Biology subjects

Kristiansen, C. K.

Publications and source records attributed to Kristiansen, C. K..

4 recordsLinked to original sources

bioRxiv2025

Metformin boosts mitochondria and neurogenesis via AMPK/mTOR/SIRT3 in POLG mutant organoids

BackgroundMutations in the POLG gene, encoding the catalytic subunit of mitochondrial DNA polymerase gamma, are the most common cause of mitochondrial diseases affecting the central nervous system. These mutations frequently result in neurodevelopmental disorders, yet the cellular and molecular mechanisms underlying POLG related encephalopathies remain poorly understood. In particular, how POLG mutations affect mitochondrial function, neural progenitor behavior, and early neurogenesis in the developing human brain has not been fully elucidated. MethodsTo investigate the impact of POLG mutations on human neurodevelopment, we generated 3D cortical brain organoids from induced pluripotent stem cells (iPSCs) derived from a patient carrying compound heterozygous POLG mutations (A467T/W748S). Organoid development was monitored using immunohistochemistry, transmission electron microscopy, and live-cell mitochondrial assays. Single-cell RNA sequencing (scRNA-seq) was performed to profile cellular diversity and transcriptional changes. Organoids were treated with metformin, a known mitochondrial modulator, and mitochondrial function was assessed by measuring membrane potential (TMRE), ATP production, and mtDNA copy number. Western blotting and immunofluorescence were used to investigate AMPK-SIRT3-mTOR signaling and markers of mitochondrial dynamics and mitophagy. Statistical analyses were performed using unpaired t-tests or ANOVA, with p-values < 0.05 considered significant. ResultsPOLG mutant cortical organoids exhibited impaired neural differentiation, with expansion of stress-associated neural progenitors and reduced neuronal populations. scRNA seq analysis revealed transcriptional signatures of oxidative stress, mitochondrial dysfunction, and suppressed neurogenesis, particularly in a specific neural progenitor subpopulation. Metformin treatment significantly improved mitochondrial membrane potential, ATP output, and mtDNA copy number in POLG organoids. It also promoted neuronal differentiation and reduced reactive progenitor states. Mechanistically, metformin activated AMPK and SIRT3, inhibited mTOR, enhanced expression of mitochondrial fusion and biogenesis markers (OPA1, PGC-1), and increased autophagic and mitophagic activity (LC3B, BNIP3). ConclusionsOur study demonstrates that POLG mutations disrupt early cortical development by impairing mitochondrial function and skewing progenitor fate. Metformin mitigates these effects by restoring mitochondrial homeostasis and promoting neurogenesis through the AMPK/SIRT3/mTOR axis. These findings offer mechanistic insights into POLG-related encephalopathy and support metformin as a candidate for therapeutic intervention in mitochondrial neurodevelopmental disorders.

neuroscience↗

bioRxiv2023

POLG genotype influences degree of mitochondrial dysfunction in iPSC derived neural progenitors, but not the parent iPSC or derived glia

Diseases caused by POLG mutations are the most common form of mitochondrial disease and associated with phenotypes of varying severity. Clinical studies have shown that patients with compound heterozygous POLG mutations have a lower survival rate than patients with homozygous mutations, but the molecular mechanisms behind this remain unexplored. Using an induced pluripotent stem cell (iPSC) model, we investigate differences between homozygous and compound heterozygous genotypes in different cell types, including patient-specific fibroblasts, iPSCs, and iPSC-derived neural stem cells (NSCs) and astrocytes. We found that compound heterozygous lines exhibited greater impairment of mitochondrial function in NSCs than homozygous NSCs, but not in fibroblasts, iPSCs, or astrocytes. Compared with homozygous NSCs, compound heterozygous NSCs exhibited more severe functional defects, including reduced ATP production, loss of mitochondrial DNA (mtDNA) copy number and complex I expression, disturbance of NAD+ metabolism, and higher ROS levels, which further led to cellular senescence and activation of mitophagy. RNA sequencing analysis revealed greater downregulation of mitochondrial and metabolic pathways, including the citric acid cycle and oxidative phosphorylation, in compound heterozygous NSCs. Our iPSC-based disease model can be widely used to understand the genotype-phenotype relationship of affected brain cells in mitochondrial diseases, and further drug discovery applications. Graphic Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=95 SRC="FIGDIR/small/526021v1_ufig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@ce1761org.highwire.dtl.DTLVardef@610ed3org.highwire.dtl.DTLVardef@1460b0org.highwire.dtl.DTLVardef@1d7fd53_HPS_FORMAT_FIGEXP M_FIG C_FIG

neuroscience↗

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↗

bioRxiv2019

Neuropathy-related mutations alter the membrane binding properties of the human myelin protein P0 cytoplasmic tail

Schwann cells myelinate selected axons in the peripheral nervous system (PNS) and contribute to fast saltatory conduction via the formation of compact myelin, in which water is excluded from between tightly adhered lipid bilayers. Peripheral neuropathies, such as Charcot-Marie-Tooth disease (CMT) and Dejerine-Sottas syndrome (DSS), are incurable demyelinating conditions that result in pain, decrease in muscle mass, and functional impairment. Many Schwann cell proteins, which are directly involved in the stability of compact myelin or its development, are subject to mutations linked to these neuropathies. The most abundant PNS myelin protein is protein zero (P0); point mutations in this transmembrane protein cause CMT subtype 1B and DSS. P0 tethers apposing lipid bilayers together through its extracellular immunoglobulin-like domain. Additionally, P0 contains a cytoplasmic tail (P0ct), which is membrane-associated and contributes to the physical properties of the lipid membrane. Six CMT- and DSS-associated missense mutations have been reported in P0ct. We generated recombinant disease mutant variants of P0ct and characterized them using biophysical methods. Compared to wild-type P0ct, some mutants have negligible differences in function and folding, while others highlight functionally important amino acids within P0ct. For example, the D224Y variant of P0ct induced tight membrane multilayer stacking. Our results show a putative molecular basis for the hypermyelinating phenotype observed in patients with this particular mutation and provide overall information on the effects of disease-linked mutations in a flexible, membrane-binding protein segment.

biophysics↗