Linda Söderberg

Amyloid-β protofibril levels correlate with spatial learning in Arctic Alzheimer’s disease transgenic mice

Oligomeric assemblies of amyloid‐β (Aβ) are suggested to be central in the pathogenesis of Alzheimer’s disease because levels of soluble Aβ correlate much better with the extent of cognitive dysfunctions than do senile plaque counts. Moreover, such Aβ species have been shown to be neurotoxic, to interfere with learned behavior and to inhibit the maintenance of hippocampal long‐term potentiation. The tg‐ArcSwe model (i.e. transgenic mice with the Arctic and Swedish Alzheimer mutations) expresses elevated levels of Aβ protofibrils in the brain, making tg‐ArcSwe a highly suitable model for investigating the pathogenic role of these Aβ assemblies. In the present study, we estimated Aβ protofibril levels in the brain and cerebrospinal fluid of tg‐ArcSwe mice, and also assessed their role with respect to cognitive functions. Protofibril levels, specifically measured with a sandwich ELISA, were found to be elevated in young tg‐ArcSwe mice compared to several transgenic models lacking the Arctic mutation. In aged tg‐ArcSwe mice with considerable plaque deposition, Aβ protofibrils were approximately 50% higher than in younger mice, whereas levels of total Aβ were exponentially increased. Young tg‐ArcSwe mice showed deficits in spatial learning, and individual performances in the Morris water maze were correlated inversely with levels of Aβ protofibrils, but not with total Aβ levels. We conclude that Aβ protofibrils accumulate in an age‐dependent manner in tg‐ArcSwe mice, although to a far lesser extent than total Aβ. Our findings suggest that increased levels of Aβ protofibrils could result in spatial learning impairment.

The Murine Version of BAN2401 (mAb158) Selectively Reduces Amyloid-β Protofibrils in Brain and Cerebrospinal Fluid of tg-ArcSwe Mice

Amyloid-β (Aβ) immunotherapy for Alzheimers disease (AD) has good preclinical support from transgenic mouse models and clinical data suggesting that a long-term treatment effect is possible. Soluble Aβ protofibrils have been shown to exhibit neurotoxicity in vitro and in vivo, and constitute an attractive target for immunotherapy. Here, we demonstrate that the humanized antibody BAN2401 and its murine version mAb158 exhibit a strong binding preference for Aβ protofibrils over Aβ monomers. Further, we confirm the presence of the target by showing that both antibodies efficiently immunoprecipitate soluble Aβ aggregates in human AD brain extracts. mAb158 reached the brain and reduced the brain protofibril levels by 42% in an exposure-dependent manner both after long-term and short-term treatment in tg-ArcSwe mice. Notably, a 53% reduction of protofibrils/oligomers in cerebrospinal fluid (CSF) that correlated with reduced brain protofibril levels was observed after long-term treatment, suggesting that CSF protofibrils/oligomers could be used as a potential biomarker. No change in native monomeric Aβ42 could be observed in brain TBS extracts after mAb158-treatment in tg-ArcSwe mice. By confirming the specific ability of mAb158 to selectively bind and reduce soluble Aβ protofibrils, with minimal binding to Aβ monomers, we provide further support in favor of its position as an attractive new candidate for AD immunotherapy. BAN2401 has undergone full phase 1 development, and available data indicate a favorable safety profile in AD patients.

Accumulation of amyloid-β by astrocytes result in enlarged endosomes and microvesicle-induced apoptosis of neurons

BackgroundDespite the clear physical association between activated astrocytes and amyloid-β (Aβ) plaques, the importance of astrocytes and their therapeutic potential in Alzheimer’s disease remain elusive. Soluble Aβ aggregates, such as protofibrils, have been suggested to be responsible for the widespread neuronal cell death in Alzheimer’s disease, but the mechanisms behind this remain unclear. Moreover, ineffective degradation is of great interest when it comes to the development and progression of neurodegeneration. Based on our previous results that astrocytes are extremely slow in degrading phagocytosed material, we hypothesized that astrocytes may be an important player in these processes. Hence, the aim of this study was to clarify the role of astrocytes in clearance, spreading and neuronal toxicity of Aβ.ResultsTo examine the role of astrocytes in Aβ pathology, we added Aβ protofibrils to a co-culture system of primary neurons and glia. Our data demonstrates that astrocytes rapidly engulf large amounts of Aβ protofibrils, but then store, rather than degrade the ingested material. The incomplete digestion results in a high intracellular load of toxic, partly N-terminally truncated Aβ and severe lysosomal dysfunction. Moreover, secretion of microvesicles containing N-terminally truncated Aβ, induce apoptosis of cortical neurons.ConclusionsTaken together, our results suggest that astrocytes play a central role in the progression of Alzheimer’s disease, by accumulating and spreading toxic Aβ species.

Characterization of the Alzheimer's disease-associated CLAC protein and identification of an amyloid β-peptide-binding site

Amyloid β-peptide (Aβ) deposition into amyloid plaques is one of the invariant neuropathological features of Alzheimers disease. Other proteins co-deposit with Aβ in plaques, and one recently identified amyloid-associated protein is the collagen-like Alzheimer amyloid plaque component CLAC. It is not known how CLAC deposition affects Aβ plaque genesis and the progress of the disease. Here, we studied the in vitro properties of CLAC purified from a mammalian expression system. CLAC displays features characteristic of a collagen protein, e.g. it forms a partly protease-resistant triple-helical structure, exhibits an intermediate affinity for heparin, and is glycosylated. Purified CLAC was also used to investigate the interaction between CLAC and Aβ. Using a solid-phase binding assay, we show that CLAC bound with a similar affinity to aggregates formed by Aβ-(1–40) and Aβ-(1–42) and that the interaction was impaired by increasing salt concentrations. An 8-residue-long sequence located in non-collagenous domain 2 of CLAC was found to be crucial for the interaction with Aβ. These findings may be useful for future therapeutic interventions aimed at finding compounds that modulate the binding of CLAC to Aβ deposits.

Collagenous Alzheimer amyloid plaque component assembles amyloid fibrils into protease resistant aggregates

Recently, a novel plaque‐associated protein, collagenous Alzheimer amyloid plaque component (CLAC), was identified in brains from patients with Alzheimers disease. CLAC is derived from a type II transmembrane collagen precursor protein, termed CLAC‐P (collagen XXV). The biological function and the contribution of CLAC to the pathogenesis of Alzheimers disease and plaque formation are unknown. In vitro studies indicate that CLAC binds to fibrillar, but not to monomeric, amyloid β‐peptide (Aβ). Here, we examined the effects of CLAC on Aβ fibrils using assays based on turbidity, thioflavin T binding, sedimentation analysis, and electron microscopy. The incubation of CLAC with preformed Aβ fibrils led to increased turbidity, indicating that larger aggregates were formed. In support of this contention, more Aβ was sedimented in the presence of CLAC, as determined by gel electrophoresis. Moreover, electron microscopy revealed an increased amount of Aβ fibril bundles in samples incubated with CLAC. Importantly, the frequently used thioflavin T‐binding assay failed to reveal these effects of CLAC. Digestion with proteinase K or trypsin showed that Aβ fibrils, incubated together with CLAC, were more resistant to proteolytic degradation. Therefore, CLAC assembles Aβ fibrils into fibril bundles that have an increased resistance to proteases. We suggest that CLAC may act in a similar way in vivo.

Specific Uptake of an Amyloid-β Protofibril-Binding Antibody-Tracer in AβPP Transgenic Mouse Brain

Evidence suggests that amyloid-β (Aβ) protofibrils/oligomers are pathogenic agents in Alzheimers disease (AD). Unfortunately, techniques enabling quantitative estimates of these species in patients or patient samples are still rather limited. Here we describe the in vitro and ex vivo characteristics of a new antibody-based radioactive ligand, [125I]mAb158, which binds to Aβ protofibrils with high affinity. [125I]mAb158 was specifically taken up in brain of transgenic mice expressing amyloid-β protein precursor (AβPP) as shown ex vivo. This was in contrast to [125I]mAb-Ly128 which does not bind to Aβ. The uptake of intraperitoneally-administered [125I]mAb158 into the brain was age- and time-dependent, and saturable in AβPP transgenic mice with modest Aβ deposition. Brain uptake was also found in young AβPP transgenic mice that were devoid of Aβ deposits, suggesting that [125I]mAb158 targets soluble Aβ protofibrils. The radioligand was diffusely located in the parenchyma, sometimes around senile plaques and only occasionally colocalized with cerebral amyloid angiopathy. A refined iodine-124-labeled version of mAb158 with much improved blood-brain barrier passage and a shorter plasma half-life might be useful for PET imaging of Aβ protofibrils.

Rapid amyloid-β oligomer and protofibril accumulation in traumatic brain injury.

Deposition of amyloid‐β (Aβ) is central to Alzheimers disease (AD) pathogenesis and associated with progressive neurodegeneration in traumatic brain injury (TBI). We analyzed predisposing factors for Aβ deposition including monomeric Aβ40, Aβ42 and Aβ oligomers/protofibrils, Aβ species with pronounced neurotoxic properties, following human TBI. Highly selective ELISAs were used to analyze N‐terminally intact and truncated Aβ40 and Aβ42, as well as Aβ oligomers/protofibrils, in human brain tissue, surgically resected from severe TBI patients (n = 12; mean age 49.5 ± 19 years) due to life‐threatening brain swelling/hemorrhage within one week post‐injury. The TBI tissues were compared to post‐mortem AD brains (n = 5), to post‐mortem tissue of neurologically intact (NI) subjects (n = 4) and to cortical biopsies obtained at surgery for idiopathic normal pressure hydrocephalus patients (iNPH; n = 4). The levels of Aβ40 and Aβ42 were not elevated by TBI. The levels of Aβ oligomers/protofibrils in TBI were similar to those in the significantly older AD patients and increased compared to NI and iNPH controls (P < 0.05). Moreover, TBI patients carrying the AD risk genotype Apolipoprotein E epsilon3/4 (APOE ε3/4; n = 4) had increased levels of Aβ oligomers/protofibrils (P < 0.05) and of both N‐terminally intact and truncated Aβ42 (P < 0.05) compared to APOE ε3/4‐negative TBI patients (n = 8). Neuropathological analysis showed insoluble Aβ aggregates (commonly referred to as Aβ plaques) in three TBI patients, all of whom were APOE ε3/4 carriers. We conclude that soluble intermediary Aβ aggregates form rapidly after TBI, especially among APOE ε3/4 carriers. Further research is needed to determine whether these aggregates aggravate the clinical short‐ and long‐term outcome in TBI.

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

BackgroundCurrently, several amyloid beta (Aβ) 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 Aβ and prevent Aβ 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 Aβ42 protofibrils, but store rather than degrade the ingested Aβ aggregates. In a co-culture set-up, the incomplete degradation of Aβ42 protofibrils by astrocytes results in increased neuronal cell death, due to the release of extracellular vesicles, containing N-truncated, neurotoxic Aβ.MethodsThe aim of the present study was to investigate if the accumulation of Aβ in astrocytes can be affected by the Aβ protofibril selective antibody mAb158. Co-cultures of astrocytes, neurons, and oligodendrocytes, derived from embryonic mouse cortex, were exposed to Aβ42 protofibrils in the presence or absence of mAb158.ResultsOur results demonstrate that the presence of mAb158 almost abolished Aβ accumulation in astrocytes. Consequently, mAb158 treatment rescued neurons from Aβ-induced cell death.ConclusionBased on these findings, we conclude that astrocytes may play a central mechanistic role in anti-Aβ immunotherapy.

Efficient clearance of Aβ protofibrils in AβPP-transgenic mice treated with a brain-penetrating bifunctional antibody

BackgroundAmyloid-β (Aβ) immunotherapy is one of the most promising disease-modifying strategies for Alzheimer’s disease (AD). Despite recent progress targeting aggregated forms of Aβ, low antibody brain penetrance remains a challenge. In the present study, we used transferrin receptor (TfR)-mediated transcytosis to facilitate brain uptake of our previously developed Aβ protofibril-selective mAb158, with the aim of increasing the efficacy of immunotherapy directed toward soluble Aβ protofibrils.MethodsAβ protein precursor (AβPP)-transgenic mice (tg-ArcSwe) were given a single dose of mAb158, modified for TfR-mediated transcytosis (RmAb158-scFv8D3), in comparison with an equimolar dose or a tenfold higher dose of unmodified recombinant mAb158 (RmAb158). Soluble Aβ protofibrils and total Aβ in the brain were measured by enzyme-linked immunosorbent assay (ELISA). Brain distribution of radiolabeled antibodies was visualized by positron emission tomography (PET) and ex vivo autoradiography.ResultsELISA analysis of Tris-buffered saline brain extracts demonstrated a 40% reduction of soluble Aβ protofibrils in both RmAb158-scFv8D3- and high-dose RmAb158-treated mice, whereas there was no Aβ protofibril reduction in mice treated with a low dose of RmAb158. Further, ex vivo autoradiography and PET imaging revealed different brain distribution patterns of RmAb158-scFv8D3 and RmAb158, suggesting that these antibodies may affect Aβ levels by different mechanisms.ConclusionsWith a combination of biochemical and imaging analyses, this study demonstrates that antibodies engineered to be transported across the blood-brain barrier can be used to increase the efficacy of Aβ immunotherapy. This strategy may allow for decreased antibody doses and thereby reduced side effects and treatment costs.

Clearance of beta-amyloid protofibrils/oligomers in the brain and CSF of TG-APP ARCSWE mice following treatment with the protofibril-selective antibody mAb158

APP-transgenic mice from the age of 4 months were fed custom-mix diets (pellets) containing 4% figs. These experimental and control mice were examined at the age of 4-5 months and 10-11 months by Morris water maze test (for spatial memory & learning ability), T maze test (for position discrimination learning ability), rota rod test (for psychomotor coordination), elevated plus maze test (for anxiety-related behavior) and open field test to analyze the effect of diet rich in figs on memory, anxiety and learning skills. Results: APPsw/Tg2576 at the age of 4-5 months and 10-11 months that were fed control diet without figs showed memory deficit, anxiety-related behavior, and severe impairment in spatial learning ability, position discrimination learning ability and motor coordination compared to the wild type mice on the same diet. Diets rich in 4% figs when fed to tg mice showed a significant improvement above factors compared to the AD tg mice on diet without figs. Conclusions: Our results suggest that dietary supplementation of figs may have the beneficial effect in reducing the risk, delaying the onset or slowing the progression of Alzheimer’s disease and also our results suggest that further studies needed to validate and determine the mechanism of action of these fruits against AD.