The extracellular chaperone clusterin potently inhibits human lysozyme amyloid formation by interacting with prefibrillar species.

We have studied the effects of the extracellular molecular chaperone, clusterin, on the in vitro aggregation of mutational variants of human lysozyme, including one associated with familial amyloid disease. The aggregation of the amyloidogenic variant I56T is inhibited significantly at clusterin to lysozyme ratios as low as 1:80 (i.e. one clusterin molecule per 80 lysozyme molecules). Experiments indicate that under the conditions where inhibition of aggregation occurs, clusterin does not bind detectably to the native or fibrillar states of lysozyme, or to the monomeric transient intermediate known to be a key species in the aggregation reaction. Rather, it seems to interact with oligomeric species that are present at low concentrations during the lag (nucleation) phase of the aggregation reaction. This behavior suggests that clusterin, and perhaps other extracellular chaperones, could have a key role in curtailing the potentially pathogenic effects of the misfolding and aggregation of proteins that, like lysozyme, are secreted into the extracellular environment.

Electrical Stimulation Using Conductive Polymer Polypyrrole Counters Reduced Neurite Outgrowth of Primary Prefrontal Cortical Neurons from NRG1-KO and DISC1-LI Mice.

Deficits in neurite outgrowth, possibly involving dysregulation of risk genes neuregulin-1 (NRG1) and disrupted in schizophrenia 1 (DISC1) have been implicated in psychiatric disorders including schizophrenia. Electrical stimulation using conductive polymers has been shown to stimulate neurite outgrowth of differentiating human neural stem cells. This study investigated the use of the electroactive conductive polymer polypyrrole (Ppy) to counter impaired neurite outgrowth of primary pre-frontal cortical (PFC) neurons from NRG1-knock out (NRG1-KO) and DISC1-locus impairment (DISC1-LI) mice. Whereas NRG1-KO and DISC1-LI exhibited reduced neurite length and number of neurite branches compared to wild-type controls, this was not apparent for cultures on electroactive Ppy. Additionally, the use of the Ppy substrate normalised the synaptophysin and PSD95 protein and mRNA expression whereas both are usually reduced by NRG1-KO or DISC1-LI. Our findings support the utility of Ppy mediated electrical stimulation to prevent the reduction of neurite outgrowth and related synaptic protein expression in the primary PFC neurons from NRG1-KO and DISC1-LI mice, providing proof-of-concept for treating neurodevelopmental diseases including schizophrenia.

Use of conducting polymers to facilitate neurite branching in schizophrenia-related neuronal development.

Schizophrenia (SCZ) is a debilitating mental disorder which results in high healthcare and loss of productivity costs to society. This disease remains poorly understood, however there is increasing evidence suggesting a role for oxidative damage in the disease etiology. We aimed to examine the effect of the conducting polymer polypyrrole on the growth and morphology of both wildtype and neuregulin-1 knock out (NRG-1 +/-) explant cells. Polypyrrole is an organic conducting polymer known to be cytocompatible and capable of acting as a platform for effective stimulation of neurons. Here we demonstrate for the first time the ability of this material to mediate processes occurring in disease affected neurons: schizophrenic model cortical neurons. Prefrontal cortical cells were grown on conducting polymer scaffolds of specific composition and showed significantly increased neurite branching and outgrowth length on the polymers compared to controls. Concurrently, a more significant enhancement was seen in both parameters in the NRG-1 +/- model cells. This finding implies that conducting polymers such as polypyrrole may be utilised to overcome neuro-functional deficits associated with neurological disease in humans.

Electrical stimulation enhances the acetylcholine receptors available for neuromuscular junction formation.

Neuromuscular junctions (NMJ) are specialized synapses that link motor neurons with muscle fibers. These sites are fundamental to human muscle activity, controlling swallowing and breathing amongst many other vital functions. Study of this synapse formation is an essential area in neuroscience; the understanding of how neurons interact and control their targets during development and regeneration are fundamental questions. Existing data reveals that during initial stages of development neurons target and form synapses driven by biophysical and biochemical cues, and during later stages they require electrical activity to develop their functional interactions. The aim of this study was to investigate the effect of exogenous electrical stimulation (ES) electrodes directly in contact with cells, on the number and size of acetylcholine receptor (AChR) clusters available for NMJ formation. We used a novel in vitro model that utilizes a flexible electrical stimulation system and allows the systematic testing of several stimulation parameters simultaneously as well as the use of alternative electrode materials such as conductive polymers to deliver the stimulation. Functionality of NMJs under our co-culture conditions was demonstrated by monitoring changes in the responses of primary myoblasts to chemical stimulants that specifically target neuronal signaling. Our results suggest that biphasic electrical stimulation at 250Hz, 100µs pulse width and current density of 1mA/cm2 for 8h, applied via either gold-coated mylar or the conductive polymer PPy, significantly increased the number and size of AChRs clusters available for NMJ formation. This study supports the beneficial use of direct electrical stimulation as a strategic therapy for neuromuscular disorders. STATEMENT OF SIGNIFICANCE: The beneficial effects of electrical stimulation (ES) on human cells in vitro and in vivo have long been known. Although the effects of stimulation are clear and the therapeutic benefits are known, no uniform parameters exist with regard to the duration, frequency and amplitude of the ES. To this end, we are answering several important questions on the parameters for ES of nerve and muscle monocultures and co-cultures by probing the effects on the enhancement of acetylcholine receptors (AChR) clustering available for neuromuscular junction formation using a conductive platform. This work opens the possibility to combine electrical stimulus delivered via conductive polymer substrates, from which biomolecules could also be delivered, providing opportunities to further enhance the therapeutic effect.

3D printing of layered brain-like structures using peptide modified gellan gum substrates.

The brain is an enormously complex organ structured into various regions of layered tissue. Researchers have attempted to study the brain by modeling the architecture using two dimensional (2D) in vitro cell culturing methods. While those platforms attempt to mimic the in vivo environment, they do not truly resemble the three dimensional (3D) microstructure of neuronal tissues. Development of an accurate in vitro model of the brain remains a significant obstacle to our understanding of the functioning of the brain at the tissue or organ level. To address these obstacles, we demonstrate a new method to bioprint 3D brain-like structures consisting of discrete layers of primary neural cells encapsulated in hydrogels. Brain-like structures were constructed using a bio-ink consisting of a novel peptide-modified biopolymer, gellan gum-RGD (RGD-GG), combined with primary cortical neurons. The ink was optimized for a modified reactive printing process and developed for use in traditional cell culturing facilities without the need for extensive bioprinting equipment. Furthermore the peptide modification of the gellan gum hydrogel was found to have a profound positive effect on primary cell proliferation and network formation. The neural cell viability combined with the support of neural network formation demonstrated the cell supportive nature of the matrix. The facile ability to form discrete cell-containing layers validates the application of this novel printing technique to form complex, layered and viable 3D cell structures. These brain-like structures offer the opportunity to reproduce more accurate 3D in vitro microstructures with applications ranging from cell behavior studies to improving our understanding of brain injuries and neurodegenerative diseases.

SOD1 protein aggregates stimulate macropinocytosis in neurons to facilitate their propagation.

BACKGROUND: Amyotrophic Lateral Sclerosis is characterized by a focal onset of symptoms followed by a progressive spread of pathology that has been likened to transmission of infectious prions. Cell-to-cell transmission of SOD1 protein aggregates is dependent on fluid-phase endocytosis pathways, although the precise molecular mechanisms remain to be elucidated. RESULTS: We demonstrate in this paper that SOD1 aggregates interact with the cell surface triggering activation of Rac1 and subsequent membrane ruffling permitting aggregate uptake via stimulated macropinocytosis. In addition, other protein aggregates, including those associated with neurodegenerative diseases (TDP-43, Httex146Q, α-synuclein) also trigger membrane ruffling to gain entry into the cell. Aggregates are able to rupture unstructured macropinosomes to enter the cytosol allowing propagation of aggregation to proceed. CONCLUSION: Thus, we conclude that in addition to basic proteostasis mechanisms, pathways involved in the activation of macropinocytosis are key determinants in the spread of pathology in these misfolding diseases.

Multifunctional conducting fibres with electrically controlled release of ciprofloxacin.

We hereby present a new method of producing coaxial conducting polymer fibres loaded with an antibiotic drug that can then be subsequently released (or sustained) in response to electrical stimulation. The method involves wet-spinning of poly(3,4-ethylenedioxythiophene) poly(styrenesulfonate) (PEDOT:PSS) fibre, which served as the inner core to the electropolymerised outer shell layer of polypyrrole (Ppy). Ciprofloxacin hydrochloride (Cipro) was selected as the model drug and as the dopant in the Ppy synthesis. The release of Cipro in phosphate buffered saline (PBS) from the fibres was controlled by switching the redox state of Ppy.Cipro layer. Released Cipro under passive and stimulated conditions were tested against Gram positive (Streptococcus pyogenes) and Gram negative (Escherichia coli) bacteria. Significant inhibition of bacterial growth was observed against both strains tested. These results confirm that Cipro retains antibacterial properties during fibre fabrication and electrochemically controlled release. In vitro cytotoxicity testing utilising the neural B35 cell line confirmed the cytocompatibility of the drug loaded conducting fibres. Electrical conductivity, cytocompatibility and tuning release profile from this flexible fibre can lead to promising bionic applications such as neuroprosthetics and localised drug delivery.

Cell attachment and proliferation on high conductivity PEDOT-glycol composites produced by vapour phase polymerisation.

High conductivity poly(3,4-ethylene dioxythiophene) (PEDOT) was synthesised using vacuum vapour phase polymerization (VVPP). The process produces PEDOT composites which incorporate glycol within the polymer. To assess biocompatibility, a suite of analytical techniques were utilised in an effort to characterise the level of glycol present and its impact on cell attachment and proliferation. A small decrease in fibroblast cell attachment and proliferation was observed with increasing glycol content within the PEDOT composite. Keratinocyte cell attachment and proliferation by comparison showed an increase. As such, the results herein indicate that cell attachment and proliferation depends on the individual cell lines used and that the impact of glycol within the PEDOT composite is negligible. This positive outcome prompted investigation of this polymer as a platform for electro-stimulation work. Application of oxidising and reducing potentials to the PEDOT composite were utilised to examine the effect on biocompatibility. Significant effects were seen with altered protein presentation on the reduced surface, and lower mass adsorbed at the oxidised surface. Keratinocytes interacted significantly better on the reduced surface whereas fibroblasts displayed dependence on protein density, with significantly lower spreading on the oxidised surface. Understanding how proteins interact at electrically biased polymer surfaces and in turn affect cell behaviour, underpins the utilisation of such tunable surfaces in biomedical devices.

Effects of glycosylation on the structure and function of the extracellular chaperone clusterin.

Clusterin is the first well characterized, constitutively secreted extracellular chaperone that binds to exposed regions of hydrophobicity on non-native proteins. It may help control the folding state of extracellular proteins by targeting them for receptor-mediated endocytosis and intracellular lysosomal degradation. A notable feature of secreted clusterin is its heavy glycosylation. Although carbohydrate comprises approximately 20-25% of the total mass of the mature molecule, its function is unknown. Results from the current study demonstrate that deglycosylation of human serum clusterin had little effect on its overall secondary structure content but produced a small increase in solvent-exposed hydrophobicity and enhanced the propensity of the molecule to aggregate in solution. These changes were associated with increased binding to a variety of ligands but did not substantially impact the ability of clusterin to inhibit heat-induced precipitation of citrate synthase. Evidence suggesting that the normally conjugated sugars are important in the interaction of secreted clusterin with a lectin-type receptor on liver cells is also presented. Bulk expression of fully processed, glycosylated clusterin in mammalian cells is difficult, often producing inappropriately disulfide-bonded high molecular weight aggregates; this has hampered previous studies aimed at identifying those regions of the molecule important in its chaperone action. The current results suggest that it may be possible in the future to study the structure and chaperone function of clusterin using recombinant protein (lacking sugars) conveniently bulk-expressed in bacteria.

Quality control of protein folding in extracellular space.

The pathologies of many serious human diseases are thought to develop from the effects of intra- or extracellular aggregates of non-native proteins. Inside cells, chaperone and protease systems regulate protein folding; however, little is known about any corresponding mechanisms that operate extracellularly. The identification of these mechanisms is important for the development of new disease therapies. This review briefly discusses the consequences of protein misfolding, the intracellular mechanisms that control folding and the potential corresponding extracellular control processes. Finally, a new speculative model is described, which proposes that newly discovered extracellular chaperones bind to exposed regions of hydrophobicity on non-native, extracellular proteins to target them for receptor-mediated endocytosis and intracellular, lysosomal degradation.