Biology subjects

Gao, R.

Publications and source records attributed to Gao, R..

100 records · Page 6Linked to original sources

bioRxiv2019

Precision calcium imaging of dense neural populations via a cell body-targeted calcium indicator

Methods for one-photon fluorescent imaging of calcium dynamics in vivo are popular due to their ability to simultaneously capture the dynamics of hundreds of neurons across large fields of view, at a low equipment complexity and cost. In contrast to two-photon methods, however, one-photon methods suffer from higher levels of crosstalk between cell bodies and the surrounding neuropil, resulting in decreased signal-to-noise and artifactual correlations of neural activity. Here, we address this problem by engineering cell body-targeted variants of the fluorescent calcium indicator GCaMP6f. We screened fusions of GCaMP6f to both natural as well as engineered peptides, and identified fusions that localized GCaMP6f to within approximately 50 microns of the cell body of neurons in live mice and larval zebrafish. One-photon imaging of soma-targeted GCaMP6f in dense neural circuits reported fewer artifactual spikes from neuropil, increased signal-to-noise ratio, and decreased artifactual correlation across neurons. Thus, soma-targeting of fluorescent calcium indicators increases neuronal signal fidelity and may facilitate even greater usage of simple, powerful, one-photon methods of population imaging of neural calcium dynamics.

neuroscience↗

bioRxiv2019

Ankyrin-G-190 palmitoylation mediates dendrite and spine morphogenesis and is altered in response to lithium

AnkG, encoded by the ANK3 gene, is a multifunctional scaffold protein with complex isoform expression: the 480 kDa and 270 kDa isoforms have roles at the axon initial segment and node of Ranvier, whereas the 190 kDa isoform (AnkG-190) has an emerging role in the dendritic shaft and spine heads. All isoforms of AnkG undergo palmitoylation, a post-translational modification regulating protein attachment to lipid membranes. However, palmitoylation of AnkG-190 has not been investigated in dendritic spines. The ANK3 gene and altered expression of AnkG proteins are associated with a variety of neuropsychiatric and neurodevelopmental disorders including bipolar disorders and are implicated in the lithium response, a commonly used mood stabilizer for bipolar disorder patients, although the precise mechanisms involved are unknown. Here, we showed that Cys70 palmitoylation stabilizes the localization of AnkG-190 in spine heads and at dendritic plasma membrane nanodomains. Mutation of Cys70 impairs AnkG-190 function in dendritic spines and alters PSD-95 scaffolding. Interestingly, we find that lithium reduces AnkG-190 palmitoylation thereby increasing its mobility in dendritic spines. Finally, we demonstrate that the palmitoyl acyl transferase ZDHHC8, but not ZDHHC5, increases AnkG-190 stability in spine heads and is inhibited by lithium. Together, our data reveal that palmitoylation is critical for AnkG-190 localization and function and a potential ZDDHC8/AnkG-190 mechanism linking AnkG-190 mobility to the neuronal effects of lithium.

neuroscience↗

bioRxiv2019

Mutant p53 Drives Clonal Hematopoiesis through Modulating Epigenetic Pathway

Clonal hematopoiesis of indeterminate potential (CHIP) increases with age and is associated with increased risks of hematological malignancies. While TP53 mutations have been identified in CHIP, the molecular mechanisms by which mutant p53 promotes hematopoietic stem and progenitor cell (HSPC) expansion are largely unknown. We discovered that mutant p53 confers a competitive advantage to HSPCs following transplantation and promotes HSPC expansion after radiation-induced stress. Mechanistically, mutant p53 interacts with EZH2 and enhances its association with the chromatin, thereby increasing the levels of H3K27me3 in genes regulating HSPC self-renewal and differentiation. Further, genetic and pharmacological inhibition of EZH2 decrease the repopulating potential of p53 mutant HSPCs. Thus, we have uncovered an epigenetic mechanism by which mutant p53 drives clonal hematopoiesis. Our work will likely establish epigenetic regulator EZH2 as a novel therapeutic target for preventing CHIP progression and treating hematological malignancies with TP53 mutations.

cancer biology↗

bioRxiv2019

CNTNAP2 ectodomain, detected in neuronal and CSF sheddomes, modulates Ca2+ dynamics and network synchrony

SUMMARYWhile many neuronal membrane-anchored proteins undergo proteolytic cleavage, little is known about the biological significance of neuronal ectodomain shedding. Using mass spectrometry (MS)-based proteomics, we showed that the neuronal sheddome mirrors human cerebrospinal fluid (hCSF). Among shed synaptic proteins in hCSF was the ectodomain of CNTNAP2 (CNTNAP2-ecto), a risk factor for neurodevelopmental disorders (NDD). Using structured-illumination microscopy (SIM), we mapped the spatial organization of neuronal CNTNAP2-ecto shedding. Using affinity chromatography followed by MS, we identified the ATP2B/PMCA Ca2+ extrusion pumps as novel CNTNAP2-ecto binding partners. CNTNAP2-ecto coimmunoprecipitates with PMCA2, a known autism risk factor, and enhances its activity, thereby modulating neuronal Ca2+ levels. Finally, we showed that CNTNAP2-ecto regulates neuronal network synchrony in primary cultures and brain slices. These data provide new insights into the biology of synaptic ectodomain shedding and reveal a novel mechanism of regulation of Ca2+ homeostasis and neuronal network synchrony.

neuroscience↗

bioRxiv2019

CNTNAP2 is targeted to endosomes by the polarity protein Par3

A decade of genetic studies has established Contactin-associated protein-like 2 (CNTNAP2) as a prominent susceptibility gene associated with multiple neurodevelopmental disorders. The development and characterization of Cntnap2 knockout models in multiple species have bolstered this claim by establishing clear connections with certain endophenotypes. Despite these remarkable in vivo findings, CNTNAP2s molecular functions are relatively unexplored, highlighting the need to identify novel protein partners. Here, we characterized an interaction between CNTNAP2 and Partitioning-defective 3 (Par3) - a polarity molecule we isolated in a yeast-two hybrid screen with CNTNAP2s C-terminus. We provide evidence that the two proteins interact via PDZ domain-mediated binding, that CNTNAP2+/Par3+ complexes are largely associated with clathrin-coated endocytic vesicles, and that Par3 causes an enlargement of these structures. Live imaging and fluorescence recovery after photobleaching (FRAP) reveals that Par3 limits the mobility of CNTNAP2 at endosomes, thus stabilizing it at that location. Finally, expression of Par3 but not Par3{Delta}PDZ can cluster endogenous CNTNAP2 in primary neurons. Collectively, we conclude that Par3 regulates CNTNAP2 spatial localization to endocytic compartments.

neuroscience↗

bioRxiv2018

Measuring the average power of neural oscillations

BackgroundNeural oscillations are often quantified as average power relative to a cognitive, perceptual, and/or behavioral task. This is commonly done using Fourier-based techniques, such as Welchs method for estimating the power spectral density, and/or by estimating narrowband oscillatory power across trials, conditions, and/or groups. The core assumption underlying these approaches is that the mean is an appropriate measure of central tendency. Despite the importance of this assumption, it has not been rigorously tested.\n\nNew methodWe introduce extensions of common approaches that are better suited for the physiological reality of how neural oscillations often manifest: as nonstationary, high-power bursts, rather than sustained rhythms. Log-transforming, or taking the median power, significantly reduces erroneously inflated power estimates.\n\nResultsAnalyzing 101 participants worth of human electrophysiology, totaling 3,560 channels and over 40 hours data, we show that, in all cases examined, spectral power is not Gaussian distributed. This is true even when oscillations are prominent and sustained, such as visual cortical alpha. Power across time, at every frequency, is characterized by a substantial long tail, which implies that estimates of average power are skewed toward large, infrequent high-power oscillatory bursts.\n\nComparison with existing methodsIn a simulated event-related experiment we show how introducing just a few high-power oscillatory bursts, as seen in real data, can, perhaps erroneously, cause significant differences between conditions using traditional methods. These erroneous effects are substantially reduced with our new methods.\n\nConclusionsThese results call into question the validity of common statistical practices in neural oscillation research.\n\nHighlightsO_LIAnalyses of oscillatory power often assume power is normally distributed.\nC_LIO_LIAnalyzing >40 hours of human M/EEG and ECoG, we show that in all cases it is not.\nC_LIO_LIThis effect is demonstrated in simple simulation of an event-related task.\nC_LIO_LIOverinflated power estimates are reduced via log-transformation or median power.\nC_LI

neuroscience↗

bioRxiv2018

Cortical Column and Whole Brain Imaging of Neural Circuits with Molecular Contrast and Nanoscale Resolution

Optical and electron microscopy have made tremendous inroads in understanding the complexity of the brain, but the former offers insufficient resolution to reveal subcellular details and the latter lacks the throughput and molecular contrast to visualize specific molecular constituents over mm-scale or larger dimensions. We combined expansion microscopy and lattice light sheet microscopy to image the nanoscale spatial relationships between proteins across the thickness of the mouse cortex or the entire Drosophila brain, including synaptic proteins at dendritic spines, myelination along axons, and presynaptic densities at dopaminergic neurons in every fly neuropil domain. The technology should enable statistically rich, large scale studies of neural development, sexual dimorphism, degree of stereotypy, and structural correlations to behavior or neural activity, all with molecular contrast.\n\nOne Sentence SummaryCombined expansion and lattice light sheet microscopy enables high speed, nanoscale molecular imaging of neural circuits over large volumes.

neuroscience↗

bioRxiv2018

Nested oscillatory dynamics in cortical organoids model early human brain network development

Structural and transcriptional changes during early brain maturation follow fixed developmental programs defined by genetics. However, whether this is true for functional network activity remains unknown, primarily due to experimental inaccessibility of the initial stages of the living human brain. Here, we developed cortical organoids that spontaneously display periodic and regular oscillatory network events that are dependent on glutamatergic and GABAergic signaling. These nested oscillations exhibit cross-frequency coupling, proposed to coordinate neuronal computation and communication. As evidence of potential network maturation, oscillatory activity subsequently transitioned to more spatiotemporally irregular patterns, capturing features observed in preterm human electroencephalography (EEG). These results show that the development of structured network activity in the human neocortex may follow stable genetic programming, even in the absence of external or subcortical inputs. Our approach provides novel opportunities for investigating and manipulating the role of network activity in the developing human cortex.\n\nHIGHLIGHTSO_LIEarly development of human functional neural networks and oscillatory activity can be modeled in vitro.\nC_LIO_LICortical organoids exhibit phase-amplitude coupling between delta oscillation (2 Hz) and high-frequency activity (100-400 Hz) during network-synchronous events.\nC_LIO_LIDifferential role of glutamate and GABA in initiating and maintaining oscillatory network activity.\nC_LIO_LIDevelopmental impairment of MECP2-KO cortical organoids impacts the emergence of oscillatory activity.\nC_LIO_LICortical organoid network electrophysiological signatures correlate with human preterm neonatal EEG features.\nC_LI\n\neTOCBrain oscillations are a candidate mechanism for how neural populations are temporally organized to instantiate cognition and behavior. Cortical organoids initially exhibit periodic and highly regular nested oscillatory network events that eventually transition to more spatiotemporally complex activity, capturing features of late-stage preterm infant electroencephalography. Functional neural circuitry in cortical organoids exhibits emergence and development of oscillatory network dynamics similar to those found in the developing human brain.

neuroscience↗

bioRxiv2018

Deep learning achieves super-resolution in fluorescence microscopy

AbtsractWe present a deep learning-based method for achieving super-resolution in fluorescence microscopy. This data-driven approach does not require any numerical models of the imaging process or the estimation of a point spread function, and is solely based on training a generative adversarial network, which statistically learns to transform low resolution input images into super-resolved ones. Using this method, we super-resolve wide-field images acquired with low numerical aperture objective lenses, matching the resolution that is acquired using high numerical aperture objectives. We also demonstrate that diffraction-limited confocal microscopy images can be transformed by the same framework into super-resolved fluorescence images, matching the image resolution acquired with a stimulated emission depletion (STED) microscope. The deep network rapidly outputs these super-resolution images, without any iterations or parameter search, and even works for types of samples that it was not trained for.

bioengineering↗

bioRxiv2018

Parameterizing neural power spectra

Electrophysiological signals across species and recording scales exhibit both periodic and aperiodic features. Periodic oscillations have been widely studied and linked to numerous physiological, cognitive, behavioral, and disease states, while the aperiodic \"background\" 1/f component of neural power spectra has received far less attention. Most analyses of oscillations are conducted on a priori, canonically-defined frequency bands without consideration of the underlying aperiodic structure, or verification that a periodic signal even exists in addition to the aperiodic signal. This is problematic, as recent evidence shows that the aperiodic signal is dynamic, changing with age, task demands, and cognitive state. It has also been linked to the relative excitation/inhibition of the underlying neuronal population. This means that standard analytic approaches easily conflate changes in the periodic and aperiodic signals with one another because the aperiodic parameters--along with oscillation center frequency, power, and bandwidth--are all dynamic in physiologically meaningful, but likely different, ways. In order to overcome the limitations of traditional narrowband analyses and to reduce the potentially deleterious effects of conflating these features, we introduce a novel algorithm for automatic parameterization of neural power spectral densities (PSDs) as a combination of the aperiodic signal and putative periodic oscillations. Notably, this algorithm requires no a priori specification of band limits and accounts for potentially-overlapping oscillations while minimizing the degree to which they are confounded with one another. This algorithm is amenable to large-scale data exploration and analysis, providing researchers with a tool to quickly and accurately parameterize neural power spectra.

neuroscience↗