Biology subjects

Chang, M.

Publications and source records attributed to Chang, M..

At least 37 records · Page 2Linked to original sources

bioRxiv2025

Oxidative stress mediates cardiac electrophysiological injury in inhalation exposure to flavored vaping products.

BackgroundElectronic nicotine delivery systems (ENDS) heat "E-liquids" to generate "E-vapor", an aerosolized mixture containing nicotine and flavors. Flavored ENDS are popular among teens who vape, however, the possible cardiac electrophysiological harm of inhalation exposure to flavored ENDS are not fully understood. ObjectiveTo test if inhalation exposure to flavoring carbonyls in E-liquids compromises mitochondrial integrity, increases oxidative stress, and leads to cardiac electrophysiological toxicity. MethodsGas chromatography mass spectrometry (GC/MS), flow cytometry, oxygen consumption rate (OCR) measurement, in-vivo programmed electrical stimulation (PES), and multielectrode array (MEA) were used in atrial like HL-1 myocytes, hiPSC derived cardiomyocytes, and mice overexpressing mitochondrial catalase (mCAT). ResultsWe compared the toxicity of E-vapor exposure from 30 differently flavored E-liquids in HL-1 cells using apoptotic annexin V flow cytometry. Most E-liquids were toxic. We identified the major flavoring carbonyls in these E-liquids and quantified their concentrations using GC/MS. Linear regression analysis showed that toxicity correlated with carbonyls concentration. Using flow cytometry of CellROX and TMRE staining, HL-1 cells exposed to flavored E-vapor showed increased reactive oxygen species and depolarized mitochondrial membrane potential. Additionally, exposure decreased OCR in these cells. In-vivo inhalation exposure to flavored E-vapor increased the inducible ventricular tachycardia duration in WT but not in mCAT mice compared to controls. MEA recordings in hiPSC derived cardiomyocytes exposed to flavored E-vapor, with or without nicotine, resulted in changes in the spontaneous beating rate. ConclusionsInhalation exposure to flavored ENDS negatively affects ventricular electrophysiology, in part via adverse mitochondrial remodeling, and increased oxidative stress.

physiology↗

bioRxiv2025

Intercellular communication in the brain via dendritic nanotubular network

Recent studies have identified intercellular networks for material exchange by bridge-like nanotubular structures, yet their existence in neurons remains unexplored within the brain. Here, we identified long, thin dendritic filopodia that establish direct dendrite-to-dendrite contacts, forming dendritic nanotubes (DNTs) in mammalian brains. Using super-resolution microscopy, we characterized their unique molecular composition and dynamics in dissociated neurons, enabling Ca2+ propagation over distances. Utilizing imaging and machine-learning-based analysis, we confirmed the in situ presence of DNTs connecting dendrites to other dendrites whose anatomical features are distinguished from synaptic dendritic spines. DNTs mediate the active transport of small molecules or human amyloid-beta (A{beta}), implicating the role of DNT network in AD pathology. Notably, DNT levels increased prior to the onset of amyloid plaque deposits in the mPFC of APP/PS1 mice. Computational simulations predicted the progression of amyloidosis, providing insight into the mechanisms underlying neurodegeneration through these DNTs. This study unveils a previously unrecognized nanotubular network, highlighting another dimension of neuronal connectivity beyond synapses.

neuroscience↗

bioRxiv2025

Mitochondrial Translation Inhibition Uncovers a Critical Metabolic-Epigenetic Interface in Renal Cell Carcinoma

Background/ObjectivesRenal cell carcinoma (RCC) exhibits distinctive metabolic vulnerabilities that may be therapeutically targeted. This study investigates how tigecycline, an FDA-approved antibiotic that inhibits mitochondrial translation, affects RCC cells and explores potential combinatorial approaches to enhance its efficacy. MethodsWe employed comprehensive metabolomic profiling, subcellular proteomics, and functional assays to characterize the effects of tigecycline on RCC cell lines, patient-derived organoids, and xenograft models. The synergistic potential of tigecycline with the histone deacetylase inhibitor entinostat was evaluated using combination index analysis. ResultsTigecycline selectively inhibited mitochondrial translation in RCC cells, reducing mitochondrially-encoded proteins while sparing nuclear-encoded components, profoundly disrupting mitochondrial bioenergetics and reducing tumor growth in xenograft models. Subcellular proteomic analyses revealed that tigecycline treatment triggered significant accumulation of multiple histone variants concurrent with cell cycle arrest. Based on this discovery, combined treatment with tigecycline and entinostat demonstrated remarkable synergism across RCC cell lines and patient derived organoids. ConclusionsOur findings identify a promising therapeutic opportunity by targeting the crosstalk between mitochondrial function and epigenetic homeostasis in RCC, with potential for rapid clinical translation given the established pharmacological profiles of both agents.

cancer biology↗

bioRxiv2025

Synaptic changes contribute to persistent extra-motor behaviour deficits in the rNLS8 TDP-43 mouse model of amyotrophic lateral sclerosis

Extra-motor symptoms are increasingly recognised in amyotrophic lateral sclerosis (ALS), encompassing cognitive, social, and behavioural deficits that can substantially impact quality of life. TAR DNA binding protein 43 (TDP-43) pathology is the central disease marker of almost all cases of ALS and approximately half of all frontotemporal dementia (FTD). However, the mechanisms linking TDP-43 pathology with extra-motor symptoms in TDP-43-associated neurodegenerative diseases remain unresolved. In this study, we used the rNLS8 mouse model, which expresses human TDP-43 with an ablated nuclear localisation sequence (hTDP-43∆NLS) in a doxycycline-regulatable manner causing progressive motor decline reminiscent of ALS, to delineate the molecular changes associated with disease-relevant phenotypes. We found that in addition to previously reported dramatic motor decline, rNLS8 mice also develop extra-motor phenotypes consistent with FTD, including disinhibition-like and anxiety-like behaviours, and social interaction impairments. These changes began in the earliest disease stages and remained readily detectable even when rNLS8 mice became severely motor impaired. Notably, extra-motor deficits persisted in rNLS8 mice that had recovered motor function upon hTDP-43∆NLS transgene suppression, regardless of whether recovery was initiated at timepoints prior to or after overt neurodegeneration begins. Transcriptomic analysis of rNLS8 mouse cortex tissues revealed early alterations in expression of 321 genes, most notably involving neuroinflammatory-related pathway activation, and all but 2 of these genes returned to control levels upon suppression of hTDP-43∆NLS expression. Further, 814 genes showed differential exon usage, indicating changes in alternative splicing, in rNLS8 mouse cortex. Of these, differential exon usage of 10 neuronal genes persisted after hTDP-43∆NLS transgene suppression, including synapse component genes Nrxn1, Unc13a, and Gls. Similarly, proteomics analysis of the cortex of rNLS8 mice revealed depletion of synaptic proteins, particularly those involved in glutamatergic signalling pathways, which also persisted following hTDP-43∆NLS transgene suppression. Similar glutamatergic pathway changes were detected in human ALS and FTD post-mortem cortex tissues. Our findings indicate that extra-motor phenotypes emerge early in disease in rNLS8 mice and remain evident despite progressive motor impairments. Further, extra-motor phenotypes persist even upon motor recovery, correlating with specific synaptic gene expression and splicing changes. Overall, this study suggests the potential utility of an expanded suite of behavioural paradigms in preclinical testing using rNLS8 mice, with enhanced relevance to the diversity of TDP-43 proteinopathies including FTD. Our findings further suggest that targeting glutamatergic synaptic components may be an avenue to correct extra-motor deficits associated with TDP-43 pathology.

neuroscience↗

bioRxiv2025

Ambient temperature structural studies of Penicillin-binding Protein 2a of Methicillin-Resistant Staphylococcus aureus with XFELs and synchrotrons

Penicillin-binding protein 2a (PBP2a) is a transpeptidase responsible for the {beta}-lactam resistance in methicillin-resistant Staphylococcus aureus (MRSA), posing significant challenges to antibiotic therapy. PBP2as unique structural features, including its highly flexible active site and allosteric regulation, enable it to maintain catalytic activity even in the presence of {beta}-lactam antibiotics. Despite extensive characterization using cryogenic crystallography, key questions remain about its dynamic properties and conformational changes under near-physiological conditions. Room-temperature crystallography methods, particularly serial femtosecond X-ray crystallography (SFX) at XFELs, provide a powerful approach to capture these dynamics. Unlike cryogenic conditions that can constrain protein flexibility, SFX enables the study of conformational variability and interaction networks closer to physiological states. Here, we present the first room-temperature structures of PBP2a obtained using SFX to uncover insights into the enzymes flexibility, allosteric communication, and catalytic mechanisms. These findings are built upon optimized large-scale production and crystallization protocols for PBP2a, ensuring high-quality microcrystals suitable for SFX data collection. The room-temperature structures reveal novel interaction patterns, including unique salt bridge networks and dynamic structural elements absent in cryogenic studies. Furthermore, comparative analyses highlight how environmental conditions influence the conformational states of PBP2a, providing new perspectives on its resistance mechanisms. By integrating structural data from EuXFEL and LCLS, this study not only enhances our understanding of PBP2as functional dynamics but also underscores the value of room-temperature crystallography in studying antibiotic resistance. These insights could guide the design of next-generation {beta}-lactam antibiotics capable of overcoming PBP2a-mediated resistance.

biochemistry↗

bioRxiv2025

The shortest telomere in telomerase-negative cells triggers replicative senescence at a critical threshold length and also fuels genomic instability

In the absence of telomerase, telomere shortening triggers replicative senescence, a tumor suppressor mechanism that is also associated with oncogenic genomic instability. Yet, the precise mechanism that connects these seemingly opposing forces remains poorly understood. To directly study the complex interplay between senescence, telomere dynamics, and genomic instability, we developed a system in Saccharomyces cerevisiae to generate and track telomeres of precise length in the absence of telomerase. Using single-telomere and single-cell analyses combined with mathematical modeling, we identify a threshold length at which telomeres switch into dysfunction. A single shortest telomere below the threshold length is necessary and sufficient to trigger the onset of replicative senescence in a majority of cells. At population level, fluctuation assays establish that rare genomic instability arises predominantly in cis to the shortest telomere as Pol32-dependent non-reciprocal translocations that result in re-elongation of the shortest telomere and likely transient escape from senescence. The switch of the shortest telomere into dysfunction and subsequent processing in telomerase-negative cells thus serves as the mechanistic link between replicative senescence onset, genomic instability and the initiation of post-senescence survival.

molecular biology↗

bioRxiv2025

Single-cell spatial transcriptome reveals pathological features of human hippocampus with sclerosis

More than half of epileptic patients ultimately turned to intractable epilepsy. Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE-HS), the most common type of intractable epilepsies, whose pathological mechanism remains elusive. Here, using 42 human hippocampal samples from surgical donors of MTLE (32 with and 10 without HS) through single-cell resolution Stereo-seq and histologcial experiments, we revealed spatially pathological changes of gene expression and cell type composition with HS systematically. After precise parcellation of hippocampal subregions and differentially expressed gene (DEG) analysis between each region with and without sclerosis, we found Cornu Ammonis (CA) subregions with higher number of DEGs were vulnerable to sclerosis, especially CA1 and CA3. Within CA1, we found that CA1-superficial and proximal areas were more vulnerable than CA1-deep and distal areas in sclerosis. Meanwhile, after analyzing 350,795 segmented cells from Stereo-seq, we found dramatically increasing density of astrocyte accompanied with significantly decreasing density of excitatory neurons in CA1, especially superficial and proximal CA1, and CA3 in sclerosis. In these two vulnerable subregions, proliferative astrocyte (P_astrocyte) and reactive astrocyte (R_astrocyte) were found to be enriched whereas apoptotic subtype of astrocyte (A_astrocyte), related with apoptotic pathway, was mainly located in alveus, which strengthened cell communication with reactive microglia (R_microglia) in HS, revealing the novel pathological feature in our work. The pseudotime analysis indicated that CA excitatory neurons underwent synaptic impairment, energy dysfunction, aging, and finally losing cell identity until death through autophagy or apoptosis. Besides, we also found a resilient subtype, EX_CA2-4.3, highly expressed extracellular matrix related genes including PDYN, and was increasing the interaction of BDNF-NTRK, NFASC-CNTN1 to withstand the damage from sclerosis. Together, our study provided a reference of human hippocampus with and without HS caused by MTLE, and highlighted the potential pathological mechanism on molecular and cellular level of MTLE-HS.

neuroscience↗

bioRxiv2025

Decoding Gene Networks Controlling Hypothalamic and Prethalamic Neuron Development.

Neuronal subtypes derived from the embryonic hypothalamus and prethalamus regulate many essential physiological processes, yet the gene regulatory networks controlling their development remain poorly understood. Using single-cell RNA- and ATAC-sequencing, we analyzed mouse hypothalamic and prethalamic development from embryonic day 11 to postnatal day 8, profiling 660,000 cells in total. This identified key transcriptional and chromatin dynamics driving regionalization, neurogenesis, and differentiation. This identified multiple distinct neural progenitor populations, as well as gene regulatory networks that control their spatial and temporal identities, and their terminal differentiation into major neuronal subtypes. Integrating these results with large-scale genome-wide association study data, we identified a central role for transcription factors controlling supramammillary hypothalamic development in a broad range of metabolic and cognitive traits. Recurring cross-repressive regulatory relationships were observed between transcription factors that induced prethalamic and tuberal hypothalamic identity on the one hand and mammillary and supramammillary hypothalamic identity on the other. In postnatal animals, Dlx1/2 was found to severely disrupt GABAergic neuron specification in both the hypothalamus and prethalamus, resulting in a loss of inhibition of thalamic neurons, hypersensitivity to cold, and behavioral hyperactivity. By identifying core gene regulatory networks controlling the specification and differentiation of major hypothalamic and prethalamic neuronal cell types, this study provides a roadmap for future efforts aimed at preventing and treating a broad range of homeostatic and cognitive disorders.

neuroscience↗

bioRxiv2024

Combined inhibition of SHP2 overcomes adaptive resistance to type 1 BRAF inhibitors in BRAF V600E-driven high-grade glioma

BRAF-mutant gliomas can be therapeutically targeted with BRAF mutant-selective inhibitors, yet responses are often transient due to short-term adaptive or long-term treatment-emergent resistance. We hypothesized that vertical inhibition of multiple signaling nodes could improve the durability of BRAF inhibition and prevent or overcome adaptive resistance. Using human tissue samples, we identified frequent RAS pathway reactivation in gliomas resistant to BRAF inhibitors, suggesting a common escape mechanism. Using patient-derived cell lines, we observed that upregulation of RAS activity was an adaptive response to BRAFi and that knockdown of SHP2, a central regulator of RAS activity, resulted in enhanced sensitivity to BRAF or MEK inhibition. Moreover, combined small molecule inhibition with SHP2 and BRAF or MEK inhibitors increased the depth and durability of ERK pathway inhibition, as well as prevented paradoxical upregulation of RAS activity. RNA sequencing analysis revealed deeper suppression of ERK transcriptional output with combined therapy, along with decreased reactivation of EGFR. Combined SHP2/BRAF small molecule inhibitors prevented growth and induced cell death in some cell line models. In cell lines with treatment-emergent resistance, moreover, combined SHP2 and BRAF inhibition overcame resistance to BRAF inhibitor monotherapy. In vivo orthotopic and patient-derived xenograft models confirmed enhanced tumor growth inhibition with combined therapy. Together, our findings demonstrate the critical role of RAS/ERK signaling reactivation in driving resistance to BRAF inhibition in glioma, and demonstrate the potential utility for adding SHP2 inhibitors to overcome resistance in BRAF V600E mutant glioma. SignificanceThe addition of a SHP2i to BRAFi in BRAF-V600E glioma cells prevents tumor growth and can overcome resistance to BRAFi in preclinical models in vitro and in vivo.

cancer biology↗

bioRxiv2024

Expanding the capillarics toolbox: 3D-printed microfluidic phaseguides and self-coalescence modules

Capillarics are microfluidic circuits that are assembled from individual fluidic elements, powered by surface tension forces encoded by microchannel geometry and surface chemistry, and enable instrument-free pre-programmed automation of multi-step liquid handling processes. 3D printing has recently transformed capillarics by enabling rapid and cost-effective prototyping, provided addition geometric degrees of freedom in multi-level fabrication, and facilitated new design paradigms with greater capabilities than traditional cleanroom fabrication. Despite widespread interest in 3D printing and development of custom high-resolution stereolithography printers for microfluidic applications, fluidic elements that require precise and tunable control over capillary pinning lines -- such as fluidic phaseguides and self-coalescence modules (SCMs) -- have so far only been manufactured with centralized and expensive cleanroom methods. Not only does this limit access to versatile capillaric features to only well-resourced settings, but it also slows innovation and widespread application of these fluid handling technologies. Here we expand the toolbox of 3D-printed capillarics to include phaseguides and SCMs, demonstrating their potential for precise instrument-free control over multi-step liquid handling and reagent rehydration. We employed benchtop stereolithography printers to prototype (up to 50X) scaled-up phaseguides and SCMs and integrated them into a capillaric circuit for inline reagent reconstitution, dynamic fluid control, and sequential drainage. We showcased scalable designs, customizable geometries, and robust self-coalescing flow for larger liquid volumes -- up to 50 L compared with 1.25 L in cleanroom-fabricated SCMs. This work represents a significant advance in democratizing access to microfluidics, with potential for broad applications in diagnostics, assay automation, and organ-on-chip systems.

bioengineering↗

bioRxiv2024

CD226+ adipose tissue macrophages arise from MDP-derived monocytes and regulate lipid metabolism.

Macrophages are innate immune cells present in all tissues, in which they participate in immune responses and maintenance of tissue homeostasis. They develop either from embryonic precursors or from circulating monocytes, and their origin impacts their functions. We previously observed robust recruitment of monocytes to brown adipose tissue in which they could differentiation into two distinct macrophage subsets identifiable by CD206 or CD226 expression. In the present study, we investigated monocyte differentiation pathways in brown adipose tissue and the function of monocyte-derived macrophages. Fate mapping analysis revealed a low contribution of GMP- and a high contribution of MDP-derived monocytes to the CD226high macrophage subset. Importantly, adoptive transfer experiments demonstrate that MDP- but not GMP-derived monocytes are pre-conditioned to give rise to CD226high macrophages. We found that MDP-derived CD226high macrophages were also present in other tissues including peritoneal cavity, adrenal glands and all adipose depots. CD226high macrophages were regulated by both GM-CSF and CSF1R. Genetic depletion of CD226high macrophages caused increased BAT and plasma triglyceride content. We thus identify CD226high MDP-derived macrophages as a new myeloid cell type conserved across tissues and tied to lipid metabolism homeostasis.

immunology↗

bioRxiv2024

Identification of conserved and tissue-restricted transcriptional profiles for lipid associated macrophages (LAMs)

Macrophages are essential immune cells present in all tissues, and are vital for maintaining tissue homeostasis, immune surveillance, and immune responses. Considerable efforts have identified shared and tissue-specific gene programs for macrophages across organs during homeostasis. This information has dramatically enhanced our understanding of tissue-restricted macrophage programming and function. However, few studies have addressed the overlapping and tissue-specific responses of macrophage subsets following inflammatory responses. One subset of macrophages that has been observed across several studies, lipid-associated macrophages (LAMs), have gained interest due to their unique role in lipid metabolism and potential as a therapeutic target. LAMs have been associated with regulating disease outcomes in metabolically related disorders including atherosclerosis, obesity, and nonalcoholic fatty liver disease (NAFLD). In this study, we utilized single-cell RNA sequencing (scRNAseq) data to profile LAMs across multiple tissues and sterile inflammatory conditions in mice and humans. Integration of data from various disease models revealed that LAMs share a set of conserved transcriptional profiles, including Trem2 and Lpl, but also identified key sets of tissue-specific LAM gene programs. Importantly, the shared LAM markers were highly conserved with human LAM populations that also emerge in chronic inflammatory settings. Overall, this analysis provides a detailed transcriptional landscape of tissue-restricted and shared LAM gene programs and offers insights into their roles in metabolic and chronic inflammatory diseases. These data may help instruct appropriate targets for broad or tissue-restricted therapeutic interventions to modulate LAM populations in disease.

immunology↗

bioRxiv2024

Joint Gray and White Matter Networks Predict Mindfulness and Mind Wandering Traits: A Data Fusion Machine Learning Approach

ObjectivesThe main aim of our study was to find out which gray matter (GM) and white matter (WM) brain features are associated with mindfulness and mind wandering, and to investigate how mindfulness mediates deliberate and spontaneous mind wandering in terms of these associated brain components. MethodsStructural MRI scans of 76 individuals and self-reported questionnaires were included in this analysis. We applied unsupervised machine learning algorithms to the brain imaging data to decompose the fused GM and WM into naturally grouping covarying networks. We then conducted a mediation analysis to assess the influence of mindfulness on deliberate and spontaneous mind wandering based on this data fusion networks. Additionally, we investigated if certain mindfulness facets mediate the two forms of mind wandering traits. ResultsWe found GM and WM networks composed of structures that have been consistently linked to mindfulness (e.g., the cingulate, insula, basal ganglia) and fronto-parietal attentive regions exert direct effects on mindfulness, as well as on deliberate and spontaneous mind wandering. We also found an indirect mediating effect of the mindfulness facet acting with awareness on spontaneous mind wandering through one of the significant brain networks, we identified. ConclusionsThis study elicited the link between mind wandering and mindfulness, and expands our knowledge on the neural bases of these two psychological constructs.

neuroscience↗

bioRxiv2024

Interstitial telomeric sequences promote gross chromosomal rearrangement via multiple mechanisms

Telomeric DNA sequences are difficult to replicate. Replication forks frequently pause or stall at telomeres, which can lead to telomere truncation and dysfunction. In addition to being at chromosome ends, telomere repeats are also present at internal locations within chromosomes, known as interstitial telomeric sequences (ITSs). These sequences are unstable and prone to triggering gross chromosomal rearrangements (GCRs). In this study, we quantitatively examined the effect of ITSs on GCR rate in Saccharomyces cerevisiae using a genetic assay. We find that GCR rate increases exponentially with ITS length. This increase can be attributed to the telomere repeat binding protein Rap1 impeding DNA replication and a bias of repairing DNA breaks at or distal to the ITS via de novo telomere addition. Additionally, we performed a genome-wide screen for genes that modulate the rate of ITS-induced GCRs. We find that mutation of core components of the DNA replication machinery leads to an increase in GCRs, but many mutants known to increase GCR rate in the absence of an ITS do not significantly affect GCR rate when an ITS is present. We also identified genes that promote the formation of ITS-induced GCRs, including genes with roles in telomere maintenance, nucleotide excision repair, and transcription. Our work thus uncovers multiple mechanisms by which an ITS promotes GCR. Significance statementTelomeric DNA repeats are found at the ends of linear chromosomes where they, together with specialized proteins that bind to them, protect chromosome ends from degradation and unwanted DNA repair activities. Telomeric repeats can also be found at internal locations in the genome, where they are called interstitial telomeric sequences (ITSs). ITSs are prone to breakage and are associated with human diseases. In this study, using bakers yeast as a model organism, we show that instability at ITSs is driven by multiple factors, and identify genes that either promote or suppress gross chromosomal rearrangements induced by the presence of an ITS.

molecular biology↗

bioRxiv2024

Revisiting the role of the spindle assembly checkpoint in the formation of gross chromosomal rearrangements in Saccharomyces cerevisiae

Multiple pathways are known to suppress the formation of gross chromosomal rearrangements (GCRs), which can cause human diseases including cancer. In contrast, much less is known about pathways that promote their formation. The spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during mitosis, has been reported to promote GCR, possibly by delaying mitosis to allow GCR-inducing DNA repair to occur. Here we show that this conclusion is the result of an experimental artifact arising from the synthetic lethality caused by disruption of the SAC and loss of the CIN8 gene, which is often lost in the genetic assay used to select for GCRs. After correcting for this artifact, we find no role of the SAC in promoting GCR. Significance statementA gross chromosomal rearrangement (GCR) is an abnormal structural change of a native chromosome. Examples of GCRs include deletions, duplications, inversions, and translocations. GCRs can lead to genetic diseases such as cancer. A previous study implicated the spindle assembly checkpoint (SAC), which ensures the proper separation of chromosomes during cell division, in facilitating the formation of GCRs. In this study, we show that this is not the case; the SAC does not promote GCR.

molecular biology↗

bioRxiv2024

Skeletal muscle effects of antisense oligonucleotides targeting glycogen synthase 1 in a mouse model of Pompe disease

Pompe disease (PD) is a progressive myopathy caused by the aberrant accumulation of glycogen in skeletal and cardiac muscle resulting from the deficiency of the enzyme acid alpha-glucosidase (GAA). Administration of recombinant human GAA as enzyme replacement therapy (ERT) works well in alleviating the cardiac manifestations of PD but loses sustained benefit in ameliorating the skeletal muscle pathology. The limited efficacy of ERT in skeletal muscle is partially attributable to its inability to curb the accumulation of new glycogen produced by the muscle enzyme glycogen synthase 1 (GYS1). Substrate reduction therapies aimed at knocking down GYS1 expression represent a promising avenue to improve Pompe myopathy. However, finding specific inhibitors for GYS1 is challenging given the presence of the highly homologous GYS2 in the liver. Antisense oligonucleotides (ASOs) are chemically modified oligomers that hybridize to their complementary target RNA to induce their degradation with exquisite specificity. In the present study, we show that ASO-mediated Gys1 knockdown in the Gaa-/- mouse model of PD led to a robust reduction in glycogen accumulation in skeletal and cardiac muscle. In addition, combining Gys1 ASO with ERT further reduced glycogen content in muscle, eliminated autophagic buildup and lysosomal dysfunction, and improved motor function in Gaa-/- mice. Our results provide a strong foundation for further validation of the use of Gys1 ASO, alone or in combination with ERT, as a therapy for PD. We propose that early administration of Gys1 ASO in combination with ERT may be the key to preventative treatment options in PD.

molecular biology↗

bioRxiv2023

Ras Activation by Hydrostatic Pressure is Enhanced by GAP and GEF in vitro

Hydrostatic pressure (HP) is a necessary stimulus for cell differentiation and growth in cultured chondrocytes. Assuming that Ras activation is involved in HP-induced reactions, if cellular Ras activity is increased by HP, Ras itself, Raf, Ras regulators including GTPase activating proteins (GAP) and guanine nucleotide exchange factors (GEF), then the upstream receptor and/or interactions between them should have HP sensitivity. Based on this hypothesis, we first attempted to examine whether Ras is activated by HP, and in the affirmative, identify which factors displayed HP sensitivity using an in vitro system to measure Ras activity. This in vitro system included mRaichu, a FRET-based Ras activity probe, to which two-point mutations were introduced to reduce Ras-independent signals. This improved Raichu was used to investigate the HP sensitivity of two components, the GAP domain (GAPd) derived from p120GAP and the GEF domain (GEFd) derived from hSOS-1. It was found that HP weakly activated Ras activity in the absence of GAPd and GEFd, presumably by facilitating GDP dissociation from Ras. A low concentration of GAPd enhanced HP-induced Ras activation by 16.3% whereas high concentrations of GAPd removed HP sensitivity, suggesting that HP partially dissociates GAPd from the GAPd-Ras-GDP complex and reduces the fraction of inactive Ras. Moreover, a broad concentration range of GEFd also enhanced HP-induced Ras activation. Given that HP also increased Ras activity under a condition mimicking cellular Ras activity, we propose that Ras activation is involved in the differentiation and growth stimulation of chondrocytes by HP. Statement of SignificanceThe Ras-cycle has been implicated in the regulation of growth and differentiation of eukaryotic cells. Here, we investigated the relationship between hydrostatic pressure (HP) and components of the Ras-cycle: Ras-Raf, the GAP domain, and the GEF domain. Generally, HP tends to weaken protein-protein and protein-ligand interactions, but in this study, a seemingly positive response was observed: HP-induced Ras activation. Additionally, this response was enhanced by the GEF domain and the GAP domain. Thus, given the multiple cellular functions of Ras and the responses of the Ras-cycle to HP, this study will help clarify the molecular mechanism by which HP modulates cellular functions, particularly in chondrocytes, which are subjected to repetitive HP stimuli in vivo.

biophysics↗

bioRxiv2023

Targeting the Hippo pathway in cancers via ubiquitination dependent TEAD degradation

The Hippo pathway is among the most frequently altered key signaling pathways in cancer. TEAD1-4 are essential transcription factors and key downstream effectors in the Hippo pathway in human cells. Here, we identified RNF146 as a ubiquitin ligase (E3) of TEADs, which negatively regulates their stability in cells through proteasome-mediated degradation. We show that RNF146-mediated TEAD ubiquitination is dependent on the TEAD PARylation state. We further validated the genetic interaction between RNF146 and the Hippo pathway in cancer cell lines and the model organism Drosophila melanogaster. Despite the RNF146 and proteasome-mediated degradation mechanisms, TEADs are stable proteins with a long half-life in cells. We demonstrate that degradation of TEADs can be greatly enhanced pharmacologically with heterobifunctional chemical inducers of protein degradation (CIDEs). These TEAD-CIDEs can effectively suppress activation of YAP/TAZ target genes in a dose-dependent manner and exhibit significant anti-proliferative effects in YAP/TAZ-dependent tumor cells, thus phenocopying the effect of genetic ablation of TEAD protein. Collectively, this study demonstrates that the ubiquitin-proteasome system plays an important role in regulating TEAD functions and provides a proof-of-concept demonstration that pharmacologically induced TEAD ubiquitination could be leveraged to target YAP/TAZ-driven cancers.

cancer biology↗