Multishank Thin-Film Neural Probes and Implantation System for High-Resolution Neural Recording Applications

Abstract Silicon probes have played a key role in studying the brain. However, the stark mechanical mismatch between these probes and the brain leads to chronic damage in the surrounding neural tissue, limiting their application in research and clinical translation. Mechanically flexible probes made of thin plastic shanks offer an attractive tissue‐compatible alternative but are difficult to implant into the brain. They also struggle to achieve the electrode density and layout necessary for the high‐resolution applications their silicon counterparts excel at. Here, a multishank high‐density flexible neural probe design is presented, which emulates the functionality of stiff silicon arrays for recording from neural population across multiple sites within a given region. The flexible probe is accompanied by a detachable 3D printed implanter, which delivers the probe by means of hydrophobic‐coated shuttles. The shuttles can then be retracted with minimal movement and the implanter houses the electronics necessary for in vivo recording applications. Validation of the probes through extracellular recordings from multiple brain regions and histological evidence of minimal foreign body response opens the path to long‐term chronic monitoring of neural ensembles

A Highly Porous Metal-Organic Framework System to Deliver Payloads for Gene Knockdown

© 2019 Elsevier Inc. Gene knockdown is an advantageous therapeutic strategy to lower dangerous genetic over-expression. However, the molecules responsible for initiating this process are unstable. Porous nanoparticles called metal-organic frameworks can encapsulate, protect, and deliver these compounds efficaciously without the need for chemical modifications—commonly done to enhance stability. By applying this platform technology, this work demonstrates the successful reduction in expression of a gene by avoiding retention and subsequent degradation in cellular compartments.This project has received funding from the European Research Council (ERC) under the European Union’s Horizon 2020 research and innovation programme (NanoMOFdeli), ERC-2016-COG 726380, and (SUPUVIR) no. 722380. M.H.T. thanks the Gates Cambridge Trust for funding, S. Haddad for helpful discussions, and A. Li for assistance with data visualization. D.F.-J. thanks the Royal Society for funding through a University Research Fellowship. S.B.d.Q.F., F.M.R., and D.I.J. were funded by Cancer Research UK Senior Group Leader Grant CRUK/A15678. O.K.F. gratefully acknowledges DTRA for financial support (grant HDTRA-1-14-1-0014). C.F.K. acknowledges funding from the UK Engineering and Physical Sciences Research Council (grants EP/L015889/1 and EP/H018301/1), the Wellcome Trust (grants 3-3249/Z/16/Z and 089703/Z/09/Z) and the UK Medical Research Council (grants MR/K015850/1 and MR/K02292X/1), and Infinitus (China) Ltd. Computational work was supported by the Cambridge High Performance Computing Cluster, Darwin

Als mutations in FUS cause neuronal dysfunction and death in caenorhabditis elegans by a dominant gain-of-function mechanism

It is unclear whether mutations in fused in sarcoma (FUS) cause familial amyotrophic lateral sclerosis via a loss-of-function effect due to titrating FUS from the nucleus or a gain-of-function effect from cytoplasmic overabundance. To investigate this question, we generated a series of independent Caenorhabditis elegans lines expressing mutant or wild-type (WT) human FUS. We show that mutant FUS, but not WT-FUS, causes cytoplasmic mislocalization associated with progressive motor dysfunction and reduced lifespan. The severity of the mutant phenotype in C. elegans was directly correlated with the severity of the illness caused by the same mutation in humans, arguing that this model closely replicates key features of the human illness. Importantly, the mutant phenotype could not be rescued by overexpression of WT-FUS, even though WTFUS had physiological intracellular localization, and was not recruited to the cytoplasmic mutant FUS aggregates. Our data suggest that FUS mutants cause neuronal dysfunction by a dominant gain-of-function effect related either to neurotoxic aggregates of mutant FUS in the cytoplasm or to dysfunction in its RNA-binding functions

FUS Phase Separation Is Modulated by a Molecular Chaperone and Methylation of Arginine Cation-π Interactions.

Reversible phase separation underpins the role of FUS in ribonucleoprotein granules and other membrane-free organelles and is, in part, driven by the intrinsically disordered low-complexity (LC) domain of FUS. Here, we report that cooperative cation-π interactions between tyrosines in the LC domain and arginines in structured C-terminal domains also contribute to phase separation. These interactions are modulated by post-translational arginine methylation, wherein arginine hypomethylation strongly promotes phase separation and gelation. Indeed, significant hypomethylation, which occurs in FUS-associated frontotemporal lobar degeneration (FTLD), induces FUS condensation into stable intermolecular β-sheet-rich hydrogels that disrupt RNP granule function and impair new protein synthesis in neuron terminals. We show that transportin acts as a physiological molecular chaperone of FUS in neuron terminals, reducing phase separation and gelation of methylated and hypomethylated FUS and rescuing protein synthesis. These results demonstrate how FUS condensation is physiologically regulated and how perturbations in these mechanisms can lead to disease

ALS/FTD mutation-induced phase transition of FUS liquid droplets and reversible hydrogels into irreversible hydrogels impairs RNP granule function

The mechanisms by which mutations in FUS and other RNA binding proteins cause ALS and FTD remain controversial. We propose a model in which low-complexity (LC) domains of FUS drive its physiologically reversible assembly into membrane-free, liquid droplet and hydrogel-like structures. ALS/FTD mutations in LC or non-LC domains induce further phase transition into poorly soluble fibrillar hydrogels distinct from conventional amyloids. These assemblies are necessary and sufficient for neurotoxicity in a C. elegans model of FUS-dependent neurodegeneration. They trap other ribonucleoprotein (RNP) granule components and disrupt RNP granule function. One consequence is impairment of new protein synthesis by cytoplasmic RNP granules in axon terminals, where RNP granules regulate local RNA metabolism and translation. Nuclear FUS granules may be similarly affected. Inhibiting formation of these fibrillar hydrogel assemblies mitigates neurotoxicity and suggests a potential therapeutic strategy that may also be applicable to ALS/FTD associated with mutations in other RNA binding proteins

Thioflavine-T and Congo Red reveal the polymorphism of insulin amyloid fibrils when probed by polarization-resolved fluorescence microscopy.

International audienceAmyloid fibrils are protein misfolding structures that involve a β-sheet structure and are associated with the pathologies of various neurodegenerative diseases. Here we show that Thioflavine-T and Congo Red, two major dyes used to image fibrils by fluorescence assays, can provide deep structural information when probed by means of polarization-resolved fluorescence microscopy. Unlike fluorescence anisotropy or fluorescence detected linear dichroism imaging, this technique allows to retrieve simultaneously both mean orientation and orientation dispersion of the dye, used here as a reporter of the fibril structure. We have observed that insulin amyloid fibrils exhibit a homogeneous behavior over the fibrils' length, confirming their structural uniformity. In addition, these results reveal the existence of various structures among the observed fibrils' population, in spite of a similar aspect when imaged with conventional fluorescence microscopy. This optical nondestructive technique opens perspectives for in vivo structural analyses or high throughput screening

Transmission electron microscopy characterization of fluorescently labelled amyloid β 1-40 and α-synuclein aggregates

<p>Abstract</p> <p>Background</p> <p>Fluorescent tags, including small organic molecules and fluorescent proteins, enable the localization of protein molecules in biomedical research experiments. However, the use of these labels may interfere with the formation of larger-scale protein structures such as amyloid aggregates. Therefore, we investigate the effects of some commonly used fluorescent tags on the morphologies of fibrils grown from the Alzheimer's disease-associated peptide Amyloid β 1-40 (Aβ40) and the Parkinson's disease-associated protein α-synuclein (αS).</p> <p>Results</p> <p>Using transmission electron microscopy (TEM), we verify that N-terminal labeling of Aβ40 with AMCA, TAMRA, and Hilyte-Fluor 488 tags does not prevent the formation of protofibrils and amyloid fibrils of various widths. We also measure the two-photon action cross-section of Aβ40 labelled with Hilyte Fluor 488 and demonstrate that this tag is suitable for use with two-photon fluorescence techniques. Similarly, we find that Alexa Fluor 488 labelling of αS variant proteins near either the N or C terminus (position 9 or 130) does not interfere with the formation of amyloid and other types of αS fibrils. We also present TEM images of fibrils grown from αS C-terminally labelled with enhanced green fluorescent protein (EGFP). Near neutral pH, two types of αS-EGFP fibrils are observed via TEM, while denaturation of the EGFP tag leads to the formation of additional species.</p> <p>Conclusions</p> <p>We demonstrate that several small extrinsic fluorescent tags are compatible with studies of amyloid protein aggregation. However, although fibrils can be grown from αS labelled with EGFP, the conformation of the fluorescent protein tag affects the observed aggregate morphologies. Thus, our results should assist researchers with label selection and optimization of solution conditions for aggregation studies involving fluorescence techniques.</p

A new era for understanding amyloid structures and disease

The aggregation of proteins into amyloid fibrils and their deposition into plaques and intracellular inclusions is the hallmark of amyloid disease. The accumulation and deposition of amyloid fibrils, collectively known as amyloidosis, is associated with many pathological conditions that can be associated with ageing, such as Alzheimer disease, Parkinson disease, type II diabetes and dialysis-related amyloidosis. However, elucidation of the atomic structure of amyloid fibrils formed from their intact protein precursors and how fibril formation relates to disease has remained elusive. Recent advances in structural biology techniques, including cryo-electron microscopy and solid-state NMR spectroscopy, have finally broken this impasse. The first near-atomic-resolution structures of amyloid fibrils formed in vitro, seeded from plaque material and analysed directly ex vivo are now available. The results reveal cross-β structures that are far more intricate than anticipated. Here, we describe these structures, highlighting their similarities and differences, and the basis for their toxicity. We discuss how amyloid structure may affect the ability of fibrils to spread to different sites in the cell and between organisms in a prion-like manner, along with their roles in disease. These molecular insights will aid in understanding the development and spread of amyloid diseases and are inspiring new strategies for therapeutic intervention