Also see Q&A below with Masuo Ohno, Robert Vassar, and John Disterhoft.

In yesterday’s Neuron, researchers presented fresh in-vivo support for the amyloid hypothesis, and they bolster the status of the APP protease BACE as the current favorite target for AD therapy development. Led by John Disterhoft and Robert Vassar at Northwestern University Feinberg School of Medicine in Chicago, with collaborators elsewhere, the study suggests that the cognitive deficits described in APP-transgenic mice are due to the Aβ peptide, not its parent protein APP, because depleting Aβ in transgenic mice reverses their memory deficits. The work further suggests that excess Aβ may wreak havoc on memory function by somehow interfering with cholinergic excitation of hippocampal neurons. The study focuses on soluble forms of Aβ. The paper also hints at a physiological function of this peptide in spatial memory. “This is an excellent and important study,” comments Mark Mattson.

The paper is the first in what will be a wave of studies examining crosses of BACE knockout mice with older transgenic strains that overproduce mutant human APP, and interpreting it fully will become easier once these other studies are available, as well. In 2001, three teams of scientists—one at Amgen including some of the present authors (Luo et al., 2001), one at Johns Hopkins’ University (Cai et al., 2001), and one at Elan Pharmaceuticals (Roberds et al., 2001)—each had created BACE knockout strains. All three strains appeared normal, raising hope that inhibiting BACE might have fewer side effects than inhibiting γ-secretase (see related New Orleans news story). Since then, these groups have bred their BACE-less mice to APP-transgenics, reasoning that such mice would have high levels of APP (and presumably the products of α-secretase cleavage) but no Aβ. This might allow scientists to finally answer the lingering question of whether the behavioral deficits described in APP-transgenic mice result from elevated Aβ levels or indeed from overexpression of the APP transgene itself. Despite abundant evidence implicating Aβ in Alzheimer's pathogenesis, direct proof of its role in cognitive decline, and any mechanism thereof, remains scant (see ARF related news story). Initial data of three studies similar to the Disterhoft/Vassar study were presented at the annual meeting of the Society for Neuroscience in New Orleans last November; brief summaries follow below.

In the present study, first author Masuo Ohno and colleagues crossed their BACE knockout mice with Tg2576 mice. ELISA analysis showed that, as expected, the bigenic mice indeed had almost no Aβ in their brains despite massive overproduction of APP, though it did detect residual Aβ40 and 42 levels that the authors suspect may be Aβ variants generated by other proteases. Ohno et al. compared the performance of their bigenic mice to that of Tg2576, BACE1 knockouts, and wild-type mice in two tests of hippocampus-based learning. First, they used a social recognition task (Kogan et al., 2000), which measures how well a mouse remembers the smell of a previously encountered littermate (a bit like the sniffing of one dog meeting another). Second, they used spontaneous alteration in the Y maze (Lalonde, 2002), which the authors believe is sensitive to early onset cognitive decline. Reflecting a recent shift in attention to soluble species of Aβ, the authors performed all their memory tests in mice at four to six months of age, well before plaques form in their brains. In both tests, the bigenic mice performed as well as wild-type mice, but Tg2576 mice had deficits, suggesting that accumulation of soluble Aβ caused these deficits, the scientists report.

Intriguingly, the BACE knockout mice, which had much less brain Aβ than did wild-type mice, also showed a mild deficit in the Y maze. This implies that Aβ may play a physiological role in learning and memory, a widely recognized but sparsely studied question (see Kamenetz et al., 2003; see also ARF related conference story). In terms of drug treatment, this result cautions that any future drug would require careful dosing to avoid depleting Aβ. Current research into additional targets of BACE besides APP also suggests as much (see ARF Live Discussion on BACE). “Nevertheless, this potential issue does not diminish BACE1 as a therapeutic target,…” the authors write.

Soluble Aβ and Cholinergic Function in Vivo
To get a sense of the mechanism by which high Aβ concentrations might impair social recognition and spatial memory, Ohno and colleagues performed electrophysiology in hippocampal slices of their bigenic mice. They picked up previous research by Rene Quirion, Mark Mattson, and others who had shown that Aβ applied to cultured neurons or slices inhibits cholinergic signal transduction. Further inspiration came from more recent work suggesting that two-month-old Tg2576 mice already have defects in protein kinase C activation downstream of muscarinic acetylcholine receptors, and that this in turn impairs the normal cholinergic modulation of GABAergic transmission in prefrontal cortex (Zhong et al., 2003). Cholinergic deficits in AD have been described as early as the 1970s, and most current AD drugs elevate acetylcholine temporarily (see Live Discussion). To test for cholinergic dysfunction in their mice, Ohno et al. measured changes in an acetylcholine-dependent consequence of learning called post-burst after hyperpolarization (AHP). The scientists report that in hippocampal slices from Tg2576 mice, they recorded a weakening of the normal stimulation of neuronal excitability by the acetylcholine receptor agonist carbachol, but that this stimulation reverted to the level of wild-type controls in the bigenic mice. Moreover, hippocampal slices from the BACE knockout mice responded like those of wild-type mice, suggesting that a different mechanism must account for the memory deficit these mice show in the Y maze, the authors add.

In summary, the authors show that genetically removing Aβ rescues two hippocampal learning and memory defects of Tg2576 mice, demonstrating that these deficits are due to Aβ, not APP. The authors also suggest that the rescue of these memory deficits occurs because purging the hippocampus of Aβ restores the normal regulation of neuronal excitability via cholinergic receptors.

In their discussion, Ohno and colleagues raise a caveat to the conclusion that Aβ is the only offending APP fragment. The bigenic mice lack not only Aβ, but also a product of BACE’s APP cleavage called β-CTF. This fragment might also contribute to memory dysfunction; prior work has indeed implicated it already (Nalbantoglu et al., 1997). Moreover, neuron-specific conditional knockouts of γ-secretase, which lead to lack of Aβ but presence of the β-CTF, do not rescue certain cognitive deficits (DeWachter et al., 2002; see Shen section of ARF New Orleans news story). Future experiments will need to sort out the contributions of various APP fragments to synaptic function and learning. This question grows even more complicated by the finding that BACE1 cleaves not only APP, but also its cousins APLP1 and APLP2 (Li and Sudhof, 2003).

Others Corroborate—or Do They?
Three other groups that are independently investigating what eliminating BACE does to APP-transgenic mice presented some of their unpublished data in New Orleans. Researchers led by Philip Wong at Johns Hopkins University presented a poster showing that breeding their BACE1 knockout mice with the APPswe/PS1 strain rescued the reference memory deficits observed in APPswe/PS1 transgenics (SfN abstract 524.11). A team led by John Morley at St. Louis University School of Medicine, Missouri, reported that reducing both Aβ and BACE levels by repeated injection of an antisense RNA construct into the brain ventricles of adult SAMP8 mice reversed the learning and memory deficits seen in these mice with the T-maze and lever press tests (525.2). (Harvard researchers are developing a parallel mRNAi approach; see Kao et al., 2003.) A third group appears to obtain somewhat different results. Researchers led by Mark Buttini and Lisa McConlogue of Elan Pharmaceuticals reported that removing BACE1 from PDAPP mice by breeding them to Elan’s BACE1 knockout strain indeed prevented progressive plaque pathology and hippocampal synaptic deficits described in PDAPP mice, but that their hippocampal atrophy remained (SfN abstract 524.7). What’s more, their colleagues Dione Kobayashi, Karen Chen, and others, tested spatial memory of these bigenic mice with the serial water maze paradigm, a variation of the original Morris water maze described in Chen et al., 2000. Like Ohno et al., this group found a spatial memory deficit in their homozygous BACE knockout. But unlike in the Ohno et al. study, this deficit was comparable to the deficit of the PDAPP mice. In addition, the Elan group saw in their BACE knockout/PDAPP mice no rescue of the PDAPP deficit, but instead the greatest memory impairment of all strains studied in this experiment (SfN abstract 524.4). These scientists also propose that Aβ may be required for normal working memory. Interestingly, this group also noted that calbindin levels changed in the mice, and that the degree of deviation from wild-type calbindin levels correlated with the memory impairment (see ARF related news story). You can view abstracts mentioned in this story at the SfN/ScholarOne website.

What Else Is New? More BACE Tidbits
BACE1 behavior, or rather, how BACE affects mouse behavior, has become an intriguing topic with a paper by researchers at GlaxoSmithKline in England, who reported last November in Molecular and Cellular Biology that they have made neuron-specific BACE1 transgenic mice that are especially bold and exploratory, whereas the corresponding BACE1 knockouts are notably timid (Harrison et al., 2003). In this month’s issue of the same journal, researchers led by Weihong Song of the University of British Columbia, Vancouver, characterize the BACE1 promoter. They fingered the transcription factor Sp1 in their attempt to understand the regulation of BACE expression (Christensen et al., 2004). On the genetics front, Chinese researchers report in the January 1 American Journal of Medical Genetics that they see a weak association of the single nucleotide polymorphism 1239G/C of the BACE1 gene in two cohorts of Han Chinese (Shi et al., 2004). And finally, a flurry of papers on experimental BACE inhibitors has just come online (Hom et al. 2004; Chen et al., 2004; Lamar et al., 2004; Park and Lee, 2003; Hu et al., 2003; Owens et al., 2003). Has your favorite experiment not made it into this snapshot of the BACE landscape? Send a comment and help complete the story.—Gabrielle Strobel

Q&A with Masuo Ohno, Robert Vassar, and John Disterhoft. Questions by Gabrielle Strobel.

Q: In your bigenic mice, what happens to the cleavage products of α-secretase? What are their levels? Might they influence the effects you see?

A: BACE1-/-/Tg2576+ mice have about twofold higher levels of brain APPs-α and C83 (the α-secretase cleavage products) as compared to BACE1+/+/Tg2576+ (Luo et al., 2001). Therefore, although unlikely, we cannot exclude the possibility that the effects we observed may involve α-secretase cleavage products to some extent. However, the vast majority of previous work by our group and others makes it very likely that reduced cerebral levels of Aβ were the reason for the rescue of the memory deficits that we observed in BACE1-/-/Tg2576+ mice.

Q: Why did you not use the water maze or the radial arm maze? That would have made it easier to compare the bigenic mice's behavioral phenotype with the results of others.

A: Recent work has focused on soluble Aβ assemblies (e.g., “ADDLs” and protofibrils) as potential neurotoxic species in AD. Therefore, we wanted to assay memory in young (4-6 months of age) Tg2576 mice before amyloid deposits developed so we would be able to analyze the effects of soluble, non-plaque-associated Aβ species on learning and memory. We chose social recognition and spontaneous alternation in the Y maze because these paradigms are very sensitive for measuring early, pre-deposit deficits in hippocampus-dependent memory in APP transgenic mice. Water maze or radial arm maze tests are possibly more standard assays of hippocampal learning. However, these tests appear less sensitive for measuring early memory deficits in young Tg2576 mice, although these paradigms become more robust for measuring memory deficits after Tg2576 mice begin to develop amyloid plaques at ~9 months of age. In addition, social recognition and spontaneous alternation in the Y maze are hippocampus-dependent tasks that rely on the natural social and exploratory behavior of mice.

Q: Why did you measure AHP? Again, would not commonly used measures of LTP have made it easier to interpret the results?

A: Emerging evidence suggests that cholinergic signaling is impaired by low concentrations of Aβ in younger Tg2576 mice, which might contribute to the early phase of cognitive impairments we observed in 4-6-month-old Tg2576 mice. Therefore, we tested the possible cholinergic mechanisms underlying the memory rescue in BACE1-/-/Tg2576+ bigenic mice. We investigated increased hippocampal neuronal excitability, as assessed by a reduction in the slow post-burst after hyperpolarization (AHP) in the bigenic mice. Our research group has done extensive investigation into AHP, which is modulated by acetylcholine, and we have shown it to be a cellular mechanism of learning. LTP, which is an electrophysiological model of synaptic plasticity and is mainly regulated by glutamatergic signals, is not a direct measure of the excitability change that we hypothesize underlies the learning rescue in the bigenic mice. Previous data showed that hippocampal LTP is normal in Tg2576 mice at 2-8 months of age, although LTP becomes impaired by the age of 15-17 months. Since we observed learning deficits during the time before plaques were formed and at ages when LTP was not impaired in the transgenic, this was further reason to concentrate on the AHP measure that we used.

Q: In New Orleans, scientists from Elan presented behavioral data on BACE ko/PDAPP bigenic mice, and these mice appear to have serious memory problems that progress with age when tested in the serial water maze. What might explain this apparent discrepancy?

A: We currently do not have an explanation for the discrepancy between Elan’s results and our own. We can only speculate that the discrepancy may possibly be related to differences in the behavioral paradigms or in the specific mice used by our two groups. For example, the Elan group used water maze while we used social recognition and Y maze tests; therefore, it is difficult to determine directly how their behavioral results relate to our own. In addition, Elan used a different APP transgenic (PDAPP) having the London APP FAD mutation (rather than the Swedish mutation in Tg2576), and the PDAPP transgene is driven by the PDGF promoter (rather than by the prion promoter in Tg2576). The mice used by our two groups also had different strain backgrounds, which together with the different APP transgenes may influence behavioral results. Clearly, more research will be needed to determine the cause of this discrepancy.

Q: There has been a flurry of papers on BACE1 inhibitors in the past couple of months. Are these all experimental tools, or do you consider any of them promising leads for drug discovery. If so, which ones?

A: The majority of BACE1 inhibitors that have been published to date are peptide-based, and therefore do not exhibit ideal drug-like properties. As such, they are mainly useful as research tools. Importantly, however, they also provide a starting point for rational drug design of small molecule BACE1 inhibitors with more favorable drug-like characteristics that may be developed into lead compounds.

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  1. This is exactly what we predicted (Dewachter and Van Leuven, 2002) or, as the Americans would put it, "what the doctor ordered…"

    The overall message of this study is loud and clear: BACE is hereby proven to be the favorite target for drug-makers. That message rings even more clearly because γ-secretase inhibitors now prove bad not only for the brain, as we predicted, as well (Dewachter et al., 2002), but also in vivo for the immune system, the intestine, and likely for any, or even all, biological subsystems in our complex bodies that depend on intramembranous proteolysis for essential functions (Wong et al., 2004).

    Some points remain somewhat worrying or startling in this study. First, why not use the "classic" cognitive test, i.e., the water maze? Second, why not measure "classic" LTP instead of the somewhat exotic cholinergic AHP? Third, and most important, is the lack of any data on APP biochemistry. I, for one, would want to know what happens to APPs-α and to the α-CTF; the discussion circumvents this issue. Indeed, the lowering of Aβ by BACE-deficiency is spectacular and likely to be a major contributor to the "rescue." Nevertheless, APPs-α is claimed by many to be beneficial for brain, but only circumstantially demonstrated to be essential for "neuronal well-being."

    Here was a chance to increase the experimental proof for that action.

    References:

    . Secretases as targets for the treatment of Alzheimer's disease: the prospects. Lancet Neurol. 2002 Nov;1(7):409-16. PubMed.

    . Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci. 2002 May 1;22(9):3445-53. PubMed.

    . Chronic treatment with the gamma-secretase inhibitor LY-411,575 inhibits beta-amyloid peptide production and alters lymphopoiesis and intestinal cell differentiation. J Biol Chem. 2004 Mar 26;279(13):12876-82. PubMed.

  2. By Dione Kobayashi and Karen Chen The paper by Disterhoft et al. reporting cognitive and cholinergic rescue with their BACE1 knockout mice on a mutant hAPP background is extremely exciting and is a strong validation of BACE inhibitory strategies for Alzheimer's disease therapeutic efforts. Other recent works over the past three years have also reported on the behavioral effects of complete genetic removal of BACE1 from mice (Harrison et al., 2003; Kobayashi et al., in SFN abstracts 2002, 2003). In addition to their novel cholinergic function results, Disterhoft et al. found mild phenotypes in exploration in BACE1 -/- mice similar to other groups, although there is some discrepancy regarding the ability of BACE1 deletion to rescue cognitive deficits due to overexpression of mutant hAPP.

    It must be emphasized that not only do the various BACE1 -/- mice differ in their hAPP mutations, level of APP overexpression, and thus their subsequent cognitive deficits, but these mice also have been subjected to widely divergent behavioral tasks. While the Y maze and social recognition tasks used by Disterhoft et al. are both nonaversive and rely on hippocampal function, these tasks also depend on the motivational state of the animals, which is admittedly impaired in spontaneous exploration.

    In studies done by our group, BACE1 -/- PDAPP mice were tested in an aversive serial spatial memory water maze paradigm that utilizes working memory (Chen et al., 2000). This modified water maze protocol was in effect designed to detect subtle spatial impairments in the PDAPP mouse that could be missed when using other spatial memory tasks, including the classic reference memory task described by Morris in 1981. While we do report significant progressive spatial memory deficits in our BACE1 -/- PDAPP mice, their impairments can also be viewed as subtle and specific. Thus, our seemingly opposing BACE1 -/- hAPP data may simply represent the dynamic range of changes in BACE1 -/- hAPP mice tested with different cognitive tasks. Alternatively, these results may be entirely independent, underlining the murkiness inherent in working with transgenic animal models.

    It will be interesting to watch this promising line of research develop, as I agree with the authors wholeheartedly that further study with other transgenic mouse lines, as well as other behavioral assessments, will be illuminating, particularly if a conditional BACE1 -/- is developed like that reported with presenilin-1 (Yu, 2001). In addition, the mild behavioral phenotypes consistently reported with BACE1 -/- alone should not be overlooked from a perspective of understanding the role of the constituents of the APP processing pathway in normal learning and memory.

    References:

    . BACE1 (beta-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes. Mol Cell Neurosci. 2003 Nov;24(3):646-55. PubMed.

    . A learning deficit related to age and beta-amyloid plaques in a mouse model of Alzheimer's disease. Nature. 2000 Dec 21-28;408(6815):975-9. PubMed.

    . APP processing and synaptic plasticity in presenilin-1 conditional knockout mice. Neuron. 2001 Sep 13;31(5):713-26. PubMed.

  3. This elegant report further supports BACE1 as a rational therapeutic target for treating the cerebral amyloidosis of Alzheimer's disease (AD). Ohno and colleagues eliminated BACE1 function in a mouse model of AD by crossing Tg2576 mice—which overexpress the Swedish mutant of amyloid precursor protein (APPSwe)—with BACE1 knockout mice. They found that genetic elimination of BACE1 blocked cerebral amyloid β-protein (Aβ) production, ameliorated the cognitive deficits of the APP-transgenic mice in hippocampal-based learning tasks, and improved the cholinergic electrophysiologic deficits in hippocampal slice preparations.

    The Tg2576 APPSwe mouse model of Alzheimer's disease develops cerebral amyloid deposits by the age of nine to 11 months [1]. In the current study, soluble Aβ in brain was increased by 25-fold relative to nontransgenic mice at four to six months—the age at which cognitive deficits and electrophysiologic deficits were detected. The findings that certain cognitive [2] and electrophysiologic deficits occur prior to cerebral amyloid deposition suggest that soluble species of Aβ adversely affect physiologic processes in the brain. The rapid amelioration of behavioral deficits in APP-transgenic mice by passive immunization with anti-Aβ antibodies [3,4] further supports soluble forms of Aβ as a functionally toxic species in these mice.

    Ohno and colleagues tested the effects of eliminating brain Aβ in the Tg2576 APPSwe mice by knockout of BACE1, which is the principle enzyme responsible for the β-secretase cleavage of APP, the initial step of Aβ synthesis. Overexpression of BACE1 accelerates Aβ production in mouse brain [5,6], and BACE1 protein levels and enzymatic activity are increased in the AD brain [7-9]. Targeting BACE1 therapeutically is potentially less toxic than reducing γ-secretase activity since BACE1 knockout mice do not exhibit overt neurological or medical problems [10]. (However, BACE1 null mice alone did exhibit impaired performance in a spatial working memory task, a deficit that resolved in the APPSwe/BACE1 null mice [1]). This paper demonstrates that behavioral and electrophysiologic deficits in APPSwe mice can be ameliorated by elimination of Aβ production in the APPSwe/BACE1 null mice, implicating Aβ (or, less likely, β-cleaved APP fragments) as the source of these deficits.

    While very encouraging for BACE1 therapeutic strategies, the usual caveats regarding translating behavioral tests in mice to the cognitive deficits in human AD apply. Cognitive deficits in AD are most consistently associated with the development of neuronal loss and neurofibrillary tangle formation in corticolimbic regions, pathological features which are not reproduced in the Tg2576 APPSwe mice. Prolonged exposure to high levels of toxic forms of Aβ may initiate distinct downstream effects in mice (behavioral deficits, cholinergic electrophysiological deficits) and humans (neuronal and synaptic loss, cholinergic deficits, neurofibrillary tangle formation). The amyloid hypothesis postulates that reducing Aβ would prevent these downstream effects; the results presented in this paper support this idea in the case of certain deficits in the APPSwe transgenic mice.

    References:

    . Correlative memory deficits, Abeta elevation, and amyloid plaques in transgenic mice. Science. 1996 Oct 4;274(5284):99-102. PubMed.

    . The relationship between Abeta and memory in the Tg2576 mouse model of Alzheimer's disease. J Neurosci. 2002 Mar 1;22(5):1858-67. PubMed.

    . Immunization reverses memory deficits without reducing brain Abeta burden in Alzheimer's disease model. Nat Neurosci. 2002 May;5(5):452-7. PubMed.

    . Reversible memory loss in a mouse transgenic model of Alzheimer's disease. J Neurosci. 2002 Aug 1;22(15):6331-5. PubMed.

    . Expression of human beta-secretase in the mouse brain increases the steady-state level of beta-amyloid. J Neurochem. 2002 Mar;80(5):799-806. PubMed.

    . Transgenic BACE expression in mouse neurons accelerates amyloid plaque pathology. J Neural Transm. 2004 Mar;111(3):413-25. PubMed.

    . Increased expression of the amyloid precursor beta-secretase in Alzheimer's disease. Ann Neurol. 2002 Jun;51(6):783-6. PubMed.

    . Beta-secretase protein and activity are increased in the neocortex in Alzheimer disease. Arch Neurol. 2002 Sep;59(9):1381-9. PubMed.

    . Elevated beta-secretase expression and enzymatic activity detected in sporadic Alzheimer disease. Nat Med. 2003 Jan;9(1):3-4. PubMed.

    . Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001 Mar;4(3):231-2. PubMed.

  4. Ohno et al. bred the BACE1 knockout (BACE1-/-) mice onto the Tg2576 APP transgenic background and tested the effects of BACE1 deficiency on Aβ production, behavioral performance, and cholinergic function at a young age (4-6 months), prior to amyloid plaque deposition. The authors showed, quite convincingly, that inhibition of Aβ production, as a result of BACE1 deficiency, rescued the behavioral deficit and cholinergic impairment present in Tg2576 transgenic mice.

    This result has several important implications: 1) It lends strong support to the amyloid hypothesis; 2) it strengthens the notion that BACE1 is a valid therapeutic target for AD intervention; and 3) it establishes that the behavioral abnormality seen in Tg2576 mice is caused by APP processing/Aβ production rather than APP overexpression.

    However, as the authors pointed out, BACE1 deficiency leads not only to inhibition of Aβ, but also to changes in other APP fragments (e.g., β-CTF). Therefore, a definitive link between Aβ and functional deficits cannot be established. In addition, the therapeutic potential of BACE1 inhibitors has to be interpreted with caution since BACE1 knockout mice alone show impaired performance in the Y maze test (Fig. 1B). Although this impairment is neutralized on the APP-overexpressing background, it represents an artificial condition as AD individuals do not overexpress APP. In this regard, it is prudent to test the BACE1 knockout and BACE1-/- Tg2576 animals in other behavioral paradigms, such as the Morris water maze or fear conditioning.

  5. This is an interesting paper, and these mice are very useful for addressing a number of issues.

    The behavioral paradigms the authors chose to use are not the strongest learning and memory tests available; more robust and better established hippocampal-dependent learning and memory paradigms, such as the Morris water maze and the contextual fear conditioning tests, might have been preferable. This could be the reason that in the spontaneous alternation Y maze, even the Tg2576 APP-transgenic mice did not perform that poorly compared to the control. More importantly, the Y maze results require more explanation: I wonder how the double-mutant mice could have behaved "normally" while BACE-/- and APP-Tg mice performed poorly (Figure 1B)? It seems a bit premature to conclude "rescue" from such results.

    Without going into details of the physiology result, Figure 2C seems to show lower values in the double-mutant group compared to the control, as well. Nevertheless, it is interesting and welcome that the authors looked at the cholinergic input.

    Overall, from the data shown in the paper, it appears that the story is more complicated, and more thought-provoking, than a simple rescue story. BACE-/- mice appear to exhibit a memory deficit (due to loss of Aβ or other BACE
    substrates?) in one behavioral test but not the other, whereas BACE-/-xTg2586 bigenic mice appear to perform better than BACE-/- mice (due to ???). If this result can be confirmed by other behavioral tests, it warrants further investigation to characterize the underlying mechanism.

    Together with APP-transgenic mice crossed into the PS1 conditional KO background, the Ohno et al. mice stand to be great models to tease out the different contributions of various fragments of APP in brain function. This would require crossing the same APP transgenic line into either PS1 cKO or BACE null background. More importantly, a battery of robust learning and memory tests should be used to test these groups of mice together using identical protocols; only then we can compare results and make meaningful conclusions.

  6. Previous studies showed that the deletion of BACE1 abolished the production of Aβ and BACE1 knockout mice are apparently normal. In this current paper, Ohno et al tested whether cognitive deficits occurring in the mutant APP mice (Tg2576) can be ameliorated in the absence of BACE1, results that have important implication for the potential therapeutic value of BACE1 in AD. Since developmental cognitive (as assessed by either social recognition task or spontaneous alternation in Y maze) and electrophysiological (hippocampal cholinergic dysfunction) abnormalities occur prior to Ab deposition in 4-6 months old Tg2576 mice, Ohno et al. elected to examine whether such memory deficits can be rescued in Tg2576 mice lacking BACE1. Their results demonstrating that the deletion of BACE1 prevented these early onset behavioral abnormalities strongly support their conclusion that increased levels of Aβ (as opposed to increased APP levels) causes the cognitive deficits occurring in Tg2576 animals.

    These authors also interpreted their findings to support the view that the inhibition of BACE1 has therapeutic value in reversing AD-associated cognitive deficits. However, it will be critical to test whether age-associated spatial memory deficits (as assessed by Morris water maze) occurring in aged Tg2576 mice can be rescued in the absence of BACE1. It is unclear at present whether Tg2576 mice lacking BACE1 will display age-associated spatial memory abnormalities; if they don't, such a positive outcome would strongly favor the idea that BACE1 inhibitors have the potential to ameliorate age-associated cognitive deficits occurring in AD.

    It is interesting to note that young (4-6 months of age) BACE1 knockout mice exhibit poor performance in spontaneous alternation in the Y maze, a deficit that is also observed in young Tg2576 mice. The reasons for this deficit are unclear. Because Tg2576 mice apparently can rescue this cognitive abnormality in BACE1 null mice and the levels of Aβ peptides in Tg2576;BACE1-/- mice were claimed to be similar to wild-type levels, these authors infer that the lack of Aβ in BACE1 null mice presumably is responsible for the behavioral deficits in the Y maze. This interpretation would raise the critical issue as to the origins of Ab peptides in the Tg2576;BACE1-/- mice. One uncertainty is whether the Aβ detected from brain extracts by the sandwich ELISA came from Aβ peptides derived from the processing of APP, or from APP-related fragments containing epitopes recognized by the antibodies used. That no Aβ peptides can be detected even when APP wild-type or APPSwe is expressed highly in BACE1-/- neurons, as previously demonstrated, would support the latter possibility.

    In this case, the lack of Aβ may not be sufficient to explain the behavioral deficits observed in these young BACE1 knockout mice. It is plausible that the lack of Aβ could affect other substrates of BACE1 to elicit the poor Y maze performance in the BACE1 null mice. However, because the behavioral abnormality observed in the BACE1 knockout mice can be rescued by the expression of APP in Tg2576 mice, this deficit is more likely related to the APP pathway rather than due directly to other BACE1 substrates. At any rate, further studies are necessary to clarify the behavioral abnormalities observed in the BACE1 null mice.

    These results raise the possibility that the therapeutic inhibition of BACE1 may not necessarily be free of mechanism-based toxicities, particularly if age-associated behavioral abnormalities are observed in BACE1-deficient mice. Thus, it would be critical to determine whether age-associated spatial memory deficits are observed in aged BACE1 null animals. Furthermore, the behavioral analysis of conditional BACE1 knockout mice should further clarify this important issue.

    In summary, while the paper by Ohno et al provides compelling evidence that early onset cognitive and cholinergic abnormalities occurring in young Tg2576 mice can be rescued by deleting BACE1, critical issues regarding the impact of BACE1 on age-associated cognitive deficits in aged mice remain to be established, as these results will have important implications for the development of potential therapeutics designed to inhibit BACE1 and ameliorate Aβ amyloidosis in AD.

  7. This new report by Ohno et al. demonstrates further that BACE1, an
    enzyme involved in the maturation of the APP precursor and the
    generation of amyloid peptides, is a potential therapeutic target
    toward the treatment of Alzheimer's disease. In mice in which the BACE1
    gene was deleted, the overexpression of the human APP-695 Swedish
    familial mutation failed to result in memory deficits and altered
    cholinergic functions. Hence, the expression of BACE1 resulting in the
    production of pathogenic amyloid peptides is apparently key to inducing
    cognitive and neurochemical deficits in the model studied. Together,
    these data suggest that BACE1 inhibitors could prove useful in the
    treatment of AD by reducing the production of amyloid peptides and
    ensuing cholinergic deficits and learning impairments. Of course, the
    safety of such inhibitors would have to be established, but data
    obtained in the mouse model are promising. Moreover, these data link
    some of the key features of the AD brain, including amyloid peptides,
    cholinergic dysfunction, and memory deficits. It would now be of interest
    to investigate the effects of clinically used acetylcholinesterase
    inhibitors in this animal model, as well as subtype selective
    nicotinic and muscarinic receptor ligands.

  8. Jeon et al. (1) suspect that the pyrogallol moiety on C-2 and/or C-3 of the catechin skeleton is responsible for the increased inhibition of BACE1 by these green tea catechins.

    It seems of interest that Bain et al. (2) have found that epigallocatechin-3-gallate inhibits DYRK1A, one the the genes considered responsible for the mental retardation of Down's syndrome.

    Could we expect that therapeutic intervention with epigallocatechin 3-gallate may be beneficial for those with Down's syndrome?

    Basi et al. (3) find that BACE2 suppresses Abeta production in cells that also express BACE1.

    Motonaga et al. (4) report increased BACE2 levels in those with Down's syndrome with Alzheimer's-type pathology and suggest that BACE2 is involved in this neuropathology.

    May there be reason to expect that the increased BACE2 may actually be beneficial?

    References:

    . Green tea catechins as a BACE1 (beta-secretase) inhibitor. Bioorg Med Chem Lett. 2003 Nov 17;13(22):3905-8. PubMed.

    . The specificities of protein kinase inhibitors: an update. Biochem J. 2003 Apr 1;371(Pt 1):199-204. PubMed.

    . Antagonistic effects of beta-site amyloid precursor protein-cleaving enzymes 1 and 2 on beta-amyloid peptide production in cells. J Biol Chem. 2003 Aug 22;278(34):31512-20. PubMed.

    . Elevated expression of beta-site amyloid precursor protein cleaving enzyme 2 in brains of patients with Down syndrome. Neurosci Lett. 2002 Jun 21;326(1):64-6. PubMed.

References

News Citations

  1. New Orleans: Aβ Oligomers and Memory: …Now They Are Bad
  2. New Orleans: Aβ Oligomers and Memory: Now They Are Good…
  3. New Orleans: Symposium Probes Why Synapses Are Suffering
  4. Calbindin Study: Is Calcium the Molecular Handle on Dysfunction in AD?

Paper Citations

  1. . Mice deficient in BACE1, the Alzheimer's beta-secretase, have normal phenotype and abolished beta-amyloid generation. Nat Neurosci. 2001 Mar;4(3):231-2. PubMed.
  2. . BACE1 is the major beta-secretase for generation of Abeta peptides by neurons. Nat Neurosci. 2001 Mar;4(3):233-4. PubMed.
  3. . BACE knockout mice are healthy despite lacking the primary beta-secretase activity in brain: implications for Alzheimer's disease therapeutics. Hum Mol Genet. 2001 Jun 1;10(12):1317-24. PubMed.
  4. . Long-term memory underlying hippocampus-dependent social recognition in mice. Hippocampus. 2000;10(1):47-56. PubMed.
  5. . The neurobiological basis of spontaneous alternation. Neurosci Biobehav Rev. 2002 Jan;26(1):91-104. PubMed.
  6. . APP processing and synaptic function. Neuron. 2003 Mar 27;37(6):925-37. PubMed.
  7. . Impaired modulation of GABAergic transmission by muscarinic receptors in a mouse transgenic model of Alzheimer's disease. J Biol Chem. 2003 Jul 18;278(29):26888-96. PubMed.
  8. . Impaired learning and LTP in mice expressing the carboxy terminus of the Alzheimer amyloid precursor protein. Nature. 1997 May 29;387(6632):500-5. PubMed.
  9. . Neuronal deficiency of presenilin 1 inhibits amyloid plaque formation and corrects hippocampal long-term potentiation but not a cognitive defect of amyloid precursor protein [V717I] transgenic mice. J Neurosci. 2002 May 1;22(9):3445-53. PubMed.
  10. . Cleavage of amyloid-beta precursor protein and amyloid-beta precursor-like protein by BACE 1. J Biol Chem. 2004 Mar 12;279(11):10542-50. PubMed.
  11. . BACE1 suppression by RNA interference in primary cortical neurons. J Biol Chem. 2004 Jan 16;279(3):1942-9. PubMed.
  12. . BACE1 (beta-secretase) transgenic and knockout mice: identification of neurochemical deficits and behavioral changes. Mol Cell Neurosci. 2003 Nov;24(3):646-55. PubMed.
  13. . Transcriptional regulation of BACE1, the beta-amyloid precursor protein beta-secretase, by Sp1. Mol Cell Biol. 2004 Jan;24(2):865-74. PubMed.
  14. . The 1239G/C polymorphism in exon 5 of BACE1 gene may be associated with sporadic Alzheimer's disease in Chinese Hans. Am J Med Genet B Neuropsychiatr Genet. 2004 Jan 1;124B(1):54-7. PubMed.
  15. . Design and synthesis of hydroxyethylene-based peptidomimetic inhibitors of human beta-secretase. J Med Chem. 2004 Jan 1;47(1):158-64. PubMed.
  16. . P3 cap modified Phe*-Ala series BACE inhibitors. Bioorg Med Chem Lett. 2004 Jan 5;14(1):245-50. PubMed.
  17. . Phe*-Ala-based pentapeptide mimetics are BACE inhibitors: P2 and P3 SAR. Bioorg Med Chem Lett. 2004 Jan 5;14(1):239-43. PubMed.
  18. . Determination of the active site protonation state of beta-secretase from molecular dynamics simulation and docking experiment: implications for structure-based inhibitor design. J Am Chem Soc. 2003 Dec 31;125(52):16416-22. PubMed.
  19. . Design and synthesis of statine-containing BACE inhibitors. Bioorg Med Chem Lett. 2003 Dec 15;13(24):4335-9. PubMed.
  20. . High affinity, bioavailable 3-amino-1,4-benzodiazepine-based gamma-secretase inhibitors. Bioorg Med Chem Lett. 2003 Nov 17;13(22):4143-5. PubMed.

Other Citations

  1. Also see Q&A below with Masuo Ohno, Robert Vassar, and John Disterhoft.

External Citations

  1. SfN/ScholarOne

Further Reading

Primary Papers

  1. . BACE1 deficiency rescues memory deficits and cholinergic dysfunction in a mouse model of Alzheimer's disease. Neuron. 2004 Jan 8;41(1):27-33. PubMed.