Formation of amyloid loops in brain tissues is controlled by the flexibility of protofibril chains.

Neurodegenerative diseases, such as Alzheimer's disease (AD), are associated with protein misfolding and aggregation into amyloid fibrils. Increasing evidence suggests that soluble, low-molecular-weight aggregates play a key role in disease-associated toxicity. Within this population of aggregates, closed-loop pore-like structures have been observed for a variety of amyloid systems, and their presence in brain tissues is associated with high levels of neuropathology. However, their mechanism of formation and relationship with mature fibrils have largely remained challenging to elucidate. Here, we use atomic force microscopy and statistical theory of biopolymers to characterize amyloid ring structures derived from the brains of AD patients. We analyze the bending fluctuations of protofibrils and show that the process of loop formation is governed by the mechanical properties of their chains. We conclude that ex vivo protofibril chains possess greater flexibility than that imparted by hydrogen-bonded networks characteristic of mature amyloid fibrils, such that they are able to form end-to-end connections. These results explain the diversity in the structures formed from protein aggregation and shed light on the links between early forms of flexible ring-forming aggregates and their role in disease.

The Amyloid Fibril-Forming ß-Sheet Regions of Amyloid ß and α-Synuclein Preferentially Interact with the Molecular Chaperone 14-3-3ζ.

14-3-3 proteins are abundant, intramolecular proteins that play a pivotal role in cellular signal transduction by interacting with phosphorylated ligands. In addition, they are molecular chaperones that prevent protein unfolding and aggregation under cellular stress conditions in a similar manner to the unrelated small heat-shock proteins. In vivo, amyloid ß (Aß) and α-synuclein (α-syn) form amyloid fibrils in Alzheimer's and Parkinson's diseases, respectively, a process that is intimately linked to the diseases' progression. The 14-3-3ζ isoform potently inhibited in vitro fibril formation of the 40-amino acid form of Aß (Aß40) but had little effect on α-syn aggregation. Solution-phase NMR spectroscopy of 15N-labeled Aß40 and A53T α-syn determined that unlabeled 14-3-3ζ interacted preferentially with hydrophobic regions of Aß40 (L11-H21 and G29-V40) and α-syn (V3-K10 and V40-K60). In both proteins, these regions adopt ß-strands within the core of the amyloid fibrils prepared in vitro as well as those isolated from the inclusions of diseased individuals. The interaction with 14-3-3ζ is transient and occurs at the early stages of the fibrillar aggregation pathway to maintain the native, monomeric, and unfolded structure of Aß40 and α-syn. The N-terminal regions of α-syn interacting with 14-3-3ζ correspond with those that interact with other molecular chaperones as monitored by in-cell NMR spectroscopy.

Short-term memory span in aphasia: Insights from speech-timing measures.

Auditory-verbal short-term memory impairments are part and parcel of aphasia and interfere with linguistic processing. To date, the science about short-term memory impairments in aphasia has been generated and dominated by studying measures of accuracy, that is, span length. Because accuracy is expressed through speech, examining the speech-timing characteristics of persons with aphasia as they engage in spoken recall could reveal insights about the manner in which accuracy is achieved. Six speech-timing measures (e.g., response durations, pause durations) were elicited from the speech waveform of word span tasks from twelve people with aphasia. Speech-timing measures were compared to neuro-typical control participants. Speech-timing performance between erroneous and correct responses in the aphasia group was also examined. Across all measures, people with aphasia produced considerably longer speech-timing patterns in comparison to control participants. Memory load affected some measures in people with aphasia and control participants. Speech-timing in correct response trials was shorter than responses in erroneous trials. Memory span correlated only with one measure, namely, speech time (defined as the sum of each individual word duration in a response). Speech time also correlated with the following measures: Aphasia severity (Aphasia Quotient of the Western Aphasia Battery), spontaneous speech, and language comprehension (also measured by the Western Aphasia Battery). Some protracted speech-timing patterns in the aphasia group may be explained by a deregulation of activation-decay patterns. However, in the absence of further evidence from people with aphasia, possible issues around the sensitivity of some speech-timing measures limit firmer conclusions. Speech-timing measures are response-time measures, which have not been systematically studied in studies of short-term or working memory in aphasia and as such, can push the current boundaries of knowledge of short-term and working memory impairments in aphasia, not only in stroke related aphasia but also other neurological conditions.

Small heat-shock proteins: important players in regulating cellular proteostasis.

Small heat-shock proteins (sHsps) are a diverse family of intra-cellular molecular chaperone proteins that play a critical role in mitigating and preventing protein aggregation under stress conditions such as elevated temperature, oxidation and infection. In doing so, they assist in the maintenance of protein homeostasis (proteostasis) thereby avoiding the deleterious effects that result from loss of protein function and/or protein aggregation. The chaperone properties of sHsps are therefore employed extensively in many tissues to prevent the development of diseases associated with protein aggregation. Significant progress has been made of late in understanding the structure and chaperone mechanism of sHsps. In this review, we discuss some of these advances, with a focus on mammalian sHsp hetero-oligomerisation, the mechanism by which sHsps act as molecular chaperones to prevent both amorphous and fibrillar protein aggregation, and the role of post-translational modifications in sHsp chaperone function, particularly in the context of disease.

Molecular mechanisms used by chaperones to reduce the toxicity of aberrant protein oligomers.

Chaperones are the primary regulators of the proteostasis network and are known to facilitate protein folding, inhibit protein aggregation, and promote disaggregation and clearance of misfolded aggregates inside cells. We have tested the effects of five chaperones on the toxicity of misfolded oligomers preformed from three different proteins added extracellularly to cultured cells. All the chaperones were found to decrease oligomer toxicity significantly, even at very low chaperone/protein molar ratios, provided that they were added extracellularly rather than being overexpressed in the cytosol. Infrared spectroscopy and site-directed labeling experiments using pyrene ruled out structural reorganizations within the discrete oligomers. Rather, confocal microscopy, SDS-PAGE, and intrinsic fluorescence measurements indicated tight binding between oligomers and chaperones. Moreover, atomic force microscopy imaging indicated that larger assemblies of oligomers are formed in the presence of the chaperones. This suggests that the chaperones bind to the oligomers and promote their assembly into larger species, with consequent shielding of the reactive surfaces and a decrease in their diffusional mobility. Overall, the data indicate a generic ability of chaperones to neutralize extracellular misfolded oligomers efficiently and reveal that further assembly of protein oligomers into larger species can be an effective strategy to neutralize such extracellular species.

A radish seed antifungal peptide with a high amyloid fibril-forming propensity.

The amyloid fibril-forming ability of two closely related antifungal and antimicrobial peptides derived from plant defensin proteins has been investigated. As assessed by sequence analysis, thioflavin T binding, transmission electron microscopy, atomic force microscopy and X-ray fiber diffraction, a 19 amino acid fragment from the C-terminal region of Raphanus sativus antifungal protein, known as RsAFP-19, is highly amyloidogenic. Further, its fibrillar morphology can be altered by externally controlled conditions. Freezing and thawing led to amyloid fibril formation which was accompanied by loss of RsAFP-19 antifungal activity. A second, closely related antifungal peptide displayed no fibril-forming capacity. It is concluded that while fibril formation is not associated with the antifungal properties of these peptides, the peptide RsAFP-19 is of potential use as a controllable, highly amyloidogenic small peptide for investigating the structure of amyloid fibrils and their mechanism of formation.

A systematic review of acoustic change complex (ACC) measurements and applicability in children for the assessment of the neural capacity for sound and speech discrimination.

OBJECTIVE: The acoustic change complex (ACC) is a cortical auditory evoked potential (CAEP) and can be elicited by a change in an otherwise continuous sound. The ACC has been highlighted as a promising tool in the assessment of sound and speech discrimination capacity, and particularly for difficult-to-test populations such as infants with hearing loss, due to the objective nature of ACC measurements. Indeed, there is a pressing need to develop further means to accurately and thoroughly establish the hearing status of children with hearing loss, to help guide hearing interventions in a timely manner. Despite the potential of the ACC method, ACC measurements remain relatively rare in a standard clinical settings. The objective of this study was to perform an up-to-date systematic review on ACC measurements in children, to provide greater clarity and consensus on the possible methodologies, applications, and performance of this technique, and to facilitate its uptake in relevant clinical settings. DESIGN: Original peer-reviewed articles conducting ACC measurements in children (< 18 years). Data were extracted and summarised for: (1) participant characteristics; (2) ACC methods and auditory stimuli; (3) information related to the performance of the ACC technique; (4) ACC measurement outcomes, advantages, and challenges. The systematic review was conducted using PRISMA guidelines for reporting and the methodological quality of included articles was assessed. RESULTS: A total of 28 studies were identified (9 infant studies). Review results show that ACC responses can be measured in infants (from < 3 months), and there is evidence of age-dependency, including increased robustness of the ACC response with increasing childhood age. Clinical applications include the measurement of the neural capacity for speech and non-speech sound discrimination in children with hearing loss, auditory neuropathy spectrum disorder (ANSD) and central auditory processing disorder (CAPD). Additionally, ACCs can be recorded in children with hearing aids, auditory brainstem implants, and cochlear implants, and ACC results may guide hearing intervention/rehabilitation strategies. The review identified that the time taken to perform ACC measurements was often lengthy; the development of more efficient ACC test procedures for children would be beneficial. Comparisons between objective ACC measurements and behavioural measures of sound discrimination showed significant correlations for some, but not all, included studies. CONCLUSIONS: ACC measurements of the neural capacity to discriminate between speech and non-speech sounds are feasible in infants and children, and a wide range of possible clinical applications exist, although more time-efficient procedures would be advantageous for clinical uptake. A consideration of age and maturational effects is recommended, and further research is required to investigate the relationship between objective ACC measures and behavioural measures of sound and speech perception for effective clinical implementation.

On alert for Ebola: public health risk assessment of travellers from Uganda to the USA during the 2022 outbreak.

BACKGROUND: On 20 September 2022, the Ugandan Ministry of Health declared an outbreak of Ebola disease caused by Sudan ebolavirus. METHODS: From 6 October 2022 to 10 January 2023, Centers for Disease Control and Prevention (CDC) staff conducted public health assessments at five US ports of entry for travellers identified as having been in Uganda in the past 21 days. CDC also recommended that state, local and territorial health departments ('health departments') conduct post-arrival monitoring of these travellers. CDC provided traveller contact information, daily to 58 health departments, and collected health department data regarding monitoring outcomes. RESULTS: Among 11 583 travellers screened, 132 (1%) required additional assessment due to potential exposures or symptoms of concern. Fifty-three (91%) health departments reported receiving traveller data from CDC for 10 114 (87%) travellers, of whom 8499 (84%) were contacted for monitoring, 1547 (15%) could not be contacted and 68 (1%) had no reported outcomes. No travellers with high-risk exposures or Ebola disease were identified. CONCLUSION: Entry risk assessment and post-arrival monitoring of travellers are resource-intensive activities that had low demonstrated yield during this and previous outbreaks. The efficiency of future responses could be improved by incorporating an assessment of risk of importation of disease, accounting for individual travellers' potential for exposure, and expanded use of methods that reduce burden to federal agencies, health departments, and travellers.

Single molecule characterization of the interactions between amyloid-ß peptides and the membranes of hippocampal cells.

Oligomers of the 40 and 42 residue amyloid-ß peptides (Aß40 and Aß42) have been implicated in the neuronal damage and impaired cognitive function associated with Alzheimer's disease. However, little is known about the specific mechanisms by which these misfolded species induce such detrimental effects on cells. In this work, we use single-molecule imaging techniques to examine the initial interactions between Aß monomers and oligomers and the membranes of live cells. This highly sensitive method enables the visualization of individual Aß species on the cell surface and characterization of their oligomerization state, all at biologically relevant, nanomolar concentrations. The results indicate that oligomers preferentially interact with cell membranes, relative to monomers and that the oligomers become immobilized on the cell surface. Additionally, we observe that the interaction of Aß species with the cell membrane is inhibited by the presence of ATP-independent molecular chaperones. This study demonstrates the power of this methodology for characterizing the interactions between protein aggregates and the membranes of live neuronal cells at physiologically relevant concentrations and opens the door to quantitative studies of the cellular responses to potentially pathogenic oligomers.

Amyloid-ß oligomers are sequestered by both intracellular and extracellular chaperones.

The aberrant aggregation of the amyloid-ß peptide into ß-sheet rich, fibrillar structures proceeds via a heterogeneous ensemble of oligomeric intermediates that have been associated with neurotoxicity in Alzheimer's disease (AD). Of particular interest in this context are the mechanisms by which molecular chaperones, part of the primary biological defenses against protein misfolding, influence Aß aggregation. We have used single-molecule fluorescence techniques to compare the interactions between distinct aggregation states (monomers, oligomers, and amyloid fibrils) of the AD-associated amyloid-ß(1-40) peptide, and two molecular chaperones, both of which are upregulated in the brains of patients with AD and have been found colocalized with Aß in senile plaques. One of the chaperones, αB-crystallin, is primarily found inside cells, while the other, clusterin, is predominantly located in the extracellular environment. We find that both chaperones bind to misfolded oligomeric species and form long-lived complexes, thereby preventing both their further growth into fibrils and their dissociation. From these studies, we conclude that these chaperones have a common mechanism of action based on sequestering Aß oligomers. This conclusion suggests that these chaperones, both of which are ATP-independent, are able to inhibit potentially pathogenic Aß oligomer-associated processes whether they occur in the extracellular or intracellular environment.

Iron promotes the toxicity of amyloid beta peptide by impeding its ordered aggregation.

We have previously shown that overexpressing subunits of the iron-binding protein ferritin can rescue the toxicity of the amyloid ß (Aß) peptide in our Drosophila model system. These data point to an important pathogenic role for iron in Alzheimer disease. In this study, we have used an iron-selective chelating compound and RNAi-mediated knockdown of endogenous ferritin to further manipulate iron in the brain. We confirm that chelation of iron protects the fly from the harmful effects of Aß. To understand the pathogenic mechanisms, we have used biophysical techniques to see how iron affects Aß aggregation. We find that iron slows the progression of the Aß peptide from an unstructured conformation to the ordered cross-ß fibrils that are characteristic of amyloid. Finally, using mammalian cell culture systems, we have shown that iron specifically enhances Aß toxicity but only if the metal is present throughout the aggregation process. These data support the hypothesis that iron delays the formation of well ordered aggregates of Aß and so promotes its toxicity in Alzheimer disease.

Twisting transition between crystalline and fibrillar phases of aggregated peptides.

We study two distinctly ordered condensed phases of polypeptide molecules, amyloid fibrils and amyloidlike microcrystals, and the first-order twisting phase transition between these two states. We derive a single free-energy form which connects both phases. Our model identifies relevant degrees of freedom for describing the collective behavior of supramolecular polypeptide structures, reproduces accurately the results from molecular dynamics simulations as well as from experiments, and sheds light on the uniform nature of the dimensions of different peptide fibrils.

Binding of the molecular chaperone αB-crystallin to Aß amyloid fibrils inhibits fibril elongation.

The molecular chaperone αB-crystallin is a small heat-shock protein that is upregulated in response to a multitude of stress stimuli, and is found colocalized with Aß amyloid fibrils in the extracellular plaques that are characteristic of Alzheimer's disease. We investigated whether this archetypical small heat-shock protein has the ability to interact with Aß fibrils in vitro. We find that αB-crystallin binds to wild-type Aß(42) fibrils with micromolar affinity, and also binds to fibrils formed from the E22G Arctic mutation of Aß(42). Immunoelectron microscopy confirms that binding occurs along the entire length and ends of the fibrils. Investigations into the effect of αB-crystallin on the seeded growth of Aß fibrils, both in solution and on the surface of a quartz crystal microbalance biosensor, reveal that the binding of αB-crystallin to seed fibrils strongly inhibits their elongation. Because the lag phase in sigmoidal fibril assembly kinetics is dominated by elongation and fragmentation rates, the chaperone mechanism identified here represents a highly effective means to inhibit fibril proliferation. Together with previous observations of αB-crystallin interaction with α-synuclein and insulin fibrils, the results suggest that this mechanism is a generic means of providing molecular chaperone protection against amyloid fibril formation.

Metastability of native proteins and the phenomenon of amyloid formation.

An experimental determination of the thermodynamic stabilities of a series of amyloid fibrils reveals that this structural form is likely to be the most stable one that protein molecules can adopt even under physiological conditions. This result challenges the conventional assumption that functional forms of proteins correspond to the global minima in their free energy surfaces and suggests that living systems are conformationally as well as chemically metastable.

The interaction of alphaB-crystallin with mature alpha-synuclein amyloid fibrils inhibits their elongation.

alphaB-Crystallin is a small heat-shock protein (sHsp) that is colocalized with alpha-synuclein (alphaSyn) in Lewy bodies-the pathological hallmarks of Parkinson's disease-and is an inhibitor of alphaSyn amyloid fibril formation in an ATP-independent manner in vitro. We have investigated the mechanism underlying the inhibitory action of sHsps, and here we establish, by means of a variety of biophysical techniques including immunogold labeling and nuclear magnetic resonance spectroscopy, that alphaB-crystallin interacts with alphaSyn, binding along the length of mature amyloid fibrils. By measurement of seeded fibril elongation kinetics, both in solution and on a surface using a quartz crystal microbalance, this binding is shown to strongly inhibit further growth of the fibrils. The binding is also demonstrated to shift the monomer-fibril equilibrium in favor of dissociation. We believe that this mechanism, by which a sHsp interacts with mature amyloid fibrils, could represent an additional and potentially generic means by which at least some chaperones protect against amyloid aggregation and limit the onset of misfolding diseases.

The interaction of unfolding α-lactalbumin and malate dehydrogenase with the molecular chaperone αB-crystallin: a light and X-ray scattering investigation.

PURPOSE: The molecular chaperone αB-crystallin is found in high concentrations in the lens and is present in all major body tissues. Its structure and the mechanism by which it protects its target protein from aggregating and precipitating are not known. METHODS: Dynamic light scattering and X-ray solution scattering techniques were used to investigate structural features of the αB-crystallin oligomer when complexed with target proteins under mild stress conditions, i.e., reduction of α-lactalbumin at 37 °C and malate dehydrogenase when heated at 42 °C. In this investigation, the size, shape and particle distribution of the complexes were determined in real-time following the induction of stress. RESULTS: Overall, it is observed that the mass distribution, hydrodynamic radius, and spherical shape of the αB-crystallin oligomer do not alter significantly when it complexes with its target protein. CONCLUSIONS: The data are consistent with the target protein being located in the outer protein shell of the αB-crystallin oligomer where it is readily accessible for possible refolding via the action of other molecular chaperones.

Effects of Verb Overlap on Structural Priming in Dialogue: Implications for Syntactic Learning in Aphasia.

Purpose Although there is increasing interest in using structural priming as a means to ameliorate grammatical encoding deficits in persons with aphasia (PWAs), little is known about the precise mechanisms of structural priming that are associated with robust and enduring effects in PWAs. Two dialogue-like comprehension-to-production priming experiments investigated whether lexically independent (abstract structural) priming and/or lexically (verb) specific priming yields immediate and longer, lasting facilitation of syntactic production in PWAs. Method Seventeen PWAs and 20 healthy older adults participated in a collaborative picture-matching task where participant and experimenter took turns describing picture cards using transitive and dative sentences. In Experiment 1, a target was elicited immediately following a prime. In Experiment 2, 2 unrelated utterances intervened between a prime and target, thereby allowing us to examine lasting priming effects. In both experiments, the verb was repeated for half of the prime-target pairs to examine the lexical (verb) boost on priming. Results Healthy older adults demonstrated abstract priming in both transitives and datives not only in the immediate (Experiment 1) but also in the lasting (Experiment 2) priming condition. They also showed significantly enhanced priming by verb overlap (lexical boost) in transitives during immediate priming. PWAs demonstrated abstract priming in transitives in both immediate and lasting priming conditions. However, the magnitude of priming was not enhanced by verb overlap. Conclusions Abstract structural priming, but not lexically specific priming, is associated with reliable and lasting facilitation of message-structure mapping in aphasia. The findings also suggest that implicit syntactic learning via a dialogue-like comprehension-to-production task remains preserved in aphasia.

Priming sentence comprehension in aphasia: Effects of lexically independent and specific structural priming.

PURPOSE: Impaired message-structure mapping results in deficits in both sentence production and comprehension in aphasia. Structural priming has been shown to facilitate syntactic production for persons with aphasia (PWA). However, it remains unknown if structural priming is also effective in sentence comprehension. We examined if PWA show preserved and lasting structural priming effects during interpretation of syntactically ambiguous sentences and if the priming effects occur independently of or in conjunction with lexical (verb) information. METHODS: Eighteen PWA and 20 healthy older adults (HOA) completed a written sentence-picture matching task involving the interpretation of prepositional phrases (PP; the chef is poking the solider with an umbrella) that were ambiguous between high (verb modifier) and low attachment (object noun modifier). Only one interpretation was possible for prime sentences, while both interpretations were possible for target sentences. In Experiment 1, the target was presented immediately after the prime (0-lag). In Experiment 2, two filler items intervened between the prime and the target (2-lag). Within each experiment, the verb was repeated for half of the prime-target pairs, while different verbs were used for the other half. Participants' off-line picture matching choices and response times were measured. RESULTS: After reading a prime sentence with a particular interpretation, HOA and PWA tended to interpret an ambiguous PP in a target sentence in the same way and with faster response times. Importantly, both groups continued to show this priming effect over a lag (Experiment 2), although the effect was not as reliable in response times. However, neither group showed lexical (verb-specific) boost on priming, deviating from robust lexical boost seen in the young adults of prior studies. CONCLUSIONS: PWA demonstrate abstract (lexically-independent) structural priming in the absence of a lexically-specific boost. Abstract priming is preserved in aphasia, effectively facilitating not only immediate but also longer-lasting structure-message mapping during sentence comprehension.

Characterisation of amyloid fibril formation by small heat-shock chaperone proteins human alphaA-, alphaB- and R120G alphaB-crystallins.

AlphaB-Crystallin is a ubiquitous small heat-shock protein (sHsp) renowned for its chaperone ability to prevent target protein aggregation. It is stress-inducible and its up-regulation is associated with a number of disorders, including those linked to the deposition of misfolded proteins, such as Alzheimer's and Parkinson's diseases. We have characterised the formation of amyloid fibrils by human alphaB-crystallin in detail, and also that of alphaA-crystallin and the disease-related mutant R120G alphaB-crystallin. We find that the last 12 amino acid residues of the C-terminal region of alphaB-crystallin are predicted from their physico-chemical properties to have a very low propensity to aggregate. (1)H NMR spectroscopy reveals that this hydrophilic C-terminal region is flexible both in its solution state and in amyloid fibrils, where it protrudes from the fibrillar core. We demonstrate, in addition, that the equilibrium between different protofilament assemblies can be manipulated and controlled in vitro to select for particular alphaB-crystallin amyloid morphologies. Overall, this study suggests that there could be a fine balance in vivo between the native functional sHsp state and the formation of amyloid fibrils.

The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures.

Clusterin is an extracellular chaperone present in all disease-associated extracellular amyloid deposits, but its roles in amyloid formation and protein deposition in vivo are poorly understood. The current study initially aimed to characterize the effects of clusterin on amyloid formation in vitro by a panel of eight protein substrates. Two of the substrates (Alzheimer's beta peptide and a PI3-SH3 domain) were then used in further experiments to examine the effects of clusterin on amyloid cytotoxicity and to probe the mechanism of clusterin action. We show that clusterin exerts potent effects on amyloid formation, the nature and extent of which vary greatly with the clusterin:substrate ratio, and provide evidence that these effects are exerted via interactions with prefibrillar species that share common structural features. Proamyloidogenic effects of clusterin appear to be restricted to conditions in which the substrate protein is present at a very large molar excess; under these same conditions, clusterin coincorporates with substrate protein into insoluble aggregates. However, when clusterin is present at much higher but still substoichiometric levels (e.g., a molar ratio of clusterin:substrate=1:10), it potently inhibits amyloid formation and provides substantial cytoprotection. These findings suggest that clusterin is an important element in the control of extracellular protein misfolding.