Sullivan, G. J. | Search bioRxiv

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↗