Biology subjects

Chen, J. L.

Publications and source records attributed to Chen, J. L..

15 recordsLinked to original sources

bioRxiv2025

Design of an orally bioavailable small molecule that modulates the microtubule-associated protein tau's pre-mRNA splicing

Frontotemporal dementia with parkinsonism linked to chromosome 17 (FTDP-17) is caused by the aberrant alternative pre-mRNA splicing of microtubule-associated protein tau (MAPT) exon 10, the inclusion of which encodes for a toxic tau protein harboring four microtube domains (4R tau). Here, we describe the design of an RNA-targeted small molecule that thermodynamically stabilizes the structure of a pre-mRNA splicing regulator element in the MAPT pre-mRNA exon 10-intron junction to reduce the inclusion of exon 10 and hence 4R tau abundance. Structure-based drug design was used to obtain compounds that form a network of specific interactions to the RNA including multiple interactions between a one nucleotide A-bulge and the Hoogsteen face of a closing GC base pair, the latter of which was enabled by the design of base triple interactions. A battery of assays revealed that the compound binds the target in vitro and in cells and affects pre-mRNA splicing in various cellular models including primary neurons from a human tau (htau) knock-in mouse model. The orally bioavailable compound was administered per os (p.o.), where treatment diminished exon 10 inclusion, and reduced the 4R tau protein isoform. Further, the molecule mitigated cellular pathologies and behavioral phenotypes observed in the htau transgenic mouse model. This study provides a potentially general pipeline to design compounds that target RNAs and affect disease pathways and deliver compounds that have oral bioavailability and blood-brain barrier penetrance.

biochemistry↗

bioRxiv2024

NMR structures and magnetic force spectroscopy studies of small molecules binding to models of an RNA CAG repeat expansion

RNA repeat expansions fold into stable structures and cause microsatellite diseases such as Huntingtons disease (HD), myotonic dystrophy type 1 (DM1), and spinocerebellar ataxias (SCAs). The trinucleotide expansion of r(CAG), or r(CAG)exp, causes both HD and SCA3, and the RNAs toxicity has been traced to its translation into polyglutamine (polyQ; HD) as well as aberrant pre-mRNA alternative splicing (SCA3 and HD). Previously, a small molecule, 1, was discovered that binds to r(CAG)exp and rescues aberrant pre-mRNA splicing in patient-derived fibroblasts by freeing proteins bound to the repeats. Here, we report the structures of single r(CAG) repeat motif (5CAG/3GAC where the underlined adenosines form a 1x1 nucleotide internal loop) in complex with 1 and two other small molecules via nuclear magnetic resonance (NMR) spectroscopy combined with simulated annealing. Compound 2 was designed based on the structure of 1 bound to the RNA while 3 was selected as a diverse chemical scaffold. The three complexes, although adopting different 3D binding pockets upon ligand binding, are stabilized by a combination of stacking interactions with the internal loops closing GC base pairs, hydrogen bonds, and van der Waals interactions. Molecular dynamics (MD) simulations performed with NMR-derived restraints show that the RNA is stretched and bent upon ligand binding with significant changes in propeller-twist and opening. Compound 3 has a distinct mode of binding by insertion into the helix, displacing one of the loop nucleotides into the major groove and affording a rod-like shape binding pocket. In contrast, 1 and 2 are groove binders. A series of single molecule magnetic force spectroscopy studies provide a mechanistic explanation for how bioactive compounds might rescue disease-associated cellular phenotypes. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=87 SRC="FIGDIR/small/608150v1_ufig1.gif" ALT="Figure 1"> View larger version (22K): org.highwire.dtl.DTLVardef@145398org.highwire.dtl.DTLVardef@7c37caorg.highwire.dtl.DTLVardef@132cbbeorg.highwire.dtl.DTLVardef@1de59d3_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2024

NMR structures of small molecules bound to a model of an RNA CUG repeat expansion

Trinucleotide repeat expansions fold into long, stable hairpins and cause a variety of incurable RNA gain-of-function diseases such as Huntingtons disease, the myotonic dystrophies, and spinocerebellar ataxias. One approach for treating these diseases is to bind small molecules to the structured RNAs. Both Huntingtons disease-like 2 (HDL2) and myotonic dystrophy type 1 (DM1) are caused by a r(CUG) repeat expansion, or r(CUG)exp. The RNA folds into a hairpin structure with a periodic array of 1x1 nucleotide UU loops (5CUG/3GUC; where the underlined nucleotides indicate the Us in the internal loop) that sequester various RNA-binding proteins (RBP) and hence the source of its gain-of-function. Here, we report NMR-refined structures of single 5CUG/3GUC motifs in complex with three different small molecules, a diguandinobenzoate (1), a derivative of 1 where the guanidino groups have been exchanged for imidazole (2), and a quinoline with improved drug-like properties (3). These structures were determined using nuclear magnetic resonance (NMR) spectroscopy and simulated annealing with restrained molecular dynamics (MD). Compounds 1, 2, and 3 formed stacking and hydrogen bonding interactions with the 5CUG/3GUC motif. Compound 3 also formed van der Waals interactions with the internal loop. The global structure of each RNA-small molecule complexes retains an A-form conformation, while the internal loops are still dynamic but to a lesser extent compared to the unbound form. These results aid our understanding of ligand-RNA interactions and enable structure-based design of small molecules with improved binding affinity for and biological activity against r(CUG)exp. As the first ever reported structures of RNA r(CUG) repeats bound to ligands, these structures can enable virtual screening campaigns combined with machine learning assisted de novo design.

biochemistry↗

bioRxiv2024

Prevalence of errors in lab-made plasmids across the globe

Plasmids are indispensable in life sciences research and therapeutics development. Currently, most labs custom-build their plasmids. As yet, no systematic data on the quality of lab-made plasmids exist. Here, we report a broad survey of plasmids from hundreds of academic and industrial labs worldwide. We show that nearly half of them contained design and/or sequence errors. For transfer plasmids used in making AAV vectors, which are widely used in gene therapy, about 40% carried mutations in the inverted terminal repeat (ITR) regions due to their inherent instability, which is influenced by flanking GC content. We also list genes difficult to clone into plasmid or package into virus due to their toxicity. Our finding raises serious concerns over the trustworthiness of lab-made plasmids, which parallels the underappreciated mycoplasma contamination and misidentified mammalian cell lines reported previously, and highlights the need for community-wide standards to uphold the quality of this ubiquitous reagent in research and medicine. Accordingly, we propose the concept of good vector practice (GVP) that covers the proper design, construction, in-process QC, final QC, banking and management of plasmids in research and medicine to uphold their quality.

molecular biology↗

bioRxiv2024

DeepVID v2: Self-Supervised Denoising with Decoupled Spatiotemporal Enhancement for Low-Photon Voltage Imaging

SignificanceVoltage imaging is a powerful tool for studying the dynamics of neuronal activities in the brain. However, voltage imaging data are fundamentally corrupted by severe Poisson noise in the low-photon regime, which hinders the accurate extraction of neuronal activities. Self-supervised deep learning denoising methods have shown great potential in addressing the challenges in low-photon voltage imaging without the need for ground truth, but usually suffer from the tradeoff between spatial and temporal performance. AimWe present DeepVID v2, a novel self-supervised denoising framework with decoupled spatial and temporal enhancement capability to significantly augment low-photon voltage imaging. ApproachDeepVID v2 is built on our original DeepVID framework,1,2 which performs frame-based denoising by utilizing a sequence of frames around the central frame targeted for denoising to leverage temporal information and ensure consistency. The network further integrates multiple blind pixels in the central frame to enrich the learning of local spatial information. Additionally, DeepVID v2 introduces a new edge extraction branch to capture fine structural details in order to learn high spatial resolution information. ResultsWe demonstrate that DeepVID v2 is able to overcome the tradeoff between spatial and temporal performance, and achieve superior denoising capability in resolving both high-resolution spatial structures and rapid temporal neuronal activities. We further show that DeepVID v2 is able to generalize to different imaging conditions, including time-series measurements with various signal-to-noise ratios (SNRs) and in extreme low-photon conditions. ConclusionsOur results underscore DeepVID v2 as a promising tool for enhancing voltage imaging. This framework has the potential to generalize to other low-photon imaging modalities and greatly facilitate the study of neuronal activities in the brain.

neuroscience↗

bioRxiv2023

Development of MAPT S305 mutation models exhibiting elevated 4R tau expression, resulting in altered neuronal and astrocytic function

Due to the importance of 4R tau in the pathogenicity of primary tauopathies, it has been challenging to model these diseases in iPSC-derived neurons, which express very low levels of 4R tau. To address this problem we have developed a panel of isogenic iPSC lines carrying the MAPT splice-site mutations S305S, S305I or S305N, derived from four different donors. All three mutations significantly increased the proportion of 4R tau expression in iPSC-neurons and astrocytes, with up to 80% 4R transcripts in S305N neurons from as early as 4 weeks of differentiation. Transcriptomic and functional analyses of S305 mutant neurons revealed shared disruption in glutamate signaling and synaptic maturity, but divergent effects on mitochondrial bioenergetics. In iPSC-astrocytes, S305 mutations induced lysosomal disruption and inflammation and exacerbated internalization of exogenous tau that may be a precursor to the glial pathologies observed in many tauopathies. In conclusion, we present a novel panel of human iPSC lines that express unprecedented levels of 4R tau in neurons and astrocytes. These lines recapitulate previously characterized tauopathy-relevant phenotypes, but also highlight functional differences between the wild type 4R and mutant 4R proteins. We also highlight the functional importance of MAPT expression in astrocytes. These lines will be highly beneficial to tauopathy researchers enabling a more complete understanding of the pathogenic mechanisms underlying 4R tauopathies across different cell types.

neuroscience↗

bioRxiv2023

Perirhinal Cortex Learns A Predictive Map of The Task Environment

Goal-directed tasks involve acquiring an internal model, known as a predictive map, of relevant stimuli and associated outcomes to guide behavior. Here, we identified neural signatures of a predictive map of task behavior in perirhinal cortex (Prh). Mice learned to perform a tactile working memory task by classifying sequential whisker stimuli over multiple training stages. Chemogenetic inactivation demonstrated that Prh is involved in task learning. Chronic two-photon calcium imaging, population analysis, and computational modeling revealed that Prh encodes stimulus features as sensory prediction errors. Prh forms stable stimulus-outcome associations that expand in a retrospective manner and generalize as animals learn new contingencies. Stimulus-outcome associations are linked to prospective network activity encoding possible expected outcomes. This link is mediated by cholinergic signaling to guide task performance, demonstrated by acetylcholine imaging and perturbation. We propose that Prh combines error-driven and map-like properties to acquire a predictive map of learned task behavior.

neuroscience↗

bioRxiv2023

Design and fabrication of a low-cost microfluidic cartridge with integrated pressure-driven check valve for molecular diagnostics platforms

This paper describes the design, fabrication, and preliminary testing of a low-cost, easy to manufacture microfluidics cartridge capable of fluid storage and manipulation through a custom pressure-driven check valve. Cartridge components are fabricated using a desktop CNC and laser cutter, the check valve is fabricated using PDMS in a custom acrylic mold, and the components are assembled using a thermal diffusion welder. Following assembly, preliminary testing of the cartridge, including fluid manipulation and use for molecular diagnostics, was performed. To pull a sample into the lysing chamber, a vacuum over 1.4PSI was required. No opening of the valve to the reaction chamber was observed. Moving fluid across the custom valve from the lysing chamber to the reaction chamber then required a vacuum over 4.5PSI. Finally, a proof-of-concept demonstration of one potential application was performed using a custom benchtop LAMP system for molecular diagnostic testing. The low-cost nature of the design, ease of manufacturing, fluid storage and manipulation demonstrated make this design ideal for research and high-volume testing in low resource environments. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=72 SRC="FIGDIR/small/522222v1_ufig1.gif" ALT="Figure 1"> View larger version (27K): org.highwire.dtl.DTLVardef@13ebeb5org.highwire.dtl.DTLVardef@5a8ffaorg.highwire.dtl.DTLVardef@1a27cd5org.highwire.dtl.DTLVardef@b1bc2e_HPS_FORMAT_FIGEXP M_FIG C_FIG Graphical abstractCustom cartridge is fabricated using a combination of a) benchtop laser cutter and b) benchtop micromilling machine. c) Components are then assembled with a 0.22{micro}m micron filter and custom plug-style check valve. d) The cartridge assembly is then bonded using a thermal diffusion bonder. e) By pulling a vacuum through the first air trap, a sample can be pulled into the pre-filled lysing chamber. f) Pulling a vacuum through the second air trap, the lysed sample can then be pulled into the pre-filled reaction chamber. g) For a proof of concept, the filled cartridge was then tested in a custom benchtop Loop Mediated Isothermal System. Following a heating cycle, reaction fluoresce can be monitored. h) An S-Curve observed through the custom LAMP system, thus demonstrating feasibility of the cartridge for use with molecular diagnostic platforms.

bioengineering↗

bioRxiv2021

High-Speed Low-Light In Vivo Two-Photon Voltage Imaging of Large Neuronal Populations

Monitoring spiking activity across large neuronal populations at behaviorally relevant timescales is critical for understanding neural circuit function. Unlike calcium imaging, voltage imaging requires kilohertz sampling rates which reduces fluorescence detection to near shot noise levels. High-photon flux excitation can overcome photon-limited shot noise but photo-bleaching and photo-damage restricts the number and duration of simultaneously imaged neurons. We investigated an alternative approach aimed at low two-photon flux, voltage imaging below the shot noise limit. This framework involved developing: a positive-going voltage indicator with improved spike detection (SpikeyGi); an ultra-fast two-photon microscope for kilohertz frame-rate imaging across a 0.4x0.4mm2 field of view, and; a self-supervised denoising algorithm (DeepVID) for inferring fluorescence from shot-noise limited signals. Through these combined advances, we achieved simultaneous high-speed, deep-tissue imaging of more than one hundred densely-labeled neurons over one hour in awake behaving mice. This demonstrates a scalable approach for voltage imaging across increasing neuronal populations.

neuroscience↗

bioRxiv2021

Flexible Simultaneous Mesoscale Two-Photon Imaging of Neural Activity at High Speeds

Understanding brain function requires monitoring local and global brain dynamics. Two-photon imaging of the brain across mesoscopic scales has presented trade-offs between imaging area and acquisition speed. We describe a flexible cellular resolution two-photon microscope capable of simultaneous video rate acquisition of four independently targetable brain regions spanning an approximate five-millimeter field of view. With this system, we demonstrate the ability to measure calcium activity across mouse sensorimotor cortex at behaviorally relevant timescales.

neuroscience↗

bioRxiv2021

Dense Functional and Molecular Readout of a Circuit Hub in Sensory Cortex

Information processing in the neocortex is carried out by neuronal circuits composed of different cell types. Recent census of the neocortex using single cell transcriptomic profiling has uncovered more than 100 putative cell types which subdivide major classes of excitatory and inhibitory neurons into distinct subclasses. The extent to which this molecular classification predicts distinct functional roles during behavior is unclear. Here, we combined population recordings using two-photon calcium imaging with spatial transcriptomics using multiplexed fluorescent in situ hybridization to achieve dense functional and molecular readout of cortical circuits during behavior. We characterized task-related responses across major transcriptomic neuronal subclasses and types in layer 2/3 of primary somatosensory cortex as mice performed a tactile working memory task. We find that as neurons are segregated into increasingly discrete molecular types, their task-related properties continue to differentiate. We identify an excitatory cell type, Baz1a, that is highly driven by tactile stimuli. Baz1a neurons homeostatically maintain stimulus responsiveness during altered sensory experience and show persistent enrichment of subsets of immediately early genes including Fos. Measurements of functional and anatomical connectivity reveal that upper layer 2/3 Baz1a neurons preferentially innervate somatostatin-expressing inhibitory neurons. We propose that this connection motif reflects a sensory-driven circuit hub that orchestrates local sensory processing in superficial layers of the neocortex.

neuroscience↗

bioRxiv2020

The Prevalence of the Val66Met Polymorphism in Musicians: Possible Evidence for Compensatory Neuroplasticity from a pilot study.

It has been reported that MET carriers may express deficits in motor learning and neuroplasticity, possibly deterring musicianship. Here, we compared the prevalence of the Val66Met Brain-derived Neurotrophic Factor single nucleotide polymorphism (rs6265) in a sample of musicians (N=50) and an ethnically matched subset from the 1000 Human Genome Project (N-424). We report no differences in genotype or allele frequencies. Results are consistent with the view that hypothesized Met-dependent deficits in neuroplasticity are either mild or can be overcome by long-term practice.

neuroscience↗

bioRxiv2020

Counterfactual Hypothesis Testing of Tumor Microenvironment Scenarios Through Semantic Image Synthesis

AO_SCPLOWBSTRACTC_SCPLOWRecent multiplexed protein imaging technologies characterize cells, their spatial organization, and interactions within microenvironments at an unprecedented resolution. Although observational data can reveal spatial associations, it does not allow users to infer salient biological relationships and cellular interactions. To address this challenge, we develop a generative model that allows users to test hypotheses about the effect of cell-cell interactions on protein expression through in silico perturbation. Our Cell-Cell Interaction GAN (CCIGAN) model employs a generative adversarial network (GAN) architecture to generate high fidelity synthetic multiplexed images from semantic cell segmentations. Our approach is unique in that it learns relationships between all imaging channels simultaneously and yields biological insights from multiple imaging technologies in silico, capturing known tumor-immune cell interactions missed by other state-of-the-art GAN models.

cancer biology↗

bioRxiv2020

A small molecule that binds an RNA repeat expansion stimulates its decay via the exosome complex

We describe the design of a small molecule that binds the structure of a r(CUG) repeat expansion [r(CUG)exp] and reverses molecular defects in two diseases mediated by the RNA - myotonic dystrophy type 1 (DM1) and Fuchs endothelial corneal dystrophy (FECD). Thus, a single structure-specific ligand has potential therapeutic benefit for multiple diseases, in contrast to oligonucleotide-based modalities that are customized for each disease by nature of targeting the gene that harbors the repeat. Indeed, the small molecule binds the target with nanomolar affinity and >100-fold specificity vs. many other RNAs and DNA. Interestingly, the compounds downstream effects are different in the two diseases, owing to the location of the repeat expansion. In DM1, r(CUG)exp is harbored in the 3 untranslated region (UTR) of and mRNA, and the compound has no effect on the RNAs abundance. In FECD, however, r(CUG)exp is located in an intron, and the small molecule, by binding the repeat expansion, facilitates excision of the intron, which is then degraded by the exosome complex exonuclease, hRRP6. Thus, structure-specific, RNA-targeting small molecules can act disease-specifically to affect biology, either by disabling its gain-of-function mechanism (DM1) or by stimulating quality control pathways to rid a disease-affected cell of a toxic RNA (FECD). Significance statementMany different diseases are caused by toxic structured RNAs. Herein, we designed a lead small molecule that binds a toxic structure and rescues disease biology. We show that a structure-specific small molecule can improve disease-associated defects in two diseases that share the common toxic RNA structure. In one disease, the toxic structure is harbored in an intron and causes its retention. The compound facilitates processing of a retained intron, enabling the disease-affected cell to remove the toxic RNA.

cell biology↗

bioRxiv2019

The RNA encoding the microtubule-associated protein tau has extensive structure that affects its biology

Tauopathies are neurodegenerative diseases that affect millions of people worldwide including those with Alzheimers disease. While many efforts have focused on understanding the role of tau protein in neurodegeneration, there has been little done to systematically analyze and study the structures within taus encoding RNA and their connection to disease pathology. Knowledge of RNA structure can provide insights into disease mechanisms and how to affect protein production for therapeutic benefit. Using computational methods based on thermodynamic stability and evolutionary conservation, we identified structures throughout the tau pre-mRNA, especially at exon-intron junctions and within the 5' and 3' untranslated regions (UTRs). In particular, structures were identified at twenty exon-intron junctions. The 5' UTR contains one structured region, which lies within a known internal ribosome entry site. The 3' UTR contains eight structured regions, including one that contains a polyadenylation signal. A series of functional experiments were carried out to assess the effects of mutations associated with mis-regulation of alternative splicing of exon 10 and to identify regions of the 3' UTR that contain cis-regulatory elements. These studies defined novel structural regions within the mRNA that affect stability and pre-mRNA splicing and may lead to new therapeutic targets for treating tau-associated diseases.

bioinformatics↗