Distinct neurotoxic TDP-43 fibril polymorphs are generated by heterotypic interactions with α-Synuclein.

Amyloid aggregates of specific proteins constitute important pathological hallmarks in many neurodegenerative diseases, defining neuronal degeneration and disease onset. Recently, increasing numbers of patients show comorbidities and overlaps between multiple neurodegenerative diseases, presenting distinct phenotypes. Such overlaps are often accompanied by colocalizations of more than one amyloid protein, prompting the question of whether direct interactions between different amyloid proteins could generate heterotypic amyloids. To answer this question, we investigated the effect of α-synuclein (αS) on the DNA-binding protein TDP-43 aggregation inspired by their coexistence in pathologies such as Lewy body dementia and limbic predominant age-related TDP-43 encephalopathy. We previously showed αS and prion-like C-terminal domain (PrLD) of TDP-43 synergistically interact to generate toxic heterotypic aggregates. Here, we extend these studies to investigate whether αS induces structurally and functionally distinct polymorphs of PrLD aggregates. Using αS-PrLD heterotypic aggregates generated in two different stoichiometric proportions, we show αS can affect PrLD fibril forms. PrLD fibrils show distinctive residue level signatures determined by solid state NMR, dye-binding capability, proteinase K (PK) stability, and thermal stability toward SDS denaturation. Furthremore, by gold nanoparticle labeling and transmission electron microscopy, we show the presence of both αS and PrLD proteins within the same fibrils, confirming the existence of heterotypic amyloid fibrils. We also observe αS and PrLD colocalize in the cytosol of neuroblastoma cells and show that the heterotypic PrLD fibrils selectively induce synaptic dysfunction in primary neurons. These findings establish the existence of heterotypic amyloid and provide a molecular basis for the observed overlap between synucleinopathies and TDP-43 proteinopathies.

De novo amyloid peptides with subtle sequence variations differ in their self-assembly and nanomechanical properties.

Proteinaceous amyloids are well known for their widespread pathological roles but lately have emerged also as key components in several biological functions. The remarkable ability of amyloid fibers to form tightly packed conformations in a cross ß-sheet arrangement manifests in their robust enzymatic and structural stabilities. These characteristics of amyloids make them attractive for designing proteinaceous biomaterials for various biomedical and pharmaceutical applications. In order to design customizable and tunable amyloid nanomaterials, it is imperative to understand the sensitivity of the peptide sequence for subtle changes based on amino acid position and chemistry. Here we report our results from four rationally-designed amyloidogenic decapeptides that subtly differ in hydrophobicity and polarity at positions 5 and 6. We show that making the two positions hydrophobic renders the peptide with enhanced aggregation and material properties while introducing polar residues in position 5 dramatically changes the structure and nanomechanical properties of the fibrils formed. A charged residue at position 6, however, abrogates amyloid formation. In sum, we show that subtle changes in the sequence do not make the peptide innocuous but rather sensitive to aggregation, reflected in the biophysical and nanomechanical properties of the fibrils. We conclude that tolerance of peptide amyloid for changes in the sequence, however small they may be, should not be neglected for the effective design of customizable amyloid nanomaterials.

Charge and redox states modulate granulin-TDP-43 coacervation toward phase separation or aggregation.

Cytoplasmic inclusions containing aberrant proteolytic fragments of TDP-43 are associated with frontotemporal lobar degeneration (FTLD) and other related pathologies. In FTLD, TDP-43 is translocated into the cytoplasm and proteolytically cleaved to generate a prion-like domain (PrLD) containing C-terminal fragments (C25 and C35) that form toxic inclusions. Under stress, TDP-43 partitions into membraneless organelles called stress granules (SGs) by coacervating with RNA and other proteins. To study the factors that influence the dynamics between these cytoplasmic foci, we investigated the effects of cysteine-rich granulins (GRNs 1-7), which are the proteolytic products of progranulin, a protein implicated in FTLD, on TDP-43. We show that extracellular GRNs, typically generated during inflammation, internalize and colocalize with PrLD as puncta in the cytoplasm of neuroblastoma cells but show less likelihood of their presence in SGs. In addition, we show GRNs and PrLD coacervate to undergo liquid-liquid phase separation (LLPS) or form gel- or solid-like aggregates. Using charge patterning and conserved cysteines among the wild-type GRNs as guides, along with specifically engineered mutants, we discover that the negative charges on GRNs drive LLPS while the positive charges and the redox state of cysteines modulate these phase transitions. Furthermore, RNA and GRNs compete and expel one another from PrLD condensates, providing a basis for GRN's absence in SGs. Together, the results help uncover potential modulatory mechanisms by which extracellular GRNs, formed during chronic inflammatory conditions, could internalize and modulate cytoplasmic TDP-43 inclusions in proteinopathies.

Ganglioside-Enriched Phospholipid Vesicles Induce Cooperative Aß Oligomerization and Membrane Disruption.

A major hallmark of Alzheimer's disease (AD) is the accumulation of extracellular aggregates of amyloid-ß (Aß). Structural polymorphism observed among Aß fibrils in AD brains seem to correlate with the clinical subtypes suggesting a link between fibril polymorphism and pathology. Since fibrils emerge from a templated growth of low-molecular-weight oligomers, understanding the factors affecting oligomer generation is important. Membrane lipids are key factors to influence early stages of Aß aggregation and oligomer generation, which cause membrane disruption. We have previously demonstrated that conformationally discrete Aß oligomers can be generated by modulating the charge, composition, and chain length of lipids and surfactants. Here, we extend our studies into liposomal models by investigating Aß oligomerization on large unilamellar vesicles (LUVs) of total brain extracts (TBE), reconstituted lipid rafts (LRs), or 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC). Varying the vesicle composition by specifically increasing the amount of GM1 gangliosides as a constituent, we found that only GM1-enriched liposomes induce the formation of toxic, low-molecular-weight oligomers. Furthermore, we found that the aggregation on liposome surface and membrane disruption are highly cooperative and sensitive to membrane surface characteristics. Numerical simulations confirm such a cooperativity and reveal that GM1-enriched liposomes form twice as many pores as those formed in the absence GM1. Overall, this study uncovers mechanisms of cooperativity between oligomerization and membrane disruption under controlled lipid compositional bias, and refocuses the significance of the early stages of Aß aggregation in polymorphism, propagation, and toxicity in AD.

Biophysical characteristics of lipid-induced Aß oligomers correlate to distinctive phenotypes in transgenic mice.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder that affects cognition and memory. Recent advances have helped identify many clinical sub-types in AD. Mounting evidence point toward structural polymorphism among fibrillar aggregates of amyloid-ß (Aß) to being responsible for the phenotypes and clinical manifestations. In the emerging paradigm of polymorphism and prion-like propagation of aggregates in AD, the role of low molecular weight soluble oligomers, which are long known to be the primary toxic agents, in effecting phenotypes remains inconspicuous. In this study, we present the characterization of three soluble oligomers of Aß42, namely 14LPOs, 16LPOs, and GM1Os with discreet biophysical and biochemical properties generated using lysophosphatidyl glycerols and GM1 gangliosides. The results indicate that the oligomers share some biophysical similarities but display distinctive differences with GM1Os. Unlike the other two, GM1Os were observed to be complexed with the lipid upon isolation. It also differs mainly in detection by conformation-sensitive dyes and conformation-specific antibodies, temperature and enzymatic stability, and in the ability to propagate morphologically-distinct fibrils. GM1Os also show distinguishable biochemical behavior with pronounced neuronal toxicity. Furthermore, all the oligomers induce cerebral amyloid angiopathy (CAA) and plaque burden in transgenic AD mice, which seems to be a consistent feature among all lipid-derived oligomers, but 16LPOs and GM1Os displayed significantly higher effect than the others. These results establish a correlation between molecular features of Aß42 oligomers and their distinguishable effects in transgenic AD mice attuned by lipid characteristics, and therefore help bridge the knowledge gap in understanding how oligomer conformers could elicit AD phenotypes.

Granulins modulate liquid-liquid phase separation and aggregation of the prion-like C-terminal domain of the neurodegeneration-associated protein TDP-43.

TAR DNA-binding protein 43 (TDP-43) has emerged as a key player in many neurodegenerative pathologies, including frontotemporal lobar degeneration (FTLD) and amyotrophic lateral sclerosis (ALS). Hallmarks of both FTLD and ALS are the toxic cytoplasmic inclusions of the prion-like C-terminal fragments of TDP-43 CTD (TDP-43 C-terminal domain), formed upon proteolytic cleavage of full-length TDP-43 in the nucleus and subsequent transport to the cytoplasm. Both full-length TDP-43 and its CTD are also known to form stress granules by coacervating with RNA in the cytoplasm during stress and may be involved in these pathologies. Furthermore, mutations in the PGRN gene, leading to haploinsufficiency and diminished function of progranulin (PGRN) protein, are strongly linked to FTLD and ALS. Recent reports have indicated that proteolytic processing of PGRN to smaller protein modules called granulins (GRNs) contributes to FTLD and ALS progression, with specific GRNs exacerbating TDP-43-induced cytotoxicity. Here we investigated the interactions between the proteolytic products of both TDP-43 and PGRN. Based on structural disorder and charge distributions, we hypothesized that GRN-3 and GRN-5 could interact with the TDP-43 CTD. We show that, under both reducing and oxidizing conditions, GRN-3 and GRN-5 interact with and differentially modulate TDP-43 CTD aggregation and/or liquid-liquid phase separation in vitro GRN-3 promoted insoluble aggregates of the TDP-43 CTD while GRN-5 mediated liquid-liquid phase separation. These results constitute the first observation of an interaction between GRNs and TDP-43, suggesting a mechanism by which attenuated PGRN function could lead to familial FTLD or ALS.

Are granulins copper sequestering proteins?

Granulins (GRN 1-7) are short (~6 kDa), cysteine-rich proteins that are generated upon the proteolytic processing of progranulin (PGRN). These peptides, along with their precursor, have been implicated in multiple pathophysiological roles, especially in neurodegenerative diseases. Previously we showed that GRN-3 and GRN-5 are fully disordered in the reduced form implicating redox sensitive attributes to the proteins. Redox-based modulations are often carried out by metalloproteins in mitigating oxidative stress and maintaining metal-homeostasis within cells. To probe whether GRNs play a role in metal sequestration, we tested the metal binding propensity of the reduced forms of GRNs -3 and - 5 under neutral and acidic pH mimicking cytosolic and lysosomal conditions, respectively. We found, at neutral pH, both GRNs selectively bind Cu and no other divalent metal cations, with a greater specificity for Cu(I). Binding of Cu did not result in a disorder-to-order structural transition but partly triggered the multimerization of GRNs via uncoordinated cystines at both pH conditions. Overall, the results indicate that GRNs -3 and - 5 have surprisingly strong affinity for Cu in the pM range, comparable to other known copper sequestering proteins. The results also hint at a potential of GRNs to reduce Cu(II) to Cu(I), a process that has significance in mitigating Cu-induced ROS cytotoxicity in cells. Together, this report uncovers metal-coordinating property of GRNs for the first time, which may have profound significance in their structure and pathophysiological functions.

Cloning, expression and purification of the low-complexity region of RanBP9 protein.

Recombinant expression and purification of proteins is key for biochemical and biophysical investigations. Although this has become a routine and standard procedure for many proteins, intrinsically disordered ones and those with low complexity sequences pose difficulties. Proteins containing low complexity regions (LCRs) are increasingly becoming significant for their roles in both normal and pathological processes. Here, we report cloning, expression and purification of N-terminal LCR of RanBP9 protein (Nt-RanBP9). RanBP9 is a scaffolding protein present in both cytoplasm and nucleus that is implicated in many cellular processes. Nt-RanBP9 is a poorly understood region of the protein perhaps due to difficulties posed by the LCR. Indeed, conventional methods presented difficulties in Nt-RanBP9 cloning due to its high GC content resulting in insignificant protein expression. These led us to use a different approach of cloning by expressing the protein as a fusion construct containing mCherry or mEGFP using in vivo DNA recombination methods. Our results indicate that expression of mEGFP-tagged Nt-RanBP9 followed by thrombin cleavage of the tag was the most effective method to obtain the protein with >90% purity and good yields. We report and discuss the challenges in obtaining the N-terminal region of RanBP9, a protein with functional implications in multiple biological processes and neurodegenerative diseases.

Effects of Stereochemistry and Hydrogen Bonding on Glycopolymer-Amyloid-ß Interactions.

Saccharide stereochemistry plays an important role in carbohydrate functions such as biological recognition processes and protein binding. Synthetic glycopolymers with pendant saccharides of controlled stereochemistry provide an attractive approach for the design of polysaccharide-inspired biomaterials. Acrylamide-based polymers containing either ß,d-glucose or ß,d-galactose pendant groups, designed to mimic GM1 ganglioside saccharides, and their small-molecule analogues were used to evaluate the effect of stereochemistry on glycopolymer solution aggregation processes alone and in the presence of Aß42 peptide using dynamic light scattering, gel permeation chromatography-multiangle laser light scattering, and fluorescence assays. Fourier transform infrared and nuclear magnetic resonance (NMR) were employed to determine hydrogen bonding patterns of the systems. The galactose-containing polymer displayed significant intramolecular hydrogen bonding and self-aggregation and minimal association with Aß42, while the glucose-containing glycopolymers showed intermolecular interactions with the surrounding environment and association with Aß42. Saturation transfer difference NMR spectroscopy demonstrated different binding affinities for the two glycopolymers to Aß42 peptide.

Cysteine-rich granulin-3 rapidly promotes amyloid-ß fibrils in both redox states.

Granulins (GRNs 1-7) are cysteine-rich proteolytic products of progranulin (PGRN) that have recently been implicated in neurodegenerative diseases including frontotemporal dementia (FTD) and Alzheimer's disease (AD). Their precise mechanism in these pathologies remains uncertain, but both inflammatory and lysosomal roles have been observed for GRNs. Among the seven GRNs, GRN-3 is well characterized and is implicated within the context of FTD. However, the relationship between GRN-3 and amyloid-ß (Aß), a protein relevant in AD pathology, has not yet been explored. To gain insight into this mechanism, we investigated the effect of both oxidized and reduced GRN-3 on Aß aggregation and found that both GRN-3 (oxidized) and rGRN-3 (reduced) bind to monomeric and oligomeric Aß42 to promote rapid fibril formation with subtle rate differences. As low molecular weight oligomers of Aß are well-established neurotoxins, rapid promotion of fibrils by GRN-3 mitigates Aß42-induced cellular apoptosis. These data provide valuable insights in understanding GRN-3's ability to modulate Aß-induced toxicity under redox control and presents a new perspective toward AD pathology. These results also prompt further investigation into the role(s) of other GRNs in AD pathogenesis.

Large fatty acid-derived Aß42 oligomers form ring-like assemblies.

As the primary toxic species in the etiology of Alzheimer disease (AD) are low molecular weight oligomers of Aß, it is crucial to understand the structure of Aß oligomers for gaining molecular insights into AD pathology. We have earlier demonstrated that in the presence of fatty acids, Aß42 peptides assemble as 12-24mer oligomers. These Large Fatty Acid-derived Oligomers (LFAOs) exist predominantly as 12mers at low and as 24mers at high concentrations. The 12mers are more neurotoxic than the 24mers and undergo self-replication, while the latter propagate to morphologically distinct fibrils with succinct pathological consequences. In order to glean into their functional differences and similarities, we have determined their structures in greater detail by combining molecular dynamic simulations with biophysical measurements. We conjecture that the LFAO are made of Aß units in an S-shaped conformation, with the 12mers forming a double-layered hexamer ring (6 × 2) while the structure of 24mers is a double-layered dodecamer ring (12 × 2). A closer inspection of the (6 × 2) and (12 × 2) structures reveals a concentration and pH dependent molecular reorganization in the assembly of 12 to 24mers, which seems to be the underlying mechanism for the observed biophysical and cellular properties of LFAOs.

Propagation of an Aß Dodecamer Strain Involves a Three-Step Mechanism and a Key Intermediate.

Proteinaceous deposits composed of fibrillar amyloid-ß (Aß) are the primary neuropathological hallmarks in Alzheimer disease (AD) brains. The nucleation-dependent aggregation of Aß is a stochastic process with frequently observed heterogeneity in aggregate size, structure, and conformation that manifests in fibril polymorphism. Emerging evidence indicates that polymorphic variations in Aß fibrils contribute to phenotypic diversity and the rate of disease progression in AD. We recently demonstrated that a dodecamer strain derived from synthetic Aß42 propagates to morphologically distinct fibrils and selectively induces cerebral amyloid angiopathy phenotype in transgenic mice. This report supports the growing contention that stable oligomer strains can influence phenotypic outcomes by faithful propagation of their structures. Although we determined the mechanism of dodecamer propagation on a mesoscopic scale, the molecular details of the microscopic reactions remained unknown. Here, we have dissected and evaluated individually the kinetics of macroscopic phases in aggregation to gain insight into the process of strain propagation. The bulk rates determined experimentally in each phase were used to build an ensemble kinetic simulation model, which confirmed our observation that dodecamer seeds initially grow by monomer addition toward the formation of a key intermediate. This is followed by conversion of the intermediate to fibrils by oligomer elongation and association mechanisms. Overall, this report reveals important insights into the molecular details of oligomer strain propagation involved in AD pathology.

Aqueous RAFT Synthesis of Glycopolymers for Determination of Saccharide Structure and Concentration Effects on Amyloid ß Aggregation.

GM1 ganglioside is known to promote amyloid-ß (Aß) peptide aggregation in Alzheimer's disease. The roles of the individual saccharides and their distribution in this process are not understood. Acrylamide-based glycomonomers with either ß-d-glucose or ß-d-galactose pendant groups were synthesized to mimic the stereochemistry of saccharides present in GM1 and characterized via 1H NMR and electrospray ionization mass spectrometry. Glycopolymers of different molecular weights were synthesized by aqueous reversible addition-fragmentation chain transfer (aRAFT) polymerization and characterized by NMR and GPC. The polymers were used as models to investigate the effects of molecular weight and saccharide unit type on Aß aggregation via thioflavin-T fluorescence and PAGE. High molecular weight (∼350 DP) glucose-containing glycopolymers had a profound effect on Aß aggregation, promoting formation of soluble oligomers of Aß and limiting fibril production, while the other glycopolymers and negative control had little effect on the Aß propagation process.

Conformational Dynamics of Specific Aß Oligomers Govern Their Ability To Replicate and Induce Neuronal Apoptosis.

Oligomers of amyloid-ß (Aß) have emerged as the primary toxic agents responsible for early synaptic dysfunction and neuronal death in Alzheimer's disease (AD). Characterization of oligomers is an important step in the progress toward delineating the complex molecular mechanisms involved in AD pathogenesis. In our previous reports, we established that a distinct 12-24mer neurotoxic oligomer of Aß42, called Large Fatty Acid derived Oligomers (LFAOs), exhibits a unique property of replication in which LFAOs directly duplicate to quantitatively larger amounts upon interacting with monomers. This self-propagative process of replication is somewhat reminiscent of prion propagation. In this report, we sought to investigate the concentration-dependent conformational dynamics LFAOs undergo and how such transitions manifest in their ability to replicate and induce neuronal apoptosis. The results indicate that LFAOs undergo a concentration-dependent transition between 12mers and disperse 12-24mers with a dissociation constant (Kd) of 0.1 µM. The two species differ in their respective tertiary/quaternary structures but not their secondary structures. This conformational dynamics of LFAOs correlates with their ability to replicate and to induce apoptosis in SH-SY5Y human neuroblastoma cells, with 12mers being more neurotoxic and prone to replication than 12-24mers. The latter result implicates the replication process dominates at low physiological concentrations. The observations made in this report may have profound significance in deciphering the elusive roles of Aß oligomer phenotypes and in determining their prion-type behavior in AD pathology.

Substrate-targeting gamma-secretase modulators.

Selective lowering of Abeta42 levels (the 42-residue isoform of the amyloid-beta peptide) with small-molecule gamma-secretase modulators (GSMs), such as some non-steroidal anti-inflammatory drugs, is a promising therapeutic approach for Alzheimer's disease. To identify the target of these agents we developed biotinylated photoactivatable GSMs. GSM photoprobes did not label the core proteins of the gamma-secretase complex, but instead labelled the beta-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-beta peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP gamma-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28-36 of amyloid-beta, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-beta act as GSMs, and some GSMs alter the production of cell-derived amyloid-beta oligomers. Furthermore, mutation of the GSM binding site in the APP alters the sensitivity of the substrate to GSMs. These findings indicate that substrate targeting by GSMs mechanistically links two therapeutic actions: alteration in Abeta42 production and inhibition of amyloid-beta aggregation, which may synergistically reduce amyloid-beta deposition in Alzheimer's disease. These data also demonstrate the existence and feasibility of 'substrate targeting' by small-molecule effectors of proteolytic enzymes, which if generally applicable may significantly broaden the current notion of 'druggable' targets.

Specific soluble oligomers of amyloid-ß peptide undergo replication and form non-fibrillar aggregates in interfacial environments.

Aggregates of amyloid-ß (Aß) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aß aggregates, low molecular weight species ranging between ~2- and 50-mers, also called "soluble oligomers," have emerged as the species responsible for early synaptic dysfunction and neuronal loss. Emerging evidence suggests that the neurotoxic oligomers need not be formed along the obligatory nucleation-dependant fibril formation pathway. In our earlier work, we reported the isolation of one such "off-pathway" 12-18-mer species of Aß42 generated from fatty acids called large fatty acid-derived oligomers (LFAOs) (Kumar, A., Bullard, R. L., Patel, P., Paslay, L. C., Singh, D., Bienkiewicz, E. A., Morgan, S. E., and Rangachari, V. (2011) PLoS One 6, e18759). Here, we report the physiochemical aspects of LFAO-monomer interactions as well as LFAO-LFAO associations in the presence of interfaces. We discovered that LFAOs are a replicating strain of oligomers that recruit Aß42 monomers and quantitatively convert them into LFAO assemblies at the expense of fibrils, a mechanism similar to prion propagation. We also found that in the presence of hexane-buffer or chloroform-buffer interfaces LFAOs are able to associate with themselves to form larger but non-fibrillar aggregates. These results further support the hypothesis that low molecular weight oligomers can be generated via non-fibril formation pathways. Furthermore, the unique replicating property of off-pathway oligomers may hold profound significance for Alzheimer disease pathology.

Biomolecular condensates - extant relics or evolving microcompartments?

Unprecedented discoveries during the past decade have unearthed the ubiquitous presence of biomolecular condensates (BCs) in diverse organisms and their involvement in a plethora of biological functions. A predominant number of BCs involve coacervation of RNA and proteins that demix from homogenous solutions by a process of phase separation well described by liquid-liquid phase separation (LLPS), which results in a phase with higher concentration and density from the bulk solution. BCs provide a simple and effective means to achieve reversible spatiotemporal control of cellular processes and adaptation to environmental stimuli in an energy-independent manner. The journey into the past of this phenomenon provides clues to the evolutionary origins of life itself. Here I assemble some current and historic discoveries on LLPS to contemplate whether BCs are extant biological hubs or evolving microcompartments. I conclude that BCs in biology could be extant as a phenomenon but are co-evolving as functionally and compositionally complex microcompartments in cells alongside the membrane-bound organelles.

Sugar distributions on gangliosides guide the formation and stability of amyloid-ß oligomers.

Aggregation of Aß peptides has been known as a key contributor to the etiology of Alzheimer's disease. Being intrinsically disordered, the monomeric Aß is susceptible to conformational excursions, especially in the presence of key interacting partners such as membrane lipids, to adopt specific aggregation pathways. Furthermore, key components such as gangliosides in membranes and lipid rafts are known to play important roles in the adoption of pathways and the generation of discrete neurotoxic oligomers. Yet, what roles the carbohydrates on gangliosides play in this process remains unknown. Here, using GM1, GM3, and GD3 ganglioside micelles as models, we show that the sugar distributions and cationic amino acids within Aß N-terminal region modulate oligomerization of Aß temporally, and dictate the stability and maturation of oligomers.

α-Synuclein emulsifies TDP-43 prion-like domain-RNA liquid droplets to promote heterotypic amyloid fibrils.

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and α-synuclein (αS) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and αS synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear if αS can modulate TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale AFM-IR spectroscopy, and biophysical analyses, we uncover the interactions of αS with phase-separated droplets. We learn that αS acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets. The aggregates of αS on these clusters emulsify the droplets by nucleating the formation of heterotypic TDP-43PrLD amyloid fibrils, structures of which are distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of αS to act as a Pickering agent while interacting with SGs and unmask the hitherto unknown role of αS in modulating TDP-43 proteinopathies.

α-Synuclein emulsifies TDP-43 prion-like domain - RNA liquid droplets to promote heterotypic amyloid fibrils.

Many neurodegenerative diseases including frontotemporal lobar degeneration (FTLD), Lewy body disease (LBD), multiple system atrophy (MSA), etc., show colocalized deposits of TDP-43 and α-synuclein (αS) aggregates. To understand whether these colocalizations are driven by specific molecular interactions between the two proteins, we previously showed that the prion-like C-terminal domain of TDP-43 (TDP-43PrLD) and αS synergistically interact to form neurotoxic heterotypic amyloids in homogeneous buffer conditions. However, it remains unclear whether and how αS modulates TDP-43 present within liquid droplets and biomolecular condensates called stress granules (SGs). Here, using cell culture and in vitro TDP-43PrLD - RNA liquid droplets as models along with microscopy, nanoscale spatially-resolved spectroscopy, and other biophysical analyses, we uncover the interactions of αS with phase-separated droplets. We learn that αS acts as a Pickering agent by forming clusters on the surface of TDP-43PrLD - RNA droplets and emulsifying them. The 'hardening' of the droplets that follow by αS aggregates on the periphery, nucleates the formation of heterotypic TDP-43PrLD amyloid fibrils with structures distinct from those derived from homogenous solutions. Together, these results reveal an intriguing property of αS as a Pickering agent in interacting with SGs and unmask the hitherto unknown role of αS in modulating TDP-43 proteinopathies.