Copper enhances aggregational toxicity of mutant huntingtin in a Drosophila model of Huntington's Disease.

Huntington's disease (HD) is a progressive neurodegenerative disorder with clinical presentations of moderate to severe cognitive, motor, and psychiatric disturbances. HD is caused by the trinucleotide repeat expansion of CAG of the huntingtin (HTT) gene. The mutant HTT protein containing pathological polyglutamine (polyQ) extension is prone to misfolding and aggregation in the brain. It has previously been observed that copper and iron concentrations are increased in the striata of post-mortem human HD brains. Although it has been shown that the accumulation of mutant HTT protein can interact with copper, the underlying HD progressive phenotypes due to copper overload remains elusive. Here, in a Drosophila model of HD, we showed that copper induces dose-dependent aggregational toxicity and enhancement of Htt-induced neurodegeneration. Specifically, we found that copper increases mutant Htt aggregation, enhances the accumulation of Thioflavin S positive ß-amyloid structures within Htt aggregates, and consequently alters autophagy in the brain. Administration of copper chelator D-penicillamine (DPA) through feeding significantly decreases ß-amyloid aggregates in the HD pathological model. These findings reveal a direct role of copper in potentiating mutant Htt protein-induced aggregational toxicity, and further indicate the potential impact of environmental copper exposure in the disease onset and progression of HD.

Photo-Excited Dyes: Emerging Technique Against Tau Protein Aggregation.

Tau aggregates are considered a pathological hallmark of Alzheimer's disease. The screening of molecules against Tau aggregation is a novel strategy for Alzheimer's disease. The photo-excited molecules have proven to be effective as a therapeutic agent in several diseases. In recent studies, the photo-excited dyes showed an inhibitory effect on Alzheimer's disease-related Tau protein aggregation and toxicity. The present chapter deals with the effect of rose bengal on the aggregation of Tau. The in vitro studies carried out with the help of electron microscopy, ThS fluorescence, and circular dichroism suggested that RB attenuated the Tau aggregation under in vitro conditions, whereas PE-RB disaggregated the mature Tau fibrils. Photo-excited rose bengal and the classical rose bengal induced a low degree of toxicity in cells. Thus, for the treatment of Alzheimer's disease, the rose bengal could be considered a potential molecule.

The disordered protein SERF promotes α-Synuclein aggregation through liquid-liquid phase separation.

The aggregation of α-Synuclein (α-Syn) into amyloid fibrils is the hallmark of Parkinson's disease. Under stress or other pathological conditions, the accumulation of α-Syn oligomers is the main contributor to the cytotoxicity. A potential approach for treating Parkinson's disease involves preventing the accumulation of these α-Syn oligomers. In this study, we present a novel mechanism involving a conserved group of disorderly proteins known as small EDRK-rich factor (SERF), which promotes the aggregation of α-Syn through a cophase separation process. Using diverse methods like confocal microscopy, fluorescence recovery after photobleaching assays, solution-state NMR spectroscopy, and Western blot, we determined that the N-terminal domain of SERF1a plays a role in the interactions that occur during cophase separation. Within these droplets, α-Syn undergoes a gradual transformation from solid condensates to amyloid fibrils, while SERF1a is excluded from the condensates and dissolves into the solution. Notably, in vivo experiments show that SERF1a cophase separation with α-Syn significantly reduces the deposition of α-Syn oligomers and decreases its cellular toxicity under stress. These findings suggest that SERF1a accelerates the conversion of α-Syn from highly toxic oligomers to less toxic fibrils through cophase separation, thereby mitigating the biological damage of α-Syn aggregation.

Recombinant human HspB5-ACD structural domain inhibits neurotoxicity by regulating pathological α-Syn aggregation.

The treatment of Parkinson's disease is a global medical challenge. α-Synuclein (α-Syn) is the causative protein in Parkinson's disease and is closely linked to its progression. Therefore, inhibiting the pathological aggregation of α-Syn and its neurotoxicity is essential for the treatment of Parkinson's disease. In this study, α-Syn and recombinant human HspB5-ACD structural domain protein (AHspB5) were produced using the BL21(DE3) E. coli prokaryotic expression system, and then the role and mechanism of AHspB5 in inhibiting the pathological aggregation of α-Syn and its neurotoxicity were investigated. As a result, we expressed α-Syn and AHspB5 proteins and characterised the proteins. In vitro experiments showed that AHspB5 could inhibit the formation of α-Syn oligomers and fibrils; in cellular experiments, AHspB5 could prevent α-Syn-induced neuronal cell dysfunction, oxidative stress damage and apoptosis, and its mechanism of action was related to the TH-DA pathway and mitochondria-dependent apoptotic pathway; in animal experiments, AHspB5 could inhibit behavioural abnormalities, oxidative stress damage and loss of dopaminergic neurons. In conclusion, this work is expected to elucidate the mechanism and biological effects of AHspB5 on the pathological aggregation of α-Syn, providing a new pathway for the treatment of Parkinson's disease and laying the foundation for recombinant AHspB5.

α-Synuclein-dependent increases in PIP5K1γ drive inositol signaling to promote neurotoxicity.

Anomalous aggregation of α-synuclein (α-Syn) is a pathological hallmark of many degenerative synucleinopathies including Lewy body dementia (LBD) and Parkinson's disease (PD). Despite its strong link to disease, the precise molecular mechanisms that link α-Syn aggregation to neurodegeneration have yet to be elucidated. Here, we find that elevated α-Syn leads to an increase in the plasma membrane (PM) phosphoinositide PI(4,5)P2, which precipitates α-Syn aggregation and drives toxic increases in mitochondrial Ca2+ and reactive oxygen species leading to neuronal death. Upstream of this toxic signaling pathway is PIP5K1γ, whose abundance and localization is enhanced at the PM by α-Syn-dependent increases in ARF6. Selective inhibition of PIP5K1γ or knockout of ARF6 in neurons rescues α-Syn aggregation and cellular phenotypes of toxicity. Collectively, our data suggest that modulation of phosphoinositide metabolism may be a therapeutic target to slow neurodegeneration for PD and other related neurodegenerative disorders.

Domain-specific modulatory effects of phosphomimetic substitutions on liquid-liquid phase separation of tau protein.

Aggregation of tau is one of the major pathogenic events in Alzheimer's disease and several other neurodegenerative disorders. Recent reports demonstrated that tau can condense into liquid droplets that undergo time-dependent transition to a solid-like state, suggesting that liquid condensates may be on the pathway to pathological aggregation of tau. While hyperphosphorylation is a key feature of tau isolated from brains of patients with Alzheimer's disease and other tauopathies, the mechanistic role of phosphorylation in tau liquid-liquid phase separation (LLPS) remains largely unexplored. In an attempt to bridge this gap, here we performed systematic studies by introducing phosphomimetic substitutions of Ser/Thr residues with negatively charged Asp/Glu residues in different regions of the protein. Our data indicate that the phosphorylation patterns that increase the polarization of charge distribution in full-length tau (tau441) promote protein LLPS, whereas those that decrease charge polarization have an opposite effect. Overall, this study further supports the notion that tau LLPS is driven by attractive intermolecular electrostatic interactions between the oppositely charged domains. We also show that the phosphomimetic tau variants with low intrinsic propensity for LLPS can be efficiently recruited to droplets formed by the variants with high LLPS propensity. Furthermore, the present data demonstrate that phosphomimetic substitutions have a major effect on time-dependent material properties of tau droplets, generally slowing down their aging. The latter effect is most dramatic for the tau variant with substitutions within the repeat domain, which correlates with the decreased fibrillation rate of this variant.

Small molecule-based fluorescent probes for the detection of α-Synuclein aggregation states.

The formation of aggregates due to protein misfolding is encountered in various neurodegenerative diseases. α-Synuclein (α-Syn) aggregation is linked to Parkinson's disease (PD). It is one of the most prevalent neurodegenerative disorders after Alzheimer's disease. Aggregation of α-Syn is associated with Lewy body formation and degeneration of the dopaminergic neurons in the brain. These are the pathological hallmarks of PD progression. α-Syn aggregates in a multi-step process. The native unstructured α-Syn monomers combine to form oligomers, followed by amyloid fibrils, and finally Lewy bodies. Recent evidence suggests that α-Syn oligomerization and fibrils formation play major roles in PD development. α-Syn oligomeric species is the main contributor to neurotoxicity. Therefore, the detection of α-Syn oligomers and fibrils has drawn significant attention for potential diagnostic and therapeutic development. In this regard, the fluorescence strategy has become the most popular approach for following the protein aggregation process. Thioflavin T (ThT) is the most frequently used probe for monitoring amyloid kinetics. Unfortunately, it suffers from several significant drawbacks including the inability to detect neurotoxic oligomers. Researchers developed several small molecule-based advanced fluorescent probes compared to ThT for the detection/monitoring of α-Syn aggregates states. These are summarized here.

Hallmarks of neurodegenerative diseases.

Decades of research have identified genetic factors and biochemical pathways involved in neurodegenerative diseases (NDDs). We present evidence for the following eight hallmarks of NDD: pathological protein aggregation, synaptic and neuronal network dysfunction, aberrant proteostasis, cytoskeletal abnormalities, altered energy homeostasis, DNA and RNA defects, inflammation, and neuronal cell death. We describe the hallmarks, their biomarkers, and their interactions as a framework to study NDDs using a holistic approach. The framework can serve as a basis for defining pathogenic mechanisms, categorizing different NDDs based on their primary hallmarks, stratifying patients within a specific NDD, and designing multi-targeted, personalized therapies to effectively halt NDDs.

Structural Insights of Fe3+ Induced α-synuclein Fibrillation in Parkinson's Disease.

Amyloid aggregation of α-synuclein (α-syn) in Lewy bodies (LBs) is the pathological hallmark of Parkinson's disease (PD). Iron, especially Fe3+, is accumulated in substantia nigra of PD patients and co-deposited with α-syn in LBs. However, how Fe3+ modulates α-syn fibrillation at molecular level remains unclear. In this study, we found that Fe3+ can promote α-syn fibrillation at low concentration while inhibit its fibrillation at high concentration. NMR titration study shows poor interaction between α-syn monomer and Fe3+. Instead, we found that Fe3+ binds to α-syn fibrils. By using cryo-electron microscopy (cryo-EM), we further determined the atomic structure of α-syn fibril in complex with Fe3+ at the resolution of 2.7 Å. Strikingly, two extra electron densities adjacent to His50 and Glu57 were observed as putative binding sites of Fe3+ and water molecules, suggesting that Fe3+ binds to the negative cleft of the fibril and stabilizes the fibril structure for promoting α-syn aggregation. Further mutagenesis study shows mutation of His50 abolishes the Fe3+-facilitated fibrillation of α-syn. Our work illuminates the structural basis of the interaction of Fe3+ and α-syn in both monomeric and fibrillar forms, and sheds light on understanding the pathological role of Fe3+ in α-syn aggregation in PD.

Liquid-liquid Phase Separation of α-Synuclein: A New Mechanistic Insight for α-Synuclein Aggregation Associated with Parkinson's Disease Pathogenesis.

Aberrant aggregation of the misfolded presynaptic protein, α-Synuclein (α-Syn) into Lewy body (LB) and Lewy neuritis (LN) is a major pathological hallmark of Parkinson's disease (PD) and other synucleinopathies. Numerous studies have suggested that prefibrillar and fibrillar species of the misfolded α-Syn aggregates are responsible for cell death in PD pathogenesis. However, the precise molecular events during α-Syn aggregation, especially in the early stages, remain elusive. Emerging evidence has demonstrated that liquid-liquid phase separation (LLPS) of α-Syn occurs in the nucleation step of α-Syn aggregation, which offers an alternate non-canonical aggregation pathway in the crowded microenvironment. The liquid-like α-Syn droplets gradually undergo an irreversible liquid-to-solid phase transition into amyloid-like hydrogel entrapping oligomers and fibrils. This new mechanism of α-Syn LLPS and gel formation might represent the molecular basis of cellular toxicity associated with PD. This review aims to demonstrate the recent development of α-Syn LLPS, the underlying mechanism along with the microscopic events of aberrant phase transition. This review further discusses how several intrinsic and extrinsic factors regulate the thermodynamics and kinetics of α-Syn LLPS and co-LLPS with other proteins, which might explain the pathophysiology of α-Syn in various neurodegenerative diseases.

Study of Tau Liquid-Liquid Phase Separation In Vitro.

Aggregation of the microtubule-associated protein tau is one of the major pathogenic events in Alzheimer's disease and several other neurodegenerative disorders. Recent reports have demonstrated that purified tau can undergo liquid-liquid phase separation in vitro, forming liquid droplets. The protein within these droplets was also found to undergo accelerated transition to fibrillar aggregates, suggesting that LLPS may play an important role in pathological aggregation of tau in neurodegenerative disorders. Here, we describe several protocols for studying LLPS behavior of the recombinant full-length tau by turbidimetric and light microscopy-based methods.

Liquid-Liquid Phase Separation Promotes Protein Aggregation and Its Implications in Ferroptosis in Parkinson's Disease Dementia.

The pathological features of PDD are represented by dopaminergic neuronal death and intracellular α-synuclein (α-syn) aggregation. The interaction of iron accumulation with α-syn and tau was further explored as an essential pathological mechanism of PDD. However, the links and mechanisms between these factors remain unclear. Studies have shown that the occurrence and development of neurodegenerative diseases such as PDD are closely related to the separation of abnormal phases. Substances such as proteins can form droplets through liquid-liquid phase separation (LLPS) under normal physiological conditions and even undergo further liquid-solid phase transitions to form solid aggregates under disease or regulatory disorders, leading to pathological phenomena. By analyzing the existing literature, we propose that LLPS is the crucial mechanism causing abnormal accumulation of α-syn, tau, and other proteins in PDD, and its interaction with iron metabolism disorder is the key factor driving ferroptosis in PDD. Therefore, we believe that LLPS can serve as one of the means to explain the pathological mechanism of PDD. Determining the significance of LLPS in neurodegenerative diseases such as PDD will stimulate interest in research into treatments based on interference with abnormal LLPS.

ACTN2 Mutant Causes Proteopathy in Human iPSC-Derived Cardiomyocytes.

Genetic variants in α-actinin-2 (ACTN2) are associated with several forms of (cardio)myopathy. We previously reported a heterozygous missense (c.740C>T) ACTN2 gene variant, associated with hypertrophic cardiomyopathy, and characterized by an electro-mechanical phenotype in human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs). Here, we created with CRISPR/Cas9 genetic tools two heterozygous functional knock-out hiPSC lines with a second wild-type (ACTN2wt) and missense ACTN2 (ACTN2mut) allele, respectively. We evaluated their impact on cardiomyocyte structure and function, using a combination of different technologies, including immunofluorescence and live cell imaging, RNA-seq, and mass spectrometry. This study showed that ACTN2mut presents a higher percentage of multinucleation, protein aggregation, hypertrophy, myofibrillar disarray, and activation of both the ubiquitin-proteasome system and the autophagy-lysosomal pathway as compared to ACTN2wt in 2D-cultured hiPSC-CMs. Furthermore, the expression of ACTN2mut was associated with a marked reduction of sarcomere-associated protein levels in 2D-cultured hiPSC-CMs and force impairment in engineered heart tissues. In conclusion, our study highlights the activation of proteolytic systems in ACTN2mut hiPSC-CMs likely to cope with ACTN2 aggregation and therefore directs towards proteopathy as an additional cellular pathology caused by this ACTN2 variant, which may contribute to human ACTN2-associated cardiomyopathies.

De novo designed protein inhibitors of amyloid aggregation and seeding.

Neurodegenerative diseases are characterized by the pathologic accumulation of aggregated proteins. Known as amyloid, these fibrillar aggregates include proteins such as tau and amyloid-ß (Aß) in Alzheimer's disease (AD) and alpha-synuclein (αSyn) in Parkinson's disease (PD). The development and spread of amyloid fibrils within the brain correlates with disease onset and progression, and inhibiting amyloid formation is a possible route toward therapeutic development. Recent advances have enabled the determination of amyloid fibril structures to atomic-level resolution, improving the possibility of structure-based inhibitor design. In this work, we use these amyloid structures to design inhibitors that bind to the ends of fibrils, "capping" them so as to prevent further growth. Using de novo protein design, we develop a library of miniprotein inhibitors of 35 to 48 residues that target the amyloid structures of tau, Aß, and αSyn. Biophysical characterization of top in silico designed inhibitors shows they form stable folds, have no sequence similarity to naturally occurring proteins, and specifically prevent the aggregation of their targeted amyloid-prone proteins in vitro. The inhibitors also prevent the seeded aggregation and toxicity of fibrils in cells. In vivo evaluation reveals their ability to reduce aggregation and rescue motor deficits in Caenorhabditis elegans models of PD and AD.

Tetramerization of the S100B Chaperone Spawns a Ca2+ Independent Regulatory Surface that Enhances Anti-aggregation Activity and Client Specificity.

Alzheimer's disease (AD) hallmarks include the aggregation of amyloid-ß (Aß), tau and neuroinflammation promoted by several alarmins. Among these is S100B, a small astrocytic homodimeric protein, upregulated in AD, whose multiple biological activities depend on localization, concentration, and assembly state. S100B was reported to inhibit the aggregation and toxicity of Aß42 and tau similarly to a holdase-type chaperone. This activity is dependent of Ca2+-binding, which triggers the exposure of a regulatory binding cleft at the S100B dimer interface with which amyloidogenic clients dynamically interact. Although the dimer prevails, a significant portion of secreted S100B in the human brain occurs as higher order multimers, whose protective functions remain uncharacterized and which we here investigate. Resorting to ThT-monitored aggregation kinetics, we determined that unlike the dimer, tetrameric S100B inhibits Aß42 aggregation at sub/equimolar ratios, an effect that persists in the absence of Ca2+ binding. Structural analysis revealed that S100B tetramerization spawns a novel extended cleft accommodating an aggregation-prone surface that mediates interactions with monomeric Aß client via hydrophobic interactions, as corroborated by Bis-ANS fluorescence and docking analysis. Correspondingly, at high ionic strength that reduces solvation and favours hydrophobic contacts, the inhibition of Aß42 aggregation by tetrameric S100B is 3-fold increased. Interestingly, this extended Ca2+-independent surface favours Aß42 as substrate, as tau K18 aggregation is not inhibited by the apo tetramer. Overall, results illustrate a mechanism through which oligomerization of the S100B chaperone fine-tunes anti-aggregation activity and client specificity, highlighting the potential functional relevance of S100B multimers in the regulation of AD proteotoxicity.

Secretagogin is a Ca2+-dependent stress-responsive chaperone that may also play a role in aggregation-based proteinopathies.

Secretagogin (SCGN) is a three-domain hexa-EF-hand Ca2+-binding protein that plays a regulatory role in the release of several hormones. SCGN is expressed largely in pancreatic ß-cells, certain parts of the brain, and also in neuroendocrine tissues. The expression of SCGN is altered in several diseases, such as diabetes, cancers, and neurodegenerative disorders; however, the precise associations that closely link SCGN expression to such pathophysiologies are not known. In this work, we report that SCGN is an early responder to cellular stress, and SCGN expression is temporally upregulated by oxidative stress and heat shock. We show the overexpression of SCGN efficiently prevents cells from heat shock and oxidative damage. We further demonstrate that in the presence of Ca2+, SCGN efficiently prevents the aggregation of a broad range of model proteins in vitro. Small-angle X-ray scattering (BioSAXS) studies further reveal that Ca2+ induces the conversion of a closed compact apo-SCGN conformation into an open extended holo-SCGN conformation via multistate intermediates, consistent with the augmentation of chaperone activity of SCGN. Furthermore, isothermal titration calorimetry establishes that Ca2+ enables SCGN to bind α-synuclein and insulin, two target proteins of SCGN. Altogether, our data not only demonstrate that SCGN is a Ca2+-dependent generic molecular chaperone involved in protein homeostasis with broad substrate specificity but also elucidate the origin of its altered expression in several cancers. We describe a plausible mechanism of how perturbations in Ca2+ homeostasis and/or deregulated SCGN expression would hasten the process of protein misfolding, which is a feature of many aggregation-based proteinopathies.

A rare natural lipid induces neuroglobin expression to prevent amyloid oligomers toxicity and retinal neurodegeneration.

Most neurodegenerative diseases such as Alzheimer's disease are proteinopathies linked to the toxicity of amyloid oligomers. Treatments to delay or cure these diseases are lacking. Using budding yeast, we report that the natural lipid tripentadecanoin induces expression of the nitric oxide oxidoreductase Yhb1 to prevent the formation of protein aggregates during aging and extends replicative lifespan. In mammals, tripentadecanoin induces expression of the Yhb1 orthologue, neuroglobin, to protect neurons against amyloid toxicity. Tripentadecanoin also rescues photoreceptors in a mouse model of retinal degeneration and retinal ganglion cells in a Rhesus monkey model of optic atrophy. Together, we propose that tripentadecanoin affects p-bodies to induce neuroglobin expression and offers a potential treatment for proteinopathies and retinal neurodegeneration.

Methodological advances and strategies for high resolution structure determination of cellular protein aggregates.

Aggregation of proteins is at the nexus of molecular processes crucial to aging, disease, and employing proteins for biotechnology and medical applications. There has been much recent progress in determining the structural features of protein aggregates that form in cells; yet, owing to prevalent heterogeneity in aggregation, many aspects remain obscure and often experimentally intractable to define. Here, we review recent results of structural studies for cell-derived aggregates of normally globular proteins, with a focus on high-resolution methods for their analysis and prediction. Complementary results obtained by solid-state NMR spectroscopy, FTIR spectroscopy and microspectroscopy, cryo-EM, and amide hydrogen/deuterium exchange measured by NMR and mass spectrometry, applied to bacterial inclusion bodies and disease inclusions, are uncovering novel information on in-cell aggregation patterns as well as great diversity in the structural features of useful and aberrant protein aggregates. Using these advances as a guide, this review aims to advise the reader on which combination of approaches may be the most appropriate to apply to their unique system.

Hyperphosphorylated tau self-assembles into amorphous aggregates eliciting TLR4-dependent responses.

Soluble aggregates of the microtubule-associated protein tau have been challenging to assemble and characterize, despite their important role in the development of tauopathies. We found that sequential hyperphosphorylation by protein kinase A in conjugation with either glycogen synthase kinase 3ß or stress activated protein kinase 4 enabled recombinant wild-type tau of isoform 0N4R to spontaneously polymerize into small amorphous aggregates in vitro. We employed tandem mass spectrometry to determine the phosphorylation sites, high-resolution native mass spectrometry to measure the degree of phosphorylation, and super-resolution microscopy and electron microscopy to characterize the morphology of aggregates formed. Functionally, compared with the unmodified aggregates, which require heparin induction to assemble, these self-assembled hyperphosphorylated tau aggregates more efficiently disrupt membrane bilayers and induce Toll-like receptor 4-dependent responses in human macrophages. Together, our results demonstrate that hyperphosphorylated tau aggregates are potentially damaging to cells, suggesting a mechanism for how hyperphosphorylation could drive neuroinflammation in tauopathies.

Molecular Dynamics Simulation Studies on the Aggregation of Amyloid-ß Peptides and Their Disaggregation by Ultrasonic Wave and Infrared Laser Irradiation.

Alzheimer's disease is understood to be caused by amyloid fibrils and oligomers formed by aggregated amyloid-ß (Aß) peptides. This review article presents molecular dynamics (MD) simulation studies of Aß peptides and Aß fragments on their aggregation, aggregation inhibition, amyloid fibril conformations in equilibrium, and disruption of the amyloid fibril by ultrasonic wave and infrared laser irradiation. In the aggregation of Aß, a ß-hairpin structure promotes the formation of intermolecular ß-sheet structures. Aß peptides tend to exist at hydrophilic/hydrophobic interfaces and form more ß-hairpin structures than in bulk water. These facts are the reasons why the aggregation is accelerated at the interface. We also explain how polyphenols, which are attracting attention as aggregation inhibitors of Aß peptides, interact with Aß. An MD simulation study of the Aß amyloid fibrils in equilibrium is also presented: the Aß amyloid fibril has a different structure at one end from that at the other end. The amyloid fibrils can be destroyed by ultrasonic wave and infrared laser irradiation. The molecular mechanisms of these amyloid fibril disruptions are also explained, particularly focusing on the function of water molecules. Finally, we discuss the prospects for developing treatments for Alzheimer's disease using MD simulations.