Alzheimer's disease (AD) is a neurodegenerative disorder that develops over decades. Glial cells, including astrocytes are tightly connected to the AD pathogenesis, but their impact on disease progression is still unclear. Our previous data show that astrocytes take up large amounts of aggregated amyloid-beta (Aβ) but are unable to successfully degrade the material, which is instead stored intracellularly. The aim of the present study was to analyze the astrocytic Aβ deposits composition in detail in order to understand their role in AD propagation. For this purpose, human induced pluripotent cell (hiPSC)-derived astrocytes were exposed to sonicated Aβ42 fibrils and magnetic beads. Live cell imaging and immunocytochemistry confirmed that the ingested Aβ aggregates and beads were transported to the same lysosomal compartments in the perinuclear region, which allowed us to successfully isolate the Aβ deposits from the astrocytes. Using a battery of experimental techniques, including mass spectrometry, western blot, ELISA and electron microscopy we demonstrate that human astrocytes truncate and pack the Aβ aggregates in a way that makes them highly resistant. Moreover, the astrocytes release specifically truncated forms of Aβ via different routes and thereby expose neighboring cells to pathogenic proteins. Taken together, our study establishes a role for astrocytes in mediating Aβ pathology, which could be of relevance for identifying novel treatment targets for AD.
Abstract Alzheimer’s disease (AD) is characterized by a substantial loss of neurons and synapses throughout the brain. The exact mechanism behind the neurodegeneration is still unclear, but recent data suggests that spreading of amyloid-β (Aβ) pathology via extracellular vesicles (EVs) may contribute to disease progression. We have previously shown that an incomplete degradation of Aβ 42 protofibrils by astrocytes results in the release of EVs containing neurotoxic Aβ. Here, we describe the cellular mechanisms behind EV-associated neurotoxicity in detail. EVs were isolated from untreated and Aβ 42 protofibril exposed neuroglial co-cultures, consisting mainly of astrocytes. The EVs were added to cortical neurons for 2 or 4 days and the neurodegenerative processes were followed with immunocytochemistry, time-lapse imaging and transmission electron microscopy (TEM). Addition of EVs from Aβ 42 protofibril exposed co-cultures resulted in synaptic loss, severe mitochondrial impairment and apoptosis. TEM analysis demonstrated that the EVs induced axonal swelling and vacuolization of the neuronal cell bodies. Interestingly, EV exposed neurons also displayed pathological lamellar bodies of cholesterol deposits in lysosomal compartments. Taken together, our data show that the secretion of EVs from Aβ exposed cells induces neuronal dysfunction in several ways, indicating a central role for EVs in the progression of Aβ-induced pathology.
OBJECTIVE The authors conducted a study to test if the cortical brain tissue levels of soluble amyloid beta (Aβ) reflect the propensity of cortical Aβ aggregate formation and may be an additional factor predicting surgical outcome following idiopathic normal pressure hydrocephalus (iNPH) treatment. METHODS Highly selective ELISAs (enzyme-linked immunosorbent assays) were used to quantify soluble Aβ40, Aβ42, and neurotoxic Aβ oligomers/protofibrils, associated with Aβ aggregation, in cortical biopsy samples obtained in patients with iNPH (n = 20), sampled during ventriculoperitoneal (VP) shunt surgery. Patients underwent pre- and postoperative (3-month) clinical assessment with a modified iNPH scale. The preoperative CSF biomarkers and the levels of soluble and insoluble Aβ species in cortical biopsy samples were analyzed for their association with a favorable outcome following the VP shunt procedure, defined as a ≥ 5-point increase in the iNPH scale. RESULTS The brain tissue levels of Aβ42 were negatively correlated with CSF Aβ42 (Spearman’s r = −0.53, p < 0.05). The Aβ40, Aβ42, and Aβ oligomer/protofibril levels in cortical biopsy samples were higher in patients with insoluble cortical Aβ aggregates (p < 0.05). The preoperative CSF Aβ42 levels were similar in patients responding (n = 11) and not responding (n = 9) to VP shunt treatment at 3 months postsurgery. In contrast, the presence of cortical Aβ aggregates and high brain tissue Aβ42 levels were associated with a poor outcome following VP shunt treatment (p < 0.05). CONCLUSIONS Brain tissue measurements of soluble Aβ species are feasible. Since high Aβ42 levels in cortical biopsy samples obtained in patients with iNPH indicated a poor surgical outcome, tissue levels of Aβ species may be associated with the clinical response to shunt treatment.
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. 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). 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 (Aβ) aggregates in vivo. 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.
Abstract Positron emission tomography (PET), a medical imaging technique allowing for studies of the living human brain, has gained an important role in clinical trials of novel drugs against Alzheimer’s disease (AD). For example, PET data contributed to the conditional approval in 2021 of aducanumab , an antibody directed towards amyloid-beta (Aβ) aggregates, by showing a dose-dependent reduction in brain amyloid after treatment. In parallel to clinical studies, preclinical studies in animal models of Aβ pathology may also benefit from PET as a tool to detect target engagement and treatment effects of anti-Aβ drug candidates. PET is associated with a high level of translatability between species as similar, non-invasive protocols allow for longitudinal rather than cross-sectional studies and can be used both in a preclinical and clinical setting. This review focuses on the use of preclinical PET imaging in genetically modified animals that express human Aβ, and its present and potential future role in the development of drugs aimed at reducing brain Aβ levels as a therapeutic strategy to halt disease progression in AD.
PET imaging of amyloid-beta (Aβ) deposits in brain has become an important aid in Alzheimer's disease diagnosis, and an inclusion criterion for patient enrolment into clinical trials of new anti-Aβ treatments. Available PET radioligands visualizing Aβ bind to insoluble fibrils, i.e. Aβ plaques. Levels of prefibrillar Aβ forms, e.g. soluble oligomers and protofibrils, correlate better than plaques with disease severity and these soluble species are the neurotoxic form of Aβ leading to neurodegeneration. The goal was to create an antibody-based radioligand, recognizing not only fibrillary Aβ, but also smaller and still soluble aggregates. We designed and expressed a small recombinant bispecific antibody construct, di-scFv 3D6-8D3, targeting the Aβ N-terminus and the transferrin receptor (TfR). Natively expressed at the blood-brain barrier (BBB), TfR could thus be used as a brain-blood shuttle. Di-scFv 3D6-8D3 bound to Aβ1-40 with high affinity and to TfR with moderate affinity. Di-scFv [124I]3D6-8D3 was injected in two transgenic mouse models overexpressing human Aβ and wild-type control mice and PET scanned at 14, 24 or 72 h after injection. Di-scFv [124I]3D6-8D3 was retained in brain of transgenic animals while it was cleared from wild-type lacking Aβ. This difference was observed from 24 h onwards, and at 72 h, 18 months old transgenic animals, with high load of Aβ pathology, displayed SUVR of 2.2–3.5 in brain while wild-type showed ratios close to unity. A subset of the mice were also scanned with [11C]PIB. Again wt mice displayed ratios of unity while transgenes showed slightly, non-significantly, elevated SUVR of 1.2, indicating improved sensitivity with novel di-scFv [124I]3D6-8D3 compared with [11C]PIB. Brain concentrations of di-scFv [124I]3D6-8D3 correlated with soluble Aβ (p < 0.0001) but not with total Aβ, i.e. plaque load (p = 0.34). We have successfully created a small bispecific antibody-based radioligand capable of crossing the BBB, subsequently binding to and visualizing intrabrain Aβ in vivo. The radioligand displayed better sensitivity compared with [11C]PIB, and brain concentrations correlated with soluble neurotoxic Aβ aggregates.
Transferrin receptor 1 (TfR1) mediated brain delivery of antibodies could become important for increasing the efficacy of emerging immunotherapies in Alzheimer's disease (AD). However, age, dose, binding to TfR1 on blood cells, and pathology could influence the TfR1-mediated transcytosis of TfR1-binders across the blood-brain barrier (BBB). The aim of the study was, therefore, to investigate the impact of these factors on the brain delivery of a bispecific TfR1-transported Aβ-antibody, mAb3D6-scFv8D3, in comparison with the conventional antibody mAb3D6.Young (3-5 months) and aged (17-20 months) WT and tg-ArcSwe mice (AD model) were injected with 125I-labeled mAb3D6-scFv8D3 or mAb3D6. Three different doses were used in the study, 0.05 mg/kg (low dose), 1 mg/kg (high dose), and 10 mg/kg (therapeutic dose), with equimolar doses for mAb3D6. The dose-corrected antibody concentrations in whole blood, blood cells, plasma, spleen, and brain were evaluated at 2 h post-administration. Furthermore, isolated brains were studied by autoradiography, nuclear track emulsion, and capillary depletion to investigate the intrabrain distribution of the antibodies, while binding to blood cells was studied in vitro using blood isolated from young and aged mice.The aged WT and tg-ArcSwe mice showed significantly lower brain concentrations of TfR-binding [125I]mAb3D6-scFv8D3 and higher concentrations in the blood cell fraction compared to young mice. For [125I]mAb3D6, no significant differences in blood or brain delivery were observed between young and aged mice or between genotypes. A low dose of [125I]mAb3D6-scFv8D3 was associated with increased relative parenchymal delivery, as well as increased blood cell distribution. Brain concentrations and relative parenchymal distribution of [125I]mAb3D6-scFv8D6 did not differ between tg-ArcSwe and WT mice at this early time point but were considerably increased compared to those observed for [125I]mAb3D6.Age-dependent differences in blood and brain concentrations were observed for the bispecific antibody mAb3D6-scFv8D3 but not for the conventional Aβ antibody mAb3D6, indicating an age-related effect on TfR1-mediated brain delivery. The lowest dose of [125I]mAb3D6-scFv8D3 was associated with higher relative BBB penetration but, at the same time, a higher distribution to blood cells. Overall, Aβ-pathology did not influence the early brain distribution of the bispecific antibody. In summary, age and bispecific antibody dose were important factors determining brain delivery, while genotype was not.
Accumulating evidence indicates that soluble Aβ oligomers are responsible for the neurotoxicity in AD. It has been shown that levels of soluble Aβ in brain are better correlated to dementia than amyloid plaque load. The Arctic APP mutation (E693G) strengthens the idea that soluble Aβ oligomers are pathogenic in AD. This mutation, found in a Swedish family, is located within the Aβ sequence and enhances the formation of large soluble oligomers of Aβ, i.e. protofibrils. To be able to study protofibrils in their natural environment we have produced antibodies with specificity for this state of aggregation, and which are able to immunologically discriminate between different Aβ conformations. To investigate the protofibrils as an antigen with regard to the nature of the monoclonal antibodies produced. To create protofibril selective antibodies lots of effort has first been put into understanding the nature of this antigen. Using Size Exclusion Chromatography (SEC) on HPLC, Native gels, Western Blot and EM we have been able to create a reliable protocol for making protofibrils, both for Aβ42 Arc and Aβ42 wt. To evaluate the immunological effects of protofibrils we have immunized mice, screened their antibody response by ELISA and investigated the produced monoclonal antibodies—both considering their molecular sequences and their ability to bind Aβ. Here we show that by immunizing mice with protofibrils we have succeeded in making monoclonal antibodies with high selectivity for protofibrils. The nature of these monoclonal antibodies can be divided into three subgroups: Low affinity IgM antibodies, high affinity IgM antibodies and high affinity IgG antibodies.
