Biology subjects

Sturgis, J.

Publications and source records attributed to Sturgis, J..

4 recordsLinked to original sources

bioRxiv2025

Metabolic dysfunction promoted by mitochondrial DNA mutation burden drives retinal degeneration

Retinal degenerative diseases, such as age-related macular degeneration (AMD), retinitis pigmentosa, and glaucoma, have been linked to mitochondrial dysfunction. However, the impact of mitochondrial DNA (mtDNA) mutation accumulation in the context of these retinopathies has yet to be thoroughly explored. Our previous studies focused on the retinal phenotype observed in the PolgD257A mutator mice (D257A), revealing the effects of aging and mtDNA mutation accumulation in the retina. We have reported that this model exhibited significant morphological and functional deficits in the retina by 6 months of age, with notable alterations in the retinal pigment epithelium (RPE) occurring as early as 3 months, including changes in the cristae density and reduction in length of mitochondria. This study investigated how mtDNA mutations affect the metabolic interaction between the retina and RPE in young (3 months) and old (12 months) wild-type (WT) and D257A mice. We assessed cellular energy production using freshly dissected retina samples from both groups through Seahorse analysis, immunofluorescence, and Western blot experiments. The analysis of aged D257A retina punches revealed significantly reduced basal and maximal mitochondrial respiration, along with increased mitochondrial reserve capacity compared to WT. However, glycolytic flux, measured as a function of extracellular acidification rate (ECAR), did not differ between WT and D257A mice. Both D257A retina and RPE exhibited decreased expression of essential electron transport proteins involved in oxidative phosphorylation. Additionally, we observed a reduction in the expression of glucose transporter 1 (GLUT-1) and lactate transporter (MCT1) at the apical surface of the RPE. Enzymes associated with glycolysis, including hexokinase II and lactate dehydrogenase A, were significantly lower in the aged D257A retina, while hexokinase I and pyruvate kinase 2 were upregulated in the RPE. These findings indicate that the accumulation of mtDNA mutations leads to impaired metabolism in both the retina and RPE. Furthermore, it suggests that glucose from the choroidal blood supply is being utilized by the RPE rather than being transported to the neural retina. Mitochondrial dysfunction in RPE promotes a glycolytic state in these cells, leading to reduced availability of metabolites and, consequently, diminished overall retinal function. These results are essential for advancing our understanding of the mechanisms underlying retinal degeneration and provide a new perspective on the role of mtDNA mutations in these diseases.

cell biology↗

bioRxiv2025

A Membrane-Disruptive Action of VBIT-4 Challenges Its Role as a Widely Used VDAC Oligomerization Inhibitor

Voltage-dependent anion channel (VDAC) is the most abundant protein of the mitochondrial outer membrane and a key regulator of metabolite exchange and mitochondrial physiology. Its oligomerization has been proposed to control processes such as mitochondrial DNA release and membrane remodeling, yet the underlying mechanisms remain poorly defined. VBIT-4 has been widely used over the last decade as a putative inhibitor of VDAC1 oligomerization, despite limited mechanistic validation. Here, using high-speed atomic force microscopy (AFM), we directly visualized VDAC1 assemblies in lipid membranes and examined the effect of VBIT-4. Unexpectedly, VBIT-4 induced membrane defects and permeabilization at micromolar concentrations, independently of VDAC1. Quantitative AFM analysis further shows that VBIT-4 does not alter VDAC1 cluster organization. Complementary electrophysiology, microscale thermophoresis, and coarse-grained molecular dynamics demonstrate that VBIT-4 partitions into lipid bilayers, increases membrane permeability, and destabilizes membrane structure, without detectable effects on VDAC1 channel properties or assemblies. Consistent with this mechanism, VBIT-4 induces VDAC1-independent cytotoxicity in HeLa cells at concentrations above 10 {micro}M. Together, these results demonstrate that VBIT-4 does not act as a specific inhibitor of VDAC1 oligomerization but instead functions as a membrane-active compound. This work provides a revised framework for interpreting studies using VBIT-4 and highlights the importance of systematically assessing drug-membrane interactions when targeting membrane proteins.

biochemistry↗

bioRxiv2025

DJ-1 deficiency and aging: dual drivers of retinal mitochondrial dysfunction.

We have previously extensively characterized the role of DJ-1 in oxidative stress regulation in the retina and RPE during aging. However, the DJ-1 protein also plays a role in regulating mitochondrias response to oxidative stress by translocating to the mitochondria where it helps clear generated reactive oxygen species (ROS). To study the effects of aging and oxidative stress in the retina, the DJ-1 KO mouse was analyzed. Freshly dissected ex vivo retinal punches were analyzed for real-time live cell metabolism. Total DNA and protein were isolated from RPE, and retina of 3- and 15-month-old DJ-1 WT and DJ-1 KO mice. The mitochondrial DNA (mtDNA) genome was divided into four discrete regions (RI-RIV), and lesions/10kb were quantified using long-extension PCR. mtDNA content was analyzed using RT-qPCR. Protein levels of OXPHOS complexes, POLG, OGG1, SOD2, and PGC1 were measured by western blotting. Seahorse analysis detected significantly decreased basal and maximal OCR in 3- and 15-month-old DJ-1 KO compared to age-matched DJ-1 WT. In the RPE, a significant decrease in the protein levels of the NDUFB8 subunit of CI, the SDHB subunit of CII, and MTCO1 of CIV in 15-month-old DJ-1 KO mice compared to 15-month-old DJ-1 WT, while the ATP5A subunit of CV was significantly decreased in 3-month-old DJ-1 KO mice compared to 3-month-old DJ-1 WT. In the retina, significantly decreased levels of NDUFB8 subunit of CI and MTCO1 of CIV were detected in the in 3-month-old DJ-1 KO mice compared to 3-month-old DJ-1 WT. We observed a significant increase in mtDNA gene content in 15-month-old DJ-1 KO RPE and retina compared to age-matched DJ-1 WT. The PGC1 levels significantly decreased in 3- and 15-month-old RPE lysates compared to their age group DJ-1 WT. However, in the retina, there was only a decrease in DJ-1 WT with aging. The POLG levels increased when 15-month-old DJ-1 KO lysates were compared to 15-month-old DJ-1 WT. The mtDNA lesions in the RPE detected a trend of increase in 15-month-old DJ-1 WT and DJ-1 KO RPE in all regions compared to 3-month-old mice. In the retina, a significant increase in mtDNA lesions/10kb accumulation in the 15-month-old DJ-1 WT RIV was detected compared to 3-month-old DJ-1 WT. The OGG1 levels significantly increased in 3-month-old retinal lysates compared to the 3-month-old DJ-1 WT. Our findings suggest that DJ-1 is critical for mitochondrial regulation and function in RPE and retina. The observed changes reflect mitochondrial dysfunction related to absence of DJ-1.

neuroscience↗

bioRxiv2023

Modeling aging and retinal degeneration with mitochondrial DNA mutation burden.

Somatic mitochondrial DNA (mtDNA) mutation accumulation has been observed in individuals with retinal degenerative disorders. To study the effects of aging and mtDNA mutation accumulation in the retina, a Polymerase gamma (POLG) deficiency model, the POLGD257A mutator mice (PolgD257A), was used. POLG is an enzyme responsible for regulating mtDNA replication and repair. Retinas of young and older mice with this mutation were analyzed in vivo and ex vivo to provide new insights into the contribution of age-related mitochondrial dysfunction due to mtDNA damage. Optical coherence tomography (OCT) image analysis revealed a decrease in retinal and photoreceptor thickness starting at 6 months of age in mice with the POLGD257A mutation compared to wild-type (WT) mice. Electroretinography (ERG) testing showed a significant decrease in all recorded responses at 6 months of age. Sections labeled with markers of different types of retinal cells, including cones, rods, and bipolar cells, exhibited decreased labeling starting at 6 months. However, electron microscopy analysis revealed differences in retinal pigment epithelium (RPE) mitochondria morphology beginning at 3 months. Interestingly, there was no increase in oxidative stress observed in the retina or RPE of POLGD257A mice. Additionally, POLGD257A RPE exhibited an accelerated rate of autofluorescence cytoplasmic granule formation and accumulation. Mitochondrial markers displayed decreased abundance in protein lysates obtained from retina and RPE samples. These findings suggest that the accumulation of mitochondrial DNA mutations leads to impaired mitochondrial function and accelerated aging, resulting in retinal degeneration.

cell biology↗