent findings indicate that toxic oligomers of amyloid-beta may cause propagation of pathology in a prion-like manner, although the underlying mechanisms are incompletely understood. Here we show that small extracellular vesicles, exosomes, from Alzheimer patients' brains contain increased levels of amyloid-beta oligomers and can act as vehicles for the neuron-to-neuron transfer of such toxic species in recipient neurons in culture. Moreover, blocking the formation, secretion or uptake of exosomes was found to reduce both the spread of oligomers and the related toxicity. Taken together, our results imply that exosomes are centrally involved in Alzheimer's disease and that they could serve as targets for development of new diagnostic and therapeutic principles.

A bispecific Tribody PET radioligand for visualization of amyloid-beta protofibrils - a new concept for neuroimaging.

Antibodies are highly specific for their target molecules, but their poor brain penetrance has restricted their use as PET ligands for imaging of targets within the CNS. The aim of this study was to develop an antibody-based radioligand, using the TribodyTM format, for PET imaging of soluble amyloid-beta (Aß) protofibrils, which are suggested to cause neurodegeneration in Alzheimer's disease. Antibodies, even when expressed in smaller engineered formats, are large molecules that do not enter the brain in sufficient amounts for imaging purposes. Hence, their transport across the blood-brain barrier (BBB) needs to be facilitated, for example through interaction with the transferrin receptor (TfR). Thus, a Fab fragment of the TfR antibody 8D3 was fused with two single chain variable fragments (scFv) of the Aß protofibril selective antibody mAb158. Five TribodyTM proteins (A1-A5) were generated with different linkers between the Fab-8D3 and scFv-158. All proteins bound to TfR and Aß protofibrils in vitro. Three of the proteins (A1-A3) were radiolabeled with iodine-125 and studied ex vivo in wild-type (wt) and transgenic mice overexpressing human Aß. The systemic pharmacokinetics were similar with half-lives in blood of around 9h for all three ligands. Brain concentrations at 2h were around 1% of the injected dose per gram brain tissue, which is similar to what is observed for small molecular radioligands and at least 10-fold higher than antibodies in general. At 72h, transgenic mice showed higher concentrations of radioactivity in the brain than wt mice (12, 15- and 16-fold for A1, A2 and A3 respectively), except in the cerebellum, an area largely devoid of Aß pathology. A3 was then labelled with iodine-124 for in vivo positron emission tomography (PET) imaging. Brain concentrations were quantified in six different regions showing a clear distinction both quantitatively and visually between wt and transgenic mice and a good correlation with Aß pathology. We have thus produced a recombinant, bispecific protein, actively transported into the brain, for PET imaging within the CNS. In a longer perspective, this technique may enable imaging of other proteins involved in neurodegenerative diseases for which imaging agents are completely lacking today.

Alpha-synuclein oligomer-selective antibodies reduce intracellular accumulation and mitochondrial impairment in alpha-synuclein exposed astrocytes.

BACKGROUND: Due to its neurotoxic properties, oligomeric alpha-synuclein (α-syn) has been suggested as an attractive target for passive immunization against Parkinson's disease (PD). In mouse models of PD, antibody treatment has been shown to lower the levels of pathogenic α-syn species, including oligomers, although the mechanisms of action remain unknown. We have previously shown that astrocytes rapidly engulf α-syn oligomers that are intracellularly stored, rather than degraded, resulting in impaired mitochondria. METHODS: The aim of the present study was to investigate if the accumulation of α-syn in astrocytes can be affected by α-syn oligomer-selective antibodies. Co-cultures of astrocytes, neurons, and oligodendrocytes were derived from embryonic mouse cortex and exposed to α-syn oligomers or oligomers pre-incubated with oligomer-selective antibodies. RESULTS: In the presence of antibodies, the astrocytes displayed an increased clearance of the exogenously added α-syn, and consequently, the α-syn accumulation in the culture was markedly reduced. Moreover, the addition of antibodies rescued the astrocytes from the oligomer-induced mitochondrial impairment. CONCLUSIONS: Our results demonstrate that oligomer-selective antibodies can prevent α-syn accumulation and mitochondrial dysfunction in cultured astrocytes.

Efficient and inexpensive transient expression of multispecific multivalent antibodies in Expi293 cells.

BACKGROUND: Immunotherapy is a very fast expanding field within drug discovery and, hence, rapid and inexpensive expression of antibodies would be extremely valuable. Antibodies are, however, difficult to express. Multifunctional antibodies with additional binding domains further complicate the expression. Only few protocols describe the production of tetravalent bispecific antibodies and all with limited expression levels.. METHODS: Here, we describe a protocol that can produce functional tetravalent, bispecific antibodies at around 22 mg protein/l to a low cost. The expression system is based on the Expi293 cells, which have been adapted to grow in denser cultures than HEK293 cells and gives higher expression yields. The new protocol transfects the Expi293 cells with PEI (which has a negligible cost). RESULTS: The protocol has been used to generate multiple variants of tetra- and hexavalent bispecific antibodies with yields of around 22 mg protein/l within 10 days. All materials are commercially available and the implementation of the protocol is inexpensive and straightforward. The bispecific antibodies generated in our lab were capable of binding to all antigens with similar affinity as the original antibody. Two of the bispecific antibodies have also been used in transgenic mice as positron emission tomography (PET) ligands to successfully detect amyloid-beta (Aß) aggregates in vivo. CONCLUSIONS: This protocol is the first describing transfection of the human Expi293 cells with PEI. It can be used to generate functional multi-specific antibodies in high amounts. The use of biological drugs, and in particular multispecific antibodies, is rapidly increasing, hence improved protocols such as the one presented here are highly valuable.

Cellular Uptake of α-Synuclein Oligomer-Selective Antibodies is Enhanced by the Extracellular Presence of α-Synuclein and Mediated via Fcγ Receptors.

Immunotherapy targeting aggregated α-synuclein has emerged as a potential treatment strategy against Parkinson's disease and other α-synucleinopathies. We have developed α-synuclein oligomer/protofibril selective antibodies that reduce toxic α-synuclein in a human cell line and, upon intraperitoneal administration, in spinal cord of transgenic mice. Here, we investigated under which conditions and by which mechanisms such antibodies can be internalized by cells. For this purpose, human neuroglioma H4 cells were treated with either monoclonal oligomer/protofibril selective α-synuclein antibodies, linear epitope monoclonal α-synuclein antibodies, or with a control antibody. The oligomer/protofibril selective antibody mAb47 displayed the highest cellular uptake and was therefore chosen for additional analyses. Next, α-synuclein overexpressing cells were incubated with mAb47, which resulted in increased antibody internalization as compared to non-transfected cells. Similarly, regular cells exposed to mAb47 together with media containing α-synuclein displayed a higher uptake as compared to cells incubated with regular media. Finally, different Fcγ receptors were targeted and we then found that blockage of FcγRI and FcγRIIB/C resulted in reduced antibody internalization. Our data thus indicate that the robust uptake of the oligomer/protofibril selective antibody mAb47 by human CNS-derived cells is enhanced by extracellular α-synuclein and mediated via Fcγ receptors. Altogether, our finding lend further support to the belief that α-synuclein pathology can be modified by monoclonal antibodies and that these can target toxic α-synuclein species in the extracellular milieu. In the context of immunotherapy, antibody binding of α-synuclein would then not only block further aggregation but also mediate internalization and subsequent degradation of antigen-antibody complexes.

Mapping of Surface-Exposed Epitopes of In Vitro and In Vivo Aggregated Species of Alpha-Synuclein.

Aggregated alpha-synuclein is the main component of Lewy bodies, intraneuronal deposits observed in Parkinson's disease and dementia with Lewy bodies. The objective of the study was to identify surface-exposed epitopes of alpha-synuclein in vitro and in vivo formed aggregates. Polyclonal immunoglobulin Y antibodies were raised against short linear peptides of the alpha-synuclein molecule. An epitope in the N-terminal region (1-10) and all C-terminal epitopes (90-140) were found to be exposed in an indirect enzyme-linked immunosorbent assay (ELISA) using recombinant monomeric, oligomeric, and fibrillar alpha-synuclein. In a phospholipid ELISA, the N-terminus and mid-region of alpha-synuclein (i.e., 1-90) were associated with phosphatidylserine and thus occluded from antibody binding. The antibodies that reacted most strongly with epitopes in the in vitro aggregates (i.e., 1-10 and epitopes between positions 90-140) also labeled alpha-synuclein inclusions in brains from transgenic (Thy-1)-h[A30P] alpha-synuclein mice and Lewy bodies and Lewy neurites in brains of patients with alpha-synucleinopathies. However, differences in reactivity were observed with the C-terminal antibodies when brain tissue from human and transgenic mice was compared. Taken together, the study shows that although similar epitopes are exposed in both in vitro and in vivo formed alpha-synuclein inclusions, structural heterogeneity can be observed between different molecular species.

Predicting Cognitive Decline across Four Decades in Mutation Carriers and Non-carriers in Autosomal-Dominant Alzheimer's Disease.

OBJECTIVES: The aim of this study was to investigate cognitive performance including preclinical and clinical disease course in carriers and non-carriers of autosomal-dominant Alzheimer's disease (adAD) in relation to multiple predictors, that is, linear and non-linear estimates of years to expected clinical onset of disease, years of education and age. METHODS: Participants from five families with early-onset autosomal-dominant mutations (Swedish and Arctic APP, PSEN1 M146V, H163Y, and I143T) included 35 carriers (28 without dementia and 7 with) and 44 non-carriers. All participants underwent a comprehensive clinical evaluation, including neuropsychological assessment at the Memory Clinic, Karolinska University Hospital at Huddinge, Stockholm, Sweden. The time span of disease course covered four decades of the preclinical and clinical stages of dementia. Neuropsychological tests were used to assess premorbid and current global cognition, verbal and visuospatial functions, short-term and episodic memory, attention, and executive function. RESULTS: In carriers, the time-related curvilinear trajectory of cognitive function across disease stages was best fitted to a formulae with three predictors: years to expected clinical onset (linear and curvilinear components), and years of education. In non-carriers, the change was minimal and best predicted by two predictors: education and age. The trajectories for carriers and non-carriers began to diverge approximately 10 years before the expected clinical onset in episodic memory, executive function, and visuospatial function. CONCLUSIONS: The curvilinear trajectory of cognitive functions across disease stages was mimicked by three predictors in carriers. In episodic memory, executive and visuospatial functions, the point of diverging trajectories occurred approximately 10 years ahead of the clinical onset compared to non-carriers. (JINS, 2017, 23, 195-203).

Serial propagation of distinct strains of Aß prions from Alzheimer's disease patients.

An increasing number of studies argues that self-propagating protein conformations (i.e., prions) feature in the pathogenesis of several common neurodegenerative diseases. Mounting evidence contends that aggregates of the amyloid-ß (Aß) peptide become self-propagating in Alzheimer's disease (AD) patients. An important characteristic of prions is their ability to replicate distinct strains, the biological information for which is enciphered within different conformations of protein aggregates. To investigate whether distinct strains of Aß prions can be discerned in AD patients, we performed transmission studies in susceptible transgenic mice using brain homogenates from sporadic or heritable (Arctic and Swedish) AD cases. Mice inoculated with the Arctic AD sample exhibited a pathology that could be distinguished from mice inoculated with the Swedish or sporadic AD samples, which was judged by differential accumulation of Aß isoforms and the morphology of cerebrovascular Aß deposition. Unlike Swedish AD- or sporadic AD-inoculated animals, Arctic AD-inoculated mice, like Arctic AD patients, displayed a prominent Aß38-containing cerebral amyloid angiopathy. The divergent transmission behavior of the Arctic AD sample compared with the Swedish and sporadic AD samples was maintained during second passage in mice, showing that Aß strains are serially transmissible. We conclude that at least two distinct strains of Aß prions can be discerned in the brains of AD patients and that strain fidelity was preserved on serial passage in mice. Our results provide a potential explanation for the clinical and pathological heterogeneity observed in AD patients.