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Taking a page from research on previously identified mutations causing early-onset Alzheimer disease, Japanese scientists have now incorporated an unusual variant of amyloid precursor protein (APP) into a new mouse model. Aβ peptides produced from this APP mutant readily oligomerize in vitro, and mice that overexpress it show synaptic and cognitive impairments, tau hyperphosphorylation, glial activation, and neuron loss—all in the absence of amyloid plaques—according to a study in the April 7 Journal of Neuroscience. Despite some researchers’ concerns about the evidence for the presence of Aβ oligomers and the absence of Aβ fibrils in these mice, their striking phenotype makes them a powerful tool for understanding this APP mutation and how it causes disease.

Researchers led by Hiroshi Mori of Osaka City University, Japan, first identified the rare APP mutation (E693Δ) in patients who presented with clinically typical Alzheimer disease, yet had low brain amyloid on in vivo imaging by positron emission tomography (Tomiyama et al., 2008 and ARF related news story). The E693Δ mutation produces a form of Aβ that lacks glutamate-22 (E22Δ), and shows a recessive pattern of inheritance in affected families. Mutant Aβ arising from this APP variant forms oligomers but no detectable fibrils in vitro, and inhibits long-term potentiation more strongly than wild-type Aβ peptides when injected into rat cerebral ventricles.

To see whether this oligomerization-prone Aβ mutant could bring on other pathological features of AD in vivo, first author Takami Tomiyama and colleagues generated transgenic mice expressing the E693Δ APP under the mouse prion promoter. They put these animals through various immunohistochemical, behavioral, and electrophysiology tests, comparing them with transgenic mice expressing wild-type human APP under the same promoter. APP expression levels in the two strains were low (i.e., comparable to endogenous mouse APP and about 1/10 that of mutant APP in Tg2576 mice) and differed at most twofold (higher in wild-type) among the lines used for immunohistochemistry and behavioral analyses.

In contrast to the Tg2576 controls, which had abundant plaques in the cerebral cortex and hippocampus at 24 months, age-matched E693Δ APP mice had no discernible plaques in these brain areas. The mutant APP transgenics did, however, show extensive intraneuronal Aβ staining in these regions, whereas the wild-type APP mice did not.

Since E22Δ Aβ in their hands oligomerizes quickly in transfected cells (Nishitsuji et al., 2009), the researchers presumed the intraneuronal Aβ signal in the E693Δ APP mice corresponded to oligomers. This appears to be the case, as shown in a separate set of cortical and hippocampal stainings using an antibody (NU-1) that selectively detects Aβ oligomers (Lambert et al., 2007). E693Δ APP mice racked up oligomeric Aβ in the cortex and hippocampus in an age-dependent fashion, starting at eight months, whereas no NU-1 signal came up in 24-month-old wild-type APP transgenic or non-transgenic controls.

Strangely, Western blots of Aβ-immunoprecipitated brain extracts revealed oligomers, which are presumed to be soluble, in the detergent-insoluble fractions—more in E693Δ than wild-type APP mice, and predominantly in the most stringent extractions done with formic acid. Scientists contacted for comment on the study raised this at best as a curiosity, at worst a key flaw. Karen Hsiao, University of Minnesota, Minneapolis, and colleagues wondered whether it could be an artifact of the extraction protocol. They noted that the current study could be solidified with biochemical data showing that Aβ oligomers are compartmentalized intracellularly, to confirm the NU-1 immunohistochemical stainings. The authors acknowledge in their paper that the detection of Aβ oligomers largely in insoluble fractions flies in the face of conventional belief, and noted in an e-mail to ARF that they are performing further studies to characterize both extracellular and intracellular Aβ oligomers.

The researchers also found no evidence that the intraneuronal Aβ in E693Δ APP mice forms fibrils. Whereas brain sections from 24-month-old Tg2576 mice showed strong staining with the amyloid-binding dye thioflavin S, tissue from age-matched E693Δ APP mice showed no signal besides a weak, diffuse staining within neurons in the cortex and hippocampus. Other scientists raised the possibility that the inability of these mice to form plaques could arise from their low expression of APP. Some pointed out that the Western blot showing Aβ oligomers in the detergent-insoluble fractions was cut such that it only showed monomers, dimers, and trimers, but not Aβ*56 and other higher-weight oligomeric forms.

What’s less disputable are the striking phenotypes seen in the E693Δ APP mice. Compared with wild-type APP transgenic and non-transgenic controls, these animals show an age-dependent drop in synaptic density, measured in brain sections stained with an antibody to the presynaptic marker synaptophysin. In addition, E693Δ APP mice had defects in short- and long-term synaptic plasticity in electrophysiology studies, and did worse in the Morris water maze (which measures spatial memory). The researchers documented all these defects starting around eight months, the age at which oligomeric Aβ was first detected in the E693Δ APP mice.

Beyond the synaptic and behavioral woes, brain sections of E693Δ APP transgenic mice showed evidence of abnormal tau phosphorylation (judged by staining with antibodies to pathological tau, PHF-1, and MC1) and glial activation (measured by antibodies to astrocyte and microglia markers GFAP and Iba-1, respectively). These abnormalities showed up starting at 12 months, and worsened with age. By 24 months, E693Δ APP transgenics also showed appreciable neuronal loss (judged by staining with an antibody to the mature neuron marker NeuN) in the hippocampal CA3 region, though not in cerebral cortex.

Amid concerns about the biochemical data, the E693Δ APP mouse appears to be the first demonstration “that neuronal loss was induced by Aβ oligomers alone in vivo in the absence of amyloid plaques,” the authors write. Despite uncertainty as to whether the intraneuronal Aβ is exclusively oligomeric, “the paper provides evidence for a provocative, new role for APP and intracellular fragments and will be of great interest to the AD community,” wrote Cindy Lemere, Brigham and Women’s Hospital, Boston, in an e-mail to ARF.—Esther Landhuis.

Reference:
Tomiyama T, Matsuyama S, Iso H, Umeda T, Takuma H, Ohnishi K, Ishibashi K, Teraoka R, Sakama N, Yamashita T, Nishitsuji K, Ito K, Shimada H, Lambert MP, Klein WL, Mori H. A Mouse Mode of Amyloid Beta Oligomers: Their Contribution to Synaptic Alteration, Abnormal Tau Phosphorylation, Glial Activation, and Neuronal Loss In Vivo. J. Neurosci. 7 April 2010;30(14):4845-4856. Abstract

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  1. My overall feeling about this paper is that it is great that this group has generated a mouse model bearing the human mutation observed in the Japanese kindred. This will open the door to further studies to better understand how this mutation works and what causes the disease in these families. To my knowledge, it is unclear whether these patients show any intraneuronal Aβ staining in the brain and/or have any extracellular Aβ deposition, as I could not find a report of pathology in the literature. One patient had low PIB binding, but that only means that they did not have a lot of fibrillar amyloid at the time of imaging (although, it should be noted that they did have some). PIB does not pick up diffuse Aβ deposition (or at least not well), so it may be possible that these patients have diffuse plaque deposition.

    This mouse model is interesting and deserves further study. It is important to note that in the absence of extracellular plaque deposition, these mice undergo behavioral deficits, synaptic loss, gliosis, hyperphosphorylation of tau, and finally, neuronal loss (at two years of age). Something is going on. This paper provides evidence for a provocative, new role for APP and intracellular fragments (whether they are truly Aβ oligomers or APP β-CTFs) and will be of great interest to the AD community.

    View all comments by Cynthia Lemere
  2. The critique about Aβ oligomers in our paper is certainly an important issue since Aβ oligomers were clearly declared as the possible cause of Alzheimer disease (Selkoe, 2002). The biochemical nature of Aβ oligomers was not discussed thoroughly in our paper (Tomiyama et al., 2010) due to the limited space and to our focus on the new model mice as described in the Journal of Neuroscience news page (see “This Week in The Journal” in the journal website).

    First of all, I have to address the currently confusing nomenclature. Aβ oligomers are referred to in several ways, i.e., Aβ dimer, Aβ trimer, low-n Aβ oligomer, and ADDLs (Lambert et al., 1998), while Aβ*56 (Lesne et al., 2006) and other high-molecular-weight oligomers are claimed. I would like to discuss here all the species of Aβ oligomers published before. In addition to these Aβ oligomer species, there are other nomenclatures dedicated to non-fibrillar Aβ assemblies, such as protofibrils, annulus, or other forms (see the review by Roychaudhuri et al., 2009) that are hard to compare to one another due to the lack of biochemical characterization and mobilities on molecular weight gels. Some of the Aβ oligomers from our model mice (Tomiyama et al., ibid.) are certainly somewhat similar to those observed previously (Walsh et al., 2002). Such low-n Aβ oligomers have been confirmed worldwide, and reproduced in several laboratories with conventional Western blot analysis, although Aβ oligomer bands with the higher Mr than trimer were usually rather faint in the blot. When we used patient CSF, or synthetic peptides with or without the deletion mutation (E693Δ), we observed more clearly Aβ oligomers with higher Mr (Tomiyama et al., 2008). Despite the different names for Aβ oligomers, it is quite likely that one Aβ oligomer actually represents all the Aβ assembly species with different names, and the discrepancy of the apparent Mr comes from the diversities of the preparations and/or detection methods used in different laboratories.

    Therefore, I undoubtedly believe that our mice also contain ADDLs (Gong et al., 2003), probably Aβ*56 or other higher-molecular-weight oligomers, although the latter Aβ species seemed to be less evident with our detection methods. This must be true because we used not only β001 but also NU-1, the monoclonal antibody that specifically recognizes ADDLs, to identify Aβ oligomers (Lambert et al., 1998). This view is further supported by our previous observation (Nishitsuji et al., 2009).

    The prominent Aβ monomer was certainly observed in our blot, not in the soluble TBS-buffer fraction nor in the Triton-buffer fraction, but in the insoluble SDS-buffer fraction and highly insoluble formic-acid (FA) fraction after the fractionation by four-step ultracentrifugation. It is, I think, noteworthy that abnormal accumulation of Aβ monomer seen in the blot was observed in these cellular compartments. We do not know at this moment whether the Aβ monomer was tightly associated with Aβ oligomers or produced in the course of the experimental preparation derived from Aβ oligomers. Further studies are needed to fully characterize both extracellular and intracellular Aβ oligomers. It is, nevertheless, a critical issue that our model animal provides evidence for increased Aβ oligomers to induce Alzheimer disease (AD)-related changes such as memory disturbance, synaptic dysfunction, neuronal death, abnormal phosphorylation of tau, glial reaction, and the decreased synaptic marker, and that these pathologies are successfully reproduced in an aging-dependent manner in the absence of amyloid plaques. In short, our mice would be a crystal AD model. We are still aware of any formation of neurofibrillary tangles yet in this model. That may need more longevity or the co-occurrence of human tau. In this particular sense, our animals could be improved to get a perfect model to completely mimic human AD.

    Finally, the present view of Aβ oligomers is obviously significant for the future diagnostic and therapeutic research on Alzheimer disease. For this purpose, pathologically active Aβ oligomers must be prepared in a reproducible and stable fashion from synthetic Aβ peptides, body fluids, and/or brain tissues to help us beat this dire disease.

    View all comments by Hiroshi Mori

References

News Citations

  1. San Diego: Oligomers Live Up to Bad Reputation, Part 2

Paper Citations

  1. . A new amyloid beta variant favoring oligomerization in Alzheimer's-type dementia. Ann Neurol. 2008 Mar;63(3):377-87. PubMed.
  2. . The E693Delta mutation in amyloid precursor protein increases intracellular accumulation of amyloid beta oligomers and causes endoplasmic reticulum stress-induced apoptosis in cultured cells. Am J Pathol. 2009 Mar;174(3):957-69. PubMed.
  3. . Monoclonal antibodies that target pathological assemblies of Abeta. J Neurochem. 2007 Jan;100(1):23-35. PubMed.
  4. . A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci. 2010 Apr 7;30(14):4845-56. PubMed.

Other Citations

  1. Tg2576

Further Reading

Papers

  1. . Alzheimer disease models and human neuropathology: similarities and differences. Acta Neuropathol. 2008 Jan;115(1):5-38. PubMed.
  2. . A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci. 2010 Apr 7;30(14):4845-56. PubMed.
  3. . The E693Delta mutation in amyloid precursor protein increases intracellular accumulation of amyloid beta oligomers and causes endoplasmic reticulum stress-induced apoptosis in cultured cells. Am J Pathol. 2009 Mar;174(3):957-69. PubMed.

News

  1. San Diego: Oligomers Live Up to Bad Reputation, Part 2

Primary Papers

  1. . A mouse model of amyloid beta oligomers: their contribution to synaptic alteration, abnormal tau phosphorylation, glial activation, and neuronal loss in vivo. J Neurosci. 2010 Apr 7;30(14):4845-56. PubMed.