Plasma NAbs-Bim levels are reduced in AD patients
To investigate whether plasma NAbs-Bim levels are altered in AD patients, 55 AD patients, 28 patients with non-AD dementia, and 70 CN subjects were recruited. There was no significant difference in age, sex, education, and the frequencies of diabetes mellitus, hypertension and stroke among the three groups. The AD group had a higher proportion of ApoE ε4 carriers and lower Mini-mental State Examination (MMSE) scores than the other two groups.
The existence of NAbs-Bim in human plasma was verified by Western Blot (Fig. S1a). Through ELISA analyses, we found that plasma levels of NAbs-Bim in AD patients were significantly lower than in the CN group. AD patients also had slightly lower plasma levels of NAbs-Bim than subjects with non-AD dementia, with no significance being achieved (Fig. 1a). After adjusting for ApoE ε4 carrier status and co-existing disorders, the difference in plasma levels of NAbs-Bim between the AD and CN group was still significant.
We next investigated the associations of plasma NAbs-Bim levels with cognitive functions as determined by MMSE scores, brain amyloid burden as reflected by Standard Uptake Value Ratio (SUVR) of PiB-PET, and plasma AD biomarkers. Plasma NAbs-Bim levels had a positive correlation with MMSE scores (Fig. 1b) and a negative correlation with PiB-PET SUVR (Fig. 1c). Besides, plasma NAbs-Bim levels were significantly correlated with plasma Aβ42 levels, but not with Aβ40 or t-tau levels (Fig. 1d-f). The above findings indicated that NAbs-Bim were negatively associated with the severity of AD, suggesting a possible protective role of these antibodies in AD.
NAbs-Bim rescue behavioral deficits in APP/PS1 mice
NAbs-Bim intervention experiments were performed to investigate the effects of NAbs-Bim on behavioral performances of APP/PS1 mice. 5 μg (at a concentration of 1 μg/μL) NAbs-Bim purified from IVIg or 5 μL PBS were injected into the right lateral ventricle of mice from 8 mon of age. The mice were subjected to behavioral analyses at 9-mon old when extensive Aβ pathologies and significant behavioral deficits could be observed. APP/PS1 mice treated with NAbs-Bim had more entries into the novel arm in the Y-maze test, which reflected a better spatial recognition memory. But the mice did not show significantly different performances in the spontaneous exploration test in Y-maze (Fig. 2a). In open-field tests, NAbs-Bim treated mice showed a longer traveling distance, a higher number of rearing and grooming, and an increased ratio of time spent in the central zone to that in the peripheral zone than control APP/PS1 mice, indicating an enhanced locomotor activity and a reduced anxiety-like behavior (Fig. 2b and c). The above findings indicated that NAbs-Bim treatment could protect against behavioral deficits in APP/PS1 mice.
NAbs-Bim attenuate Aβ pathologies in APP/PS1 mice
We first confirmed that NAbs-Bim can exactly bind to Bim by immunofluorescence (IF) co-staining of Bim, NeuN, and DAPI (Fig. S1b). To explore whether NAbs-Bim treatment could reduce Aβ deposition in brains of APP/PS1 mice, we performed Aβ immunohistochemical (IHC) staining (6E10) for total Aβ plaques and Congo red staining for compact Aβ plaques. There was no significant difference in area fractions and the total plaque density of total Aβ plaques either in the neocortex or in the hippocampus between NAbs-Bim and the control group. Compared with APP/PS1 controls, mice treated with NAbs-Bim displayed a significant reduction in area fractions of compact plaque in the neocortex, but not in the hippocampus. No significant difference was observed in the compact plaque density either in the neocortex or in the hippocampus (Fig. 3a and b). Taking together, our findings indicated that NAbs-Bim could attenuate Aβ pathologies in APP/PS1 mice.
We next measured amyloid precursor protein (APP) and its metabolites in brain homogenates of APP/PS1 mice. We found that Aβ, soluble APP (sAPPα+β), and C-terminal fragments (CTF)-β levels were significantly decreased in the brain of mice treated with NAbs-Bim when compared with controls, while full-length APP (APPfl), sAPPα, and CTF-α had no differences (Fig. 3c). These results indicated that NAbs-Bim inhibited Aβ production via decreasing amyloidogenic processing of APP.
We determined levels of secretases responsible for APP processing. Beta-site amyloid precursor protein cleaving enzyme 1 (BACE1) is the major β-secretase that catalyzes β-cleavage and promotes amyloidogenic processing of APP . NAbs-Bim treatment group had significantly lower levels of BACE1 in brain homogenates in comparison with APP/PS1 controls. However, no significant differences were observed in a Disintegrin and Metalloproteinase 10 (ADAM10) or presenilin 1 (PS1, which is the catalytic subunit of γ secretase) levels (Fig. 3d). These results implied that the decrease in amyloidogenic processing of APP might be due to the reduction of BACE1. We also tested Aβ-degrading enzymes, including insulin-degrading enzyme (IDE) and neprilysin (NEP), and Aβ transporters, including low-density lipoprotein receptor-related protein-1 (LRP1) and receptor for advanced glycation end products (RAGE). RAGE was found to be decreased in NAbs-Bim treated mice in comparison with controls, while no differences were found in IDE, NEP, and LRP1 levels (Fig. 3e), suggesting that NAbs-Bim might reduce the receptor-mediated influx of Aβ through the blood-brain barrier (BBB).
Additionally, to confirm that NAbs-Bim could indeed inhibit the Bim-mediated apoptosis pathway in mice, we evaluated the abundance of Bim and its downstream protein in brain homogenates. NAbs-Bim treated group had significantly lower levels of extracellular Bim, total Bim, BAX, and BAK levels than APP/PS1 controls (Fig S1c).
NAbs-Bim attenuate neuroinflammation, Tau hyperphosphorylation, and synaptic degeneration in APP/PS1 mice
We then investigated whether NAbs-Bim could affect other AD-type pathologies. Activated microglia (CD68+) was significantly decreased after NAbs-Bim treatment both in the neocortex and in the hippocampus (Fig. 4a). NAbs-Bim treatment mildly reduced the activation of astrocytes (GFAP+) but no significance was achieved either in the neocortex or in the hippocampus (Fig. 4b). NAbs-Bim significantly reduced area fractions of pT231-positive neurons in the hippocampus of NAbs-Bim treated mice when compared to controls (Fig. 4c). Meanwhile, the levels of total tau (Tau5) and phosphorylated tau at multiple epitopes, including pS396 and pT231, were reduced in the NAbs-Bim treated group (Fig. 4d).
Furthermore, the NAbs-Bim treated mice displayed increased NeuN and Map-2 positive area fractions and a decreased activated caspase-3 positive area fraction in the hippocampus (Fig. 4e-h). Synapse-related proteins, including PSD93, PSD95, Snap, SYN1, and VAMP1, were also measured. There was a significant difference in PSD95 between NAbs-Bim treated mice and controls (Fig. 4i). Taking together, these findings suggested that NAbs-Bim protected against neuroinflammation, tau hyperphosphorylation, and synaptic degeneration in APP/PS1 mice.
NAbs-Bim antagonize Bim-induced neuronal apoptosis and amyloidogenic processing of APP in vitro
Bim is suggested to be a proapoptotic protein that actions mainly in intracellular compartments, which might not be accessible to NAbs-Bim. Furthermore, no evidence is now available regarding the role of Bim in APP metabolism. Therefore, to reveal the mechanisms of the protective effects of NAbs-Bim, we first investigated the pathological effects of extracellular Bim. SH-SY5Y-APP695 cells were treated with Bim protein, Bim protein plus NAbs-Bim, or PBS. Mitochondrial staining found that Bim treated group displayed a significantly lower positive area fraction than the other two groups (Fig. 5a), indicating that extracellular Bim protein could have pro-apoptotic effects, which can be antagonized by NAbs-Bim. Furthermore, Bim treated group showed increased levels of Aβ, CTF-β, sAPPα+β, and BACE-1 when compared to the other groups, which could be attenuated by NAbs-Bim (Fig. 5b and c). These results indicated that extracellular Bim protein could enhance Aβ generation through promoting amyloidogenic processing of APP and NAbs-Bim may exert protective effects through neutralizing extracellular Bim.
In SH-SY5Y-APP695 cell lines without extraneous Bim protein treatment, NAbs-Bim groups showed increased mitochondrial positive area fractions in a dose-dependent manner (Fig. S1d). To investigate whether NAbs-Bim would influence the activity of endogenous extracellular Bim, we performed IF co-staining for Bim and DAPI. The siRNA-Bim, which could inhibit the Bim expression, treated group was selected as a positive control. We found that the Bim-positive area fraction of the NAbs-Bim treated group was significantly lower than the control group and higher than the siRNA-Bim treated group (Fig. 6a). Furthermore, the NAbs-Bim group had a significantly higher mitochondrial positive area fraction than the control group, but had no difference with the siRNA group (Fig. 6b). These findings indicated that NAbs-Bim could inhibit the activity of Bim and increase neuronal survival.
We next measured APP metabolites and APP cleavage enzymes in cell protein extracts. NAbs-Bim group displayed lower levels of Aβ, sAPPα+β, CTF-β, and BACE-1 than control. Compared with the siRNA group, the NAbs-Bim group had higher levels of sAPPα+β. No significant differences were found in other APP metabolites and APP cleavage enzymes among groups (Fig. 6c and d). Levels of Aβ-degrading enzymes (IDE and NEP) and Aβ transport receptors across BBB (LRP-1 and RAGE) were also measured. NAbs-Bim group and siRNA group had significantly lower levels of RAGE than the control group, while there were no differences in other proteins among groups (Fig. 6e). These results were consistent with the invivo experiments and supported that NAbs-Bim promoted the survival of neurons by reducing Aβ production via decreasing Bim-induced amyloidogenic processing of APP.
Collectively, the above findings suggested that extracellular Bim could simultaneously promote neuronal apoptosis and the amyloidogenic processing of APP. NAbs-Bim might actions through antagonizing these pathological effects of extracellular Bim, thus exerting neuroprotective effects in AD.