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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]

A neuropathological hallmark of Alzheimer's disease (AD) is the presence of large numbers of senile plaques in the brain. These deposits are rich in fibrils that are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that soluble Aβ aggregates as well as fibrils are important in the etiology of AD. Low levels of endogenous soluble Aβ aggregates make them difficult to characterize, but several species in extracts of AD brains have been detected by gel electrophoresis in sodium dodecyl sulfate (SDS) and immunoblotting. Individual Aβ oligomers ranging in size from dimers through dodecamers of 4 kDa monomeric Aβ have been resolved in other laboratories as discrete species by size exclusion chromatography (SEC). In an effort to reconstitute soluble Aβ aggregates in vitro that resemble the endogenous soluble Aβ aggregates, we previously found that monomeric Aβ(1-42) rapidly forms soluble oligomers in the presence of dilute SDS micelles. Here we extend this work in two directions. First, we contrast the size and secondary structure of these oligomers with those of synthetic Aβ(1-42) fibrils. SEC and multiangle light scattering were used to obtain a molecular mass of 150 kDa for the isolated oligomers. The oligomers partially dissociated to monomers through nonamers when incubated with SDS, but in contrast to endogenous oligomers, we saw no evidence of these discrete species prior to SDS treatment. One hypothesis to explain this difference is that endogenous oligomers are stabilized by covalent cross-linking induced by unknown cellular agents. To explore this hypothesis, optimal mass spectrometry (MS) analysis procedures need to be developed for Aβ cross-linked in vitro. In our second series of studies, we began this process by treating monomeric and aggregated Aβ(1-42) with three cross-linking agents: transglutamitiase, glutaraldehyde, and Cu(II) with peroxide. We compared the efficiency of covalent cross-linking with these agents, the effect of cross-linking on peptide secondary structure, the stability of the cross-linked structures to thermal unfolding, and the sites of peptide cross-linking obtained from proteolysis and MS. [ABSTRACT FROM AUTHOR]

Alzheimer' s disease (AD) is characterized by large numbers of senile plaques in the brain that consist of fibrillar aggregates of 40- and 42-residue amyloid-β (Aβ) peptides. However, the degree of dementia in AD correlates better with the concentration of soluble Aβ species assayed biochemically than with histologically determined plaque counts, and several investigators now propose that soluble aggregates of Aβ are the neurotoxic agents that cause memory deficits and neuronal loss. These endogenous aggregates are minor components in brain extracts from AD patients and transgenic mice that express human Aβ, but several species have been detected by gel electrophoresis in sodium dodecylsulfate (SDS) and isolated by size exclusion chromatography (SEC). Endogenous Aβ aggregation is stimulated at cellular interfaces rich in lipid rafts, and anionic micelles that promote Aβ aggregation in vitro may be good models of these interfaces. We previously found that micelles formed in dilute SDS (2 mM) promote Aβ(1—40) fiber formation by supporting peptide interaction on the surface of a single micelle complex. In contrast, here we report that monomeric Aβ(1—42) undergoes an immediate conversion to a predominant β-structured conformation in 2 mM SDS which does not proceed to amyloid fibrils. The conformational change is instead rapidly followed by the near quantitative conversion of the 4 kDa monomer SDS gel band to 8—14 kDa bands consistent with dimers through tetramers. Removal of SDS by dialysis gave a shift in the predominant SDS gel bands to 30—60 kDa. While these oligomers resemble the endogenous aggregates, they are less stable. In particular, they do not elute as discrete species on SEC, and they are completed disaggregated by boiling in 1% SDS. It appears that endogenous oligomeric Aβ aggregates are stabilized by undefined processes that have not yet been incorporated into in vitro Aβ aggregation procedures. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is characterized by the presence of large numbers of fibrillar amyloid deposits in the form of senile plaques in the brain. The fibrils in senile plaques are composed of 40- and 42-residue amyloid-β (Aβ) peptides. Several lines of evidence indicate that fibrillar Aβ and especially soluble Aβ aggregates are important in the pathogenesis of AD, and many laboratories have investigated soluble Aβ aggregates generated from monomeric Aβ in vitro. Of these in vitro aggregates, the best characterized are called protofibrils. They are composed of globules and short rods, show primarily β-structure by circular dichroism (CD), enhance the fluorescence of bound thioflavin T, and readily seed the growth of long fibrils. However, one difficulty in correlating soluble Aβ aggregates formed in vitro with those in vivo is the high probability that cellular interfaces affect the aggregation rates and even the aggregate structures. Reports that focus on the features of interfaces that are important in Aβ aggregation have found that amphiphilic interactions and micellar-like Aβ structures may play a role. We previously described the formation of Aβ(1–40) aggregates at polar-nonpolar interfaces, including those generated at microdroplets formed in dilute hexafluoro-2-propanol (HFIP). Here we compared the Aβ(1–40) aggregates produced on sodium dodecyl sulfate (SDS) micelles, which may be a better model of biological membranes with phospholipids that have anionic headgroups. At both HFIP and SDS interfaces, changes in peptide secondary structure were observed by CD immediately when Aβ(1–40) was introduced. With HFIP, the change involved an increase in predominant/3-structure content and in fluorescence with thioflavin T, while with SDS, a partial α-helical conformation was adopted that gave no fluorescence. However, in both systems, initial amorphous clustered aggregates progressed to soluble fibers rich in β-structure over a roughly 2 day period. Fiber formation was much faster than in the absence of an interface, presumably because of the close intermolecular proximity of peptides at the interfaces. While these fibers resembled protofibrils, they failed to seed the aggregation of Aβ(1–40) monomers effectively. [ABSTRACT FROM AUTHOR]

The diphtheria toxin repressor (DtxR) is an Fe(II)-activated transcriptional regulator of iron homeostatic and virulence genes in Corynebacterium diphtheriae. DtxR is a two-domain protein that contains two structurally and functionally distinct metal binding sites. Here, we investigate the molecular steps associated with activation by Ni(II)Cl2 and Cd(ll)Cl2. Equilibrium binding energetic s for Ni(II) were obtained from isothermal titration calorimetry, indicating apparent metal dissociation constants of 0.2 and 1.7 μM for two independent sites. The binding isotherms for Ni(II) and Cd(II) exhibited a characteristic exothermic-endothermic pattern that was used to infer the metal binding sequence by comparing the wild-type isotherm with those of several binding site mutants. These data were complemented by measuring the distance between specific backbone amide nitrogens and the first equivalent of metal through heteronuclear NMR relaxation measurements. Previous studies indicated that metal binding affects a disordered to ordered transition in the metal binding domain. The coupling between metal binding and structure change was investigated using near-UV circular dichroism speciroscopy. Together, the data show that the first equivalent of metal is bound by the primary metal binding site. This binding orients the DNA binding helices and begins to fold the N-terminal domain. Subsequent binding at the ancillary site completes the folding of this domain and formation of the dimer interface. This model is used to explain the behavior of several mutants. [ABSTRACT FROM AUTHOR]

Diphtheria toxin repressor (DtxR) regulates the expression of iron-sensitive genes in Corynebacterium diphtheriae, including the diphtheria toxin gene. DtxR contains an N-terminal metal- and DNA-binding domain that is connected by a proline-rich flexible peptide segment (Pr) to a C-terminal src homology 3 (SH3)-like domain. We determined the solution structure of the intramolecular complex formed between the proline-rich segment and the SH3-like domain by use of NMR spectroscopy. The structure of the intramolecularly bound Pr segment differs from that seen in eukaryotic prolylpeptide- SH3 domain complexes. The prolylpeptide ligand is bound by the SH3-like domain in a deep crevice lined by aliphatic amino acid residues and passes through the binding site twice but does not adopt a polyprolyl type-II helix. NMR studies indicate that this intramolecular complex is present in the apo-state of the repressor. Isothermal equilibrium denaturation studies show that intramolecular complex formation contributes to the stability of the apo-repressor. The binding affinity of synthetic peptides to the SH3-like domain was determined using isothermal titration calorimetry. From the structure and the binding energies, we calculated the enhancement in binding energy for the intramolecular reaction and compared it to the energetics of dimerization. Together, the structural and biophysical studies suggest that the proline-rich peptide segment of DtxR functions as a switch that modulates the activation of repressor activity. [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]

Aggregates of amyloid-β (Aβ) peptides have been implicated in the etiology of Alzheimer disease. Among the different forms of Aβ 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 Aβ42 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 Aβ42 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. [ABSTRACT FROM AUTHOR]

Amyloid-β (Aβ) peptide aggregation is known to play a central role in the etiology of Alzheimer's disease (AD). Among various aggregates, low-molecular weight soluble oligomers of Aβ are increasingly believed to be the primary neurotoxic agents responsible for memory impairment. Anionic interfaces are known to influence the Aβ aggregation process significantly. Here, we report the effects of interfaces formed by medium-chain (C9-C12), saturated non-esterified fatty acids (NEFAs) on Aβ42 aggregation. NEFAs uniquely affected Aβ42 aggregation rates that depended on both the ratio of Aβ:NEFA as well the critical micelle concentration (CMC) of the NEFAs. More importantly, irrespective of the kind of NEFA used, we observed that two distinct oligomers, 12-18 mers and 4-5 mers were formed via different pathway of aggregation under specific experimental conditions: (i) 12-18 mers were generated near the CMC in which NEFAs augment the rate of Aβ42 aggregation towards fibril formation, and, (ii) 4-5 mers were formed above the CMC, where NEFAs inhibit fibril formation. The data indicated that both 12-18 mers and 4-5 mers are formed along an alternate pathway called 'off-pathway' that did not result in fibril formation and yet have subtle structural and morphological differences that distinguish their bulk molecular behavior. These observations, (i) reflect the possible mechanism of Aβ aggregation in physiological lipid-rich environments, and (ii) reiterate the fact that all oligomeric forms of Aβ need not be obligatory intermediates of the fibril formation pathway. [ABSTRACT FROM AUTHOR]

Background: In all amyloid diseases, protein aggregates have been implicated fully or partly, in the etiology of the disease. Due to their significance in human pathologies, there have been unprecedented efforts towards physiochemical understanding of aggregation and amyloid formation over the last two decades. An important relation from which hydrodynamic radii of the aggregate is routinely measured is the classic Stokes-Einstein equation. Here, we report a modification in the classical Stokes-Einstein equation using a mixture theory approach, in order to accommodate the changes in viscosity of the solvent due to the changes in solute size and shape, to implement a more realistic model for Aβ aggregation involved in Alzheimer's disease. Specifically, we have focused on validating this model in protofibrill lateral association reactions along the aggregation pathway, which has been experimentally well characterized. Results: The modified Stokes-Einstein equation incorporates an effective viscosity for the mixture consisting of the macromolecules and solvent where the lateral association reaction occurs. This effective viscosity is modeled as a function of the volume fractions of the different species of molecules. The novelty of our model is that in addition to the volume fractions, it incorporates previously published reports on the dimensions of the protofibrils and their aggregates to formulate a more appropriate shape rather than mere spheres. The net result is that the diffusion coefficient which is inversely proportional to the viscosity of the system is now dependent on the concentration of the different molecules as well as their proper shapes. Comparison with experiments for variations in diffusion coefficients over time reveals very similar trends. Conclusions: We argue that the standard Stokes-Einstein's equation is insufficient to understand the temporal variations in diffusion when trying to understand the aggregation behavior of Aβ42 proteins. Our modifications also involve inclusion of improved shape factors of molecules and more appropriate viscosities. The modification we are reporting is not only useful in Aβ aggregation but also will be important for accurate measurements in all protein aggregation systems. [ABSTRACT FROM AUTHOR]

Background: The aggregates of a protein called, 'Aβ' found in brains of Alzheimer's patients are strongly believed to be the cause for neuronal death and cognitive decline. Among the different forms of Aβ aggregates, smaller aggregates called 'soluble oligomers' are increasingly believed to be the primary neurotoxic species responsible for early synaptic dysfunction. Since it is well known that the Aβ aggregation is a nucleation dependant process, it is widely believed that the toxic oligomers are intermediates to fibril formation, or what we call the 'on-pathway' products. Modeling of Aβ aggregation has been of intense investigation during the last decade. However, precise understanding of the process, pre-nucleation events in particular, are not yet known. Most of these models are based on curve-fitting and overlook the molecular-level biophysics involved in the aggregation pathway. Hence, such models are not reusable, and fail to predict the system dynamics in the presence of other competing pathways. Results: In this paper, we present a molecular-level simulation model for understanding the dynamics of the amyloid-β (Aβ) peptide aggregation process involved in Alzheimer's disease (AD). The proposed chemical kinetic theory based approach is generic and can model most nucleation-dependent protein aggregation systems that cause a variety of neurodegenerative diseases. We discuss the challenges in estimating all the rate constants involved in the aggregation process towards fibril formation and propose a divide and conquer strategy by dissecting the pathway into three biophysically distinct stages: 1) pre-nucleation stage 2) post-nucleation stage and 3) protofibril elongation stage. We next focus on estimating the rate constants involved in the protofibril elongation stages for Aβ42 supported by in vitro experimental data. This elongation stage is further characterized by elongation due to oligomer additions and lateral association of protofibrils (73) and to properly validate the rate constants involved in these phases we have presented three distinct reaction models. We also present a novel scheme for mapping the fluorescence sensitivity and dynamic light scattering based in vitro experimental plots to estimates of concentration variation with time. Finally, we discuss how these rate constants will be incorporated into the overall simulation of the aggregation process to identify the parameters involved in the complete Aβ pathway in a bid to understand its dynamics. Conclusions: We have presented an instance of the top-down modeling paradigm where the biophysical system is approximated by a set of reactions for each of the stages that have been modeled. In this paper, we have only reported the kinetic rate constants of the fibril elongation stage that were validated by in vitro biophysical analyses. The kinetic parameters reported in the paper should be at least accurate upto the first two decimal places of the estimate. We sincerely believe that our top-down models and kinetic parameters will be able to accurately model the biophysical phenomenon of Aβ protein aggregation and identify the nucleation mass and rate constants of all the stages involved in the pathway. Our model is also reusable and will serve as the basis for making computational predictions on the system dynamics with the incorporation of other competing pathways introduced by lipids and fatty acids. [ABSTRACT FROM AUTHOR]

Inclusions of TAR DNA-binding protein-43 (TDP-43), a nuclear protein that regulates transcription and RNA splicing, are the defining histopathological feature of frontotemporal lobar degeneration with ubiquitin-positive inclusions (FTLD-Us) and sporadic and familial forms of amyotrophic lateral sclerosis (ALS). In ALS and FTLD-U, aggregated, ubiquitinated, and N-terminally truncated TDP-43 can be isolated from brain tissue rich in neuronal and glial cytoplasmic inclusions. The loss of TDP-43 function resulting from inappropriate cleavage, translocation from the nucleus, or its sequestration into inclusions could play important roles in neurodegeneration. However, it is not known whether TDP-43 fragments directly mediate toxicity and, more specifically, whether their abnormal aggregation is a cause or consequence of pathogenesis. We report that the ectopic expression of a ≈25-kDa TDP-43 fragment corresponding to the C-terminal truncation product of caspase-cleaved TDP-43 leads to the formation of toxic, insoluble, and ubiquitinand phospho-positive cytoplasmic inclusions within cells. The 25-kDa C-terminal fragment is more prone to phosphorylation at S409/S410 than full-length TDP-43, but phosphorylation at these sites is not required for inclusion formation or toxicity. Although this fragment shows no biological activity, its exogenous expression neither inhibits the function nor causes the sequestration of full-length nuclear TDP-43, suggesting that the 25-kDa fragment can induce cell death through a toxic gain-of-function. Finally, by generating a conformation-dependent antibody that detects C-terminal fragments, we show that this toxic cleavage product is specific for pathologic inclusions in human TDP-43 proteinopathies. [ABSTRACT FROM AUTHOR]

Selective lowering of Aβ42 levels (the 42-residue isoform of the amyloid-β peptide) with small-molecule γ-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 γ-secretase complex, but instead labelled the β-amyloid precursor protein (APP), APP carboxy-terminal fragments and amyloid-β peptide in human neuroglioma H4 cells. Substrate labelling was competed by other GSMs, and labelling of an APP γ-secretase substrate was more efficient than a Notch substrate. GSM interaction was localized to residues 28–36 of amyloid-β, a region critical for aggregation. We also demonstrate that compounds known to interact with this region of amyloid-β act as GSMs, and some GSMs alter the production of cell-derived amyloid-β 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 Aβ42 production and inhibition of amyloid-β aggregation, which may synergistically reduce amyloid-β 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. [ABSTRACT FROM AUTHOR]

Analyses of the biologic effects of mutations in the BRI2 (ITM2b) and the amyloid β precursor protein (APP) genes support the hypothesis that cerebral accumulation of amyloidogenic peptides in familial British and familial Danish dementias and Alzheimer's disease (AD) is associated with neurodegeneration. We have used somatic brain transgenic technology to express the BRI2 and BRI2-Aβ1- 40 transgenes in APP mouse models. Expression of BRI2-Aβ1- 40 mimics the suppressive effect previously observed using conventional transgenic methods, further validating the somatic brain transgenic methodology. Unexpectedly, we also find that expression of wild-type human BRI2 reduces cerebral Aβ deposition in an AD mouse model. Additional data indicate that the 23 aa peptide, Bri23, released from BRI2 by normal processing, is present in human CSF, inhibits Aβ aggregation in vitro and mediates its anti-amyloidogenic effect in vivo. These studies demonstrate that BRI2 is a novel mediator of Aβ deposition in vivo. [ABSTRACT FROM AUTHOR]

Neurofibrillary tangles (NFTs) are a pathological hallmark of Alzheimer's disease and other tauopathies, but recent studies in a conditional mouse model of tauopathy (rTg4510) have suggested that NFT formation can be dissociated from memory loss and neurodegeneration. This suggests that NFTs are not the major neurotoxic tau species, at least during the early stages of pathogenesis. To identify other neurotoxic tau protein species, we performed biochemical analyses on brain tissues from the rTg4510 mouse model and then correlated the levels of these tau proteins with memory loss. We describe the identification and characterization of two forms of tau multimers (140 and 170 kDa), whose molecular weight suggests an oligomeric aggregate, that accumulate early in the pathogenic cascade in this mouse model. Similar tau multimers were detected in a second mouse model of tauopathy (JNPL3) and in tissue from patients with Alzheimer's disease and FTDP-17 (frontotemporal dementia and parkinsonism linked to chromosome 17). Moreover, levels of the tau multimers correlated consistently with memory loss at various ages in the rTg4510 mouse model. Our findings suggest that accumulation of early-stage aggregated tau species, before the formation of NFT, is associated with the development of functional deficits during the pathogenic progression of tauopathy. [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]