Oligomerization by co-assembly of β-amyloid and α-synuclein

Aberrant self-assembly of an intrinsically disordered protein is a pathological hallmark of protein misfolding diseases, such as Alzheimer’s and Parkinson’s diseases (AD and PD, respectively). In AD, the 40–42 amino acid-long extracellular peptide, β-amyloid (Aβ), self-assembles into oligomers, which eventually aggregate into fibrils. A similar self-association of the 140 amino acid-long intracellular protein, α-synuclein (αS), is responsible for the onset of PD pathology. While Aβ and αS are primarily extracellular and intracellular polypeptides, respectively, there is evidence of their colocalization and pathological overlaps of AD and PD. This evidence has raised the likelihood of synergistic, toxic protein-protein interactions between Aβ and αS. This mini review summarizes the findings of studies on Aβ-αS interactions related to enhanced oligomerization via co-assembly, aiming to provide a better understanding of the complex biology behind AD and PD and common pathological mechanisms among the major neurodegenerative diseases.

The AD afflicted brain is also characterized by intraneuronal filamentous aggregates composed of phosphorylated tau (Twohig and Nielsen, 2019), a microtubule-binding protein that consists of 352-441 residues. The multiple repeat regions in tau are aggregation-prone (Jouanne et al., 2017). In AD, tau pathology generally occurs later relative to neuronal cell loss, memory deficit and Aβ plaque formation (Oddo et al., 2003) and can be induced by Aβ aggregates (Gotz et al., 2001;Vasconcelos et al., 2016).
The recent progresses in AD therapy and diagnosis have reinforced the critical role of oligomers in AD pathology, though no symptom-modifying therapeutic agents are currently available for PD (Paolini Paoletti et al., 2020). A couple of antibody-based AD drugs, aducanumab and lecanemab, have recently been approved by FDA (Walsh et al., 2021;Couzin-Frankel and Piller, 2022). Aducanumab targets Aβ aggregates, including oligomers and fibrils (Sevigny et al., 2016), whereas lecanemab does Aβ protofibrillar oligomers (van Dyck et al., 2023). These findings support that amyloid oligomers play a critical role in neurodegenerative diseases, though the efficacy of aducanumab and the safety of lecanemab are still debatable (Walsh et al., 2021;Couzin-Frankel and Piller, 2022). While oligomerizations of single amyloid proteins have been extensively examined (for example, see (Nguyen et al., 2021)), oligomer formation driven by interactions between multiple amyloid proteins (e.g., Aβ and αS) is relatively understudied, which is a major focus of this minireview.

Pathological synergy between AD and PD via interactions between Aβ and αS
A great deal of evidence indicates that both AD-and PDrelated symptoms are detected in some patients. For example, many (~50%) patients with AD develop LB pathology in addition to Aβ plaques and tau tangles (Irwin et al., 2013;Visanji et al., 2019). These patients exhibit faster cognitive decline and shorter lifespan compared to those with only AD pathology (Kraybill et al., 2005). Similarly, PD patients can be diagnosed with dementia (PDD) and PDD patients show more severe cognitive dysfunction than AD patients (Hamilton 2000;Irwin et al., 2013). Moreover, both dementia and parkinsonism are evident in Diffuse Lewy Body disease (DLB) (Hamilton 2000). Importunately, previous in vivo studies with transgenic (Tg) mice showed greater neuronal degeneration, stronger neuroinflammation, and more severe deficits in cognition and motor skill when Aβ and αS are both present (Larson et al., 2017;Khan et al., 2018;Lloyd et al., 2021). These findings serve as direct evidence that the synergistic connections of AD and PD pathologies are mediated by Aβ and αS (Twohig and Nielsen, 2019;Bassil et al., 2020;Murakami and Ono, 2022).

Oligomerization enhanced by Aβ-αS interactions: Clinical and animal studies
Strong evidence in the literature has shown that the synergistic toxic effects are associated with enhanced oligomerization via Aβ-αS interactions. For instance, the level of soluble αS oligomers is higher and amyloid plaque load is lower in human brain with AD and PD co-pathologies than in AD alone (Heyman et al., 1999;Tsigelny et al., 2008). A postmortem analysis showed greater amounts of toxic oligomeric αS in AD brains compared to healthy controls (Larson et al., 2017). Aβ and αS co-expression in double Tg mice enhances αS oligomerization compared to single Tg mice, while reducing Aβ fibrillar plaques (Larson et al., 2017;Khan et al., 2018). Moreover, intracerebral injections of αS-containing brain FIGURE 1 (A) Amino acid sequences of Aβ and αS. In Aβ, the hydrophobic Aβ17-21 (LVFFA) and the hydrophobic Aβ C-terminus are shown single-underlined and double-underlined, respectively. The two additional C-terminal residues of Aβ42 relative to Aβ40 are Ile-Ala shown in a parenthesis. In αS, the NAC domain is underlined. (B) Schematics of aggregations of Aβ and αS by self-assembly (solid lines) from their monomeric forms to oligomeric, and to fibrillar forms or via co-assembly (dotted lines). extracts into AD mice inhibit Aβ deposition into fibrillar plaques, presumably increasing the quantity of Aβ oligomers (Bachhuber et al., 2015).

Co-localization of Aβ and αS for direct protein-protein interactions
While Aβ and αS can facilitate oligomerization of each other indirectly, for example, by enhancing protein production (Roberts et al., 2017), a large body of evidence points to direct Aβ-αS interactions as a more effective cross-talk mechanism (Ren et al., 2019). Generally, Aβ and αS are considered as extracellular and intracellular proteins, respectively.
Nevertheless, a chance of these proteins being in spatial proximity is not negligible, permitting the direct proteinprotein interactions. For example, expression levels of αS are high in human brain regions where AD lesions are abundant (Mandal et al., 2006). In addition, co-localization of Aβ and αS (either its NAC fragment or full-length form) was detected within fibrillar plaques (Drummond et al., 2017;Bluhm et al., 2022) in clinical and animal studies and suggested to occur in cellular compartments, such as an mitochondrion (Hashimoto et al., 2003). Aβ once produced extracellularly can be accumulated intraneuronally (LaFerla et al., 2007). Likewise, αS initially produced intracellularly can be secreted into extracellular space (Lee et al., 2005). Thus, direct protein-protein interactions between Aβ and αS occur both intracellularly and    (Masliah et al., 2001;Hashimoto et al., 2003;Tsigelny et al., 2008;Kayed et al., 2020).

Impact of Aβ-αS interaction on aggregation: In vivo and in vitro studies
Studies on Aβ-αS interactions in vivo would be most ideal to understand the outcome of the interactions under a biological context. Several animal and cell culture studies have reported impact of direct Aβ-αS interactions on aggregations (Table 1). For example, Aβ promotes αS oligomerization and LB inclusions in Tg mice and cultured neurons (Masliah et al., 2001;Tsigelny et al., 2008;Larson et al., 2017;Khan et al., 2018;Lloyd et al., 2021). Aβ plaque deposition can be accelerated by αS in animal studies (Clinton et al., 2010), though the opposite effects of αS were reported elsewhere (Bachhuber et al., 2015;Khan et al., 2018;Lloyd et al., 2021). Unfortunately, conformations of amyloid proteins are crucial for their associations with other amyloid proteins, internalization mechanisms and pathological effects (Peng et al., 2018;Kayed et al., 2020), yet are difficult to control in samples of biological origin (Peng et al., 2018). Similarly, the collection of brain-derived amyloid aggregates requires sample treatments that can alter aggregation state (Casali and Lanreth, 2016), for example, the use of denaturing agents (e.g., Sarkosyl) for extraction (Ren and Sahara, 2013) or acidic buffer for elution of proteins (Puangmalai et al., 2020). Moreover, isolations of Aβ and αS from biological origins suffer from low quantities and the lack of concentration controls (Casali and Lanreth, 2016).
Instead, in vitro studies with synthetic Aβ and recombinant αS might be more suitable to examine Aβ-αS interactions for several reasons: 1) synthetic Aβ and recombinant αS injections into animal models have proven effective for studying the onset, progression and outcomes of AD and PD pathologies, and their co-pathology (Bloom 2014;McAllister et al., 2020); 2) conformations and aggregation states of Aβ and αS − critical for the severity and the phenotype of pathology − are relatively easier to control and characterize in vitro than in vivo (McAllister et al., 2020), permitting an understanding of molecular outcomes from specific Aβ-αS interactions. In previous in vitro studies (Table 1), Aβ enhanced αS aggregation (Ono et al., 2012;Koppen et al., 2020), including αS oligomerization (Masliah et al., 2001;Tsigelny et al., 2008). αS can either accelerate (Ono et al., 2012) or inhibit Aβ aggregation in vitro (Bachhuber et al., 2015). Unfortunately, the intrinsic complexity associated with different conformers of Aβ and αS (i.e., monomeric, oligomeric, and fibrillar forms) has precluded the identification of exact synergistic mechanisms linked to AD, PD and their copathologies. Moreover, most previous studies have provided limited insight into conformer-specific Aβ-αS interactions. This limitation arose because aggregation was frequently illdefined with no distinction between oligomerization and fibrilization. In addition, Aβ-αS interactions were often characterized under denaturing conditions (e.g., with SDS), which can introduce undesired artifacts on aggregation states, complicating data interpretation. Other histopathology and injection studies have focused on fibrils, underestimating the role of oligomers in comorbidities.

Conformer-specific Aβ-αS interactions: αS-assisted oligomerization of Aβ in vitro
Recent in vitro studies by Kim and coworkers have provided experimental evidence of Aβ-αS co-assembly into potentially toxic oligomers, revealing important insights into the nature of Aβ-αS interactions: excess soluble αS species (i.e., αS monomers and oligomers) prevent fibrillization of Aβ40 while enhancing oligomerization of Aβ40 ( Figure 2; Table 1). Though the αS monomer concentration tested in this study was 350 μM, exceeding the physiological αS concentrations (i.e., low μM range (Wilhelm et al., 2014)), oligomeric αS at 17 μM, which is slightly above the physiological αS concentrations, was effective for enhancing Aβ oligomerization in vitro. Thus, this observation suggests a possibility of oligomeric αS seeding Aβ oligomerization in vivo. Moreover, in vivo αS concentrations can be elevated locally by various mechanisms (Owen et al., 2019), which may promote the formation of αS oligomers. Once formed, these oligomers are kinetically stable at < μM concentrations (Nag et al., 2011), available for enhancing Aβ oligomerization. In the same study, the C-terminus of Aβ40 was shown to be directly involved in interactions with αS, facilitating the formation of co-assembled oligomers. The competitive immunoassay provides evidence for binding of Aβ40 in all three forms (i.e. monomeric, oligomeric and fibrillar Aβ40) to αS fibrils via the Aβ C-terminus (Candreva et al., 2020). In addition, aggregations of αS monomers and oligomers were facilitated by Aβ40 fibrils (Candreva et al., 2020), as reported elsewhere (Ono et al., 2012). Interestingly, when judged by Direct Aβ-αS interaction networks. Blue arrows: common to both Aβ40-αS and Aβ42-αS (Candreva et al., 2020;Chau and Kim, 2022). Addition of αS monomers and oligomers inhibits fibrilization of monomeric (m-) and oligomeric (o-) Aβ, while promoting oligomerization of Aβ monomers and stabilizing preformed Aβ oligomers via co-assembly. Monomeric, oligomeric, and fibrillar (f-) Aβ can be incorporated into αS fibrils. Red arrows: specific to Aβ42-αS (Chau and Kim, 2022). αS fibrils partially interferes with conversion of soluble Aβ42, but not Aβ40, into insoluble aggregates, instead possibly promoting the formation of large oligomeric Aβ42.

Frontiers in Molecular Biosciences
frontiersin.org thioflavin T fluorescence, incorporation of αS into fibrillar Aβ40 or Aβ40 into fibrillar αS was relatively minor or slow compared to co-assembled oligomerization (Candreva et al., 2020). The implication is that Aβ40-αS interactions might occur more drastically for the formation of oligomers rather than fibrils.
The subsequent study showed both similarities and dissimilarities between Aβ42-αS and Aβ40-αS interactions. Specifically, while monomeric and oligomeric αS promoted oligomerization of Aβ42, similar to Aβ40, αS fibrils induced the formation of large Aβ42 oligomers -a finding unique to Aβ42 (Figure 2; Table 1) (Chau and Kim, 2022). The C-terminus of Aβ42 is primarily utilized for its interactions with αS, similar to Aβ40, yet other regions of Aβ42 (e.g., Aβ22-35) are also involved (Chau and Kim, 2022). A molecular dynamics simulation study suggested that direct Aβ42-NAC interactions induce the formation of new β-strands in Aβ42 while the NAC domain remains structurally unchanged (Atsmon-Raz and Miller, 2016). A similar structural study with Aβ and the full-length αS will provide valuable insight into the molecular determinants crucial in Aβ-αS interactions.
Overall, these studies demonstrate that Aβ and αS can directly interact to form Aβ-αS co-assembled oligomers, possibly responsible for the enhanced oligomerization in vivo when Aβ and αS co-exist. Thus, Aβ-αS interactions provide a synergistic mechanism to produce oligomeric assemblies and may have common biological consequences in AD, PD, DLB and PDD, given that amyloid oligomers are usually neurotoxic (Ahmed et al., 2010;Giehm et al., 2011).

Aβ-αS interactions in a biological context
For a better understanding of pathogenic overlap between AD and PD, how the conformer-specific Aβ-αS interactions are manifested in a biological context would be a critical first step (Murakami and Ono, 2022). This biological examination would interrogate the pathological relevance of Aβ-αS interactions, closing a knowledge gap in a link among Aβ-αS co-assembly, oligomerization and synergistic toxic effects. Intracellular and extracellular co-localizations of Aβ and αS (Chinta et al., 2010;Drummond et al., 2017) raise a possibility of transportation across cell membranes of oligomers formed by Aβ-αS interactions, spreading their toxic effects (Kayed et al., 2020). Given αS's ubiquitous and abundant expression in brain and strong innate ability to shuttle between intra-and extracellular compartments (Irwin et al., 2013), the role of αS in orchestrating a pathophysiological ensemble of AD and PD by enhancing Aβ oligomerization is of a particular therapeutic relevance (Kayed et al., 2020). Moreover, the co-assembly of Aβ and αS may facilitate circumvention of their proteolytic clearances, possibly increasing their toxic effects in vivo. Thus, a strategy to modulate the interactions would open a new therapeutic avenue, beyond targeting self-assembly of a single amyloid protein adopted by most anti-amyloid therapeutic strategies developed against AD and PD (Murakami and Ono, 2022).

Additional complexity
While Aβ-αS interactions may provide additional routes to form oligomeric species beyond aggregation of individual amyloid proteins, heterogeneity of oligomers in size, morphology, structure, and seeding ability further increases pathological complexity. Aβ and αS oligomers of biological or synthetic origins range from dimers to~200 mers (Breydo and Uversky, 2015) and display different morphologies, such as globular and protofibrillar shapes . While many Aβ and αS oligomers possess well-defined secondary structures, usually β-sheets, others are structurally disordered (Breydo and Uversky, 2015;Gao et al., 2020). Moreover, amyloid oligomers can be either on-pathway intermediates of fibrillization or off-pathway end products (Breydo and Uversky, 2015), differing in seeding ability. Inhibitions of Aβ42 fibrillization by off-pathway αS oligomers -generated by forming covalent adducts with a dopamine metabolite or a polyunsaturated fatty acid -have recently been reported (Dhakal et al., 2021), though whether the modulations lead to Aβ-αS co-assembly into oligomers remains unclear. In this study, interactions of Aβ42 with the two structurally distinct off-pathway αS oligomers were found to promote the formation of Aβ42 assemblies that differ in toxicity, suggesting the importance of αS oligomer conformation in the observed phenotypes and clinical manifestations (Dhakal et al., 2021). Thus, testing with specific subgroups of Aβ and αS oligomers for the outcomes of their interactions would further depict the heterogeneitydriven complexity of the interactions.
The Aβ-αS interaction may extend further with other amyloidogenic and non-amyloidogenic proteins, increasing the boundaries of interaction networks (Murakami and Ono, 2022). For example, tau is an intracellular amyloid protein, whose filamentous aggregates are found in the AD afflicted brain (Buee et al., 2000;Twohig and Nielsen, 2019). Tau is highly abundant as soluble monomers and does not spontaneously aggregate under the physiological condition (Jouanne et al., 2017;Visanji et al., 2019), yet its aggregation into oligomers and filaments related to neurotoxicity can be induced by Aβ following phosphorylation-dependent and independent mechanisms (Jin et al., 2011;Vasconcelos et al., 2016;Gyparaki et al., 2021). Similar to Aβ and αS, oligomeric rather than monomeric or filamentous tau is toxic (Ghag et al., 2018) and responsible for neuronal dysfunction in AD (Spires et al., 2006). αS can also induce tau aggregation in both phosphorylationdependent (Twohig and Nielsen, 2019) and independent manners (Giasson et al., 2003). αS oligomers rather than αS monomers and fibrils catalyze tau oligomerization (Lasagna-Reeves et al., 2010) and αS and tau can promote their mutual aggregations (Giasson et al., 2003;Dasari et al., 2019). Thus, the direct Aβ-αS interactions extend further with tau through the shared release, trafficking and uptake mechanisms (Twohig and Nielsen, 2019;Visanji et al., 2019), that allow intra-and intercellular propagation of pathological seeds of Aβ, αS and tau (Frost et al., 2009;Irwin et al., 2013;Kayed et al., 2020), initiating an autocatalytic cycle of aggregation (Vasconcelos et al., 2016) and spreading pathology (Lloyd et al., 2021). The protein-protein interaction network centered on Aβ-αS can also extend with a nonamyloidogenic protein, such as DNA-binding protein TDP-43, known to bind to αS (Dhakal et al., 2022).

Conclusion
Aβ-αS interactions leading to the formation of co-assembled oligomers and their propagation are deemed responsible for pathological complexity, overlap, and heterogeneity of AD, PD and other major neurodegenerative diseases. Thus, a better understanding of Aβ-αS interactions will be required to close current knowledge gaps and point to new therapeutic strategies targeting oligomerization.

Author contributions
JRK collected materials, wrote the manuscript and approved its submission.

Funding
This work was supported by the National Institutes of Health [grant number R21AG049137].

Conflict of interest
The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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