Biology subjects

Sullivan, G. J.

Publications and source records attributed to Sullivan, G. J..

11 recordsLinked to original sources

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

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

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↗

bioRxiv2023

A method to correct for local alterations in DNA copy number that bias functional genomics assays applied to antibiotic-treated bacteria

Functional genomics techniques, such as transposon insertion sequencing and RNA sequencing, are key to studying relative differences in mutant fitness or gene expression under selective conditions. However, certain stress conditions, mutations, or antibiotics can directly interfere with DNA synthesis, resulting in systematic changes in local DNA copy number along the chromosome. This can lead to artefacts in sequencing-based functional genomics data when comparing antibiotic treatment to an unstressed control, with relative differences in gene-wise read counts being the result of alterations in chromosomal replication dynamics rather than selection or direct gene regulation. We term this artefact chromosomal location bias and implement a principled statistical approach to correct for it by calculating local normalization factors along the chromosome. These normalization factors are then directly incorporated in statistical analyses using standard RNA-sequencing analysis methods without modifying the read counts themselves, preserving important information about the mean-variance relationship in the data. We illustrate the utility of this approach by generating and analysing a ciprofloxacin-treated transposon insertion sequencing dataset in Escherichia coli as a case study. We show that ciprofloxacin treatment generates chromosomal location bias in the resulting data, and we further demonstrate that failing to correct for this bias leads to false predictions of mutant drug sensitivity as measured by minimum inhibitory concentrations. We have developed an R package and user-friendly graphical Shiny application, ChromoCorrect, that detects and corrects for chromosomal bias in read count data, enabling the application of functional genomics technologies to the study of antibiotic stress. IMPORTANCEAltered gene dosage due to changes in DNA replication has been observed under a variety of stresses with a variety of experimental techniques. However, the implications of changes in gene dosage for sequencing-based functional genomics assays are rarely considered. We present a statistically principled approach to correcting for the effect of changes in gene dosage, enabling testing for differences in the fitness effects or regulation of individual genes in the presence of confounding differences in DNA copy number. We show that failing to correct for these effects can lead to incorrect predictions of resistance phenotype when applying functional genomics assays to investigate antibiotic stress, and we provide a user-friendly application to detect and correct for changes in DNA copy number.

genomics↗

bioRxiv2023

The NAD+ Precursor Nicotinamide Riboside Rescues Mitochondrial Defects and Neuronal Loss in iPSC derived Cortical Organoid of Alpers' Disease

Alpers syndrome is an early-onset neurodegenerative disorder usually caused by biallelic pathogenic variants in the gene encoding the catalytic subunit of polymerase-gamma (POLG), which is essential for mitochondrial DNA (mtDNA) replication. The disease is progressive, incurable, and inevitably it leads to death from drug-resistant status epilepticus. The neurological features of Alpers syndrome are intractable epilepsy and developmental regression, with no effective treatment; the underlying mechanisms are still elusive, partially due to lack of good experimental models. Here, we generated the patient-derived induced pluripotent stem cells from one Alpers patient carrying the compound heterozygous mutations of A467T (c.1399G>A) and P589L (c.1766C>T), and further differentiated them into cortical organoids and neural stem cells (NSCs) for mechanistic studies of neural dysfunction in Alpers syndrome. Patient cortical organoids exhibited a phenotype that faithfully replicated the molecular changes found in patient postmortem brain tissue, as evidenced by cortical neuronal loss and depletion of mtDNA and complex I (CI). Patient NSCs showed mitochondrial dysfunction leading to ROS overproduction and downregulation of the NADH pathway. More importantly, the NAD+ precursor nicotinamide riboside (NR) significantly ameliorated mitochondrial defects in patient brain organoids. Our findings demonstrate that the iPSC model and brain organoids are good in vitro models of Alpers disease; this first-in-its-kind stem cell platform for Alpers syndrome enables therapeutic exploration and has identified NR as a viable drug candidate for Alpers disease and, potentially, other mitochondrial diseases with similar causes.

neuroscience↗

bioRxiv2022

Loss-of-function mutation in human Oxidation Resistance gene 1 disrupts the spatial-temporal regulation of histone arginine methylation in early brain development

We report a loss-of-function mutation in the TLDc domain of human Oxidation Resistance 1 (OXR1) gene, resulting in early-onset epilepsy, developmental delay, cognitive disabilities, and cerebellar atrophy. Patient lymphoblasts show impaired cell survival, proliferation, and hypersensitivity to oxidative stress. These phenotypes are rescued by TLDc domain replacement. We generated patient derived induced pluripotent stem cells (iPSCs) revealing impaired neural differentiation along with dysregulation of genes essential for neurodevelopment. We identified that OXR1 influences histone arginine methylation by activating protein arginine methyltransferases (PRMTs), suggesting OXR1 dependent mechanisms regulating gene expression during neurodevelopment. We modeled the function of OXR1 in early human brain development using patient derived brain organoids revealing that OXR1 contributes to the spatial-temporal regulation of histone arginine methylation in specific brain regions. Our work provides new insights into pathological features and molecular underpinnings associated with OXR1 deficiency, highlighting the therapeutic potential of OXR1 in numerous neurodegenerative and neurodevelopmental disorders.

pathology↗

bioRxiv2022

No evidence for detectable direct effects of magnetic field on cellular autofluorescence

Dramatically increased levels of electromagnetic radiation in the environment have raised concerns over the potential health hazards of electromagnetic fields. Various biological effects of magnetic fields have been proposed. Despite decades of intensive research, the molecular mechanisms procuring cellular responses remain largely unknown. The current literature is conflicting with regards to evidence that magnetic fields affect functionality directly at cellular level. Therefore, a search for potential direct cellular effects of magnetic fields represents a cornerstone that may propose an explanation for potential health hazards associated with magnetic fields. Recently, it was postulated that autofluorescence of HeLa cells is magnetic field sensitive, relying on single-cell imaging kinetic measurements. Here, we explore the utility of this approach by undertaking a screen for magnetic sensitivity of cellular autofluorescence in statistically relevant numbers (90-107) of HeLa cells. We did not observe any changes in cellular autofluorescence decay, when a modulated magnetic field was applied. We present a number of arguments indicating weak points in the analysis of magnetic field effects based on the imaging of cellular autofluorescence decay. Our work indicates that new methods are required to elucidate the effects of magnetic fields at the cellular level.

cell biology↗

bioRxiv2022

Species identification of early colonial bone artefacts excavated from Pyrmont, Australia, by mass spectrometric identification of collagen peptides

Zooarchaeology by Mass Spectrometry (ZooMS) is a rapidly developing and increasingly utilised peptide mass fingerprinting (PMF) technique that analyses Collagen 1A1 and 1A2 marker peptides for the genus- or species-level identification of fragmentary bones in the archaeological record. Traditionally, this analysis is performed using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-ToF-MS) to identify characteristic m/z values of known marker peptides. Here we present data on the application of a modified ZooMS approach, using nanoflow liquid chromatography - tandem mass spectrometry proteomics, to the analysis of a collection of six early colonial Australian (early to mid-19th Century CE) worked bone artefacts, believed to be mostly knife handles, excavated from a site in Pyrmont, Sydney, Australia in 2017. We were successfully able to identify characteristic marker peptides for bovine COL1A1 and COL1A2 in all six bone artefacts.

biochemistry↗

bioRxiv2020

Stem cell derived astrocytes with POLG mutations and mitochondrial dysfunction including abnormal NAD+ metabolism is toxic for neurons

The inability to reliably replicate mitochondrial DNA (mtDNA) by mitochondrial DNA polymerase gamma (POLG) leads to a subset of common mitochondrial diseases associated with neuronal death and depletion of neuronal mtDNA. Defining disease mechanisms remains difficult due to the limited access to human tissue. Astrocytes are highly abundant in the brain, playing a crucial role in the support and modulation of neuronal function. Astrocytes also respond to insults affecting the brain. Following damage to the center neural system, which can be hypoxia, inflammation or neurodegeneration, astrocytes become activated and lose their supportive role and gain toxic functions that induce rapid death of neurons and oligodendrocytes. The role of astrocyte reactivation and the consequences this has for neuronal homeostasis in mitochondrial diseases has not been explored. Here, using patient cells carrying POLG mutations, we generated iPSCs and then differentiated into astrocytes. We demonstrated that POLG-astrocytes exhibited both mitochondrial dysfunctions, including loss of mitochondrial membrane potential, energy failure, complex I and IV defects, disturbed NAD+/NADH metabolism, and mtDNA depletion. Further, POLG derived astrocytes presented an A1-like reactive phenotype with increased proliferation, invasion, upregulation of pathways involved in response to stimulus, immune system process, cell proliferation and cell killing. Under direct and indirect co-culture with neurons, POLG-astrocytes exhibited a toxic effect leading to the death of neurons. Our findings demonstrate that mitochondrial dysfunction caused by POLG mutations leads not only to intrinsic defects in energy metabolism affecting both neurons and astrocytes, but also to neurotoxic damage driven by astrocytes. Our studies provide a robust astroglia-neuronal interaction model for future investigation of mitochondrial involvement in neurogenesis and neurodegenerative diseases. Highlights{blacksquare} Patient-specific astrocytes harbouring a POLG mutation showed lower mitochondrial membrane potential and mtDNA depletion. {blacksquare}POLG-astrocytes generated elevated L-lactate as the end glycolytic product. {blacksquare}Patient-specific astrocytes with POLG mutations exhibited mitochondrial respiratory chain disruption accompanied with abnormal UCP2/SirT1/SirT3 mediated NAD+ metabolism. {blacksquare}Suppressed complex I and IV-driven respiration contributed to the pathological mechanisms in POLG-related disease. {blacksquare}POLG-astrocytes exhibit A1-reactive phenotype and neurotoxic potential. Graphic abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=166 SRC="FIGDIR/small/423652v1_ufig1.gif" ALT="Figure 1"> View larger version (61K): org.highwire.dtl.DTLVardef@18995fdorg.highwire.dtl.DTLVardef@1d615d4org.highwire.dtl.DTLVardef@334aa8org.highwire.dtl.DTLVardef@16a84ba_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

bioRxiv2020

Scalable production of tissue-like vascularised liver organoids from human PSCs.

A lack of physiological parity between 2D cell culture and in vivo, has paved the way towards more organotypic models. Organoids exist for a number of tissues, including the liver. However, current approaches to generate hepatic organoids suffer drawbacks, including a reliance on extracellular matrices (ECM), the requirement to pattern in 2D culture, costly growth factors and a lack of cellular diversity, structure and organisation. Current hepatic organoid models are generally simplistic, composed of hepatocytes or cholangiocytes, which renders them less physiologically relevant when compared to native tissue. Here we aim to address these drawbacks. To address this, we have developed an approach that does not require 2D patterning, is ECM independent combined with small molecules to mimic embryonic liver development that produces massive quantities of liver like organoids. Using single-cell RNA sequencing and immunofluorescence we demonstrate a liver-like cellular repertoire, a higher order cellular complexity, presenting with vascular luminal structures, innervation and a population of resident macrophage - the Kupffer cells. The organoids exhibit key liver functions including drug metabolism, serum protein production, coagulation factor production, bilirubin uptake and urea synthesis. The organoids can be transplanted and maintained in mice producing human albumin long term. The organoids exhibit a complex cellular repertoire reflective of the organ, have de novo vascularization and innervation, enhanced function and maturity. This is a pre-requisite for a myriad of applications from cellular therapy, tissue engineering, drug toxicity assessment, disease modeling, to basic developmental biology.

cell biology↗

bioRxiv2020

Electromembrane extraction and mass spectrometry for liver organoid drug metabolism studies

Liver organoids are emerging tools for precision drug development and toxicity screening. We demonstrate that electromembrane extraction (EME) based on electrophoresis across an oil membrane is suited for segregating selected organoid-derived drug metabolites prior to mass spectrometry (MS)-based measurements. EME, allowed drugs and drug metabolites to be separated from cell medium components (albumin, etc.) that could interfere with subsequent measurements. Multi-well EME (Parallel-EME) holding 100 L solutions allowed for simple and repeatable monitoring of heroin phase I metabolism kinetics. Organoid Parallel-EME extracts were compatible with ultrahigh-performance liquid chromatography (UHPLC) used to separate the analytes prior to detection. Taken together, liver organoids are well-matched with EME followed by MS-based measurements.

biochemistry↗