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. 2025 Aug 4;17:180. doi: 10.1186/s13195-025-01812-9

Aducanumab binds high molecular weight soluble Aβ oligomers and restores intracellular calcium levels

Lu Yu 1,2,#, Xueying Wang 1,3,#, Tri H Doan 1, Yutian Fan 4, Thierry Bussiere 5, Brian J Bacskai 1, Ksenia V Kastanenka 1,✉
PMCID: PMC12323110  PMID: 40760501

Abstract

Background

Alzheimer's disease (AD) is characterized by amyloid-beta (Aβ) accumulation, leading to the formation of neurotoxic soluble oligomers (AβOs) that impair calcium homeostasis in neurons and astrocytes. Aducanumab, a fully human monoclonal antibody targeting aggregated Aβ, has been approved for AD treatment due to its ability to reduce amyloid plaque burden. However, its specificity toward different AβO species and its functional impact on calcium homeostasis remain unclear.

Methods

We investigated aducanumab's ability to recognize and immunodeplete low-molecular-weight (LMW) and high-molecular-weight (HMW) AβOs using three Aβ preparations: (1) transgenic conditioned media (TgCM) from cultured Tg2576 neurons, (2) synthetic Aβ42-derived diffusible ligands (ADDLs), and (3) TBS-soluble fractions from aged Tg2576 mouse brain. Size exclusion chromatography and ELISA were used to characterize AβO species. Multiphoton calcium imaging of neuron-astrocyte co-cultures was performed to assess the impact of aducanumab on AβO-induced calcium overload.

Results

Aducanumab preferentially bound and immunodepleted HMW AβOs in ADDLs and the TBS-soluble fraction of Tg2576 mouse brain extracts but did not recognize LMW AβOs in TgCM. In calcium imaging experiments, all three AβO preparations induced calcium overload in neuron-astrocyte co-cultures. Immunodepletion with aducanumab prevented calcium overload in cultures exposed to ADDLs and Tg2576 brain extracts but not in those treated with immunodepleted TgCM, indicating that aducanumab selectively neutralizes HMW AβOs.

Conclusions

Our findings demonstrate that aducanumab specifically targets HMW AβOs, mitigating their neurotoxic effects by restoring intracellular calcium homeostasis. These results provide mechanistic insight into aducanumab’s therapeutic action and support its potential role in modifying AD pathology by selectively neutralizing Aβ species.

Introduction

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by the accumulation of amyloid-beta (Aβ) peptides in the brain, leading to the formation of soluble Aβ oligomers (AβOs) and insoluble fibrils [1–3]. AβOs disrupt calcium homeostasis and lead to calcium elevations, or calcium overload, in neurons and astrocytes. Calcium overload is associated with neurotoxicity and AD progression [4–6]. Therefore, amelioration of calcium overload serves as a functional indicator of treatment efficacy. Aducanumab, a fully human antibody that binds to the aggregated forms of Aβ, has been shown to slow cognitive decline in AD patients [7–9] as well as in AD animal models [10–12]. The U.S. Food and Drug Administration (FDA) has approved aducanumab for AD treatment based on its ability to decrease amyloid burden in the brain. It was the first disease-modifying therapy to be licensed since 2003 and the first drug specifically directed at the pathophysiology of the disease [13, 14].

Our previous research has shown that aducanumab is able to cross the blood–brain barrier and engage the target, amyloid plaques. A single application of aducanumab directly to the brains, cleared amyloid plaques yet did not prevent deposition of new plaques in aged Tg2576 mice, a mouse model of amyloidosis. 6-month long treatment with aducanumab administered intraperitoneally weekly did not clear amyloid plaques yet restored neuronal calcium homeostasis in aged Tg2576 mice. We hypothesized that aducanumab targeted and neutralized neurotoxic soluble Aβ oligomers (AβOs) and thus restored neuronal calcium homeostasis in absence of plaque clearance in aged Tg2576 mice [15]. To test this hypothesis, we investigated aducanumab's propensity to recognize low-molecular-weight (LMW) and high-molecular-weight (HMW) species of AβOs. We determined aducanumab's ability to target and neutralize LMW and HMW AβOs using in vitro experiments.

We utilized size exclusion chromatography followed by ELISA measurements to analyze the LMW and HMW Aβ species present in the following preparations: 1) transgenic conditioned media collected from cultured Tg2576 neurons (TgCM), 2) synthetic Aβ42-derived diffusible ligands (ADDLs), and 3) TBS-soluble fraction from an aged Tg2576 mouse brain. TgCM primarily contains low molecular weight (LMW) AβOs. ADDLs and Tg2576 brain extracts contain some LMW and mostly high molecular weight (HMW) AβOs. Aducanumab preferentially binds to HMW Aβ species, not LMW AβOs. These three AβO preparations were immunodepleted with aducanumab and a range of additional antibodies. Immunodepleted supernatants were applied to primary neuron-astrocyte co-cultures loaded with the Indo-1 calcium indicator [16, 17]. Basal intracellular calcium was monitored using multiphoton microscopy. AβOs present in all three preparations elicited calcium elevations, calcium overload, in neuron-astrocyte co-cultures. Immunodepletion with aducanumab prevented calcium overload elicited by AβOs present in ADDLs and Tg2576 mouse brain extract, since immunodepleted ADDLs and Tg2576 mouse brain extract failed to elicit calcium overload. However, immunodepletion with aducanumab of TgCM did not prevent calcium overload in co-cultures. Thus, aducanumab targets and neutralizes HMW AβOs and restores neuronal as well as astrocytic functions by normalizing calcium homeostasis.

Methods

Primary neuronal culture

All animal procedures were performed following the Massachusetts General Hospital animal care committee’s regulations. Primary neurons and astrocytes were isolated from cerebral cortices of E14-15 CD1 mouse embryos (Charles River Laboratories) as described previously [18]. Following manufacturer's protocol, cortical tissue was dissected and mechanically dissociated using a Papain Dissociation System (Worthington Biochemical Corp). Neurons and astrocytes were cultured and maintained in a Neurobasal medium with 2% B27 supplement, 2 mM Glutamax, 100U/mL penicillin, and 100 g/mL streptomycin at 37 °C with 5% CO2 for 12–14 days in vitro (DIV).

Preparation of AβOs

Transgenic conditioned media (TgCM) and wildtype media (WtM)

We isolated primary cortical neurons and astrocytes from E13-E15 transgenic Tg2576 or nontransgenic mice of same background as described previously [16]. Tg2576 mice are an amyloidosis model of Alzheimer's disease. These mice overexpress a mutant form of the human amyloid precursor protein (APP) that leads to elevated Aβ production [19]. Primary cortical neurons and astrocytes were plated and maintained in culture for 14DIV. The culture media enriched with human Aβ oligomers (TgCM) was collected from Tg2576 cultures. Culture media lacking human Aβ oligomers (WtM) was derived from nontransgenic cultures. The levels of human Aβ40 and Aβ42 in the TgCM and immunodepleted samples were measured using a human-specific ELISA kit (Wako), according to the manufacturer's instructions. The levels of rat/human Aβ40 were measured using rat/human ELISA kits (Wako) and were determined to be 0.6 nM.

ADDLs

Synthetic ADDLs were prepared from human Aβ1-42 (AnaSpec Inc.; Catalog # AS-20276) as described in previous work [18]. Briefly, 1 mg of synthetic Aβ1–42 was dissolved in 222 mL cold hexafluoro-2-propanol, vortexed, and incubated at 37 °C for 1 h. Solvent was evaporated with a Speedvac for 20 min and the resulting film was stored at − 20 °C overnight. Anhydrous DMSO was dissolved to make a ~ 100 µM stock solution. The ADDL sample was ran at 280 nm using a spectrophotometer, and ADDL concentrations were determined according to the Beer-Lambert law. Stock was diluted in 50 mL Ringer’s Solution (B. Broun Medical Inc.) at 37 °C to the desired final concentration of 3 nM. The final AβO solution was incubated at 37 °C for 15 min before use.

TBS soluble fraction of mouse brains

A brain from a 22-month-old Tg2576 mouse was harvested and frozen with liquid nitrogen [20, 21]. Both hemispheres were homogenized in Tris-Buffered Saline (TBS) with protease inhibitors, 0.7 mg/ml pepstatin A, and 1 mM phenylmethylsulphonyl fluoride. The homogenate was centrifuged at 55,000 g for 30 min at 4 °C, and the supernatant was collected as the TBS-soluble fraction. TBS-soluble fraction of the brain was used in the experiments.

Size exclusion chromatography

AβOs were prepared as described above and incubated for 1 h at 37 °C. 1 mL of AβO solution was separated by SEC on single Superdex 75 columns (GE Healthcare) in 50 mM ammonium acetate (pH 8.5) at a flow rate of 0.5 mL/min, with an AKTA purifier 10 (GE Healthcare) and dialyzed against PBS. Aβ40 and Aβ42 levels were quantified in individual SEC fractions after dilution using the human specific Aβ40 and Aβ42 ELISA kits (Wako Chemicals) as suggested by the manufacturer.

Antibodies tested

This work utilized the following anti-Aβ antibodies: 3D6, gantenerumab, solanezumab, aducanumab or isotype matched P1.17 control antibody that does not recognize the Aβ.

Immunodepletion of TgCM

Immunodepletion of human Aβ from TgCM was performed overnight using the recombinant protein G beads (Dynabeads, Life Technologies) in low protein bunding microcentrifuge tubes. Briefly, to avoid non-specific binding, protein G beads were washed with cold Neurobasal medium and incubated with 1 ml of TgCM and each experimental antibody for 30 min at 4 °C while shaking. Then supernatant media was removed from the bead-antibody-Aβ complex using a magnet. Bead-antibody-Aβ complexes were dissociated and Aβ was collect in eluates. Aβ content in the supernatants and eluates was measured using the human specific Aβ ELISA kits (Wako).

Immunodepletion of ADDLs

The recombinant protein G beads were pre-washed in Neurobasal media and then added to 1 mL of 3 nM ADDLs with 9 µg of experimental antibody. The mixture was placed into low protein binding microcentrifuge tubes and rotated overnight at 4 °C. Then supernatant media was removed from the bead-antibody-Aβ complex using a magnet. Bead-antibody-Aβ complexes were dissociated and Aβ was collect in eluates. Aβ content was measured using the human specific Aβ42 ELISA kit (Wako). ADDLs are Aβ42-specific preparations that lack Aβ40.

Immunodepletion of TBS-soluble brain extract

Similarly, recombinant protein G beads were pre-washed in Neurobasal medium and then incubated with 1 mg of anti-Aβ antibodies for 10 min at room temperature with rotation. After washing, the bead-antibody complex was incubated with 1 ml of Tg2576 brain extract for 30 min at 4 °C while shaking. Then supernatant media was removed from the bead-antibody-Aβ complex using a magnet. Bead-antibody-Aβ complexes were dissociated and Aβ was collected in eluates. Human Aβ was measured using human specific Aβ ELISA kits (Wako).

Calcium imaging using multiphoton microscopy

12–14 DIV cultures, containing neurons and astrocytes, were incubated with 6 μM Indo-1/AM dye (Invitrogen) at 37 °C for 45 min. A Zeiss LSM 510 multiphoton live imaging system was used to image cells, which were maintained at 37 °C in a humidified environment containing 5% CO2:O2. Multiphoton excitation of Indo-1 was performed according to our previous report [18]. Multiple fields of view were acquired randomly and included 300–400 cells per dish. After obtaining images at baseline, the cells were treated with immunodepleted AβOs or AβOs alone for 45 min. The cultures were then reimaged.

Image processing and analysis

Image analysis to measure the ratio of fluorescence intensity of bound calcium to unbound calcium using Indo-1 reporter was done as described elsewhere [15]. The images were pseudo-colored according to calcium concentrations derived from the Indo-1 ratios. Calcium overload was defined as a ratio of Indo-1 greater than two standard deviations above the average Indo-1 ratio in images acquired at baseline, prior to treatments with supernatants.

Statistics

GraphPad Prism, Microsoft Excel and custom programs written in MATLAB were used to perform statistical analyses. Data were expressed as mean ± SEM. Datasets were tested for normality (Shapiro–Wilk normality test, D’Agostino and Pearson omnibus normality test, or Kolmogorov–Smirnov test). Kruskal–Wallis test was used to test for statistical differences in Aβ levels across groups, followed by Dunn's Multiple Comparison Test to identify significant differences between conditions. For calcium imaging experiments, statistical analyses were conducted using a mixed effects model to account for hierarchical data structure, with treatment condition as a mixed effect and individual culture dishes as random effects due to multiple cells imaged per dish. Specifically, calcium overload was first calculated by averaging percentages of overloaded cells within each dish, then dish-level averages were compared statistically across groups. Comparisons with a confidence interval of 95% (p < 0.05) were considered significant.

Results

Aducanumab did not immunodeplete LMW Aβ in TgCM collected from cultured Tg2576 neurons

The experimental procedure for the research study is presented in Fig. 1. We utilized three preparations containing AβOs: 1) transgenic conditioned media collected from cultured Tg2576 neurons (TgCM), 2) synthetic Aβ42-derived diffusible ligands (ADDLs), and 3) TBS-soluble fraction from an aged Tg2576 mouse brain. The AβO-enriched preparations were immunodepleted with a range of anti-Aβ antibodies: 1) aducanumab originates from a human B-cell clone and binds to soluble Aβ aggregates and insoluble fibrils, with weak binding to monomers [7, 22]. 2) 3D6 is a murine-derived antibody targeting the N-terminal region of Aβ, binding to monomers, dimers, trimers, and oligomers [23, 24]. 3) Gantenerumab, a human monoclonal antibody, binds to the N-terminus of Aβ monomers and aggregated forms including dimers, oligomers, as well as amyloid plaques [25, 26]. 4) Solanezumab, a humanized antibody from murine fragments, targets the central region of Aβ and binds primarily to soluble monomers and dimers, with limited affinity for larger aggregates [27, 28]. 5) P1.17 serves as a non-specific isotype control for aducanumab antibody. P1.17 does not bind Aβ [15].

Fig. 1.

Fig. 1

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Schematic of the experimental procedures. A, B The immunodepletion process. A Beads Only: AβOs were added to a tube with magnetic beads. After separation with a magnet, the supernatant and eluates were collected for ELISA analysis. B Treated: AβOs were added to a tube with antibody-conjugated beads. After separation with a magnet, the supernatant and eluates were collected for ELISA analysis. C: Experimental timeline for calcium imaging experiments. Cells were incubated with Indo-1 for 45 min, followed by baseline imaging of cultures. Then, immunodepleted supernatants were added and incubated for 60 min, followed by reimaging (treated imaging) of cultures

Thus, preparations containing AβOs were added to low protein binding microcentrifuge tubes containing uncoated magnetic beads in absence (Fig. 1A) or presence (Fig. 1B) of anti-Aβ antibodies. Eluates containing magnetic bead-antibody-Aβ complex were separated from supernatants using a magnet. The magnetic bead-antibody-Aβ complexes were dissociated. Human Aβ levels were measured in the supernatants and eluates using ELISAs. In absence of antibodies, the eluate contained the unbound magnetic beads with little to no Aβ. The supernatant contained high levels of Aβ (Fig. 1A). If anti-Aβ antibodies bound to AβOs were present in preparations, then eluates would contain high levels of Aβ, leaving supernatants with lower Aβ levels (Fig. 1B). Finally the effect of AβOs in preparations immunodepleted with antibodies was determined on calcium homeostasis in primary neuron-astrocyte co-cultures (Fig. 1C). To monitor basal calcium levels, neuron-astrocyte co-cultures were incubated with the calcium indicator Indo-1 and imaged to establish baseline intracellular calcium levels. After a 60 min treatment with immunodepleted supernatants, the cultures were reimaged to monitor changes in basal calcium levels.

Prior to immunodepletion, TgCM was subjected to size exclusion chromatography (SEC) followed by human Aβ level measurements with ELISA to determine the size of AβOs present. As shown in Fig. 2A, TgCM contained negligible Aβ levels in fractions 1–13, which corresponded to AβOs greater than 13.7 kDa. TgCM contained high levels of AβOs detected in fractions 15–19, corresponding to 9–13.7 kDa. Since Aβ monomer was 4.5 kDa [29], 9–13.5 kDa AβOs corresponded to dimers and trimers. Thus, TgCM contained high levels of Aβ dimers and trimers consistent with earlier reports [4]. TgCM had negligible levels of Aβ in fractions 21–29, which corresponded to 4.5 kDa monomers. Thus, TgCM was composed primarily of Aβ dimers as well as trimers and had negligible levels of HMW AβOs. This suggests TgCM contained primarily LMW species of Aβ.

Fig. 2.

Fig. 2

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Aducanumab did not immunodeplete LMW Aβ in TgCM. A SEC revealed the prevalence of LMW Aβ in transgenic conditioned media (TgCM) collected from cultured Tg2576 neurons. B, C Human Aβ40 levels in supernatants (B) and eluates (C) measured with ELISA after immunodepletion with antibodies. D, E Human Aβ42 levels measured in supernatants (D) and eluates (E). Data were presented as mean ± SEM. Kruskal–Wallis test was used to test for statistical significance. n = 4–8 samples/group. *p < 0.05, **p < 0.01, ***p < 0.001. Statistical comparisons were made between each experimental condition and bead group

Subsequently, TgCM was immunodepleted with aducanumab as well as three other anti-Aβ antibodies, 3D6, gantenerumab, and solanezumab. Immunodepletion with P1.17 antibody that was isotype matched to aducanumab and did not recognize an epitope in mouse served as the control. Uncoated magnetic beads served as an additional control (Fig. 1A). After immunodepletion with the above antibodies, the supernatants and eluates were collected to measure human specific Aβ40 and Aβ42 concentrations using ELISAs (Fig. 1B).

Magnetic beads alone in absence of antibodies pulled negligible Aβ from supernatants, thus Aβ levels were high in supernatants (Fig. 2B, D) and low in eluates (Fig. 2C, E). The control antibody P1.17, when used in conjunction with magnetic beads, also demonstrated limited efficacy in immunodepleting Aβ, as evidenced by the high Aβ levels in the supernatants (Fig. 2B, D) and low levels in the eluates (Fig. 2C, E). When aducanumab was added with magnetic beads, we observed the high Aβ levels in the supernatants (Fig. 2B, D) and low levels in the eluates (Fig. 2C, E), showing that aducanumab did not effectively immunodeplete Aβ from TgCM. Immunodepletion with gantenerumab resulted in high Aβ concentrations in the supernatants (Fig. 2B, D) and low levels in the eluates (Fig. 2C, E), indicating that gantenerumab also did not effectively immunodeplete Aβ from TgCM. In contrast, 3D6 and solanezumab led to reduced Aβ levels in the supernatants (Fig. 2B, D) and increased levels in the eluates (Fig. 2C, E), demonstrating that both 3D6 and solanezumab effectively immunodepleted Aβ from TgCM.

Our analyses revealed no statistically significant differences in supernatant Aβ40 levels after immunodepletion with the P1.17, aducanumab, or gantenerumab when compared to the bead group (Fig. 2B). Likewise, we observed no statistically significant differences in supernatant Aβ42 levels after immunodepletion with the P1.17, aducanumab, or gantenerumab when compared to the bead group (Fig. 2D). We detected significant increases in eluate Aβ40 levels after immunodepletion with 3D6 and solanezumab (Fig. 2C). We also detected significant increases in eluate Aβ42 levels after immunodepletion with 3D6 and solanezumab (Fig. 2E). Since aducanumab did not immunodeplete TgCM, aducanumab did not recognize LMW AβOs present in TgCM.

Immunodepletion with aducanumab did not prevent calcium overload after application of TgCM to wildtype primary cortical cultures

Neurons and astrocytes maintain their intracellular calcium levels at ~ 100 nM to support a myriad of neuronal and astrocytic functions [30]. Mouse models of amyloidosis contain a small yet vulnerable population of neurons and astrocytes with significant elevations in basal calcium, calcium overload [15, 31]. Calcium overload is elicited due to impairments in calcium homeostasis. Our earlier work showed that aducanumab administration to Tg2576 mice resulted in restoration of calcium homeostasis and normalization of basal calcium levels in neurons in vivo [15]. However, it is unclear whether aducanumab targeted LMW or HMW species of AβOs in vivo. Application of LMW AβOs present in TgCM directly to the brains of healthy nontransgenic mice elicited calcium overload in vivo [4]. Furthermore, application of TgCM AβOs to primary neuron-astrocyte co-cultures elicited calcium overload in vitro [16]. Thus, restoration of calcium homeostasis and rescue of calcium overload could serve as functional redout of treatment efficacy.

To assess the effect of aducanumab on TgCM-induced neurotoxicity in primary neuron-astrocyte co-cultures, we monitored basal intracellular calcium levels ([Ca2+]i) in neuron-astrocyte co-cultures. We performed intracellular calcium imaging of neurons and astrocytes loaded with a calcium indicator, Indo-1. Cultured cells were imaged at baseline (pre-treatment) (Fig. 3A, C, E, G, I, K, M). Then cultures were treated with the supernatants of TgCM immunodepleted with P1.17, 3D6, aducanumab, gantenerumab, solanezumab, as well as TgCM and wildtype media (WtM) in absence of immunodepletion for 60 min before a second round of imaging. Cell bodies were selected and analyzed in images obtained before and after treatment to determine the proportion of cells exhibiting calcium overload after treatment. We observed a significant increase in the percentage of cultured cells exhibiting calcium overload after the application of P1.17-immunodepleted supernatants (Fig. 3B, O), compared to baseline (Fig. 3A, O). Cultures treated with 3D6-immunodepleted supernatants (Fig. 3D, O) did not exhibit a significant increase in calcium overload relative to the baseline (Fig. 3C, O). A significant elevation in calcium overload was observed following the application of aducanumab-immunodepleted supernatants (Fig. 3E, F, O). Gantenerumab-immunodepleted supernatants elicited a significant increase in calcium overload (Fig. 3G, H, O). Solanezumab-immunodepleted supernatants did not cause a significant increase in calcium overload (Fig. 3I, J, O). The application of TgCM alone elicited a significant increase in calcium overload (Fig. 3K, L, O), similar to prior reports [16]. The application of WtM, which contains mouse Aβ but lacks human AβOs, did not result in significant calcium overload (Fig. 3M, N, O). Thus, human but not mouse Aβ oligomers elicited calcium elevations in cultures. Overall, these findings suggested that aducanumab did not immunodeplete TgCM containing LMW Aβ nor did it prevent TgCM-elicited calcium overload in cultures.

Fig. 3.

Fig. 3

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TgCM immunodepletion with aducanumab did not prevent calcium overload in primary neuron-astrocyte co-cultures. Representative multiphoton images at baseline (A, C, E, G, I, K, M) and after application of immunodepleted supernatants (B, D, F, H, J, L, N). O Percentage of nontransgenic cell bodies exhibiting calcium overload before (baseline) and after application of immunodepleted supernatants across conditions (treated). Scale bar: 50 μm. Data are presented as mean ± SEM. n = 4–8 dishes (621–1910 cells). Mixed Effect Model used to test for statistical significance, **p < 0.01, ***p < 0.001. Statistical comparisons were made between each experimental condition and baseline

Aducanumab bound preferentially to high molecular weight species of Aβ42 aggregates from ADDLs

ADDLs are a well-characterized soluble oligomeric form of Aβ42 found in vivo that play a critical role in AD pathogenesis [32]. ADDLs lack Aβ40. ADDLs can be prepared synthetically following a standardized and well-adopted protocol [33–36]. Our published work reported that ADDLs contain both LMW and HMW species of Aβ42, with HMW species being more prevalent [18]. Here, we used P1.17, aducanumab, and 3D6 to immunodeplete ADDLs. The supernatants and eluates were collected, and human Aβ42 concentrations were measured with ELISAs. Immunodepletion with magnetic beads alone left high Aβ42 levels in the supernatants (Fig. 4A) and low levels in the eluates (Fig. 4B), indicating beads in absence of antibodies could not immunodeplete Aβ. Immunodepletion with P1.17 also left high Aβ42 levels in the supernatants (Fig. 4A) and low levels in the eluates (Fig. 4B). In contrast, immunodepletion with aducanumab resulted in a significantly lower supernatant Aβ42 levels (Fig. 4A) when compared to that in bead condition. Accordingly, immunodepletion with aducanumab resulted in a significant increase in eluate Aβ42 concentrations (Fig. 4B). Similarly, immunodepletion with 3D6 resulted in significantly lower supernatant Aβ concentrations (Fig. 4A) and significantly higher eluate levels of Aβ42 (Fig. 4B). These results indicated that aducanumab immunodepleted Aβ42 from ADDLs, targeting HMW species specifically.

Fig. 4.

Fig. 4

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Aducanumab immunodepleted HMW Aβ42 aggregates from ADDLs. A, B Human Aβ42 levels in supernatants (A) and eluates (B) measured with ELISA after immunodepletion with antibodies. Data were presented as mean ± SEM. n = 4–7 samples/group. Kruskal–Wallis used to test for statistical significance, *p < 0.05, **p < 0.01. Statistical comparisons were made between each experimental condition and bead

Immunodepletion with aducanumab prevented calcium overload after application of ADDLs to wildtype primary cortical co-cultures

We performed calcium imaging in co-cultures incubated with Indo-1 before and after applying the supernatants of ADDLs immunodepleted by aducanumab, 3D6, or the P1.17 control antibody. ADDLs immunodepleted with P1.17 resulted in calcium overload in primary cortical cultures (Fig. 5B), compared to baseline (Fig. 5A). However, immunodepletion with aducanumab prevented calcium overload (Fig. 5E, F). Similarly, 3D6-depleted supernatants did not elicit calcium overload in primary co-cultures (Fig. 5C, D). Thus, Aβ42 present in supernatants elicited calcium overload. However, immunodepletion with aducanumab neutralized HMW Aβ42 species present in ADDLs and prevented calcium overload in cortical co-cultures exposed to ADDLs.

Fig. 5.

Fig. 5

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ADDL preparations immunodepleted with aducanumab prevented calcium overload when applied to primary co-cultures. Representative multiphoton images at baseline (A, C, E) and after application of immunodepleted supernatants (B, D, F). G Percentage of nontransgenic cell bodies exhibiting calcium overload before (baseline) and after application of immunodepleted supernatants (treated). Scale bar: 50 μm. Data were presented as mean ± SEM. n = 6–8 dishes (601–1571 cells). Mixed Effect Model used to test for statistical significance, ***p < 0.001. Statistical comparisons were made between each experimental condition and baseline

Aducanumab immunodepleted HMW Aβ42 in Tg2576 brain homogenate

Tg2576 mice are an amyloidosis model of Alzheimer's disease. These mice overexpress a mutant form of the human amyloid precursor protein (APP) that leads to elevated Aβ production and deposition of amyloid plaques in brain parenchyma [19]. We harvested a single brain from a 22-month-old Tg2576 mouse and isolated a TBS-soluble fraction. We performed immunodepletion of the TBS-soluble fraction using beads alone or beads supplemented with aducanumab or 3D6. We then measured the levels of human Aβ40 and Aβ42 in the supernatant and elution fractions using ELISA. Immunodepletion with beads alone left high Aβ40 and Aβ42 levels in the supernatants and low Aβ40 and Aβ42 levels in the eluates (Fig. 6A, C). Immunodepletion with aducanumab led to a reduction in supernatant Aβ42, but not Aβ40 levels relative to the beads group (Fig. 6A, C). Conversely, the Aβ42, but not Aβ40 levels were higher in elutes than those in the beads group (Fig. 6A, C). Tg2576 mouse brain contained LMW and HMW Aβ40 and Aβ42 species (Fig. 6B, D). To elucidate the degree to which aducanumab immunodepleted LMW and HMW Aβ40 and Aβ42 species, we performed SEC on supernatants immunodepleted with beads alone or beads supplemented with aducanumab or 3D6. Thus, we determined the molecular weight distributions of Aβ40 and Aβ42 in the immunodepleted supernatants. As shown in Fig. 6B, aducanumab did not appear to significantly immunodeplete supernatants containing HMW Aβ40 (fractions 1–6) nor LMW Aβ40 (fractions 12–18). Instead, aducanumab preferentially immunodepleted HMW Aβ42 species, with some effects also observed on LMW Aβ42. In contrast, 3D6 immunodepleted both HMW and LMW Aβ40 and Aβ42 species (Fig. 6D). Altogether, our data suggest that aducanumab preferentially targeted HMW Aβ42 compared to HMW Aβ40 and LMW Aβ species.

Fig. 6.

Fig. 6

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Aducanumab immunodepleted HMW Aβ42 in TBS-soluble fraction of brain homogenate from a 22-month-old Tg2576 mouse. A Human Aβ40 levels in supernatants and eluates measured with ELISA after immunodepletion with antibodies. B SEC revealed immunodepletion of a small amount of HMW Aβ40 species with aducanumab in a Tg2576 brain homogenate. C Human Aβ42 levels in supernatants and eluates measured with ELISA after immunodepletion with antibodies. D SEC revealed immunodepletion of HMW Aβ42 species with aducanumab in Tg2576 brain homogenate

Immunodepletion with aducanumab prevented calcium overload after application of HMW Aβ in Tg2576 brain homogenate to wildtype primary cortical cultures

Given that aducanumab targeted HMW species of Aβ42 in the brain, we isolated TBS-soluble fractions 1–2 of a single 22-month old Tg2576 mouse brain. We immunodepleted the sample with beads alone, beads supplemented with aducanumab or 3D6. We then introduced immunodepleted supernatants onto wildtype primary cortical co-cultures. Immunodepletion with beads alone failed to prevent calcium overload in co-cultures (Fig. 7B, G) relative to baseline (Fig. 7A, G). However, immunodepletion with aducanumab prevented calcium overload (Fig. 7F, G) when compared to baseline (Fig. 7E, G). Similarly, immunodepletion with 3D6 prevented calcium overload in co-cultures (Fig. 7D, G) in comparison with baseline (Fig. 7C, G). Thus, immunodepletion of TBS-soluble fraction of Tg2576 mouse brain with aducanumab neutralized HMW Aβ42 species and prevented calcium overload in primary co-cultures.

Fig. 7.

Fig. 7

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TBS-soluble fraction of Tg2576 brain homogenate immunodepleted with aducanumab prevented calcium overload when applied to primary co-cultures. Representative multiphoton images at baseline (A, C, E) and after application of immunodepleted supernatants (B, D, F). G Percentage of nontransgenic cell bodies exhibiting calcium overload before (baseline) and after application of immunodepleted supernatants (treated). Scale bar: 50 μm. Data were presented as mean ± SEM. n = 20 images/group (698–2149 cells). Mixed Effect Model used to test for statistical significance, *p < 0.05. Statistical comparisons were made between each experimental condition and baseline

Discussion

Our earlier work reported that aducanumab, a fully human antibody, when administered to Tg2576 mice, was able to cross the blood–brain barrier and engage the target, amyloid plaques. A single application of aducanumab directly to the brains, cleared amyloid plaques yet did not prevent deposition of new plaques in Tg2576 mice. 6-month long treatment with aducanumab administered intraperitoneally weekly restored neuronal calcium homeostasis without clearance of amyloid plaques in Tg2576 mice. Aducanumab was shown to effectively recognize various amyloid-beta (Aβ) species, including soluble Aβ oligomers (AβOs) in addition to insoluble fibrils [37–39]. Aducanumab did not target Aβ monomers [38, 40]. We hypothesized that aducanumab targeted and neutralized neurotoxic soluble oligomers (AβOs) and thus restored neuronal calcium homeostasis in absence of plaque clearance in Tg2576 mice [15]. To test this hypothesis, we investigated aducanumab's propensity to recognize low-molecular-weight (LMW) and high-molecular-weight (HMW) species of oligomeric Aβ40 and Aβ42. We determined aducanumab's ability to target and neutralize LMW and HMW AβOs using in vitro experiments.

The AβOs were identified as particularly toxic species of Aβ disrupting neuronal and astrocytic calcium homeostasis, leading to calcium overload, sustained elevations in intracellular calcium levels as seen in Alzheimer’s mouse models [41–43]. Here, we focused on specifically examining the effects of aducanumab on different molecular weight species of AβOs and its subsequent impact on calcium homeostasis in primary cortical neuron-astrocyte co-cultures. We found aducanumab preferentially bound and immunodepleted HMW Aβ42 oligomers not LMW forms of Aβ42. Aducanumab showed limited recognition of HMW and LMW Aβ40 oligomers under the conditions tested. The differences in calcium toxicity across TgCM, ADDLs, and Tg2576 brain lysates reflect their distinct compositions of HMW and LMW Aβ oligomers. TgCM primarily contains LMW AβOs, driving its calcium toxicity, whereas the toxicity of ADDLs and brain lysates is predominantly due to HMW Aβ42, which is effectively neutralized by aducanumab.

Low molecular weight (LMW) and high molecular weight (HMW) forms of Aβ contribute differently to the disease's pathology [44]. LMW AβOs are known to interfere with synaptic plasticity and neuronal communication, contributing to early synaptic dysfunction and the onset of cognitive deficits observed in AD patients [44, 45]. However, HMW AβOs are significantly more neurotoxic and are associated with the progression of AD symptoms, including memory loss, difficulty in communication, disorientation, and changes in behavior and personality, due to their larger aggregates disrupting neuronal networks [44, 46, 47]. Aβ42 is more prone to aggregation compared to Aβ40, forming toxic oligomers and plaques that drive AD pathology[48]. Elevated Aβ42 levels relative to Aβ40 accelerate the aggregation kinetics and stabilize toxic oligomeric species with intermediate conformations, enhancing their neurotoxic effects. These oligomers disrupt synaptic function and contribute to the morphology of amyloid fibrils associated with cognitive decline and memory loss [49]. Thus, targeting HMW Aβ42 helps mitigate the most harmful effects of amyloid pathology, potentially slowing disease progression and improving clinical outcomes.

Here, we used size exclusion chromatography (SEC) to determine the size of AβO species present in the TgCM. TgCM contained primarily LMW AβOs, specifically dimers and trimers of Aβ consistent with previous reports [4]. To determine whether aducanumab could target Aβ in TgCM, we incubated TgCM with aducanumab. Aducanumab did not immunodeplete TgCM containing LMW Aβ. Then, we used aducanumab to immunodeplete an alternative source of Aβ oligomers, ADDLs. ADDLs when prepared as synthetic peptides are composed of both HMW and LMW Aβ42, with a predominance of the HMW species as our previous work and other published studies reported [18, 50–53]. Aducanumab preferentially targeted and immunodepleted HMW Aβ42 oligomers present in ADDLs, not LMW species present in TgCM. It is worth noting that although aducanumab did not significantly prevent calcium overload in TgCM-treated neuron-astrocyte cultures, there was a trend toward reduced overload relative to P1.17 controls. One possible explanation is that residual antibody in the supernatants, due to partial immunodepletion, may continue to interact with soluble Aβ during incubation. This interaction could modulate Aβ aggregation kinetics and shift the equilibrium toward less toxic intermediates, thereby partially mitigating calcium overload [54, 55]. These findings highlight the need to further investigate subtle, antibody-mediated effects on Aβ toxicity in future studies.

The Tg2576 mouse model of Alzheimer’s disease exhibited age-dependent Aβ deposition in the brain parenchyma with significant amyloid burden at 22 months of age. We used aducanumab to immunodeplete the TBS-soluble fraction of a 22-month-old Tg2576 mouse brain. Similar to ADDLs, aducanumab immunodepleted HMW Aβ42 in Tg2576 brain homogenate without targeting LMW Aβ42, nor Aβ40 oligomers. Previous studies have shown that aducanumab can slow the progression of Alzheimer's disease through its anti-Aβ properties [56–58]. Herein, we determined that aducanumab preferentially bound to HMW Aβ42 oligomers compared to LMW Aβ42 species. This preferential binding is significant because HMW Aβ42 oligomers were described as highly neurotoxic and contributed to AD progression [44–49]. By specifically targeting these toxic HMW Aβ42 species, aducanumab was able to exert its therapeutic effects not disrupting Aβ monomers that might be necessary for healthy neuronal function [38].

Calcium homeostasis is tightly regulated in neurons and astrocytes. Intracellular calcium is maintained at ~ 100 nM for proper cellular function. AβOs are known to elevate intracellular calcium, eliciting calcium overload, which contributes to neuronal dysfunction in AD [4, 15, 59]. Therefore, restoration of basal calcium could serve as a functional indicator of treatment efficacy. Our earlier work reported that aducanumab restored neuronal calcium homeostasis in Tg2576 mice in vivo [15]. Here, we determined the effect of aducanumab- immunodepleted TgCM, ADDLs, and Tg2576 mouse brain extracts on calcium overload in wildtype primary co-cultures. Since aducanumab neutralized HMW Aβ42 oligomers in ADDLs, aducanumab prevented calcium overload when immunodepleted ADDL preparations were applied to co-cultures. Similarly, since aducanumab neutralized HMW Aβ42 oligomers in a Tg2576 brain homogenate, aducanumab prevented calcium overload when immunodepleted brain homogenate was applied to co-cultures. Finally, since aducanumab did not recognized LMW Aβ40 nor Aβ42 in TgCM, aducanumab did not prevent calcium overload when immunodepleted TgCM was applied to co-cultures. Thus, immunotherapy with aducanumab may have beneficial effects on neuronal network function, likely by neutralizing highly toxic HMW Aβ42 oligomers in the brain.

In addition, we compared aducanumab with other anti-Aβ antibodies, such as 3D6 [55, 60], gantenerumab [61, 62], and solanezumab [63, 64]. These antibodies showed some efficacy in preventing calcium overload but with a distinct profile in Aβ depletion. This differential targeting highlights the potential for tailored development of immunotherapies for AD, where specific Aβ oligomers could be targeted based on their pathological roles.

Our study is limited by the small sample size, which may affect the generalizability of our findings. The TBS-soluble Aβ fraction experiments were performed using brain extracts from a single Tg2576 mouse. Future studies with larger sample sizes will be needed to further explore the therapeutic potential of aducanumab and validate its preferential binding to HMW Aβ42 oligomers.

Conclusions

In conclusion, our findings support the role of aducanumab in selectively targeting and neutralizing HMW Aβ42 oligomers thus contributing to the restoration of cellular calcium homeostasis disrupted in AD. This adds valuable insight into the development of immunotherapies aimed at specific, toxic forms of Aβ, enhancing the potential for effective AD treatments.

Abbreviations

AD

Alzheimer's disease

Aβ

Amyloid-beta

AβOs

Amyloid-beta oligomers

LMW

Low molecular weight

HMW

High molecular weight

TgCM

Transgenic conditioned media

Tg2576

A transgenic mouse model of amyloidosis

ADDLs

Aβ42-derived diffusible ligands

TBS

Tris-buffered saline

SEC

Size exclusion chromatography

DIV

Days in vitro

WtM

Wild-type media

SWA

Slow wave activity

ROI

Region of interest

Authors’ contributions

K.V.K. and B.J.B. designed research, K.V.K. and X.W. performed research, L.Y., T.H.D. and Y.F. analyzed data, L.Y. and K.V.K. wrote the initial draft, K.V.K., B.J.B., L.Y., X.W., and T.H.D. reviewed & edited the paper.

Funding

This research was funded by Biogen. L.Y.'s work was also supported by the Advanced Program of The Affiliated Hospital of Xuzhou Medical University (PYJH2024314).

Data availability

No datasets were generated or analysed during the current study.

Declarations

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Competing interests

The authors declare no competing interests.

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Lu Yu and Xueying Wang contributed equally to this work.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

No datasets were generated or analysed during the current study.

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