Introduction:

Astrocytes play key roles in maintaining homeostasis within the central nervous system, with metabolism and cellular bioenergetics emerging as key mediators of this role. Moreover, recent evidence suggests that perturbation of astrocyte metabolism is a feature of many neurodegenerative conditions. LHON is an inherited neurodegenerative condition which typically manifests as sudden onset bilateral loss of vision and retinal ganglion cells. LHON is driven by deficiencies in respiratory Complex I, with consequent neurodegeneration explained by insufficient adenosine triphosphate (ATP) and aberrant reactive oxygen species (ROS) production. Curiously, spontaneous visual recovery has been noted, for reasons that remain unclear. Moreover, the contribution of astroglia to LHON pathology remains poorly understood though post-mortem evidence indicates a degree of gliosis in affected retinae, indicating astroglial involvement. Given the growing body of evidence supporting a role for aberrant mitochondrial respiration as a key contributor to astrocyte reactivity in various conditions, here we tested the hypothesis that LHON-affected astrocytes will exhibit morphological and functional changes indicative of astrocyte reactivity.

Aims:

Our aims were to determine whether LHON-affected astrocytes demonstrated bioenergetic or morphological shifts indicative of astrocyte reactivity, and to interrogate whether these changes were related to LHON phenotype severity and visual recovery status.

Methods:

Induced pluripotent stem cells derived from either a healthy control donor (WT) or LHON-affected patients ± visual recovery (recovery, R; no recovery, NR) were differentiated into astrocytes. Immunocytochemistry was used to confirm astrocyte identity. Mean fluorescence intensity (MFI) was measured and corrected for cell area using an automated pipeline. Live extracellular metabolic flux analyses were performed to interrogate changes to astrocyte respiration.

Results:

≥99% of cells were immunopositive for the astrocyte markers glial fibrillary acidic protein (GFAP) and S100B, indicating astrocyte fate specification across all genotypes. GFAP MFI was significantly reduced in LHON-NR astrocytes relative to WT, whereas LHON-R did not differ significantly from either WT or LHON-NR. S100B MFI was significantly reduced in both LHON phenotypes relative to WT with no significant difference between LHON phenotypes. Bioenergetic analyses indicated significantly reduced mitochondrial respiration in LHON-NR astrocytes and reduced coupling of oxygen consumption to ATP production relative to both WT and LHON-R astrocytes. Oxygen-ATP coupling was also significantly reduced in LHON-R astrocytes relative to WT. Both LHON phenotypes exhibited a compensatory increase in extracellular acidification, a proxy of glycolysis, relative to WT.  

Conclusions:

Preliminary data indicate significant perturbation of mitochondrial respiration in LHON-affected astrocytes, with concomitant upregulation of glycolysis. These data are indicative of a reactive astrocyte phenotype and suggest a pro-inflammatory response. LHON-NR astrocytes exhibited greater deviation from WT astrocytes than LHON-R. How this contributes to LHON severity or recovery capacity remains unclear. The reduced GFAP and S100B MFIs may be explained by secretion of these factors or metabolic impairment impinging protein synthesis. Future work will focus on elucidating the metabolic phenotype of LHON-affected astrocytes, assessing cell motility, and characterising cytokine and ROS release profiles. Coculture assays will be used to explore how LHON-affected astrocytes affect neuronal health. Differences between LHON phenotypes will form a key part of our analyses.