Pathogenic Protein Aggregates Research Articles

Activate or Inhibit? Implications of Autophagy Modulation as a Therapeutic Strategy for Alzheimer's Disease.

Neurodegenerative diseases result in a range of conditions depending on the type of proteinopathy, genes affected or the location of the degeneration in the brain. Proteinopathies such as senile plaques and neurofibrillary tangles in the brain are prominent features of Alzheimer’s disease (AD). Autophagy is a highly regulated mechanism of eliminating dysfunctional organelles and proteins, and plays an important role in removing these pathogenic intracellular protein aggregates, not only in AD, but also in other neurodegenerative diseases. Activating autophagy is gaining interest as a potential therapeutic strategy for chronic diseases featuring protein aggregation and misfolding, including AD. Although autophagy activation is a promising intervention, over-activation of autophagy in neurodegenerative diseases that display impaired lysosomal clearance may accelerate pathology, suggesting that the success of any autophagy-based intervention is dependent on lysosomal clearance being functional. Additionally, the effects of autophagy activation may vary significantly depending on the physiological state of the cell, especially during proteotoxic stress and ageing. Growing evidence seems to favour a strategy of enhancing the efficacy of autophagy by preventing or reversing the impairments of the specific processes that are disrupted. Therefore, it is essential to understand the underlying causes of the autophagy defect in different neurodegenerative diseases to explore possible therapeutic approaches. This review will focus on the role of autophagy during stress and ageing, consequences that are linked to its activation and caveats in modulating this pathway as a treatment.

WWOX is a Risk Factor for Alzheimer’s Disease: How and Why?

A recent large genome-wide association meta-analysis revealed that the human WWOX gene is regarded as one of the five newly identified risk factors for Alzheimer’s disease (AD). However, this study did not functionally characterize how WWOX protein deficiency affects AD initiation, progression and neurodegeneration. In this review, evidence and perspectives are provided regarding how WWOX works in limiting neurodegeneration. Firstly, loss of WWOX/Wwox gene leads to severe neural diseases with degeneration, metabolic disorder and early death in the newborns. Downregulation of pY33-WWOX may start at middle ages, and this leads to slow aggregation of a cascade of proteins, namely TRAPPC6A[Formula: see text], TIAF1 and SH3GLB2, that leads to amyloid-beta (A[Formula: see text]) formation and tau tangle formation in old-aged AD patients. Secondly, functional antagonism between tumor suppressors p53 and WWOX may occur in vivo, in which p53-mediated inflammation is blocked by WWOX. Loss of balance in the functional antagonism leads to aggregation of pathogenic proteins for AD such as tau and A[Formula: see text] in the brain cortex and hippocampus. Thirdly, downregulation of pY33-WWOX is accompanied by upregulation of pS14-WWOX. The event frequently correlates with enhanced AD progression and cancer cell growth in vivo. A small peptide Zfra4-10 dramatically suppresses pS14-WWOX and restores memory loss in triple transgenic (3xTg) mice, and inhibits cancer growth in mice as well. Finally, a supporting scenario is that WWOX deficiency induces enhanced cell migration and loss of cell-to-cell recognition. This allows the generation of neuronal heterotopia and associated epileptic seizure in WWOX-deficient newborn patients.

Pesticide interactions induce alterations in secondary structure of malate dehydrogenase to cause destability and cytotoxicity

Environmental exposure to pesticides increases the risk of neurotoxicity and neurodegenerative diseases. The mechanism of pesticide-induced toxicity is attributed to the increased reactive oxygen species, mitochondrial dysfunction, inhibition of key cellular enzymes and accelerated pathogenic protein aggregation. The structural basis of pesticide-protein interaction is limited to pathogenic proteins such as α-synuclein, Tau and amyloid-beta. However, the effect of pesticides on metabolic proteins is still unexplored. Here, we used rotenone and chlorpyrifos to understand the interaction of these pesticides with a metabolic protein, malate dehydrogenase (MDH) and the consequent pesticide-induced cytotoxicity. We found that rotenone and chlorpyrifos strongly bind to MDH, interferes with protein folding and triggers alteration in its secondary structure. Both pesticides showed high binding affinities for MDH as observed by NMR and LCMS. Rotenone and chlorpyrifos induced structural alterations during MDH refolding resulting in the formation of cytotoxic conformers that generated oxidative stress and reduced cell viability. Our findings suggest that pesticides, in general, interact with proteins resulting in the formation of cytotoxic conformers that may have implications in neurotoxicity and neurodegenerative diseases.

Development of a brain-permeable peptide nanofiber that prevents aggregation of Alzheimer pathogenic proteins.

Alzheimer's disease (AD) is proposed to be induced by abnormal aggregation of amyloidβ in the brain. Here, we designed a brain-permeable peptide nanofiber drug from a fragment of heat shock protein to suppress aggregation of the pathogenic proteins. To facilitate delivery of the nanofiber into the brain, a protein transduction domain from Drosophila Antennapedia was incorporated into the peptide sequence. The resulting nanofiber efficiently suppressed the cytotoxicity of amyloid βby trapping amyloid β onto its hydrophobic nanofiber surface. Moreover, the intravenously or intranasally injected nanofiber was delivered into the mouse brain, and improved the cognitive function of an Alzheimer transgenic mouse model. These results demonstrate the potential therapeutic utility of nanofibers for the treatment of AD.

Single-vesicle imaging reveals lipid-selective and stepwise membrane disruption by monomeric α-synuclein

The interaction of the neuronal protein α-synuclein with lipid membranes appears crucial in the context of Parkinson's disease, but the underlying mechanistic details, including the roles of different lipids in pathogenic protein aggregation and membrane disruption, remain elusive. Here, we used single-vesicle resolution fluorescence and label-free scattering microscopy to investigate the interaction kinetics of monomeric α-synuclein with surface-tethered vesicles composed of different negatively charged lipids. Supported by a theoretical model to account for structural changes in scattering properties of surface-tethered lipid vesicles, the data demonstrate stepwise vesicle disruption and asymmetric membrane deformation upon α-synuclein binding to phosphatidylglycerol vesicles at protein concentrations down to 10 nM (∼100 proteins per vesicle). In contrast, phosphatidylserine vesicles were only marginally affected. These insights into structural consequences of α-synuclein interaction with lipid vesicles highlight the contrasting roles of different anionic lipids, which may be of mechanistic relevance for both normal protein function (e.g., synaptic vesicle binding) and dysfunction (e.g., mitochondrial membrane interaction).

Phagocytic glia are obligatory intermediates in transmission of mutant huntingtin aggregates across neuronal synapses.

Emerging evidence supports the hypothesis that pathogenic protein aggregates associated with neurodegenerative diseases spread from cell to cell through the brain in a manner akin to infectious prions. Here, we show that mutant huntingtin (mHtt) aggregates associated with Huntington disease transfer anterogradely from presynaptic to postsynaptic neurons in the adult Drosophila olfactory system. Trans-synaptic transmission of mHtt aggregates is inversely correlated with neuronal activity and blocked by inhibiting caspases in presynaptic neurons, implicating synaptic dysfunction and cell death in aggregate spreading. Remarkably, mHtt aggregate transmission across synapses requires the glial scavenger receptor Draper and involves a transient visit to the glial cytoplasm, indicating that phagocytic glia act as obligatory intermediates in aggregate spreading between synaptically-connected neurons. These findings expand our understanding of phagocytic glia as double-edged players in neurodegeneration-by clearing neurotoxic protein aggregates, but also providing an opportunity for prion-like seeds to evade phagolysosomal degradation and propagate further in the brain.

Probing the amyloid protein aggregates with unsymmetrical monocationic trimethine cyanine dyes

A series of novel trimethine cyanine dyes were synthesized and evaluated as potential probes for the detection of pathogenic protein aggregates, amyloid fibrils. The spectral properties of the chromophores were scrutinized in a free state as well as in the presence of non-fibrillized and fibrillar insulin emphasizing the cyanine aggregation that accompany the dye-protein interactions. The trimethines under study were found to be non-fluorescent in aqueous media and when bound to the non-fibrillized protein due to the H-aggregation of trimethines being more pronounced in the case of the control insulin. Depending on the variations with respect to the N-alkyl fragment of the benzothiazolic moiety, the dyes are increasing their emission signal in dozens of times in the presence of fibrillar insulin. The fluorescence turn-on responses observed upon binding of the AK3-11 cyanine dye to the insulin amyloid fibrils are attributed to a self-staking to non-staking transition of this trimethine. Overall, we demonstrated that trimethine cyanine dyes are prospective amyloid probes capable amyloid probes capable of operating in both fluorescence and absorption modes.

Inhibition of Amyloid β Fibrillation by Nanoparticles Composed of Ovalbumin-derived Amphiphilic Peptides

Utilizing egg white proteins and peptides as functional materials is very attractive, because they are available at low cost. Ovalbumin (OVA) is a major globular protein in egg white. Previously, we focused on the peptide fragment corresponding to the helix A region of OVA (pN1-22, acetyl-GSIGAASMEFCFDVFKELKVHH) and explored its function. Here, we report nanoparticle formation of this OVA-derived peptide and its ability to prevent the aggregation of pathogenic proteins.

Aggregation and Cellular Toxicity of Pathogenic or Non-pathogenic Proteins

More than 20 unique diseases such as diabetes, Alzheimer’s disease, Parkinson’s disease are caused by the abnormal aggregations of pathogenic proteins such as amylin, β-amyloid (Aβ), and α-synuclein. All pathogenic proteins differ from each other in biological function, primary sequences, and morphologies; however, the proteins are toxic when aggregated. Here, we investigated the cellular toxicity of pathogenic or non-pathogenic protein aggregates. In this study, six proteins were selected and they were incubated at acid pH and high temperature. The aggregation kinetic and cellular toxicity of protein species with time were characterized. Three non-pathogenic proteins, bovine serum albumin (BSA), catalase, and pepsin at pH 2 and 65 °C were stable in protein structure and non-toxic at a lower concentration of 1 mg/mL. They formed aggregates at a higher concentration of 20 mg/mL with time and they induced the toxicity in short incubation time points, 10 min and 20 min only and they became non-toxic after 30 min. Other three pathogenic proteins, lysozyme, superoxide dismutase (SOD), and insulin, also produced the aggregates with time and they caused cytotoxicity at both 1 mg/mL and 20 mg/mL after 10 min. TEM images and DSC analysis demonstrated that fibrils or aggregates at 1 mg/mL induced cellular toxicity due to low thermal stability. In DSC data, fibrils or aggregates of pathogenic proteins had low thermal transition compared to fresh samples. The results provide useful information to understand the aggregation and cellular toxicity of pathogenic and non-pathogenic proteins.

Designed Cell-Penetrating Peptide Inhibitors of Amyloid-beta Aggregation and Cytotoxicity

Summary Amyloid proteins and peptides are a major contributing factor to the development of various neurodegenerative disorders, including Alzheimer's and prion diseases. Previously, a designed cell-penetrating peptide (CPP) comprising a hydrophobic signal sequence followed by a prion protein (PrP)-derived polycationic sequence (PrP23–28: KKRPKP) was shown to have potent anti-prion properties. Here, we extend this approach toward the amyloid-beta (Aβ) peptide amyloid formation, which is associated with Alzheimer's disease. We characterized the interactions of the CPP with Aβ using complementary in vitro and in silico experiments. We report that the CPP stabilizes Aβ in a non-amyloid state and inhibits Aβ-induced neurotoxicity. Moreover, replacing PrP23–28 with a corresponding segment from Aβ results in a construct with similar CPP functionality and antagonism of Aβ aggregation and neurotoxicity. Our findings reveal a general underlying principle for inhibition of pathogenic protein aggregation that may facilitate the design of CPP-based therapeutics for amyloid diseases.

Inhibition of synucleinopathic seeding by rationally designed inhibitors.

Seeding, in the context of amyloid disease, is the sequential transfer of pathogenic protein aggregates from cell-to-cell within affected tissues. The structure of pathogenic seeds provides the molecular basis and enables rapid conversion of soluble protein into fibrils. To date, there are no inhibitors that specifically target seeding of Parkinson's disease (PD)-associated α-synuclein (α-syn) fibrils, in part, due to lack of information of the structural properties of pathological seeds. Here we design small peptidic inhibitors based on the atomic structure of the core of α-syn fibrils. The inhibitors prevent α-syn aggregation in vitro and in cell culture models with binding affinities of 0.5 μM to α-syn fibril seeds. The inhibitors also show efficacy in preventing seeding by human patient-derived α-syn fibrils. Our results suggest that pathogenic seeds of α-syn contain steric zippers and suggest a therapeutic approach targeted at the spread and progression that may be applicable for PD and related synucleinopathies.

Bacterial production and direct functional screening of expanded molecular libraries for discovering inhibitors of protein aggregation.

Protein misfolding and aggregation are associated with a many human disorders, including Alzheimer's and Parkinson's diseases. Toward increasing the effectiveness of early-stage drug discovery for these conditions, we report a bacterial platform that enables the biosynthesis of molecular libraries with expanded diversities and their direct functional screening for discovering protein aggregation inhibitors. We illustrate this approach by performing, what is to our knowledge, the largest functional screen of small-size molecular entities described to date. We generated a combinatorial library of ~200 million drug-like, cyclic peptides and rapidly screened it for aggregation inhibitors against the amyloid-β peptide (Aβ42), linked to Alzheimer's disease. Through this procedure, we identified more than 400 macrocyclic compounds that efficiently reduce Aβ42 aggregation and toxicity in vitro and in vivo. Finally, we applied a combination of deep sequencing and mutagenesis analyses to demonstrate how this system can rapidly determine structure-activity relationships and define consensus motifs required for bioactivity.

The microglial NLRP3 inflammasome is activated by amyotrophic lateral sclerosis proteins.

Microglial NLRP3 inflammasome activation is emerging as a key contributor to neuroinflammation during neurodegeneration. Pathogenic protein aggregates such as β-amyloid and α-synuclein trigger microglial NLRP3 activation, leading to caspase-1 activation and IL-1β secretion. Both caspase-1 and IL-1β contribute to disease progression in the mouse SOD1G93A model of amyotrophic lateral sclerosis (ALS), suggesting a role for microglial NLRP3. Prior studies, however, suggested SOD1G93A mice microglia do not express NLRP3, and SOD1G93A protein generated IL-1β in microglia independent to NLRP3. Here, we demonstrate using Nlrp3-GFP gene knock-in mice that microglia express NLRP3 in SOD1G93A mice. We show that both aggregated and soluble SOD1G93A activates inflammasome in primary mouse microglia leading caspase-1 and IL-1β cleavage, ASC speck formation, and the secretion of IL-1β in a dose- and time-dependent manner. Importantly, SOD1G93A was unable to induce IL-1β secretion from microglia deficient for Nlrp3, or pretreated with the specific NLRP3 inhibitor MCC950, confirming NLRP3 as the key inflammasome complex mediating SOD1-induced microglial IL-1β secretion. Microglial NLRP3 upregulation was also observed in the TDP-43Q331K ALS mouse model, and TDP-43 wild-type and mutant proteins could also activate microglial inflammasomes in a NLRP3-dependent manner. Mechanistically, we identified the generation of reactive oxygen species and ATP as key events required for SOD1G93A -mediated NLRP3 activation. Taken together, our data demonstrate that ALS microglia express NLRP3, and that pathological ALS proteins activate the microglial NLRP3 inflammasome. NLRP3 inhibition may therefore be a potential therapeutic approach to arrest microglial neuroinflammation and ALS disease progression.

USP10 is a critical factor for Tau-positive stress granule formation in neuronal cells

Tau aggregates in neurons of brain lesions is a hallmark pathology of tauopathies, including Alzheimer’s disease (AD). Recent studies suggest that the RNA-binding protein TIA1 initiates Tau aggregation by inducing the formation of stress granules (SGs) containing Tau. SGs are stress-inducible cytoplasmic protein aggregates containing many RNA-binding proteins that has been implicated as an initial site of multiple pathogenic protein aggregates in several neurodegenerative diseases. In this study, we found that ubiquitin-specific protease 10 (USP10) is a critical factor for the formation of Tau/TIA1/USP10-positive SGs. Proteasome inhibition or TIA1-overexpression in HT22 neuronal cells induced the formation of TIA1/Tau-positive SGs, and the formations were severely attenuated by depletion of USP10. In addition, the overexpression of USP10 without stress stimuli in HT22 cells induced TIA1/Tau/USP10-positive SGs in a deubiquitinase-independent manner. In AD brain lesions, USP10 was colocalized with Tau aggregates in the cell body of neurons. The present findings suggest that USP10 plays a key role in the initiation of pathogenic Tau aggregation in AD through SG formation.

Analyzing aggregation propensities of clinically relevant PTEN mutants: a new culprit in pathogenesis of cancer and other PTENopathies

While studies on pathological protein aggregation are largely limited to neurodegenerative disease, emerging evidence suggests that other diseases are also associated with pathogenic protein aggregation. For example, tumor suppressor protein p53, and its mutant conformers, undergo protein aggregation, exacerbating the cancer phenotype. These findings raise the possibility that inactivation of tumor suppressors via protein aggregation may participate in cancer and other disease pathologies. Since tumor suppressor protein PTEN has similar functions to p53, and is mutated in multiple diseases, we examined the aggregation propensity of PTEN wild-type and 1523 clinically relevant PTEN mutants. Applying computational tools to PTEN mutation databases revealed that PTEN wild-type protein can aggregate under physiological conditions, and 274 distinct PTEN mutants had increased aggregation propensity. To understand the mechanism underlying PTEN conformer aggregation, we analyzed the physicochemical properties of these 274 PTEN mutants and defined their aggregation potential. We conclude that increased aggregation propensity of select PTEN mutants may contribute to disease phenotypes. Our studies have built the foundation for interrogating the aggregation potential of these select mutants in cancers and in PTENopathies. Elucidating the pathogenic mechanisms associated with aggregation-prone PTEN conformers will aid in developing therapies that target PTEN-aggregates in multiple diseases.Communicated by Ramaswamy H. Sarma

Endogenous mouse huntingtin is highly abundant in cranial nerve nuclei, co-aggregates to Abeta plaques and is induced in reactive astrocytes in a transgenic mouse model of Alzheimer\u2019s disease

Pathogenic variants of the huntingtin (HTT) protein and their aggregation have been investigated in great detail in brains of Huntington’s disease patients and HTT-transgenic animals. However, little is known about the physiological brain region- and cell type-specific HTT expression pattern in wild type mice and a potential recruitment of endogenous HTT to other pathogenic protein aggregates such as amyloid plaques in cross seeding events. Employing a monoclonal anti-HTT antibody directed against the HTT mid-region and using brain tissue of three different mouse strains, we detected prominent immunoreactivity in a number of brain areas, particularly in cholinergic cranial nerve nuclei, while ubiquitous neuronal staining appeared faint. The region-specific distribution of endogenous HTT was found to be comparable in wild type rat and hamster brain. In human amyloid precursor protein transgenic Tg2576 mice with amyloid plaque pathology, similar neuronal HTT expression patterns and a distinct association of HTT with Abeta plaques were revealed by immunohistochemical double labelling. Additionally, the localization of HTT in reactive astrocytes was demonstrated for the first time in a transgenic Alzheimer’s disease animal model. Both, plaque association of HTT and occurrence in astrocytes appeared to be age-dependent. Astrocytic HTT gene and protein expression was confirmed in primary cultures by RT-qPCR and by immunocytochemistry. We provide the first detailed analysis of physiological HTT expression in rodent brain and, under pathological conditions, demonstrate HTT aggregation in proximity to Abeta plaques and Abeta-induced astrocytic expression of endogenous HTT in Tg2576 mice.

A Fluorogenic AggTag Method Based on Halo- and SNAP-Tags to Simultaneously Detect Aggregation of Two Proteins in Live Cells.

Protein aggregation involves the assembly of partially misfolded proteins into oligomeric and higher-order structures that have been associated with several neurodegenerative diseases. However, numerous questions relating to protein aggregation remain unanswered due to the lack of available tools for visualization of these species in living cells. We recently developed a fluorogenic method named aggregation tag (AggTag), and presented the AggTag probe P1, based on a Halo-tag ligand, to report on the aggregation of a protein of interest (POI) in live cells. However, the Halo-tag-based AggTag method only detects the aggregation of one specific POI at a time. In this study, we have expanded the AggTag method by using SNAP-tag technology to enable fluorogenic and biorthogonal detection of the aggregation of two different POIs simultaneously in live cells. A new AggTag probe-P2, based on a SNAP-tag ligand bearing a green solvatochromic fluorophore-was synthesized for this purpose. Using confocal imaging and chemical crosslinking experiments, we confirmed that P2 can also report both on soluble oligomers and on insoluble aggregates of a POI fused with SNAP-tag in live cells. Ultimately, we showed that the orthogonal fluorescence of P1 and P2 allows for simultaneous visualization of two different pathogenic protein aggregates in the same cell.

Stability Effect of Quinary Interactions Reversed by Single Point Mutations

In cells, proteins are embedded in a crowded environment that controls their properties via manifold avenues including weak protein-macromolecule interactions. A molecular level understanding of these quinary interactions and their contribution to protein stability, function, and localization in the cell is central to modern structural biology. Using a mutational analysis to quantify the energetic contributions of single amino acids to the stability of the ALS related protein superoxide dismutase I (SOD1) in mammalian cells, we show that quinary interactions destabilize SOD1 by a similar energetic offset for most of the mutants, but there are notable exceptions: Mutants that alter its surface properties can even lead to a stabilization of the protein in the cell as compared to the test tube. In conclusion, quinary interactions can amplify and even reverse the mutational response of proteins, being a key aspect in pathogenic protein misfolding and aggregation.

Adsorption Mechanism of Amyloid Fibrils to Graphene Nanosheets and Their Structural Destruction

A detailed understanding of the interaction mechanisms between pathogenic protein aggregates and nanomaterials is indispensable to the application and development in biomedical area. Here, all-atom molecular dynamics (MD) simulations were performed to characterize the interaction between a graphene nanosheet with amyloid fibrils domain, a mostly parallel β-sheet model as a typical representative. It was found that graphene had strong capabilities to interact with Aβ fibrils, which first demonstrated prominent impacts on the molecular structure of the outer side chains of Aβ fibrils, then secondary structures of amyloid fibrils partially collapsed to varying degrees, indicating that the introduction of graphene nanosheets can cause severe structural disassociation and configuration change owing to adsorption of amyloid fibrils. As for the adhesion of Aβ fibrils to graphene, van der Waals (vdW) interaction was mainly responsible for driving Aβ fibrils binding to graphene, which effectively inhibited the sel...

Relation Between Stress Granules and Cytoplasmic Protein Aggregates Linked to Neurodegenerative Diseases.

ᅟ: A hallmark of neurodegenerative diseases is the accumulation of cytoplasmic protein aggregates in neurons of affected subjects. Among recently identified elements of these aggregates are RNA-binding proteins (RBPs) involved in RNA metabolism and alternative splicing and have in common the presence of low complexity domains (LCD) that are prone to self-assemble and form aggregates. The mechanism of cytoplasmic protein aggregation remains elusive. Stress granules (SGs) that are micrometric RNA-protein assemblies located in the cytoplasm of cells exposed to environmental stress are suspected to play the role of seeds. The review sheds light on the recent experimental results that suggest a link between SGs and cytoplasmic protein aggregates but also propose other routes for the formation of these aggregates. PURPOSE OF REVIEW: To analyze the potential relationship between cytoplasmic protein aggregates in neurons of affected subjects and stress granules. RECENT FINDINGS: Liquid phase separation explains how protein and RNA could assemble in membraneless compartments, notably SGs. These results highlight the importance of RBPs with LCD in the SG assembly. Maturation of SGs and in particular the dense core is a potential source of insoluble protein aggregates. Several lines of evidence linked stress granule dynamics to pathogenic protein aggregates. (i) Proteins that accumulate in cytoplasmic aggregates are also SG components. (ii) Neurons are specifically exposed to stress events due to their high metabolism and long lifespan. (iii) Diseases linked protein mutations affect the SG dynamics. (iv) SG dense core could be a breeding ground for protein aggregates. However, we should also keep in mind that SGs are not the only RNA-protein assembly in the cytoplasm; the RNA transport granules could also play a role in the formation of insoluble protein aggregates.