br Acknowledgements This work was supported
Acknowledgements This work was supported by National Natural Science Foundation of China (Nos. 21376172, 21528601 and 21621004) and the Natural Science Foundation of Tianjin from Tianjin Municipal Science and Technology Commission (Contract No. 16JCZDJC32300).
Introduction Alzheimer’s disease (AD), the most common cause of dementia affecting the elderly, is a progressive neurodegenerative disorder characterized pathologically by the accumulation of intracellular neurofibrillary tangles and extracellular neuritic plaques (Masters and Selkoe, 2012, De Strooper and Karran, 2016). The primary components of the plaques are 39–43 amino Cy3 RNA receptor long amyloid-β (Aβ) peptides, derived from the amyloid precursor protein (APP) by sequential cleavage via β- and γ-secretases (Haass et al., 2012, Maulik et al., 2013, Andrew et al., 2016). Multiple lines of experimental evidence indicate that the accumulation of Aβ in the brain contributes to the loss of neurons and subsequent development of AD pathology (Poduslo et al., 2010, Revett et al., 2013, De Strooper and Karran, 2016). Physiologically, Aβ peptide predominantly exists in two isoforms, Aβ1–40 and Aβ1–42. Aβ1–42 is hypothesized to be the main culprit involved in AD pathology as it exhibits greater neuronal toxicity and is believed to be the principal constituent of diffuse/neuritic plaques (Irie et al., 2005, Masters and Selkoe, 2012, Zheng et al., 2015). Due to its amphiphilic nature and high hydrophobic properties, Aβ1–42 is able to self-aggregate into a variety of stable structures ranging from oligomers to amyloid fibrils (Irie et al., 2005, Masters and Selkoe, 2012, Thal et al., 2015). It is generally accepted that oligomeric forms of Aβ1–42 are the predominant isoform involved in cell toxicity (Irie et al., 2005, Hayden and Teplow, 2013, Tipping et al., 2015), but given the many different types of oligomeric Aβ (Benilova et al., 2012) it is not clear if or how their specific aggregate structure relates to cell toxicity and AD pathogenicity. The nature of an Aβ aggregate can be influenced by its primary sequence. The normal peptide contains a hydrophilic N-terminus (residues 1–16), a hydrophobic central domain (residues 17–21), a hydrophilic linker region (residues 22–30) and a hydrophobic C-terminus (residues 31–42) (Fig. 1) (Kang et al., 1987). While monomeric Aβ is largely flexible, its amyloid form contains a C-terminal core β-hairpin motif with a flexible hydrophilic N-terminus (Lührs et al., 2005, Ahmed et al., 2010). Mutations within the Aβ sequence can be associated with disease; many of these mutations are located adjacent to the central hydrophobic domain [Flemish (A21G), Dutch (E22Q), Arctic (E22G), Italian (E22K), Iowa (D23N)] where they can predictably interfere with amyloid core formation (Dahlgren et al., 2002, Irie et al., 2005). However, there are a number of N-terminal mutations that can also affect disease susceptibility and may do so by affecting oligomer formation. For example, the English (H6R) and Tottori (D7N) mutant Aβ peptides produce larger oligomers (Hayden and Teplow, 2013), and the Taiwanese (D7H) isoform has been shown to produce more stable oligomers (Chen et al., 2012). More evidence for a role of the N-terminus in Aβ pathogenesis comes from studies of mice and rats. These rodents produce Aβ but do not develop extracellular plaques or AD, possibly due to their differences in three amino acids (R5G, Y10F, and H13R) situated within the hydrophilic N-terminal domain of the peptide (De Strooper et al., 1995). Previous studies have demonstrated differences in Aβ aggregation rates and cytotoxicity between the human (hAβ1–42) and rat (rAβ1–42), where the human peptide was more prone to fibrillization and induced higher cellular toxicity (Edrey et al., 2013, Lv et al., 2013). Interestingly, single mutation studies of H13R (human toward rodent), rather than showing an intermediate aggregation profile, showed increased fibrillization of the mutant but with a reduced toxicity (Poduslo et al., 2010). Single mutation studies of Y10F (human toward rodent) also demonstrated increased aggregation in the mutant despite a lower toxicity (Dai et al., 2012). Thus, the correlation between fibrillization potential and toxicity is not always direct. To better understand whether N-terminal mutations can affect hAβ1–42 toxicity through influences on fibril formation or oligomerization, we generated double mutants (human toward rodent – Fig. 1) to contrast with the existing literature, which has largely examined single mutants (human toward rodent). Apart from determining fibrillization kinetics, oligomer sizes and toxicity profiles of these peptides, we also tested the peptides in combination with hAβ1–42, to assess for dominant negative or positive effects upon hAβ1–42 aggregation and toxicity.