Biology subjects

Kosik, K. S.

Publications and source records attributed to Kosik, K. S..

8 recordsLinked to original sources

bioRxiv2018

Narrow equilibrium window for complex coacervation of tau and RNA under cellular conditions

The conditions that lead to the liquid-liquid phase separation (LLPS) of the tau protein, a microtubule associated protein whose pathological aggregation has been implicated in neurodegenerative disorders, are not well understood. Establishing a phase diagram that delineates the boundaries of phase co-existence is key to understanding its LLPS. Using a combination of EPR, turbidity measurements, and microscopy, we show that tau and RNA form complex coacervates with lower critical solution temperature (LCST) behavior. The coacervates are reversible, and the biopolymers can be driven to the supernatant phase or coacervate phase by varying the experimental conditions (temperature, salt concentration, tau:RNA charge ratio, total polymer concentration and osmotic stress). Furthermore, the coacervates can be driven to a fibrillar state through the addition of heparin. The equilibrium phase diagram of the tau/RNA complex coacervate system can be described by a Flory-Huggins model, augmented by an approximate Voorn Overbeek electrostatic term (FH-VO), after fitting the experimental data to an empirical Flory interaction parameter divided into an entropic and enthalpic term. However, a more advanced model in which tau and RNA are treated as discrete bead-spring chains with a temperature-dependent excluded volume interaction and electrostatic interactions between charged residues, investigated through field theoretic simulations (FTS), provided direct and unique insight into the thermodynamic driving forces of tau/RNA complexation. FTS corroborated the experimental finding that the complex coacervation of tau and RNA is has an entropy-driven contribution, with a transition temperature around the physiological temperature of 37 {degrees}C and salt concentrations around 100-150 mM. Together, experiment and simulation show that LLPS of tau can occur under physiological cellular conditions, but has a narrow equilibrium window over experimentally tunable parameters including temperature, salt and tau concentrations. Guided by our phase diagram, we show that tau can be driven towards LLPS under live cell coculturing conditions with rationally chosen experimental parameters.

biophysics↗

bioRxiv2018

Regulation of Cell-Type-Specific Transcriptomes by miRNA Networks During Human Brain Development

MicroRNAs (miRNAs) regulate many cellular events by regulating hundreds of mRNA transcripts. However, it is unclear how miRNA-mRNA interactions are contextualized into the framework of transcriptional heterogeneity among closely related cells of the developing human brain. By combining the multiple complementary approaches, AGO2-HITS-CLIP, single-cell profiling and bipartite network analysis, we show that the miRNA-mRNA network operates as functional modules related to cell-type identities and undergo dynamic transitions during brain development.

developmental biology↗

bioRxiv2017

Use of a Neural Circuit Probe to Validate in silico Predictions of Inhibitory Connections

Understanding how neuronal signals propagate in local network is an important step in understanding information processing. As a result, spike trains recorded with Multi-electrode Arrays (MEAs) have been widely used to study behaviors of neural connections. Studying the dynamics of neuronal networks requires the identification of both excitatory and inhibitory connections. The detection of excitatory relationships can robustly be inferred by characterizing the statistical relationships of neural spike trains. However, the identification of inhibitory relationships is more difficult: distinguishing endogenous low firing rates from active inhibition is not obvious. In this paper, we propose an in silico interventional procedure that makes predictions about the effect of stimulating or inhibiting single neurons on other neurons, and thereby gives the ability to accurately identify inhibitory causal relationships. To experimentally test these predictions, we have developed a Neural Circuit Probe (NCP) that delivers drugs transiently and reversibly on individually identified neurons to assess their contributions to the neural circuit behavior. With the help of NCP, three inhibitory connections identified by our in silico modeling were validated through real interventional experiments. Together, these methods provide a basis for mapping complete neural circuits.

neuroscience↗

bioRxiv2017

Tau Internalization is Regulated by 6-O Sulfation on Heparan Sulfate Proteoglycans (HSPGs)

The misfolding and accumulation of tau protein into intracellular aggregates known as neurofibrillary tangles is a pathological hallmark of neurodegenerative diseases such as Alzheimers disease. However, while tau propagation is a known marker for disease progression, exactly how tau propagates from one cell to another and what mechanisms govern this spread are still unclear. Here, we report that cellular internalization of tau is regulated by quaternary structure and have developed a cellular assay to screen for genetic modulators of tau uptake. Using CRISPRi technology we have tested 3200 genes for their ability to regulate tau entry and identified enzymes in the heparan sulfate proteoglycan biosynthetic pathway as key regulators. We show that 6-O-sulfation is critical for tauheparan sulfate interactions and that this modification regulates uptake in human central nervous system cell lines, iPS-derived neurons, and mouse organotypic brain slice culture. Together, these results suggest novel strategies to halt tau transmission.

neuroscience↗

bioRxiv2017

Enhanced neuronal regeneration in the CAST/Ei mouse strain is linked to expression of differentiation markers after injury

Peripheral nerve regeneration after injury requires a broad program of transcriptional changes. We investigated the basis for the enhanced nerve regenerative capacity of the CAST/Ei mouse strain relative to C57BL/6 mice. RNA sequencing of dorsal root ganglia (DRG) showed a CAST/Ei specific upregulation of Ascl1 after injury. Ascl1 overexpression in C57BL/6 mice DRG neurons enhanced their neurite outgrowth. Ascl1 is regulated by miR-7048-3p, which is down-regulated in CAST/Ei mice. Inhibition of miR-7048-3p enhances neurite outgrowth. Following injury, CAST/Ei neurons largely retained their mature neuronal profile as determined by single cell RNAseq, whereas the C57BL/6 neurons acquired an immature profile. These findings suggest that one facet of the enhanced regenerative phenotype is preservation of neuronal identity in response to injury.

molecular biology↗

bioRxiv2017

MEA Viewer: a High-performance Interactive Application for Visualizing Electrophysiological Data

Multi-electrode arrays (MEAs) have been used for many years to measure electrical activity in ensembles of many hundreds of neurons, and are used in research areas as diverse as neuronal connectivity and drug discovery. A high sampling frequency is required to adequately capture action potentials, also known as spikes, the primary electrical event associated with neuronal activity, and the resulting raw data files are large and difficult to visualize with traditional plotting tools. Many common approaches to deal with this issue, such as extracting spikes times and solely performing spike train analysis, significantly reduce data dimensionality. Unbiased data exploration benefits from the use of tools that minimize data transforms and such tools enable the development of heuristic perspective from data prior to any subsequent processing. Here we introduce MEA Viewer, a high-performance interactive application for the direct visualization of multi-channel electrophysiological data. MEA Viewer provides many high-performance visualizations of electrophysiological data, including an easily navigable overview of all recorded extracellular signals overlaid with spike timestamp data and an interactive raster plot. Beyond the fundamental data displays, MEA Viewer can signal average and spatially overlay the extent of action potential propagation within single neurons. This view extracts information below the spike detection threshold to directly visualize the propagation of action potentials across the plane of the MEA. This entirely new method of using MEAs opens up new and novel research applications for medium density arrays. MEA Viewer is licensed under the General Public License version 3, GPLv3, and is available at http://github.com/dbridges/mea-tools.

neuroscience↗

bioRxiv2017

Recording action potential propagation in single axons using multi-electrode arrays

The small caliber of central nervous system (CNS) axons makes routine study of axonal physiology relatively difficult. However, while recording extracellular action potentials from neurons cultured on planer multi-electrode arrays (MEAs) we found activity among groups of electrodes consistent with action potential propagation in single neurons. Action potential propagation was evident as widespread, repetitive cooccurrence of extracellular action potentials (eAPs) among groups of electrodes. These eAPs occurred with invariant sequences and inter-electrode latencies that were consistent with reported measures of action potential propagation in unmyelinated axons. Within co-active electrode groups, the inter-electrode eAP latencies were temperature sensitive, as expected for action potential propagation. Our data are consistent with these signals primarily reflecting axonal action potential propagation, from axons with a high density of voltage-gated sodium channels. Repeated codetection of eAPs by multiple electrodes confirmed these eAPs are from individual neurons and averaging these eAPs revealed sub-threshold events at other electrodes. The sequence of electrodes at which eAPs co-occur uniquely identifies these neurons, allowing us to monitor spiking of single identified neurons within neuronal ensembles. We recorded dynamic changes in single axon physiology such as simultaneous increases and decreases in excitability in different portions of single axonal arbors over several hours. Over several weeks, we measured changes in inter-electrode propagation latencies and ongoing changes in excitability in different regions of single axonal arbors. We recorded action potential propagation signals in human induced pluripotent stem cell-derived neurons which could thus be used to study axonal physiology in human disease models.\n\nSignificance StatementStudying the physiology of central nervous system axons is limited by the technical challenges of recording from axons with pairs of patch or extracellular electrodes at two places along single axons. We studied action potential propagation in single axonal arbors with extracellular recording with multi-electrode arrays. These recordings were non-invasive and were done from several sites of small caliber axons and branches. Unlike conventional extracellular recording, we unambiguously identified and labelled the neuronal source of propagating action potentials. We manipulated and quantified action potential propagation and found a surprisingly high density of axonal voltage-gated sodium channels. Our experiments also demonstrate that the excitability of different portions of axonal arbors can be independently regulated on time scales from hours to weeks.

neuroscience↗

bioRxiv2017

RNA Stores Tau Reversibly in Complex Coacervates

Non-membrane-bound organelles that behave like liquid droplets are widespread among eukaryotic cells. Their dysregulation appears to be a critical step in several neurodegenerative conditions. Here we report that tau protein, the primary constituent of Alzheimer neurofibrillary tangles, can form liquid droplets and therefore has the necessary biophysical properties to undergo liquid-liquid phase separation (LLPS) in cells. Consonant with the factors that induce LLPS, tau is an intrinsically disordered protein that complexes with RNA to form droplets. Uniquely, the pool of RNAs to which tau binds in living cells are tRNAs. This phase state of tau is held in an approximately 1:1 charge balance across the protein and the nucleic acid constituents, and can thus be maximal at different RNA:tau mass ratios depending on the biopolymer constituents involved. This feature is characteristic of complex coacervation. We furthermore show that the LLPS process is directly and sensitively tuned by salt concentration and temperature, implying it is modulated by both electrostatic interactions between the involved protein and nucleic acid constituents, as well as net changes in entropy. Despite the high protein concentration within the complex coacervate phase, tau is locally freely tumbling and capable of diffusing through the droplet interior. In fact, tau in the condensed phase state does not reveal any immediate changes in local protein packing, local conformations and local protein dynamics from that of tau in the dilute solution state. In contrast, the population of aggregation-prone tau as induced by the complexation with heparin is accompanied by large changes in local tau conformations and irreversible aggregation. However, prolonged residency within the droplet state eventually results in the emergence of detectable {beta}-sheet structures according to thioflavin-T assay. These findings suggest that the droplet state can incubate tau and pre-dispose the protein toward the formation of insoluble fibrils.

biophysics↗