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    • While there is much in vivo work

    2023-01-14

    While there is much in vivo work on prion-like Aβ, it has not been shown that one can induce inclusions of Aβ in cultured cells as has been shown for tau and α-synuclein. One reason is practical; Aβ is a low molecular weight metabolite cleaved from within the larger amyloid precursor protein (APP) and it is therefore less feasible to construct a cell line expressing physiologically generated, fluorescently labeled Aβ. This makes it difficult to study Aβ inclusions and transfer in cells. There is also a theoretical reason; as Aβ plaques are extracellular, intracellular Aβ has been viewed as less relevant. However, intraneuronal Aβ42 accumulation is seen before plaques in the brain areas first affected by AD and appears to be among the earliest changes in AD (Gouras et al., 2000) and is associated with synaptic pathology (Takahashi et al., 2002). Accumulation of intraneuronal Aβ coincides with cognitive symptoms and occurs before plaques in the 3xTg AD mouse (Billings et al., 2005). Recently it was also reported that FAD mutations in presenilin 2 specifically increase the intracellular pool of Aβ42 (Sannerud et al., 2016). Furthermore, if conversion of monomeric to aggregated/prion-like Aβ is a stochastic process (Goedert, 2015), one would expect the biological conversion to happen where concentrations are high as they are in subcellular compartments (Hu et al., 2009); the acidic environment of the late endosome/lysosome also favors Aβ aggregation (Burdick et al., 1992). Thus, if Aβ has prion-like properties, one might expect intracellular prion-like conversion as is the case for prion protein and other prion-like proteins (Aguzzi and Lakkaraju, 2016; Luk et al., 2009; Sanders et al., 2014).
    Materials and methods
    Results
    Discussion A major question in the field is which forms of Aβ have seeding capacity (Jucker and Walker, 2011). Here we provide data suggesting that intracellular fibrillar oligomers of Aβ can be a seeding unit. We demonstrate that seeded nucleation of Aβ can be induced intracellularly in an APP producing cell line by treatment with mouse FAD brain extract. Native PAGE, labeling with conformation dependent Octreotide acetate sale and infrared spectroscopy support that the inclusions were oligomeric, and in the terminology introduced by Kayed et al., “fibrillar oligomers” (Kayed et al., 2007). Furthermore, SDS treatment revealed SDS-resistant low n-oligomers, indicating aggregates reminiscent of amyloid and prion aggregates (Bagriantsev et al., 2006). Moreover, cells with induced Aβ aggregates could seed Aβ aggregation in naive APP-expressing cells, thus demonstrating seeded nucleation with a purely intracellular source of Aβ. Remarkably, native PAGE and infrared spectroscopy indicated that the Aβ oligomers from the parent clone were similar to those from the daughter clone. These data are consistent with formation of fibrillar oligomeric strains of intracellular prion-like Aβ that can be transmitted both vertically and horizontally. Numerous studies underscore an early role of intraneuronal Aβ in AD pathogenesis (Billings et al., 2005; Gouras et al., 2010; Takahashi et al., 2002). According to the prion-like hypothesis this stochastic formation of prion-like Aβ would be one of the earliest steps in AD. Though most Aβ is normally secreted, the probability of stochastic conversion of physiological Aβ to prion-like/seeding Aβ should depend on concentration (Goedert, 2015) and the highest concentrations of soluble Aβ are found in intracellular vesicles (Hu et al., 2009). Furthermore, it has been reported, and we also observe, that intracellular accumulation and aggregation of added synthetic Aβ42 increased cleavage of APP into Aβ and increases levels of APP (Davis-Salinas et al., 1995; Yang et al., 1995, Yang et al., 1999), potentially creating a positive feedback loop of increased intracellular Aβ aggregation and production. Yang et al. did not investigate whether this intracellular Aβ aggregation persisted over replicative generations. It is possible that they achieved intracellular prion-like conversion with synthetic Aβ, albeit with the extremely high concentration of 25 μM Aβ42, whereas our treatment of cells with brain extract contains approximately 20 nM Aβ and with prion-like cell extract about 2 nM. Considering the results of Yang et al., the fact that in vivo seeding of Aβ can be achieved with synthetic Aβ (Kummer et al., 2011; Stöhr et al., 2012) and that α-synuclein and tau seeding can be done with synthetic fibrils, it's likely that in vitro prion-like intracellular seeding can be accomplished with some specific aggregation states and concentrations of synthetic Aβ.