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Therapies designed to clear pathogenic Aβ deposits from the brains of human patients hold great promise for the treatment and possible cure of Alzheimer's disease (AD) 1 . Previous studies in multiple mouse models of AD have demonstrated that passive immune therapies targeting Aβ can effectively reduce Aβ pathology, structural neuronal abnormalities and, in some cases, behavioral deficits 2,3 . However, these encouraging preclinical results have failed to predict clinical efficacy in recent phase 3 clinical trials 4 . A better understanding of the cellular mechanisms of the antibodies in the diseased brain may help to explain this discrepancy. Although such studies are not feasible in humans, two-photon Ca 2+ imaging allows simultaneous monitoring of the activity of large numbers of neurons in mouse models in vivo 5 . Using this method, previous work in transgenic AD models overexpressing mutant human amyloid precursor protein (APP) revealed that Aβ pathology promotes hyperexcitation and tonic hyperactivity of a large number of neurons in hippocampal 6 and neocortical 7 brain regions, thereby impairing the normal function of neuronal circuits involved in cognitive information processing 8 . Such hyperactivity may increase the risk for epileptogenic activity in both animal models 9 and human patients 10 , particularly in those carrying familial AD mutations 11 .
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We explored whether treatment with Aβ-targeting antibodies can reverse these functional neuronal impairments. First, we used twophoton Ca 2+ imaging in the PDAPP mouse model and found, consistent with previous observations in other models 7,8 , a marked increase of neuronal activity levels in the neocortex of 12-17-month-old plaque-bearing mice when compared with age-matched wild-type (WT) littermates (Supplementary Fig. 1a,b). The frequency distribution of the action potential-evoked neuronal Ca 2+ transients was markedly shifted toward higher values in PDAPP mice (Supplementary Fig. 1c,d). Furthermore, the fraction of neurons that were hyperactive (>6 transients per min) was markedly larger in PDAPP than in WT mice, while the fraction of silent neurons (0 transients per min) was not different between PDAPP and WT mice (Supplementary Fig. 1e).
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In an attempt to treat the neuronal dysfunction, we intraperitoneally injected plaque-bearing PDAPP mice with the monoclonal antibody 3D6 (the murine equivalent of bapineuzumab 4 ) at a weekly dose of 10 mg per kg of body weight over a period of 3 months, according to standard protocols 2 . Control PDAPP mice received an irrelevant, isotype-matched antibody. We analyzed two independent age cohorts of mice that had different Aβ loads in their brains before the treatment. A younger group was immunized at 12 months of age and an older group was treated beginning at 14 months of age. As expected 2 , the treatment reduced brain Aβ burden (Fig. 1a and Supplementary Fig. 2a). In marked contrast with this marked decrease of Aβ pathology, we observed in the same transgenic mice an aggravation of neuronal dysfunction instead of the expected amelioration (Fig. 1b-g and Supplementary Fig. 2b,c). This aggravation manifested itself in several ways. First, we found in both age cohorts that the average frequency of Ca 2+ transients was doubled in treated animals compared with controls (Fig. 1b and Supplementary Fig. 2b) and was almost fivefold higher than in WT mice (WT: 1.5 ± 0.24 transients per min, n = 9 mice, t = 3.68, d.f. = 6.25, P = 0.01). Second, the proportion of hyperactive neurons was three-to fourfold larger in treated than in control mice (Fig. 1c and Supplementary Fig. 2c). Finally, in a fraction of treated animals (47%), the increased hyperactivity was associated with an unusual neuronal synchrony (Fig. 1d-g). Such synchrony, which has not been observed in WT mice and is only rarely seen in untreated PDAPP mice, may further promote the epileptogenesis observed in many mouse models of AD 9 . Next, we wondered whether antibody treatment could ameliorate neuronal dysfunction at an earlier disease stage, before plaque deposition. For this purpose, we studied PDAPP mice at 5 months of age and found hyperactivity already at this young age. Notably, even a single dose of 3D6 antibody (30 mg per kg; for detailed protocol, see ref. 12), applied 24 h before imaging, was sufficient to aggravate hyperactivity (Fig. 1h-j). The treatment of WT mice with 3D6 antibodies had no significant effect (P = 0.875; Fig. 1k). These results suggested that the 'pro-excitatory' effect of the anti-Aβ antibody 3D6 was dependent on APP overexpression in the transgenic AD model and, most likely, the binding to Aβ, and that it can occur even in the absence of plaques.
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Marc Aurel Busche 1-3 , Christine Grienberger
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1,4 , Aylin D Keskin 1 , Beomjong Song 1 , Ulf Neumann 5 , Matthias Staufenbiel 6 , Hans Förstl 2 & Arthur Konnerth 1,3
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Among the most promising approaches for treating Alzheimer´s disease is immunotherapy with amyloid- (A)-targeting antibodies. Using in vivo two-photon imaging in mouse models, we found that two different antibodies to A used for treatment were ineffective at repairing neuronal dysfunction and caused an increase in cortical hyperactivity. This unexpected finding provides a possible cellular explanation for the lack of cognitive improvement by immunotherapy in human studies.
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with the steroidal anti-inflammatory agent dexamethasone. Because long-term dexamethasone treatment, comparable to the β1 treatment, is not feasible in APP transgenic mice 14 , we relied on a short-term, high-dose delivery (Online Methods), as is frequently used in humans with autoimmune and chronic inflammatory diseases 15 . The cortical activity levels in the β1-treated transgenic mice were not affected by dexamethasone treatment (Supplementary Fig. 4a). To test the consequences of an inflammatory reaction, we locally applied lipopolysaccharide (LPS; 1 mg ml -1 ) to neurons in the cortex of transgenic mice, but we did not detect any changes in activity levels (Supplementary Fig. 4b,c). Consistent with these results, the analysis of pro-inflammatory cytokines and chemokines in brain homogenates did not show detectable differences between β1-treated mice and the control group (Supplementary Table 1). Thus, under our experimental conditions, we found no evidence for a prominent role of inflammation in promoting or aggravating neuronal hyperactivity.
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In conclusion, our results from independent cohorts of AD transgenic mice demonstrate that passive immunotherapy with Aβ-targeting antibodies was not only ineffective in treating neuronal dysfunction, but actually worsened it. Administration of two different monoclonal antibodies resulted in significantly increased numbers of pathologically hyperactive neurons and promoted, in some cases, abnormal synchrony of cortical activity. Our findings indicate that the previously reported beneficial effects of antibody treatment on neuronal structure, including recovery of neuritic dystrophy 16 or prevention of synaptophysin loss 17 , are not sufficient for the repair of neuronal To validate these results with a different monoclonal antibody against Aβ, we passively immunized another APP-overexpressing model of AD that exhibits neuronal hyperactivity (Fig. 2a,b), namely the Tg2576 mouse model, with β1 antibodies 13 . It has been shown that β1 can reduce Aβ plaque loads in aged AD transgenic mice treated for 5 months, but, overall, β1 appeared to be less effective at clearing plaques than 3D6 (ref. 13). For consistency with the 3D6 antibody therapy (see above), we treated Tg2576 mice for 3 months with β1 antibodies and found that this treatment had no major effect on soluble and insoluble Aβ levels (Supplementary Fig. 3), allowing us to ask whether the aggravation of neuronal impairments depends on the reduction in Aβ burden. Indeed, after 3 months of treatment, cortical activity levels, as well as the fractions of hyperactive neurons, were massively elevated in β1-treated animals when compared with mice that received isotype-matched control antibodies (Fig. 2a-c). This experiment suggests, similar to the observations made in 3D6-treated, pre-depositing PDAPP mice (Fig. 1h-j), that the worsening of neuronal dysfunction by anti-Aβ antibodies can occur independently of the effects on amyloid plaque pathology. In control experiments, performed to validate and extend the previous result with acute injections of 3D6 antibodies in WT mice (Fig. 1k), we administered β1 antibodies chronically over a period of 3 months to WT mice. Cortical activity levels were not significantly different between β1-treated and control WT mice (P = 0.763; Fig. 2d).
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Finally, we investigated the possible contribution of inflammatory processes to the Aβ antibody-mediated aggravation of neuronal hyperactivity (Supplementary Fig. 4). First, we performed a treatment dysfunction. Some previous animal studies have shown an amelioration of behavioral deficits after antibody treatment 3 , and it is unclear, at this stage, how the discrepancy between such behavioral improvement and worsening of neuronal dysfunction can be resolved. Furthermore, it is currently not known whether the increase in neuronal hyperactivity, as measured by two-photon imaging, has any bearing on the efficacy, or lack thereof 4 , of antibodies to Aβ in human patients with AD. Our results provide evidence that the aggravation of neuronal hyperactivity is directly related to binding of the antibodies to Aβ rather than to some non-Aβ-related properties of the antibodies. Although inflammatory processes did not seem to have a prominent role under our experimental conditions, we cannot exclude the possibility that inflammatory effects of passive immunotherapy could contribute to increased neuronal hyperactivity in transgenic animals. The pro-excitatory effect was not observed in WT animals; thus, even if the increased hyperactivity involves inflammation, it must be Aβ dependent. Regardless of the precise mechanisms involved, however, the lack of improvement of neuronal function and the risk of aggravation of dysfunction by antibodies to Aβ was unexpected and highlights the urgent need to include functional in vivo assays into the toolbox of methods for the preclinical assessment of treatment strategies for AD. Together, our results suggest a cellular mechanism that, in combination with other factors [18][19][20] , may be the reason for the failure of anti-Aβ immunotherapies in repairing cognitive deficits 4 .