. Systematic in vivo analysis of the intrinsic determinants of amyloid Beta pathogenicity. PLoS Biol. 2007 Oct 30;5(11):e290. PubMed.

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  1. Homing In on the Molecular Determinants of Aβ Assembly and Toxicity: Lessons from the Computer, Test Tube, and Fly

    The paper by Luheshi et al. describes the direct comparison of in silico, in vitro, and in vivo analysis of Aβ aggregation. It builds on many years of computational modeling by Vendruscolo and Dobson and animal modelling by Crowthers and Lomas, and it strongly suggests that protofibrillar assemblies of Aβ are the primary neurotoxic species both in their fly models and perhaps in brains of patients suffering with Alzheimer disease.

    Using a previously described algorithm, the authors estimated intrinsic aggregation propensities of all 419 possible single point mutations of Aβ1-42 and all 379 single point mutations of the more toxic Aβ1-42E22G. Based on these data, they selected 15 mutations predicted to produce peptides with a broad range of aggregation propensities. The authors then generated flies transgenic for each of the 15 mutant peptides, plus flies transgenic for wild-type Aβ40 and Aβ42. In each case four to six independent lines for each of the 17 Aβ sequences were generated, i.e., a total of 68-102 different individual transgenic fly lines. After completing this gigantic task, the authors then set about characterizing the transgenic flies.

    Certain mutations, such as E22G, caused a dramatic reduction in both the life span and locomotor ability of flies, whereas other mutations had the opposite effect. Strikingly, comparison of intrinsic aggregation propensity versus longevity or locomotor activity revealed a strong correlation. This demonstrates a direct link between aggregation and decreased locomotion/longevity.

    However, the correlation was not perfect, and one particular mutation, I31E/E22G, did not fit this trend. In vitro analysis of the fibril-forming propensity of the I31E/E22G confirmed that this peptide had a similar aggregation propensity as did the E22G peptide. Moreover, when the authors examined brain from flies expressing I31E/E22G and E22G, they found highly similar levels of amyloid deposits. So if I31E/E22G flies had abundant amyloid deposits, why did they not exhibit a phenotype similar to E22G flies?

    The suggested answer came from careful immunohistochemical analysis, which indicated that the E22G flies had not only profound Aβ deposition, but also substantial vacuolation, whereas vacuoles were absent in brains of I31E/E22G flies. Since both the I31E/E22G and E22G flies had abundant amyloid deposits, this led the authors to speculate that something other than amyloid fibrils was precipitating vacuole formation. Mindful of the burgeoning evidence that non-fibrillar soluble forms of Aβ play an important role in cognitive impairment, the authors revised their approach and developed a second algorithm designed to predict the relative propensity of proteins to form protofibrils (PFs). Comparison of predicted propensity of different Aβ mutations to form PF with the relative change in longevity or locomotor ability yielded a substantially improved correlation between that predicted in silico and that observed in flies.

    This is an impressive piece of work, but like all leaps forward it raises more questions than it answers. Specifically, while the second algorithm was designed to predict PF forming propensity, it is not clear what the authors actually define as PF. Also unclear is whether the algorithm can predict the formation of structures other than PFs; for instance, what if the algorithm predicts the formation of low-n oligomers? Whatever the answer, it will be extremely interesting to isolate Aβ species from brains of the different mutant flies and attempt to identify the Aβ assembly form that precipitates their impaired locomotion and untimely death.

  2. Aggregation Propensity of Aβ Predicts Longevity of Fly Model of AD
    AD pathology is strongly associated with initial stages of amyloid-β protein (Aβ) aggregation. At different stages of Aβ assembly, Aβ oligomers, protofibrils, and fibrils are observed which differ in structure as well as toxic function. In particular, earlier assemblies, oligomers, are known to be toxic to cells in cell cultures and in a transgenic mouse model. While Aβ oligomers seem to be involved in cell death, it is a lot less clear how Aβ oligomers mediate their toxic function. There is an ongoing debate on intra- versus extracellular assembly processes that are associated with toxicity. There are numerous studies indicating strong and potentially disruptive interactions between Aβ oligomers and lipid bilayers, some suggesting that Aβ assemblies form ion channels in the cell membrane, thereby inducing abnormal calcium transport and consequently cell death. It is quite possible that there is more than one potentially toxic pathway of Aβ assembly.

    Given the complexity of Aβ oligomer-mediated toxicity in humans and in transgenic mouse models, it is thus quite surprising to encounter a study that shows a clear correlation between the protein's primary structure, which determines its aggregation propensity, and in vivo consequences of such aggregation. In an extensive in vivo-->in silico study, Leila Luheshi and colleagues link the aggregation propensity of full-length Aβ to the neuronal dysfunction in a Drosophila model of AD. Luheshi et al. used single point mutations of Aβ42 wild-type (WT) and its Arctic mutant E22G in combination with a previously reported algorithm to calculate intrinsic aggregation propensities based only on the amino acid sequence. They selected 17 mutational variants out of 798 total, and expressed them throughout the central nervous system of fruit flies. The longevity and locomotor ability of multiple lines of flies for each variant were compared to Aβ42-WT and Aβ42-E22G. Luheshi et al. found a statistically significant correlation between the propensity of a variant to aggregate and its effect on the longevity as well as locomotor ability.

    There were a few exceptions, most notably Aβ42-I31E/E22G, where there was no correlation between the predicted aggregation propensity (which was found to be similar to that of Aβ42-E22G) and its effect on longevity and locomotor ability. Further examination of the Aβ42-I31E/E22G showed that while this variant has a high propensity to aggregate into fibrils (similar to Aβ42-E22G), it does not create vacuoles in the brain of flies, which translates into a lack of neurodegeneration. When Luheshi et al. adjusted their computational approach to calculation of propensity to form protofibrils instead of fibrils, they found a significantly stronger correlation between the protofibril formation propensity and locomotor activity/longevity.

    As Dominic Walsh noted before me, the definition of a protofibril that Luheshi et al. use in their redesigned computational approach is not clearly explained, but that is crucial to a deeper understanding of processes leading to various longevities of different fly variants. It is not necessarily clear what the cause of death of the flies was. Was a reduced/enhanced longevity in all variants related to increased/decreased neuronal loss? Did Aβ soluble oligomers form at all or is Aβ oligomerization inhibited in this animal model? In a naturally occurring human mutation, i.e., the E22G Arctic, protofibril formation is enhanced [1]. However, recent work by Cheng et al. in Lennart Mucke's lab demonstrated in transgenic mice that overexpress Aβ with the Arctic mutation a significantly higher propensity to form fibrils compared to the wild-type, but reduced functional deficits and reduced levels of deficit-causing Aβ*56 oligomers [2].

    We have to be cautious when extrapolating results from one species to another, in particular because Aβ is very sensitive to relatively small changes in the environment, such as pH, and has a high propensity to interact with other proteins. The present findings by Luheshi et al. provide important insights into aberrant Aβ aggregation and its deleterious effects. They also raise a series of questions that will hopefully be addressed in future studies.

    References:

    . The 'Arctic' APP mutation (E693G) causes Alzheimer's disease by enhanced Abeta protofibril formation. Nat Neurosci. 2001 Sep;4(9):887-93. PubMed.

    . Accelerating amyloid-beta fibrillization reduces oligomer levels and functional deficits in Alzheimer disease mouse models. J Biol Chem. 2007 Aug 17;282(33):23818-28. PubMed.

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