In this work, we examine the efficacy of the diphenylpyrazole (DPP) compound anle138b in an animal model of Aβ deposition. Oral application of anle138b ameliorates Aβ‐induced deficits in synaptic plasticity and memory formation. Using in vivo and in vitro approaches, we provide evidence that this effect is linked to the capacity of anle138b to reduce the conductivity of Aβ pores in lipid bilayer membranes. Although other mechanisms likely contribute to this effect, our data suggest the functional modulation of the membrane bound Aβ‐oligomers as a mechanism for neuroprotection and support the idea that anle138b should be taken into clinical trials to treat aggregopathies, including AD.

Alzheimer's disease (AD) is the most common neurodegenerative disorder causing a severe emotional and economic burden to our societies. Due to increased life expectancies, the number of those afflicted with AD is expected to double by 2025. Despite intensive research, effective therapeutic approaches are still not available. The pathogenesis of AD has been linked to protein aggregation, namely the aggregation of amyloid‐beta peptides (Aβ) and tau protein. The accumulation of pathogenic aggregates of Aβ peptides in the brain appears to be a key event in the pathogenesis of AD (Iversen et al , 1995 ; Tanzi, 2005 ; Jakob‐Roetne & Jacobsen, 2009 ; Goate & Hardy, 2012 ), and targeting amyloid pathology still represents a promising therapeutic strategy (Haass & Selkoe, 2007 ; Sevigny et al , 2016 ). The precise molecular events that trigger amyloid‐induced decline of synaptic plasticity and neuronal cell death are still not entirely resolved and are likely to be multifactorial. One of the first explanations of neuronal dysfunction and toxicity in AD is the channel hypothesis first proposed by Arispe and coworkers (Arispe et al , 1993 ), which postulates that unregulated Aβ ion channels result in a loss of ionic homeostasis (primarily through a gain of Ca 2+ ) that eventually triggers neuronal dysfunction and cell death . In vivo evidence for this mechanism is, however, still rare, and a compound that would block pores and be active in mammalian animal models has not been reported yet. Thus, the original request by Arispe and coworkers that a useful strategy for drug discovery for treatment of AD should include screening compounds for their ability to block or otherwise modify Aβ channels is still left unsatisfied (Arispe et al , 1993 ).

To provide in vivo evidence for pore formation, we treated primary hippocampal neurons with Aβ 1‐40 monomers or oligomers in the absence or presence of anle138b and assayed membrane integrity. While the addition of Aβ 1‐40 oligomers but not Aβ 1‐40 monomers significantly damaged membrane integrity, this effect was reversed by anle138b (Fig 5 C). This restoration is not simply due to an anle138b‐mediated reduction in cell death, since the cell viability of hippocampal neurons measured in the MTT assay was identically affected by Aβ 1‐40 monomer and oligomer treatment in our experimental setting (Fig 5 D). In this context, it is important to state that different protocols for Aβ treatment of neurons have been used to study various effects of Aβ on neuronal plasticity and integrity. The concentration of Aβ oligomers employed in our study is within the range of these studies. Nevertheless, such data always need to be interpreted with care since a cell culture system cannot not fully recapitulate the situation observed in human patients. Interestingly, anle138b treatment was also able to ameliorate the effect of toxic Aβ species on membrane integrity if added after neurons had been incubated with Aβ oligomers and already exhibited impaired viability, which is in line with our data showing that anle138b completely ameliorated LTP and partially restored memory defects in the post‐plaque group ( Appendix Fig S7A and B ). We also employed another experimental system to test the effect of Aβ 1‐42 oligomers and anle138b on biological membranes. To this end, we employed the cytochrome release assay (CRA) on isolated mitochondria that were exposed to either α‐synuclein, tau, or Aβ 1‐42 oligomer in the presence or absence on anle138b. Our data reveal a substantial damage to mitochondrial membranes in all conditions that is attenuated by anle138b ( Appendix Fig S7C ), suggesting that anle138b has general effect on membrane integrity.

When we examined the effect of Aβ 1–42 in DOPS/POPE (1:1) membranes, we observed a stepwise growth of bulk membrane conductance. Such activity is indicative of the combined action of many individual pores (Fig 5 A). Pore “stacking” is likely the result of prolonged open lifetimes and the formation and/or opening of additional conducting pores. Anle138b‐doped membranes demonstrated fewer simultaneously active pores and significantly reduced bulk conductance (Fig 5 A and B, Appendix Fig S5 ) compared to membranes lacking the anle138b compound (Fig 5 A and B, Appendix Fig S5 ). Our results indicate that treatment with anle138b alters the pore dynamics, resulting in less stable and shorter lived “open” pores. Decreased pore stability leads to a reduction in the total number of simultaneously conducting pores and significantly decreased conductance across the membrane. AFM data revealed that anle138b treatment did not affect the surface structure of Aβ 1‐42 pores (Fig EV2 ), suggesting that anle138b does not simply prevent Aβ 1‐42 from entering lipid bilayer membranes and forming pores. Rather anle138b appears to render conducting Aβ pores to non‐conducting ones—likely through structural change to the membrane embedded region of Aβ 1–42 —thereby providing one possible mechanism by which anle138b ameliorates LTP and learning deficits in APPPS1ΔE9 mice. Similar effects were observed when the conductance measurements were repeated in oxidized cholesterol ( Appendix Fig S5B ).

To better understand the mechanisms that underlie the therapeutic effect of anle138b, we analyzed its impact on Aβ‐induced pore formation (Arispe et al , 1993 ). To this end, we employed black lipid membranes (BLM) using a mixture of POPE and DOPS in a 1:1 ratio or oxidized cholesterol/n‐decane, an assay in which the current passing through a membrane is a measure of membrane integrity ( Appendix Fig S5A ). We first established that the conductance and the morphology of POPE and DOPS lipids, as measured by atomic force microscopy (AFM), lipid bilayers are not affected in the presence of anle138b or Aβ peptides ( Appendix Fig S6 ).

Given that anle138b was shown to counter aggregation in models for α‐synuclein and prion toxicity (Wagner et al , 2013 ), it is likely that at least part of the therapeutic effect observed in this study is due to anle138b interfering with amyloid aggregation. To test this hypothesis, we prepared brain slices for histochemical analysis. We used thioflavin S, a common stain to quantify Aβ plaques (Fig 4 A). We first assayed the amyloid plaques pathology in the hippocampus and cortex in the pre‐plaques group. Since no amyloid pathology was detectable in WT mice, these mice served as negative control. We observed a significant reduction in the number of plaques and the total area covered by plaques in anle138b‐treated mice (Fig 4 A and B). Similar results were obtained when we analyzed plaque load in the post‐plaques group (Fig 4 B and C), although the pathology was generally more severe in the post‐plaque group. Thus, oral administration of anle138b reduces amyloid pathology when given before or after the onset of pathology.

A previous study reported that anle138b exhibits therapeutic effect in TauP301S mice, a mouse model for Tau pathology (Wagner et al , 2015 ). Taking into account that Tau and amyloid pathology represent the two major causative factors for AD, we wondered if anle138b would affect brain homeostasis as measured by hippocampal gene expression also in TauP301S mice. To this end, we obtained hippocampal brain tissue from the same experiment using anle138b or vehicle‐treated wild‐type and TauP301S mice as described by Wagner et al ( 2015 ). The experimental design employed by Wagner et al is similar to our pre‐plaque group, since anle138b was feed to mice upon weaning. Similar to our data, feeding anle138b to wild‐type mice had a neglectable effect on hippocampal gene expression when compared to vehicle fed wild‐type mice ( Appendix Fig S4 , Dataset EV1 ). The comparison of vehicle‐treated wild‐type to vehicle‐treated TauP301S mice revealed 257 differentially expressed genes that mainly represent increased pathways linked to neuroinflammation ( Appendix Fig S4 ). Of note, this gene expression signature was significantly ameliorated in anle138b‐treated TauP301S mice and only 16 genes were differentially expressed when comparing vehicle‐ vs. anle138b‐treated TauP301S mice ( Appendix Fig S4 ). These data allow for a number of interesting conclusions. First, at the hippocampal gene expression level, the overexpression of the human Tau gene carrying the P301S mutation leads to inflammatory response but in contrast to the data observed in APPPS1ΔE9 mice has very limited impact on the expression of genes linked to synaptic plasticity and learning and memory. Second, treatment with anle138b in TauP301S mice ameliorates the gene expression changes, at least when treatment is initiated before the onset of pathology, further confirming that treatment with anle138b helps to reinstate hippocampal homeostasis.

We next analyzed hippocampal gene expression in the post‐plaque group (Fig EV1 ). When comparing WT placebo and APPPS1ΔE9 placebo mice, we found 130 differentially expressed genes of which the majority (124) were up‐regulated (Fig 3 D), a finding confirmed via qPCR ( Appendix Fig S3A ). The comparison of anle138b‐treated WT and anle138b‐treated APPPS1ΔE9 mice revealed 220 differentially expressed genes, 207 up‐regulated, and 13 down‐regulated (Fig 3 D). Around half of these, 103 were also deregulated in the comparison WT placebo vs. APPPS1ΔE9 placebo (Fig 3 E), a finding confirmed via qPCR ( Appendix Fig S3B ). Pathway analysis revealed that gene expression changes almost exclusively represent an induction of neuroinflammatory processes (Fig 3 F) in response to the APP and PS1 transgenes and this induction is not changed by treatment with anle138b. These data suggest that anle138b treatment does not have a major impact on inflammatory processes when given at a stage of advanced amyloid pathology. Since anle138b treatment nevertheless reinstated hippocampal synaptic plasticity and also partially restored memory function, these findings indicate that the therapeutic efficacy of anle138b is most likely not solely due to the dampening of amyloid‐induced inflammation.

Because anle138b restored LTP and spatial memory functions in pre‐plaque mice (see Fig 1 ), we hypothesized that the transcriptome of APPPS1ΔE9 mice treated with anle138b may more closely resemble that of wild‐type mice. Indeed, a wild‐type‐like gene expression profile is to a large extent reinstated in anle138b‐treated APPPS1ΔE9 and only 27 genes were deregulated when comparing wild‐type mice treated with anle138b vs. APPPS1ΔE9 mice treated with anle138b groups (Fig 3 A). Thus, none of the pathways deregulated in response to amyloid pathology (Fig 3 B) remained significant after anle138b treatment. Accordingly, no enrichment for any specific pathway could be detected. The possibility remained that anle138b treatment may simply reduce the expression levels of the APP and PS1 transgenes specifically in APPPS1ΔE9. However, the RNA‐seq data showed that APP and PS1 are increased in placebo‐treated APPPS1ΔE9 mice when compared to placebo‐treated wild‐type mice (see Dataset EV1 ). Similarly, APP and PS1 transgenes were elevated in anle138b‐treated APPPS1ΔE9 mice compared to anle138b‐treated wild‐type mice (see Dataset EV1 ). These data were confirmed via qPCR showing that the expression of APP and PS1 transgenes is similar in placebo‐ and anle138b‐treated APPPS1ΔE9 mice (Fig 3 C); the gene expression data are in line with the electrophysiological and behavioral findings. It also shows that in the pre‐plaque group, anle138b treatment reinstates cellular homeostasis in the hippocampus of APPPS1ΔE9 mice.

Pathological alterations often lead to aberrant changes in transcriptional plasticity indicating deregulated cellular homeostasis (Fischer, 2014a ). In support of this, numerous studies demonstrated that AD pathogenesis correlates with altered gene expression in various brain regions (Fischer, 2014b ; Benito et al , 2015 ; Matarin et al , 2015 ). Moreover, monitoring gene expression changes can inform about the efficacy of therapeutic intervention (Benito et al , 2015 ). In keeping with this idea, we performed RNA sequencing from hippocampal tissue dissected from mice in the pre‐ and post‐plaque (Fig EV1 ) cohorts. Notably, there were no differences in hippocampal gene expression when comparing WT placebo‐ vs. WT anle138b‐treated mice, suggesting that anle138b has no direct effect on transcriptome plasticity. Thus, we first compared gene expression in placebo‐treated WT and placebo‐treated APPPS1ΔE9 mice of the pre‐plaque group. We identified 202 differentially expressed genes of which 73 were up‐ and 129 were down‐regulated (Fig 3 A; Dataset EV1 ). Pathway analysis shows that down‐regulated genes are linked to reduced energy metabolism, mitochondria function, cytoskeleton integrity, and synaptic plasticity, while pathways linked to cell growth were increased (Fig 3 B). These data are in line with previous reports of gene expression changes in AD (Benito et al , 2015 ; Matarin et al , 2015 ) and were confirmed via qPCR ( Appendix Fig S3A ).

To analyze whether reinstatement of hippocampal plasticity would also correlate with improved memory function, we subjected mice to the Morris water maze test. WT mice treated with anle138b rapidly learned the task as indicated by reduced escape latency throughout the 8 days of training (Fig 2 D). Placebo‐treated 10‐month‐old APPPS1Δ9 mice display impaired spatial learning as indicated by the escape latency that did not significantly decrease during the training (Fig 2 D). When compared to the placebo group, APPPS1Δ9 mice treated with anle138b showed improved spatial learning (Fig 2 D). A probe test was performed after 8 days of training. WT mice treated with anle138b showed a significant preference for the target quadrant indicative of intact spatial reference memory (Fig 2 E). Placebo‐treated APPPS1Δ9 mice exhibited severely impaired memory function (Fig 2 E) and displayed no target preference (Fig 2 E). In APPPS1Δ9 mice treated with anle138b (Fig 2 E), target preference was improved significantly but did not reach WT levels. Of note, swim speed was not different between the experimental groups (Fig 2 F). Explorative behavior (Fig 2 G) and basal anxiety (Fig 2 H) were measured in the open field test. There was no significant difference amongst groups. We also tested whether anle138b treatment would affect memory function in wild‐type mice but did not observe any significant difference ( Appendix Fig S2 ). Thus, oral administration of anle138b partially restores hippocampal plasticity and memory function in APPPS1Δ9 mice even at an advanced stage of pathology.

Encouraged by these findings, we investigated whether anle138b could also reinstate synaptic plasticity and memory function when significant amyloid deposition had already occurred employing the post‐plaque group (Fig EV1 ). To this end, 6‐month‐old APPPS1Δ9 mice were treated with either anle138b or placebo for 4 months. Wild‐type mice treated with anle138b served as an additional control group. Analysis was performed when mice were 10 months of age. In a first cohort, we measured hippocampal LTP. WT mice treated with anle138b showed robust LTP (Fig 2 A), while LTP was significantly impaired in placebo‐treated APPPS1Δ9 mice (Fig 2 B). Notably, a complete restoration of hippocampal LTP was seen in APPPS1Δ9 mice treated with anle138b (Fig 2 C). In conclusion, similar to the pre‐plaque group treatment with anle138b had a marked ameliorating effect on LTP even after the onset of plaque deposition.

To initially test the potential of anle138b as therapeutic strategies to treat amyloid aggregation in Alzheimer's disease, we analyzed its effect in a Drosophila model for amyloid‐induced neurotoxicity. We observed that treatment with anle138b improved survival times when compared to a vehicle‐treated group ( Appendix Fig S1 ). On the basis of these data, we decided to test the efficacy of anle138b in a mouse model for amyloid deposition. We like to state that none of the currently employed animal models for AD fully recapitulate the phenotypes seen in AD patients, and thus, care has to be taken when interpreting such data. In our study, we employed APPPS1Δ9 mice (Jankowsky et al , 2001 ), a well‐established model for AD‐linked amyloid deposition. Since in the patients therapeutic intervention is normally initiated only after the onset of amyloid plaque formation, we decided to test anle138b in two experimental cohorts. In the “pre‐plaque group,” treatment was initiated before the onset of pathology when mice were 2 months of age, while in the “post‐plaque group” treatment was initiated after the onset of amyloid deposition and memory disturbances in 6‐month‐old mice (Fig EV1 ; Jankowsky et al , 2004 ; Lalonde et al , 2005 ; Reiserer et al , 2007 ). In both cohorts, anle138b was continuously provided via food pellets. Thus, in the pre‐plaque group, mice were subjected to anle138b or placebo treatment from 2 months of age, and electrophysiological, behavioral, and biochemical analyses were initiated at 6 months of age. A group of wild‐type mice (WT) treated with anle138b served as an additional control. We first measured synaptic plasticity by analyzing hippocampal long‐term potentiation (LTP). While robust hippocampal LTP at the Schaffer collateral synapse was observed in WT control mice treated with anle138b (Fig 1 A), LTP was significantly impaired in APPPS1Δ9 mice that received placebo (Fig 1 B). Notably, this LTP deficit was completely rescued in APPPS1Δ9 mice treated with anle138b (Fig 1 C). These data suggest that oral application of anle138b protects against Aβ‐induced impairment of hippocampal synaptic plasticity. To test whether the effect of anle138b on hippocampal plasticity also improved hippocampus‐dependent memory function, another group of anle138b and placebo‐treated mice were subjected to the Morris water maze test, a well‐established paradigm to assay spatial memory in rodents (Morris, 1984 ). Anle138b‐treated WT mice displayed robust spatial learning as indicated by decreasing escape latency throughout the 8 days of training (Fig 1 D). In contrast, APPPS1Δ9 mice treated with placebo showed a significantly impaired escape latency (Fig 1 D). This deficit was partially rescued in APPPS1Δ9 mice that received anle138b. Spatial reference memory was analyzed in a probe test performed after 8 days of training. While WT mice showed a significant preference for the target quadrant, no such effect was observed in placebo‐treated APPPS1Δ9 mice (Fig 1 E), confirming memory impairment in APPPS1Δ9 mice. In contrast, anle138b‐treated APPPS1Δ9 mice displayed a significant preference for the target quadrant indicating restored spatial memory (Fig 1 E). Swim speed was similar amongst the groups (Fig 1 F). We also examined if anle138b would affect basal explorative behavior (Fig 1 G) or basal anxiety (Fig 1 H). No difference was found amongst the groups suggesting that oral administration of anle138b can protect APPPS1Δ9 mice from deteriorating hippocampal synaptic plasticity and hippocampus‐dependent memory consolidation.

Discussion

In this work, we investigated the effect of anle138b in the established APPPS1ΔE9 mouse model of AD. The APPPS1ΔE9 mouse model is characterized by dysfunction of neurons and detected by memory decline after 4 months and severe plaques formation after 6 months (Kummer et al, 2014). The most important findings of the present study were that anle138b treatment completely restored hippocampal LTP in the pre‐ and post‐plaque groups of APPPS1ΔE9 mice. In line with this observation, spatial reference memory was fully or partially restored in the pre‐ and post‐plaque groups, respectively. There have been numerous pre‐clinical studies aiming to restore synaptic plasticity and memory function in mouse models for amyloid pathology. These range from therapeutic approaches that aim to modulate causative factors including Aβ‐toxicity via antibody‐based therapies (Selkoe & Hardy, 2016), small molecules that modulated APP processing (Vassar et al, 2014), small molecules that are thought to target toxic amyloid species directly (Doig & Derreumaux, 2015) to symptomatic treatments not directly targeted toward amyloids (Fischer, 2016). Anle138b belongs to the first category. Its therapeutic effect resembles that of other small molecule drugs. For example, epigallocatechin gallate (EGCG) was shown to affect Aβ toxicity by redirecting toxic Aβ‐structures into off‐pathway oligomers (Ehrnhoefer et al, 2008; Bieschke et al, 2010). This agent was able to ameliorate spatial memory deficits in APP mice after the onset of amyloid deposition (Liu et al, 2014). In this context, it is interesting to note that when administered via dry food anle138b was previously found to be taken up to reach levels of 100 μM in the brain (Wagner et al, 2015). Anle138b metabolites are very hydrophilic, and they were detected in multiple organs but were absent in brain (Wagner et al, 2013). This indicates that anle138b is not metabolized in the brain. Anle138b was also found to be non‐toxic in mice up to a dose of 2 g/kg. Mice receiving similar concentrations of anle138b as used in our study lived without any detectable toxicity, even when the drug was given longer than a year (Wagner et al, 2013). Also in our study, no negative or positive effects of anle138b on WT mice were observed.

In addition to the restoration of hippocampal LTP and spatial reference memory, we also observed restoration of physiological, wild‐type‐like gene expression profile in the pre‐plaque group, indicating that hippocampal cells reinstate homeostasis. In APPPS1ΔE9 mice that received normal chow, genes linked to metabolic function and neuronal plasticity were markedly down‐regulated. Feeding with anle138b‐containing chow completely reversed this effect on gene expression. Even in the placebo‐treated pre‐plaque group, we observed little evidence for neuroinflammatory processes, which is in contrast to the data from the post‐plaque group. In fact, the gene expression changes observed in the placebo‐treated post‐plaque group were dominated by increased inflammation. These data suggest that in APPPS1ΔE9 mice, the decline of synaptic plasticity precedes inflammatory processes. Of note, anle138b treatment had no effect on pathological gene expression pattern in the post‐plaque group, yet hippocampal LTP was completely restored and spatial memory was partially restored. These data suggest that therapeutic strategies that aim to reduce amyloid toxicity—at least in the APPPS1ΔE9 model—may have little influence on neuroinflammation when applied at an advanced stage of the disease. Nevertheless, a significant therapeutic effect could be observed in the post‐plaque group. This might be explained by removal of toxic Aβ species which can lead to the restoration of synaptic function in neurons even in a detrimental inflammatory environment. These data are highly interesting, since one argument for the failure of clinical trials targeting amyloid deposition or modulation of Aβ cleavage is that such treatments are ineffective when given at an advanced stage of the diseases. Our data suggest, however, that anle138b targets Aβ‐related pathological events that allow recovery of synaptic function even if secondary pathological events such as inflammation persist.

The finding that anle138b treatment ameliorates synaptic plasticity and learning deficits in APPPS1ΔE9 is of utmost importance. These data are in line with previous observations showing that the same compounds have beneficial effects in animal models for Creutzfeld‐Jakob, Parkinson's disease, and Tau pathology. Specifically, the latter finding is intriguing, since Tau and Aβ pathology are believed to be the main causative factors of AD pathogenesis. While Wagner et al (2015) showed that anle138b in Tau P301S mice rescues synaptic and neuronal loss, we could further substantiate these data by showing that anle138b treatment also ameliorates defects in hippocampal transcriptome plasticity in the same Tau P301S mice. Taken together, these data suggest the revealing possibility that anle138b is able to ameliorate Tau and Aβ pathology. Therefore, to the best of our knowledge, anle138b would be the first small molecule that has a direct effect on the two major hallmarks of AD.

These data are also important from a translational point of view. Taking into account that the various animal models for AD only recapitulate part of the pathogenesis observed in human patients, it is intriguing that anle138b was able to rescue disease progression in various different AD models and models for aggregopathies, raising the hope that anle138b could also be effective in human patients.

The mechanism by which toxic Aβ species induce synaptic dysfunction and neurotoxicity is still unresolved. Proposed mechanisms include NMDA receptor endocytosis (Shankar et al, 2007), α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazole propionic acid surface receptor modulation (Querfurth & LaFerla, 2010), and Aβ pore formation (Arispe et al, 1993). Moreover, the presence of amyloid plaques was shown to change structural plasticity of neurons (Spires & Hyman, 2004). Nevertheless, amyloid plaques are unlikely the sole reason for disturbed synaptic function and memory consolidation in AD and it has even been suggested that they present a compensatory mechanism of the brain in order to deal with toxic Aβ species (Selkoe & Hardy, 2016). This is further supported by our findings that anle138b treatment in the post‐plaque group was able to restore hippocampal LTP and spatial reference memory, although the plaque load was even higher when compared to placebo‐treated APPPS1ΔE9 mice of the pre‐plaque group suggesting that the reduction in amyloid plaque load cannot be the main mechanisms by which anle138b exerts its therapeutic action. The pore formation hypothesis has long been proposed but tested experimentally to a very limited degree, which is also due to the fact that an array of various in vitro and in vivo methods needs to be combined to address this issue. We focused on the question whether anle138b modulates the pore forming activity of Aβ because synaptic function relies on the integrity of membranes and their ability to modulate ion fluxes in a voltage‐dependent way. This pore‐based mechanism has been envisaged to underlie impaired neuronal function and cell death (Arispe et al, 1993). Modulation of pore formation was also implicated in cell‐based models for EGCG mode of action (Diaz et al, 2009) and of its congeners MRS2481 and MRS2485 (Diaz et al, 2009). The observed stepwise growth of bulk membrane conductance in the presence of Aβ (without anle138b) indicates that activity increases through the combined action of many individual pores.

Numerous molecular dynamics (MD) simulations (Jang et al, 2008; Arce et al, 2011; Gillman et al, 2014) and NMR spectroscopy‐based investigations (Sarkar et al, 2014) of Aβ pores have pointed to a β‐barrel structure for the intramembrane region of the pore. It was previously shown that Aβ containing a point substituted proline (F19P) showed pore structure by AFM but did not demonstrate ionic conductance (Capone et al, 2012; Connelly et al, 2012b). The chemical structure of proline introduces a “kink” in the peptide's secondary structure, which is known to disrupt β‐sheet formation. MD simulations of F19P barrel structure showed that β‐sheet destabilization led the highly charged N‐terminal regions to bind at the peptide mouth and collapsed the pore (Umehara et al, 2010; Connelly et al, 2012a). Our data are consistent with such a model and suggest that anle138b induces a conformational change within Aβ pores that greatly reduces or, in most pores eliminates, ionic flux. Steric blockage by anle138b without a conformational change of the pore is energetically unfavorable due to the hydrophobicity of anle138b, as this scenario would require anle138b to be in contact with water molecules inside the pore. The above‐mentioned mechanisms provide further insight into the method of action in preventing pore activity and reducing Alzheimer's pathogenicity (Fig 5E).

It has to be mentioned, however, that all of the above‐described experiments are based on in vitro approaches using lipid bilayers. In vivo evidence further supporting the notion that anle138b counteracts the detrimental effect of toxic Aβ species on membrane integrity stems from our finding that Aβ 1‐40 administration to cultured hippocampal neurons compromised membrane integrity, an effect which was attenuated by anle138b. Our interpretation that conversion of conducting to non‐conduction Aβ pores is one possible mode of anle138b action. It may also explain that anle138b ameliorated all tested disease phenotypes in the pre‐plaque group, while in the post‐plaque group only LTP was completely restored. Hence, in the post‐plaque group, the presence of toxic Aβ species may already have induced secondary processes such as inflammation that persistently affects memory function even if membrane integrity and LTP are restored in neurons. Yet, we like to reiterate that while our data point to the existence of Aβ pores in vivo, a definite proof is still missing. Further evidence in support of the Aβ pores stems from a study in which Aβ oligomers induce single‐channel calcium fluorescence transients in Xenopus oocytes (Demuro et al, 2011). Nevertheless, we cannot exclude that anle138b affects other cellular processes than Aβ‐mediated conducting pores in membranes. Thus, it is also possible for example that in vivo Aβ sequesters membrane lipids, thereby affecting membrane integrity, which is then prevented by the action of anle138b.

In conclusion, our data show that anle138b can reinstate synaptic plasticity and memory function in a mouse model for amyloid pathology via mechanisms that—at least in part—appear to involve the blockage of Aβ‐induced pores in membranes. Careful analysis of this activity indicates that the oligomers are still in the membrane, but pores have a changed conductivity profile, mainly staying open for shorter time and lacking the possibility of building up large currents as seen when anle138b was absent. Taking into account that anle138b was also shown to ameliorate disease phenotypes in a mouse model for Tau pathology, thus being to the best of our knowledge one of the first compounds that seems to causatively interfere with both of the two major hallmarks of AD, we suggest that anle138b to further be validated in clinical trials for its potential to treat AD and perhaps other aggregopathies.