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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. This Perspective discusses how biomolecular condensates are co-evolving as complex microcompartments alongside membrane-bound organelles in cells. [ABSTRACT FROM AUTHOR]

Self-templating propagation of protein aggregate conformations is increasingly becoming a significant factor in many neurological diseases. In Alzheimer disease (AD), intrinsically disordered amyloid-β (Aβ) peptides undergo aggregation that is sensitive to environmental conditions. High-molecular weight aggregates of Aβ that form insoluble fibrils are deposited as senile plaques in AD brains. However, low-molecular weight aggregates called soluble oligomers are known to be the primary toxic agents responsible for neuronal dysfunction. The aggregation process is highly stochastic involving both homotypic (Aβ-Aβ) and heterotypic (Aβ with interacting partners) interactions. Two of the important members of interacting partners are membrane lipids and surfactants, to which Aβ shows a perpetual association. Aβ–membrane interactions have been widely investigated for more than two decades, and this research has provided a wealth of information. Although this has greatly enriched our understanding, the objective of this review is to consolidate the information from the literature that collectively showcases the unique phenomenon of lipid-mediated Aβ oligomer generation, which has largely remained inconspicuous. This is especially important because Aβ aggregate “strains” are increasingly becoming relevant in light of the correlations between the structure of aggregates and AD phenotypes. Here, we will focus on aspects of Aβ-lipid interactions specifically from the context of how lipid modulation generates a wide variety of biophysically and biochemically distinct oligomer sub-types. This, we believe, will refocus our thinking on the influence of lipids and open new approaches in delineating the mechanisms of AD pathogenesis. This article is part of a Special Issue entitled: Protein Aggregation and Misfolding at the Cell Membrane Interface edited by Ayyalusamy Ramamoorthy. [ABSTRACT FROM AUTHOR]

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. This article presents a hitherto unknown role of a-Synuclein protein to function as a Pickering agent for TDP-43-RNA biomolecular condensates to emulsify them towards heterotypic amyloid fibrils. [ABSTRACT FROM AUTHOR]

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 toFTLD 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

As the primary toxic species in the etiology of Alzheimer disease (AD) are low molecular weight oligomers of Aβ, it is crucial to understand the structure of Aβ oligomers for gaining molecular insights into AD pathology. We have earlier demonstrated that in the presence of fatty acids, Aβ42 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 Aβ 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. [ABSTRACT FROM AUTHOR]

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‐β (Aβ) 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 Aβ42, 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 Aβ42 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. [ABSTRACT FROM AUTHOR]

Aggregation of Aβ peptides is a key contributor to the etiology of Alzheimer's disease. Being intrinsically disordered, monomeric Aβ is susceptible to conformational excursions, especially in the presence of important interacting partners such as membrane lipids, to adopt specific aggregation pathways. Furthermore, 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 do 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 Aβ N-terminal region modulate oligomerization of Aβ temporally, and dictate the stability and maturation of oligomers. These results demonstrate the selectivity of sugar distributions on the membrane surface toward oligomerization of Aβ and thus implicate cell-selective enrichment of oligomers. [Display omitted] • Gangliosides are important membrane components linked to the etiology of Alzheimer's disease. • Sugar distributions on gangliosides GM1, GM3, and GD3 differentially affect the oligomerization of Aβ42. • Aβ-sugar interactions are affected by the cationic residues in the N-terminal of Aβ. • Sugar distribution on ganglioside confers specificity of interaction for the formation and stability of oligomers. [ABSTRACT FROM AUTHOR]

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-β (Aβ), 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 Aβ aggregation and found that both GRN-3 (oxidized) and rGRN-3 (reduced) bind to monomeric and oligomeric Aβ42 to promote rapid fibril formation with subtle rate differences. As low molecular weight oligomers of Aβ are well-established neurotoxins, rapid promotion of fibrils by GRN-3 mitigates Aβ42-induced cellular apoptosis. These data provide valuable insights in understanding GRN-3's ability to modulate Aβ-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. [ABSTRACT FROM AUTHOR]

GM1 ganglioside is known to promote amyloid-β (Aβ) 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 β-d-glucose or β-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 Aβ aggregation via thioflavin-T fluorescence and PAGE. High molecular weight (∼350 DP) glucose-containing glycopolymers had a profound effect on Aβ aggregation, promoting formation of soluble oligomers of Aβ and limiting fibril production, while the other glycopolymers and negative control had little effect on the Aβ propagation process. [ABSTRACT FROM AUTHOR]

Granulins (Grns) are a family of small, cysteine-rich proteins that are generated upon proteolytic cleavage of their precursor, progranulin (Pgrn). All seven Grns (A-G) contain 12 conserved cysteines that form 6 intramolecular disulfide bonds, rendering this family of proteins unique. Grns are known to play multi-functional roles, including wound healing, embryonic growth, and inflammation and are implicated in neurodegenerative diseases. Despite their manifold functions, there exists a dearth of information regarding their structure-function relationship. Here, we sought to establish the role of disulfide bonds in promoting structure by investigating the fully reduced GrnB (rGrnB).We report that monomeric rGrnB is an intrinsically disordered protein (IDP) at lowconcentrations. rGrnB undergoes dimerization at higher concentrations to form a fuzzy complexwithout a net gain in the structure--a behavior increasingly identified as a hallmark of some IDPs. Interestingly, we show that rGrnB is also able to activate NF-μB in human neuroblastoma cells in a concentration-dependent manner. This activation correlates with the observed monomer-dimer dynamics. Collectively, the presented data establish that the intrinsic disorder of rGrnB governs conformational dynamics within the reduced form of the protein, and suggest that the overall structure of Grns could be entirely dictated by disulfide bonds. [ABSTRACT FROM AUTHOR]

Oligomers of amyloid-β (Aβ) 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 Aβ42, 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 Aβ oligomer phenotypes and in determining their prion-type behavior in AD pathology. [ABSTRACT FROM AUTHOR]

The aggregation of amyloid-β (Aβ) peptide and its deposition in parts of the brain form the central processes in the etiology of Alzheimer disease (AD). The low-molecular weight oligomers of Aβ aggregates (2 to 30 mers) are known to be the primary neurotoxic agents whose mechanisms of cellular toxicity and synaptic dysfunction have received substantial attention in the recent years. However, how these toxic agents proliferate and induce widespread amyloid deposition throughout the brain, and what mechanism is involved in the amplification and propagation of toxic oligomer species, are far from clear. Emerging evidence based on transgenic mice models indicates a transmissible nature of Aβ aggregates and implicates a prion-like mechanism of oligomer propagation, which manifests as the dissemination and proliferation of Aβ toxicity. Despite accumulating evidence in support of a transmissible nature of Aβ aggregates, a clear, molecular-level understanding of this intriguing mechanism is lacking. Recently, we reported the characterization of unique replicating oligomers of Aβ42 (12–24 mers) in vitro called arge atty cid-derived ligomers (LFAOs) (Kumar et al., 2012, J. Biol. Chem). In the current report, we establish that LFAOs possess physiological activity by activating NF-κB in human neuroblastoma cells, and determine the experimental parameters that control the efficiency of LFAO replication by self-propagation. These findings constitute the first detailed report on monomer – oligomer lateral propagation reactions that may constitute potential mechanism governing transmissibility among Aβ oligomers. These data support the previous reports on transmissible mechanisms observed in transgenic animal models. [ABSTRACT FROM AUTHOR]

[Display omitted] • α-Synuclein and prion-like domain of TDP-43 monomers synergistically promote hybrid fibrils. • Monomers and aggregates of α-Synuclein seed TDP-43 monomers. • TDP-43 fibrils fail to seed α-Synuclein monomers. • Cross-seeded hybrid aggregates are more cytotoxic than homotypic fibrils. Aberrant aggregation and amyloid formation of tar DNA binding protein (TDP-43) and α-synuclein (αS) underlie frontotemporal dementia (FTD) and Parkinson's disease (PD), respectively. Amyloid inclusions of TDP-43 and αS are also commonly co-observed in amyotrophic lateral sclerosis (ALS), dementia with Lewy bodies (DLB) and Alzheimer disease (AD). Emerging evidence from cellular and animal models show colocalization of the TDP-43 and αS aggregates, raising the possibility of direct interactions and co-aggregation between the two proteins. In this report, we set out to answer this question by investigating the interactions between αS and prion-like pathogenic C-terminal domain of TDP-43 (TDP-43 PrLD). PrLD is an aggregation-prone fragment generated both by alternative splicing as well as aberrant proteolytic cleavage of full length TDP-43. Our results indicate that two proteins interact in a synergistic manner to augment each other's aggregation towards hybrid fibrils. While monomers, oligomers and sonicated fibrils of αS seed TDP-43 PrLD monomers, TDP-43 PrLD fibrils failed to seed αS monomers indicating selectivity in interactions. Furthermore, αS modulates liquid droplets formed by TDP-43 PrLD and RNA to promote insoluble amyloid aggregates. Importantly, the cross-seeded hybrid aggregates show greater cytotoxicity as compared to the individual homotypic aggregates suggesting that the interactions between the two proteins have a discernable impact on cellular functions. Together, these results bring forth insights into TDP-43 PrLD – αS interactions that could help explain clinical and pathological presentations in patients with co-morbidities involving the two proteins. [ABSTRACT FROM AUTHOR]

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. • Recombinant expression of proteins containing low complexity sequences are difficult due to proteolytic degradation. • We bring forth a novel cloning and expression method for generating pure N-terminal low complexity region of RanBP9 protein. • We present a novel way of cloning using E. coli's endogenous DNA recombination with nearly 100% accuracy. • We show that LCRs can be effectively purified in god yields as a fusion construct with GFP or mCherry tag. [ABSTRACT FROM AUTHOR]

Low molecular weight oligomers of amyloid-β (Aβ) have emerged as the primary toxic agents in the etiology of Alzheimer disease (AD). Polymorphism observed within the aggregation end products of fibrils are known to arise due to microstructural differences among the oligomers. Diversity in aggregate morphology correlates with the differences in AD, cementing the idea that conformational strains of oligomers could be significant in phenotypic outcomes. Therefore, it is imperative to determine the ability of strains to faithfully propagate their structure. Here we report fibril propagation of an Aβ42 dodecamer called large fatty acid-derived oligomers (LFAOs). The LFAO oligomeric strain selectively induces acute cerebral amyloid angiopathy (CAA) in neonatally-injected transgenic CRND8 mice. Propagation in-vitro occurs as a three-step process involving the association of LFAO units. LFAO-seeded fibrils possess distinct morphology made of repeating LFAO units that could be regenerated upon sonication. Overall, these data bring forth an important mechanistic perspective into strain-specific propagation of oligomers that has remained elusive thus far. [ABSTRACT FROM AUTHOR]