Biology subjects

Chen, A.

Publications and source records attributed to Chen, A..

At least 145 records · Page 8Linked to original sources

bioRxiv2024

Early life adversity drives sex-dependent changes in 5-mC DNA methylation of parvalbumin cells in the prefrontal cortex in rats

Early life adversity (ELA) can result in increased risk for developing affective disorders, such as anxiety or depression, later in life, with women showing increased risk. Interactions between an individuals genes and their environment play key roles in producing, as well as mitigating, later life neuropathology. Our current understanding of the underlying epigenomic drivers of ELA associated anxiety and depression are limited, and this stems in part from the complexity of underlying biochemical processes associated with how early experiences shapes later life behavior. Epigenetic alterations, or experience-driven modifications to DNA, can be leveraged to understand the interplay between genes and the environment. The present study characterized DNA methylation patterning, assessed via evaluation of 5-methylcytosine (5-mC), following ELA in a Sprague Dawley rat model of ELA induced by early caregiver deprivation. This study utilized maternal separation to investigate sex- and age-specific outcomes of ELA on epigenetic patterning in parvalbumin (PV)-containing interneurons in the prefrontal cortex (PFC), a subpopulation of inhibitory neurons which are associated with ELA and affective dysfunction. While global analysis of 5-mC methylation and CpG site specific pyrosequencing of the PV promoter, Pvalb, showed no obvious effects of ELA, when analyses were restricted to assessing 5-mC intensity in colocalized PV cells, there were significant sex and age dependent effects. We found that ELA leads sex-specific changes in PV cell counts, and that cell counts can be predicted by 5-mC intensity, with males and females showing distinct patterns of methylation and PV outcomes. ELA also produced sex-specific effects in corticosterone reactivity, with juvenile females showing a blunted stress hormone response compared to controls. Overall, ELA led to a sex-specific developmental shift in PV profile, which is comparable to profiles that are seen at a later developmental timepoint, and this shift may be mediated in part by epigenomic alterations driven by altered DNA methylation.

neuroscience↗

bioRxiv2024

Surface Patterned Omniphobic Tiles (SPOTs): a versatile platform for scalable liquid handling

Manipulating liquids is a ubiquitous need for experiments across numerous scientific disciplines. To overcome limitations of current methods, we introduce Surface Patterned Omniphobic Tiles (SPOTs). This platform combines geometry and surface engineering, building on discontinuous wetting approaches to leverage capillarity for metering liquids. The SPOTs platform allows manipulation of hundreds to thousands of independent experiments without expensive equipment or large consumable costs. These devices can handle a wide range of liquid types and volumes (<10 nanoliters to >10 microliters) with better precision than pipetting. The platform is inexpensive and easy to fabricate, fast and intuitive to use, and cross-compatible with existing microwell plate layouts. We demonstrate how these capabilities facilitate diverse experiments including testing antibiotic combinations for synergy and antagonism, material screening of perovskites, and genotyping microbial isolates. We anticipate SPOTs will enable users from disparate domains to quickly and easily run a wide range of high-throughput experiments.

bioengineering↗

bioRxiv2024

Mutation of two intronic nucleotide alters RNA structure and dynamics, inhibiting MBNL1 and RBFOX1 regulated splicing of the Insulin Receptor

Alternative splicing (AS) of Exon 11 of the Insulin Receptor (INSR) is highly regulated and disrupted in several human disorders. To better understand INSR exon 11 AS regulation, splicing activity of an INSR exon 11 minigene reporter was measured across a gradient of the AS regulator muscleblind-like 1 protein (MBNL1). The RNA-binding protein Fox-1 (RBFOX1) was added to determine its impact on MBNL1-regulated splicing. The role of the RBFOX1 UGCAUG binding site within intron 11 was assessed across the MBNL1 gradient. Mutating the UGCAUG motif inhibited RBFOX1 regulation of exon 11 and had the unexpected effect of reducing MBNL1 regulation of this exon. Molecular dynamics simulations showed that exon 11 and the adjacent RNA adopts a dynamically stable conformation. Mutation of the RBFOX1 binding site altered RNA structure and dynamics, while a mutation that created an optimal MBNL1 binding site at the RBFOX1 site shifted the RNA back to wild type. An antisense oligonucleotide (ASO) was used to confirm the structure in this region of the pre-mRNA. This example of intronic mutations shifting pre-mRNA structure and dynamics to modulate splicing suggests RNA structure and dynamics should be taken into consideration for AS regulation and therapeutic interventions targeting pre-mRNA. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/574689v1_ufig1.gif" ALT="Figure 1"> View larger version (28K): org.highwire.dtl.DTLVardef@17e410org.highwire.dtl.DTLVardef@7deb00org.highwire.dtl.DTLVardef@cf69f9org.highwire.dtl.DTLVardef@13684e8_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOAbstract figure:C_FLOATNO Model for INSR exon 11 splicing regulation through the UGCAUG motif. We propose that the UGCAUG motif, MBNL1, and RBFOX1 cooperatively regulate INSR exon 11 splicing. Mutating this UGCAUG motif is sufficient to alter RNA structural dynamics to disrupt this regulation. C_FIG

biochemistry↗

bioRxiv2023

Genomic evidence for hybridization and introgression between blue peafowl and green peafowl and selection for white plumage

The blue peafowl (Pavo cristatus) and the green peafowl (Pavo muticus) have significant public affection due to their stunning appearance, although the green peafowl is currently endangered. Some studies have suggested introgression between these the two species, although evidence is mixed. In this study, we successfully assembled a high-quality chromosome-level reference genome of the blue peafowl, including the autosomes, Z and W sex chromosomes as well as a complete mitochondria DNA sequence. Data from 77 peafowl whole genomes, 76 peafowl mitochondrial genomes and 33 peahen W chromosomes genomes provide the first substantial genetic evidence for recent hybridization between green and blue peafowl. We found three hybrid green peafowls in zoo samples rather than in the wild samples, with blue peafowl genomic content of 16-34%. Maternal genetic analysis showed that two of the hybrid female green peafowls contained complete blue peafowl mitochondrial genomes and W chromosomes. Hybridization of endangered species with its relatives is extremely detrimental to conservation. Some animal protection agencies release captive green peafowls in order to maintain the wild population of green peafowls. Therefore, in order to better protect the endangered green peafowl, we suggest that purebred identification must be carried out before releasing green peafowls from zoos into the wild in order to preventing the hybrid green peafowl from contaminating the wild green peafowl. In addition, we also found that there were historical introgression events of green peafowl to blue peafowl in four Zoo blue peafowl individuals. The introgressed genomic regions contain IGFBP1 and IGFBP2 genes that could affect blue peafowl body size. Finally, we identified that the nonsense mutation (g.4:12583552G>A) in the EDNRB2 gene is the genetic causative mutation for white feather color of blue peafowl (also called white peafowl), which prevents melanocytes from being transported into feathers, such that melanin cannot be deposited.

genomics↗

bioRxiv2023

Cytotoxic chemotherapy potentiates the immune response and efficacy of combination CXCR4/PD-1 inhibition in models of pancreatic ductal adenocarcinoma

PurposeThe CXCL12-CXCR4 chemokine axis plays a significant role in modulating T-cell infiltration into the pancreatic tumor microenvironment. Despite promising preclinical findings, clinical trials combining inhibitors of CXCR4 (AMD3100/BL-8040) and anti-programmed death 1/ligand1 (anti-PD1/PD-L1) have failed to improve outcomes. Experimental DesignWe utilized a novel ex vivo autologous patient-derived immune/organoid (PDIO) co-culture system using human peripheral blood mononuclear cells and patient derived tumor organoids, and in vivo the autochthonous LSL-KrasG12D/+; LSL-Trp53R172H/+; Pdx-1-Cre (KPC) pancreatic cancer mouse model to interrogate the effects of either monotherapy or all combinations of gemcitabine, AMD3100, and anit-PD1 on CD8+ T cell activation and survival. ResultsWe demonstrate that disruption of the CXCL12-CXCR4 axis using AMD3100 leads to increased migration and activation of CD8+ T-cells. In addition, when combined with the cytotoxic chemotherapy gemcitabine, CXCR4 inhibition further potentiated CD8+ T-cell activation. We next tested the combination of gemcitabine, CXCR4 inhibition, and anti-PD1 in the KPC pancreatic cancer mouse model and demonstrate that this combination markedly impacted the tumor immune microenvironment by increasing infiltration of natural killer cells, the ratio of CD8+ to regulatory T-cells, and tumor cell death while decreasing tumor cell proliferation. Moreover, this combination extended survival in KPC mice. ConclusionsThese findings suggest that combining gemcitabine with CXCR4 inhibiting agents and anti-PD1 therapy controls tumor growth by reducing immunosuppression and potentiating immune cell activation and therefore may represent a novel approach to treating pancreatic cancer.

cancer biology↗

bioRxiv2023

uniLIVER: a Human Liver Cell Atlas for Data-Driven Cellular State Mapping

The liver performs several vital functions such as metabolism, toxin removal and glucose storage through the coordination of various cell types. The cell type compositions and cellular states undergo significant changes in abnormal conditions such as fatty liver, cirrhosis and liver cancer. As the recent breakthrough of the single-cell/single-nucleus RNA-seq (sc/snRNA-seq) techniques, there is a great opportunity to establish a reference cell map of liver at single cell resolution with transcriptome-wise features. In this study, we build a unified liver cell atlas uniLIVER by integrative analyzing a large-scale sc/snRNA-seq data collection of normal human liver with 331,125 cells and 79 samples from 6 datasets. Besides the hierarchical cell type annotations, uniLIVER also proposed a novel data-driven strategy to map any query dataset to the normal reference map by developing a machine learning based framework named LiverCT. Applying LiverCT on the datasets from multiple abnormal conditions (1,867,641 cells and 439 samples from 12 datasets), the alterations of cell type compositions and cellular states were systematically investigated in liver cancer.

bioinformatics↗

bioRxiv2023

Scalable Genotyping of Microbial Colonies

The sequence of the 16S region is taxonomically informative and widely used for genotyping microbes. While it is easy and inexpensive to genotype several isolates by Sanger sequencing the 16S region, this method becomes quite costly if scaled to many isolates. High throughput sequencing provides one potential avenue for obtaining 16S sequences at scale, but presents additional challenges. First, DNA purification workflows for high-throughput sample preparation are labor intensive and expensive. Second, cost-effective multiplexing and library preparation schemes are difficult to implement for many libraries on a single sequencing run. Therefore, we implemented a scalable protocol for isolate genotyping involving colony polymerase chain reaction (PCR) with simple cell lysis as well as a four barcode indexing scheme that enables scalable multiplexing and streamlined library preparation by amplifying with four primers simultaneously in a single reaction. We tested this protocol on 93 colonies cultured from environmental samples, and we were able to ascertain the identity of [~]90% of microbial isolates.

microbiology↗

bioRxiv2023

Stereopy: modeling comparative and spatiotemporal cellular heterogeneity via multi-sample spatial transcriptomics

Tracing cellular dynamic changes across conditions, time, and space is crucial for understanding the molecular mechanisms underlying complex biological systems. However, integrating multi-sample data in a unified and flexible way to explore cellular heterogeneity remains a major challenge. Here, we present Stereopy, a flexible and versatile framework for modeling and dissecting comparative and spatiotemporal patterns in multi-sample spatial transcriptomics with interactive data visualization. To optimize this flexible framework, we have developed three key components: a multi-sample tailored data container, a scope controller, and an analysis transformer. Furthermore, Stereopy showcases three transformative applications supported by pivotal algorithms. Firstly, the multi-sample cell community detection (CCD) algorithm introduces an innovative capability to detect specific cell communities and identify genes responsible for pathological changes in comparable datasets. Secondly, the spatially resolved temporal gene pattern inference (TGPI) algorithm represents a notable advancement in detecting important spatiotemporal gene patterns while concurrently considering spatial and temporal features, which enhances the identification of important genes, domains and regulatory factors closely associated with temporal datasets. Finally, the 3D niche-based regulation inference tool, named NicheReg3D, reconstructs the 3D cell niches to enable the inference of cell-gene interaction network within the spatial texture, thus bridging intercellular communications and intracellular regulations to unravel the intricate regulatory mechanisms that govern cellular behavior. Overall, Stereopy serves as both a bioinformatics toolbox and an extensible framework that provides researchers with enhanced data interpretation abilities and new perspectives for mining multi-sample spatial transcriptomics data.

bioinformatics↗

bioRxiv2023

The Effect of Pseudoknot Base Pairing on Cotranscriptional Structural Switching of the Fluoride Riboswitch

A central question in biology is how RNA sequence changes influence dynamic conformational changes during cotranscriptional folding. Here we investigated this question through the study of transcriptional fluoride riboswitches, non-coding RNAs that sense the fluoride anion through the coordinated folding and rearrangement of a pseudoknotted aptamer domain and a downstream intrinsic terminator expression platform. Using a combination of E. coli RNA polymerase in vitro transcription and cellular gene expression assays, we characterized the function of mesophilic and thermophilic fluoride riboswitch variants. We showed that only variants containing the mesophilic pseudoknot function at 37 {degrees}C. We next systematically varied the pseudoknot sequence and found that a single wobble base pair is critical for function. Characterizing thermophilic variants at 65 {degrees}C through Thermus aquaticus RNA polymerase in vitro transcription showed the importance of this wobble pair for function even at elevated temperatures. Finally, we performed all-atom molecular dynamics simulations which supported the experimental findings, visualized the RNA structure switching process, and provided insight into the important role of magnesium ions. Together these studies provide deeper insights into the role of riboswitch sequence in influencing folding and function that will be important for understanding of RNA-based gene regulation and for synthetic biology applications.

molecular biology↗

bioRxiv2023

hURAT1 Transgenic Mouse Model for Evaluating Targeted Urate-Lowering Agents

ObjectiveUrate transporter 1 (URAT1), a well-established urate-lowering therapeutic target for hyperuricemia and gout treatment, expresses in the kidney proximal tubule and is responsible for uric acid (UA) reabsorption. However, non-primate animal models currently used in pharmacological studies failed to evaluate URAT1 inhibitors effectiveness because their URAT1 has a very low UA affinity compared to human URAT1, resulting in a lag in targeting drug screening and novel therapy development for gout treatment. We established a human URAT1 (hURAT1) transgenic knock-in (KI) mouse model to assess uricosuric agents effectiveness and characterize URAT1-caused pathogenesis. MethodsWe generated hURAT1 transgenic mice using CRISPR/Cas9 knock-in technique. mUrat1 knockout was achieved by replacing exon 1 coding sequence with a human SLC22A12 CDS-pA cassette. Based on the above transgenic mice, a hyperuricemia model was further established by hypoxanthine administration. ResultsThe hURAT1-KI mice successfully expressed hURAT1 protein to the apical side of the kidney proximal tubule epithelium, where a native human URAT1 kidney localization in human body. Upon hypoxanthine challenge, the blood UA level was elevated in hURAT1-KI mice, exhibiting an approximately 37% increase compared to wild-type (WT) mice. The elevated blood UA level could be alleviated by hURAT1 inhibitor benzbromarone treatment in the hURAT1-KI mice whereas no response was observed in WT littermates. Therefore, hURAT1 transgenic mice responded well to inhibitors and can be used to evaluate the therapeutic effects. ConclusionThe hURAT1-KI hyperuricaemia mouse model would be valuable for preclinical evaluation of urate-lowering agents toward gout treatment and studying UA metabolic complexities in humans.

pharmacology and toxicology↗

bioRxiv2023

High-sensitivity whole-genome recovery of single viral species in environmental samples

Characterizing unknown viruses is essential for understanding viral ecology and preparing against viral outbreaks. Recovering complete genome sequences from environmental samples remains computationally challenging using metagenomics, especially for low-abundance species with uneven coverage. This work presents a method for reliably recovering complete viral genomes from complex environmental samples. Individual genomes are encapsulated into droplets and amplified using multiple displacement amplification. A novel gene detection assay, which employs an RNA-based probe and an exonuclease, selectively identifies droplets containing the target viral genome. Labeled droplets are sorted using a microfluidic sorter, and genomes are extracted for sequencing. Validation experiments using a sewage sample spiked with two known viruses demonstrate the methods efficacy. We achieve 100% recovery of the spiked-in SV40 (Simian virus 40, 5243bp) genome sequence with uniform coverage distribution, and approximately 99.4% for the larger HAd5 genome (Human Adenovirus 5, 35938bp). Notably, genome recovery is achieved with as few as one sorted droplet, which enables the recovery of any desired genomes in complex environmental samples, regardless of their abundance. This method enables targeted characterizations of rare viral species and whole-genome amplification of single genomes for accessing the mutational profile in single virus genomes, contributing to an improved understanding of viral ecology.

bioengineering↗

bioRxiv2023

Nicotinamide Riboside Supplementation Ameliorates Mitochondrial Dysfunction and Neuronal Loss in POLG Mutant Midbrain Organoids

Mitochondrial dysfunction is associated with many neurodegenerative disorders and is particularly prominent in conditions tied to POLG mutations. POLG encodes DNA polymerase gamma vital for mitochondrial DNA replication. Employing 3D human pluripotent stem cell-derived midbrain organoids (hMOs), harbouring POLG mutations, this study explores their differentiation, transcriptional alterations, and underlying pathways of neurodegeneration associated with POLG mutations. The generated hMOs displayed midbrain specificity and, at three months, a reduced diameter, suggesting growth challenges from POLG mutations. A reduced presence of dopaminergic neurons, particularly in DA2 and ventral midbrain classes, was evident. Intriguingly, post-treatment with 1 mM Nicotinamide Riboside (NR), an NAD+ precursor, the organoids demonstrated an increased count of DA and VMN neurons and an elevated gene expression, especially in processes crucial to mitochondrial and synaptic functions. Our findings spotlight NAD+ supplementation has potential therapeutic value in addressing POLG-associated neuronal and mitochondrial deficits. Moreover, the unique insights garnered from single-cell RNA sequencing, and enrichment analyses further emphasize the significance of mitochondrial disturbances and potential interventions for POLG-related neurodegenerative conditions. In summary, we underscore the transformative potential of NAD+ in managing neurodegenerative diseases associated with POLG mutations. It also establishes the utility of POLG mutant hMOs as a potent research model.

neuroscience↗

bioRxiv2023

Global Sensitivity Analysis of the Onset of Nasal Passage Infection by SARS-CoV-2 With Respect to Heterogeneity in Host Physiology and Host Cell-Virus Kinetic Interactions

Throughout the COVID-19 pandemic, positive nasal swab tests have revealed dramatic population heterogeneity in viral titers spanning 6 orders-of-magnitude. Our goal here is to probe potential drivers of infection outcome sensitivity arising from (i) physiological heterogeneity between hosts and (ii) host-variant heterogeneity in the detailed kinetics of cell infection and viral replication. Toward this goal, we apply global sensitivity methods (Partial Rank Correlation Coefficient analysis and Latin Hypercube Sampling) to a physiologically faithful, stochastic, spatial model of inhaled SARS-CoV-2 exposure and infection in the human respiratory tract. We focus on the nasal passage as the primary origin of respiratory infection and site of clinical testing, and we simulate the spatial and dynamic progression of shed viral load and infected cells in the immediate 48 hours post infection. We impose immune evasion, i.e., suppressed immune protection, based on the preponderance of clinical evidence that nasal infections occur rapidly post exposure, largely independent of immune status. Global sensitivity methods provide the de-correlated outcome sensitivities to each source of within-host heterogeneity, including the dynamic progression of sensitivities at 12, 24, 36, and 48 hours post infection. The results reveal a dynamic rank-ordering of the drivers of outcome sensitivity in early infection, providing insights into the dramatic population-scale outcome diversity during the COVID-19 pandemic. While we focus on SARS-CoV-2, the model and methods are applicable to any inhaled virus in the immediate 48 hours post infection.

immunology↗

bioRxiv2023

Predicting Small Molecule Ligand/RNA Binding Pocket Binding Modes Using Metadynamics

Understanding the structural dynamics of how small molecule ligand recognize its RNA binding pocket is always a crucial determinant in pharmaceutical research. Molecular dynamics (MD) simulation is often used to interpretate this process at atomic resolution. However, the insurmountable high energy barriers in the binding pathway results in the nonergodic dynamics for unbiased MD sampling. To address this limitation, we applied well-tempered metadynamics coupled with upper wall restrain in this work, therefore providing an novel modeling approach for sampling the multiple state transitions during this binding process and probing the most energy favorable binding modes through two-dimensional free energy landscape reconstructed by incorporating couple possible hydrogen binding interactions between small molecule ligand and its RNA binding pocket as collective variables (CVs). Our computational predictions of binding modes for all five cases studied are in quantitative agreement with structures solved by X-ray crystallography or NMR with RMSD less than 2.0 [A]. In addition, we presented the first molecular dynamics binding pathway and binding mechanism for the three cases of in vitro selected RNA aptamer. Our study demonstrated that metadynamics can be applied to effectively sampling state transitions of ligand binding events. By coupling with upper wall restrain, we have enabled fast free energy profile calculation and binding mode prediction for small molecule-RNA binding process, facilitating RNA-ligand binding investigation. This method therefore could be much-needed in computer-aided drug design pipelines of RNA-targeted small molecule compounds. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=115 SRC="FIGDIR/small/560960v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@8049b0org.highwire.dtl.DTLVardef@65ed27org.highwire.dtl.DTLVardef@1b4b54aorg.highwire.dtl.DTLVardef@ea2ef9_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2023

Mitochondrial Dysfunction and Neuronal Anomalies in POLG Mutant Midbrain Organoids

Human pluripotent stem cell-derived midbrain organoids offer transformative potential for elucidating brain development, disease representation, and therapeutic innovations. We introduce a novel methodology to generate midbrain-specific organoids from both embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). By employing tailored differentiation cues, notably dual-SMAD inhibition combined with FGF-8b and Sonic hedgehog agonist purmorphamine, we direct pluripotent stem cells towards a midbrain lineage. These organoids, growing beyond 2mm in diameter, house diverse neuroepithelial cell populations. Their midbrain character is affirmed by the pronounced expression of midbrain-specific markers and the absence of forebrain and hindbrain indicators. Critically, these organoids differentiate into dopaminergic neurons characteristic of the midbrain, displaying both morphological sophistication and electrophysiological vigor. Additionally, our experiments with POLG iPSC-derived midbrain organoids revealed a marked loss of dopaminergic neurons and diminished expression of genes governing mitochondrial pathways. This evidence underscores the models potential in simulating mitochondrial diseases and neurodegenerative conditions, notably Parkinsons disease. Our protocol thus emerges as a pivotal instrument for crafting functionally adept, midbrain-centric organoids, paving avenues for advanced studies in midbrain evolution, disorders like Parkinsons disease, and their interplay with mitochondrial dysfunction.

neuroscience↗

bioRxiv2023

Endolysosomal dysfunction in radial glia progenitor cells leads to defective cerebral angiogenesis and compromised Blood-Brain Barrier integrity

The neurovascular unit (NVU) is a complex structure comprising neurons, glia, and pericytes that interact with specialized endothelial cells to maintain cerebral homeostasis and blood-brain barrier (BBB) integrity. Alterations to NVU formation and function can lead to serious forms of cerebrovascular disease, including cerebral small vessel diseases (CSVDs), a range of pathological changes of cerebral capillaries within the white matter contributing to BBB dysfunction and demyelination. Despite the growing recognition of the pivotal roles played by neuro-vascular and glia-vascular interfaces in NVU formation and functioning, CSVD research has mainly focused on characterizing pericyte and EC dysfunction, leaving our understanding of the contribution of non-vascular cells of the brain parenchyma limited. Here, we use a novel zebrafish mutant to delve into the intricate interplay among NVU components and demonstrate how the compromised specification of a progenitor cell population sets off a cascade of events, ultimately leading to severe cerebrovascular abnormalities. The mutation affects Scavenger Receptor B2 (scarb2)/Lysosomal Membrane Protein 2 (limp2), a highly conserved protein residing in the membrane of late endosomes and lysosomes. We find Scarb2 to be predominantly expressed in Radial Glia Cells (RGCs), a multipotent cell giving rise to neurons and glia in both zebrafish and mammals. Through live imaging and genetic manipulations, we identify impaired Notch3 signaling in RGCs and their glial progeny as the primary consequence of Scarb2a depletion and show that this disruption causes excessive neurogenesis at the expense of glial cell differentiation. We further pinpoint compromised acidification of the endolysosomal compartment in mutant cells as the underlying cause of disrupted Notch3 processing, linking for the first time Notch3 defects in non-vascular cells of the brain parenchyma to CSVD phenotypes. Given the evolutionary conservation of SCARB2 expression and the remarkable recapitulation of CSVD phenotypes, scarb2 mutants provide a promising framework for investigating the mechanisms governing Notch3 processing in non-vascular cells and their involvement in the onset of CSVD.

developmental biology↗

bioRxiv2023

Division of labor among H3K4 Methyltransferases Defines Distinct Facets of Homeostatic Plasticity

Heterozygous mutations in any of the six H3K4 methyltransferases (KMT2s) result in monogenic neurodevelopmental disorders, indicating nonredundant yet poorly understood roles of this enzyme family in neurodevelopment. Recent evidence suggests that histone methyltransferase activity may not be central to KMT2 functions; however, the enzymatic activity is evolutionarily conserved, implicating the presence of selective pressure to maintain the catalytic activity. Here, we show that H3K4 methylation is dynamically regulated during prolonged alteration of neuronal activity. The perturbation of H3K4me by the H3.3K4M mutant blocks synaptic scaling, a form of homeostatic plasticity that buffers the impact of prolonged reductions or increases in network activity. Unexpectedly, we found that the six individual enzymes are all necessary for synaptic scaling and that the roles of KMT2 enzymes segregate into evolutionary-defined subfamilies: KMT2A and KMT2B (fly-Trx homologs) for synaptic downscaling, KMT2C and KMT2D (Trr homologs) for upscaling, and KMT2F and KMT2G (dSet homologs) for both directions. Selective blocking of KMT2A enzymatic activity by a small molecule and targeted disruption of the enzymatic domain both blocked the synaptic downscaling and interfered with the activity-dependent transcriptional program. Furthermore, our study revealed specific phases of synaptic downscaling, i.e., induction and maintenance, in which KMT2A and KMT2B play distinct roles. These results suggest that mammalian brains have co-opted intricate H3K4me installation to achieve stability of the expanding neuronal circuits.

neuroscience↗

bioRxiv2023

Hallmark molecular and pathological features of POLG disease are recapitulated in cerebral organoids

In our research, we developed a 3D brain organoid model to study POLG-related encephalopathy, a mitochondrial disease stemming from POLG gene mutations. We utilized induced pluripotent stem cells (iPSCs) derived from patients with these mutations to generate cortical organoids, which exhibited typical POLG disease features, such as altered morphology, neuronal loss, and mtDNA depletion. We also identified significant dysregulation in pathways crucial for neuronal development and function, alongside upregulated NOTCH and JAK-STAT signaling pathways. Metformin treatment ameliorated many of these abnormalities, except for the persistent affliction of inhibitory DA GLU neurons. This novel model effectively mirrors both the molecular and pathological attributes of POLG disease, providing a valuable tool for mechanistic understanding and therapeutic screening for POLG-related disorders and other conditions characterized by compromised neuronal mtDNA maintenance and complex I deficiency. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=127 SRC="FIGDIR/small/558087v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@1759de3org.highwire.dtl.DTLVardef@d0a3dborg.highwire.dtl.DTLVardef@1b58eaborg.highwire.dtl.DTLVardef@a986a8_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIWe have successfully developed cortical organoid model that represents POLG-related disease. C_LIO_LIThis model effectively replicates both histological and molecular signatures seen in the brains of patients. C_LIO_LIThe cortical organoid model displays a range of features common in POLG-related disease, including neurodegeneration, mtDNA depletion, and neuronal complex I deficiency. C_LIO_LIThe use of metformin supplementation in this model improved mitochondria protein and reduced cell death. C_LI

cell biology↗