The expanding amyloid family: Structure, stability, function, and pathogenesis.

The hidden world of amyloid biology has suddenly snapped into atomic-level focus, revealing over 80 amyloid protein fibrils, both pathogenic and functional. Unlike globular proteins, amyloid proteins flatten and stack into unbranched fibrils. Stranger still, a single protein sequence can adopt wildly different two-dimensional conformations, yielding distinct fibril polymorphs. Thus, an amyloid protein may define distinct diseases depending on its conformation. At the heart of this conformational variability lies structural frustrations. In functional amyloids, evolution tunes frustration levels to achieve either stability or sensitivity according to the fibril's biological function, accounting for the vast versatility of the amyloid fibril scaffold.

Cryo-EM structures of the D290V mutant of the hnRNPA2 low-complexity domain suggests how D290V affects phase separation and aggregation.

Heterogeneous nuclear ribonucleoprotein A2 (hnRNPA2) is a human ribonucleoprotein that transports RNA to designated locations for translation via its ability to phase separate. Its mutated form, D290V, is implicated in multisystem proteinopathy known to afflict two families, mainly with myopathy and Paget's disease of bone. Here, we investigate this mutant form of hnRNPA2 by determining cryo-EM structures of the recombinant D290V low complexity domain. We find that the mutant form of hnRNPA2 differs from the WT fibrils in four ways. In contrast to the WT fibrils, the PY-nuclear localization signals in the fibril cores of all three mutant polymorphs are less accessible to chaperones. Also, the mutant fibrils are more stable than WT fibrils as judged by phase separation, thermal stability, and energetic calculations. Similar to other pathogenic amyloids, the mutant fibrils are polymorphic. Thus, these structures offer evidence to explain how a D-to-V missense mutation diverts the assembly of reversible, functional amyloid-like fibrils into the assembly of pathogenic amyloid, and may shed light on analogous conversions occurring in other ribonucleoproteins that lead to neurological diseases such as amyotrophic lateral sclerosis and frontotemporal dementia.

TDP-43 and RNA form amyloid-like myo-granules in regenerating muscle.

A dominant histopathological feature in neuromuscular diseases, including amyotrophic lateral sclerosis and inclusion body myopathy, is cytoplasmic aggregation of the RNA-binding protein TDP-43. Although rare mutations in TARDBP-the gene that encodes TDP-43-that lead to protein misfolding often cause protein aggregation, most patients do not have any mutations in TARDBP. Therefore, aggregates of wild-type TDP-43 arise in most patients by an unknown mechanism. Here we show that TDP-43 is an essential protein for normal skeletal muscle formation that unexpectedly forms cytoplasmic, amyloid-like oligomeric assemblies, which we call myo-granules, during regeneration of skeletal muscle in mice and humans. Myo-granules bind to mRNAs that encode sarcomeric proteins and are cleared as myofibres mature. Although myo-granules occur during normal skeletal-muscle regeneration, myo-granules can seed TDP-43 amyloid fibrils in vitro and are increased in a mouse model of inclusion body myopathy. Therefore, increased assembly or decreased clearance of functionally normal myo-granules could be the source of cytoplasmic TDP-43 aggregates that commonly occur in neuromuscular disease.

Bioinformatic identification of previously unrecognized amyloidogenic proteins.

Low-complexity domains (LCDs) of proteins have been shown to self-associate, and pathogenic mutations within these domains often drive the proteins into amyloid aggregation associated with disease. These domains may be especially susceptible to amyloidogenic mutations because they are commonly intrinsically disordered and function in self-association. The question therefore arises whether a search for pathogenic mutations in LCDs of the human proteome can lead to identification of other proteins associated with amyloid disease. Here, we take a computational approach to identify documented pathogenic mutations within LCDs that may favor amyloid formation. Using this approach, we identify numerous known amyloidogenic mutations, including several such mutations within proteins previously unidentified as amyloidogenic. Among the latter group, we focus on two mutations within the TRK-fused gene protein (TFG), known to play roles in protein secretion and innate immunity, which are associated with two different peripheral neuropathies. We show that both mutations increase the propensity of TFG to form amyloid fibrils. We therefore conclude that TFG is a novel amyloid protein and propose that the diseases associated with its mutant forms may be amyloidoses.

Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA): A technology-development proof of concept diagnostic accuracy study of dielectrophoresis in patients with oral squamous cell carcinoma and dysplasia.

BACKGROUND: Delays in the identification and referral of oral cancer remain frequent. An accurate and non-invasive diagnostic test to be performed in primary care may help identifying oral cancer at an early stage and reduce mortality. Point-of-care Analysis for Non-invasive Diagnosis of Oral cancer (PANDORA) was a proof-of-concept prospective diagnostic accuracy study aimed at advancing the development of a dielectrophoresis-based diagnostic platform for oral squamous cell carcinoma (OSCC) and epithelial dysplasia (OED) using a novel automated DEPtech 3DEP analyser. METHODS: The aim of PANDORA was to identify the set-up of the DEPtech 3DEP analyser associated with the highest diagnostic accuracy in identifying OSCC and OED from non-invasive brush biopsy samples, as compared to the gold standard test (histopathology). Measures of accuracy included sensitivity, specificity, positive and negative predictive value. Brush biopsies were collected from individuals with histologically proven OSCC and OED, histologically proven benign mucosal disease, and healthy mucosa (standard test), and analysed via dielectrophoresis (index test). RESULTS: 40 individuals with OSCC/OED and 79 with benign oral mucosal disease/healthy mucosa were recruited. Sensitivity and specificity of the index test was 86.8% (95% confidence interval [CI], 71.9%-95.6%) and 83.6% (95% CI, 73.0%-91.2%). Analysing OSCC samples separately led to higher diagnostic accuracy, with 92.0% (95% CI, 74.0%-99.0%) sensitivity and 94.5% (95% CI, 86.6%-98.5%) specificity. CONCLUSION: The DEPtech 3DEP analyser has the potential to identify OSCC and OED with notable diagnostic accuracy and warrants further investigation as a potential triage test in the primary care setting for patients who may need to progress along the diagnostic pathway and be offered a surgical biopsy.

The α-synuclein hereditary mutation E46K unlocks a more stable, pathogenic fibril structure.

Aggregation of α-synuclein is a defining molecular feature of Parkinson's disease, Lewy body dementia, and multiple systems atrophy. Hereditary mutations in α-synuclein are linked to both Parkinson's disease and Lewy body dementia; in particular, patients bearing the E46K disease mutation manifest a clinical picture of parkinsonism and Lewy body dementia, and E46K creates more pathogenic fibrils in vitro. Understanding the effect of these hereditary mutations on α-synuclein fibril structure is fundamental to α-synuclein biology. We therefore determined the cryo-electron microscopy (cryo-EM) structure of α-synuclein fibrils containing the hereditary E46K mutation. The 2.5-Å structure reveals a symmetric double protofilament in which the molecules adopt a vastly rearranged, lower energy fold compared to wild-type fibrils. We propose that the E46K misfolding pathway avoids electrostatic repulsion between K46 and K80, a residue pair which form the E46-K80 salt bridge in the wild-type fibril structure. We hypothesize that, under our conditions, the wild-type fold does not reach this deeper energy well of the E46K fold because the E46-K80 salt bridge diverts α-synuclein into a kinetic trap-a shallower, more accessible energy minimum. The E46K mutation apparently unlocks a more stable and pathogenic fibril structure.

Prevalence and species distribution of the low-complexity, amyloid-like, reversible, kinked segment structural motif in amyloid-like fibrils.

Membraneless organelles (MLOs) are vital and dynamic reaction centers in cells that compartmentalize the cytoplasm in the absence of a membrane. Multivalent interactions between protein low-complexity domains contribute to MLO organization. Previously, we used computational methods to identify structural motifs termed low-complexity amyloid-like reversible kinked segments (LARKS) that promote phase transition to form hydrogels and that are common in human proteins that participate in MLOs. Here, we searched for LARKS in the proteomes of six model organisms: Homo sapiens, Drosophila melanogaster, Plasmodium falciparum, Saccharomyces cerevisiae, Mycobacterium tuberculosis, and Escherichia coli to gain an understanding of the distribution of LARKS in the proteomes of various species. We found that LARKS are abundant in M. tuberculosis, D. melanogaster, and H. sapiens but not in S. cerevisiae or P. falciparum. LARKS have high glycine content, which enables kinks to form as exemplified by the known LARKS-rich amyloidogenic structures of TDP43, FUS, and hnRNPA2, three proteins that are known to participate in MLOs. These results support the idea of LARKS as an evolved structural motif. Based on these results, we also established the LARKSdb Web server, which permits users to search for LARKS in their protein sequences of interest.

Systemic AAV9 gene therapy using the synapsin I promoter rescues a mouse model of neuronopathic Gaucher disease but with limited cross-correction potential to astrocytes.

Gaucher disease is caused by mutations in the GBA gene, which encodes for the lysosomal enzyme ß-glucocerebrosidase (GCase), resulting in the accumulation of storage material in visceral organs and in some cases the brain of affected patients. While there is a commercially available treatment for the systemic manifestations, neuropathology still remains untreatable. We previously demonstrated that gene therapy represents a feasible therapeutic tool for the treatment of the neuronopathic forms of Gaucher disease (nGD). In order to further enhance the therapeutic affects to the central nervous system, we systemically delivered an adeno-associated virus (AAV) serotype 9 carrying the human GBA gene under control of a neuron-specific promoter to an nGD mouse model. Gene therapy increased the life span of treated animals, rescued the lethal neurodegeneration, normalized the locomotor behavioural defects and ameliorated the visceral pathology. Together, these results provided further indication of gene therapy as a possible effective treatment option for the neuropathic forms of Gaucher disease.

Transcriptome-based screening of ion channels and transporters in a migratory chondroprogenitor cell line isolated from late-stage osteoarthritic cartilage.

Chondrogenic progenitor cells (CPCs) may be used as an alternative source of cells with potentially superior chondrogenic potential compared to mesenchymal stem cells (MSCs), and could be exploited for future regenerative therapies targeting articular cartilage in degenerative diseases such as osteoarthritis (OA). In this study, we hypothesised that CPCs derived from OA cartilage may be characterised by a distinct channelome. First, a global transcriptomic analysis using Affymetrix microarrays was performed. We studied the profiles of those ion channels and transporter families that may be relevant to chondroprogenitor cell physiology. Following validation of the microarray data with quantitative reverse transcription-polymerase chain reaction, we examined the role of calcium-dependent potassium channels in CPCs and observed functional large-conductance calcium-activated potassium (BK) channels involved in the maintenance of the chondroprogenitor phenotype. In line with our very recent results, we found that the KCNMA1 gene was upregulated in CPCs and observed currents that could be attributed to the BK channel. The BK channel inhibitor paxilline significantly inhibited proliferation, increased the expression of the osteogenic transcription factor RUNX2, enhanced the migration parameters, and completely abolished spontaneous Ca2+ events in CPCs. Through characterisation of their channelome we demonstrate that CPCs are a distinct cell population but are highly similar to MSCs in many respects. This study adds key mechanistic data to the in-depth characterisation of CPCs and their phenotype in the context of cartilage regeneration.

Neonatal brain-directed gene therapy rescues a mouse model of neurodegenerative CLN6 Batten disease.

The neuronal ceroid lipofuscinoses (NCLs), more commonly referred to as Batten disease, are a group of inherited lysosomal storage disorders that present with neurodegeneration, loss of vision and premature death. There are at least 13 genetically distinct forms of NCL. Enzyme replacement therapies and pre-clinical studies on gene supplementation have shown promising results for NCLs caused by lysosomal enzyme deficiencies. The development of gene therapies targeting the brain for NCLs caused by defects in transmembrane proteins has been more challenging and only limited therapeutic effects in animal models have been achieved so far. Here, we describe the development of an adeno-associated virus (AAV)-mediated gene therapy to treat the neurodegeneration in a mouse model of CLN6 disease, a form of NCL with a deficiency in the membrane-bound protein CLN6. We show that neonatal bilateral intracerebroventricular injections with AAV9 carrying CLN6 increase lifespan by more than 90%, maintain motor skills and motor coordination and reduce neuropathological hallmarks of Cln6-deficient mice up to 23 months post vector administration. These data demonstrate that brain-directed gene therapy is a valid strategy to treat the neurodegeneration of CLN6 disease and may be applied to other forms of NCL caused by transmembrane protein deficiencies in the future.

DNA Facilitates Oligomerization and Prevents Aggregation via DNA Networks.

Previous studies have shown that nucleic acids can nucleate protein aggregation in disease-related proteins, but in other cases, they can act as molecular chaperones that prevent protein aggregation, even under extreme conditions. In this study, we describe the link between these two behaviors through a combination of electron microscopy and aggregation kinetics. We find that two different proteins become soluble under harsh conditions through oligomerization with DNA. These DNA/protein oligomers form "networks," which increase the speed of oligomerization. The cases of DNA both increasing and preventing protein aggregation are observed to stem from this enhanced oligomerization. This observation raises interesting questions about the role of nucleic acids in aggregate formation in disease states.

Epilepsy Gene Therapy Using an Engineered Potassium Channel.

Refractory focal epilepsy is a devastating disease for which there is frequently no effective treatment. Gene therapy represents a promising alternative, but treating epilepsy in this way involves irreversible changes to brain tissue, so vector design must be carefully optimized to guarantee safety without compromising efficacy. We set out to develop an epilepsy gene therapy vector optimized for clinical translation. The gene encoding the voltage-gated potassium channel Kv1.1, KCNA1, was codon optimized for human expression and mutated to accelerate the recovery of the channels from inactivation. For improved safety, this engineered potassium channel (EKC) gene was packaged into a nonintegrating lentiviral vector under the control of a cell type-specific CAMK2A promoter. In a blinded, randomized, placebo-controlled preclinical trial, the EKC lentivector robustly reduced seizure frequency in a male rat model of focal neocortical epilepsy characterized by discrete spontaneous seizures. When packaged into an adeno-associated viral vector (AAV2/9), the EKC gene was also effective at suppressing seizures in a male rat model of temporal lobe epilepsy. This demonstration of efficacy in a clinically relevant setting, combined with the improved safety conferred by cell type-specific expression and integration-deficient delivery, identify EKC gene therapy as being ready for clinical translation in the treatment of refractory focal epilepsy.SIGNIFICANCE STATEMENT Pharmacoresistant epilepsy affects up to 0.3% of the population. Although epilepsy surgery can be effective, it is limited by risks to normal brain function. We have developed a gene therapy that builds on a mechanistic understanding of altered neuronal and circuit excitability in cortical epilepsy. The potassium channel gene KCNA1 was mutated to bypass post-transcriptional editing and was packaged in a nonintegrating lentivector to reduce the risk of insertional mutagenesis. A randomized, blinded preclinical study demonstrated therapeutic effectiveness in a rodent model of focal neocortical epilepsy. Adeno-associated viral delivery of the channel to both hippocampi was also effective in a model of temporal lobe epilepsy. These results support clinical translation to address a major unmet need.

AAV9 intracerebroventricular gene therapy improves lifespan, locomotor function and pathology in a mouse model of Niemann-Pick type C1 disease.

Niemann-Pick type C disease (NP-C) is a fatal neurodegenerative lysosomal storage disorder. It is caused in 95% of cases by a mutation in the NPC1 gene that encodes NPC1, an integral transmembrane protein localized to the limiting membrane of the lysosome. There is no cure for NP-C but there is a disease-modifying drug (miglustat) that slows disease progression but with associated side effects. Here, we demonstrate in a well-characterized mouse model of NP-C that a single administration of AAV-mediated gene therapy to the brain can significantly extend lifespan, improve quality of life, prevent or ameliorate neurodegeneration, reduce biochemical pathology and normalize or improve various indices of motor function. Over-expression of human NPC1 does not cause adverse effects in the brain and correctly localizes to late endosomal/lysosomal compartments. Furthermore, we directly compare gene therapy to licensed miglustat. Even at a low dose, gene therapy has all the benefits of miglustat but without adverse effects. On the basis of these findings and on-going ascendency of the field, we propose intracerebroventricular gene therapy as a potential therapeutic option for clinical use in NP-C.

High-throughput, low-loss, low-cost, and label-free cell separation using electrophysiology-activated cell enrichment.

Currently, cell separation occurs almost exclusively by density gradient methods and by fluorescence- and magnetic-activated cell sorting (FACS/MACS). These variously suffer from lack of specificity, high cell loss, use of labels, and high capital/operating cost. We present a dielectrophoresis (DEP)-based cell-separation method, using 3D electrodes on a low-cost disposable chip; one cell type is allowed to pass through the chip whereas the other is retained and subsequently recovered. The method advances usability and throughput of DEP separation by orders of magnitude in throughput, efficiency, purity, recovery (cells arriving in the correct output fraction), cell losses (those which are unaccounted for at the end of the separation), and cost. The system was evaluated using three example separations: live and dead yeast; human cancer cells/red blood cells; and rodent fibroblasts/red blood cells. A single-pass protocol can enrich cells with cell recovery of up to 91.3% at over 300,000 cells per second with >3% cell loss. A two-pass protocol can process 300,000,000 cells in under 30 min, with cell recovery of up to 96.4% and cell losses below 5%, an effective processing rate >160,000 cells per second. A three-step protocol is shown to be effective for removal of 99.1% of RBCs spiked with 1% cancer cells while maintaining a processing rate of ∼170,000 cells per second. Furthermore, the self-contained and low-cost nature of the separator device means that it has potential application in low-contamination applications such as cell therapies, where good manufacturing practice compatibility is of paramount importance.

Dielectrophoretic analysis of treated cancer cells for rapid assessment of treatment efficacy.

Whilst personalized medicine (where interventions are precisely tailored to a patient's genotype and phenotype, as well as the nature and state of the disease) is regarded as an optimal form of treatment, the time and cost associated with it means it remains inaccessible to the greater public. A simpler alternative, stratified medicine, identifies groups of patients who are likely to respond to a given treatment. This allows appropriate treatments to be selected at the start of therapy, avoiding the common "trial and error" approach of replacing a therapy only once it is demonstrated to be ineffective in the patient. For stratification to be effective, tests are required that rapidly predict treatment effectiveness. Most tests use genetic analysis to identify drug targets, but these can be expensive and may not detect changes in the phenotype that affect drug sensitivity. An alternative method is to assess the whole-cell phenotype by evaluating drug response using cells from a biopsy. We assessed dielectrophoresis to assess drug efficacy on short timescales and at low cost. To explore the principle of assessing drug efficacy we examined two cell lines (one expressing EGFR, one not) with the drug Iressa. We then further explored the sensitive cells using combinations of chemotherapeutic and radiotherapeutic therapies. Our results compare with known effects of these cell/treatment combination, and offer the additional benefit over methods such as TUNEL of detecting drug effects such as cell cycle arrest, which do not cause cell death.

Accurate quantification of apoptosis progression and toxicity using a dielectrophoretic approach.

A loss of ability of cells to undergo apoptosis (programmed cell death, whereby the cell ceases to function and destroys itself) is commonly associated with cancer, and many anti-cancer interventions aim to restart the process. Consequently, the accurate quantification of apoptosis is essential in understanding the function and performance of new anti-cancer drugs. Dielectrophoresis has previously been demonstrated to detect apoptosis more rapidly than other methods, and is low-cost, label-free and rapid, but has previously been unable to accurately quantify cells through the apoptotic process because cells in late apoptosis disintegrate, making cell tracking impossible. In this paper we use a novel method based on light absorbance and multi-population tracking to quantify the progress of apoptosis, benchmarking against conventional assays including MTT, trypan blue and Annexin-V. Analyses are performed on suspension and adherent cells, and using two apoptosis-inducing agents. IC50 measurements compared favourably to MTT and were superior to trypan blue, whilst also detecting apoptotic progression faster than Annexin-V.

Effects of cell detachment methods on the dielectric properties of adherent and suspension cells.

Cell analyses such as flow cytometry, dielectrophoresis, and some patch-clamp techniques require that cells be in monodisperse suspension in order for analysis to occur. Where cells have a normally adherent phenotype in vivo, this requires that cells be removed from the surface of the culture flask or vessel. This can be achieved in many ways, but most commonly either by the use of a dissociation medium of some form, or by scraping the cells from the surface. Both methods have potential drawbacks; chemical methods may alter the properties of the cells to such a degree that the measurement might be regarded as meaningless, whilst scraping could cause physical damage to the structure of the cells. In this paper, we use dielectrophoresis to analyse the electrical properties of two adherent cell lines detached by multiple methods, and compare these against a control cell line of suspension cells, examined in both the native state and when subject to the same chemical treatments. The results indicate that most chemical agents do not alter the electrophysiology of cells directly, though they may trigger some potential cell deterioration processes such as apoptosis in the cells. This can be observed in the production of apoptotic body-like particles and the alteration of cytoplasmic conductivity (which has been associated with apoptotic water efflux). However, cells detached by scraping exhibited statistically significant differences in their electrophysiological properties when compared to those detached by the chemical methods, indicating that this method is unsuitable for detachment of adherent cells prior to analysis of isolates suspension cells.

Epithelial cancer cells exhibit different electrical properties when cultured in 2D and 3D environments.

BACKGROUND: Many drug development and toxicology studies are performed using cells grown in monolayers in well-plates and flasks, despite the fact that these are widely held to be different to cells found in the native environment. 3D, tissue engineered, organotypical tissue culture systems have been developed to be more representative of the native tissue environment than standard monolayer cultures. Whilst the biochemical differences between cells grown in 2D and 3D culture have been explored, the changes on the electrophysiological properties of the cells have not. METHODS: We compared the electrophysiological properties of primary normal oral keratinocytes (nOK) and cancerous abnormal oral keratinocytes (aOK), cultured in standard monolayer and reconstituted 3D organotypical tissue cultures. The electrophysiological properties of populations of the cells were analysed using dielectrophoresis. The intracellular conductivity of aOK was significantly increased when grown in organotypical cultures compared to counterpart cells grown in monolayer cultures. RESULTS: 3D cultured aOK showed almost identical intracellular conductivity to nOK also grown in organotypical cultures, but significantly different to aOK grown in monolayers. The effective membrane capacitance of aOK grown in 3D was found to be significantly higher than nOK, but there was no significant difference between the electrophysiological properties of nOK grown in 2D and 3D cultures. GENERAL SIGNIFICANCE: This work suggests that factors such as cell shape and cytoplasmic trafficking between cells play an important role in their electrophysiology, and highlights the need to use in vitro models more representative of native tissue when studying cell electrophysiological properties.

Factors affecting particle collection by electro-osmosis in microfluidic systems.

Alternating-current electro-osmosis, a phenomenon of fluid transport due to the interaction between an electrical double layer and a tangential electric field, has been used both for inducing fluid movement and for the concentration of particles suspended in the fluid. This offers many advantages over other phenomena used to trap particles, such as placing particles at an electrode centre rather than an edge; benefits of scale, where electrodes hundreds of micrometers across can trap particles from the molecules to cells at the same rate; and a trapping volume limited by the vortex height, a phenomenon thus far unstudied. In this paper, the collection of particles due to alternating-current electro-osmosis driven collection is examined for a range of particle concentrations, inter-electrode gap widths, chamber heights and media viscosity and density. A model of collection behaviour is described where particle collection over time is governed by two processes, one driven by the vortices and the other by sedimentation, allowing the determination of the maximum height of vortex-driven collection, but also indicates how trapping is limited by high particle concentrations and fluid velocities. The results also indicate that viscosity, rather than density, is a significant governing factor in determining the trapping behaviour of particles.

Continuous flow nanoparticle concentration using alternating current-electroosmotic flow.

Achieving real-time detection of environmental pathogens such as viruses and bacterial spores requires detectors with both rapid action and a suitable detection threshold. However, most biosensors have detection limits of an order of magnitude or more above the potential infection threshold, limiting their usefulness. This can be improved through the use of automated sample preparation techniques such as preconcentration. In this paper, we describe the use of AC electroosmosis to concentrate nanoparticles from a continuous flow. Electrodes at an optimized angle across a flow cell, and energized by a 1 kHz signal, were used to push nanoparticles to one side of a flow cell, and to extract the resulting stream with a high particle concentration from that side of the flow cell. A simple model of the behavior of particles in the flow cell has been developed, which shows good agreement with experimental results. The method indicates potential for higher concentration factors through cascading devices.