Biology subjects

Hagstrom, S.

Publications and source records attributed to Hagstrom, S..

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↗

bioRxiv2020

Mutations in BBS2 Cause Apparent Nonsyndromic Retinitis Pigmentosa

PurposeTo identify and functionally test the causative mutations in the BBS2 gene in a family presenting with retinitis pigmentosa and infertility and to generate a bbs2-/- mutant zebrafish. MethodsA female proband and her male sibling were clinically evaluated and genetic testing with targeted next-generation sequencing was performed. Mutations were verified by Sanger sequencing. Protein localization was examined by transient expression and immunocytochemistry in cultured HEK-293T cells. Mutations in the zebrafish bbs2 gene were generated by CRISPR/Cas9 and retinal phenotypes were examined by immunohistochemistry. ResultsThe proband and her brother exhibited reduced visual fields, retinal degeneration, and bone spicule deposits, consistent with retinitis pigmentosa. The brother also reported symptoms consistent with infertility. Compound heterozygous mutations in the BBS2 gene; namely NM_031885.4 (BBS2):c.823C>T (p.R275X) and NM_031885.4 (BBS2):c.401C>G (p.P134R), were identified in the proband and her brother. Both mutations interfered with ciliary localization of Bbs2 in cell culture. Mutation of the zebrafish bbs2 gene resulted in progressive cone degeneration and rhodopsin mislocalization. ConclusionMissense mutations of BBS2 leads to non-syndromic retinitis pigmentosa, but not Bardet-Biedl Syndrome, even though Bbs2 fails to localize to cilia. In zebrafish, the complete loss of bbs2 results in cone degeneration and ciliopathy phenotypes, indicating a requirement for Bbs2 in photoreceptor survival.

genetics↗

bioRxiv2019

The urinary microbiota composition remains stable over time and under various storage conditions

BackgroundNew sensitive techniques have revealed a large population of bacteria in the human urinary tract, challenging the perception of the urine of healthy humans being sterile. While the role of this urinary microbiota is unknown, dysbiosis has been linked to disorders like urgency urinary incontinence and interstitial cystitis. When comparing studies it is crucial to account for possible confounders introduced due to methodological differences. Here we investigated whether storage condition or time of collection, had any impact on the urinary microbial composition. ResultsFor comparison of different storage conditions, urine was collected from five healthy adult female donors, and analyzed by 16S rRNA gene sequencing. Using the same methods, the daily or day-to-day variation in urinary microbiota was investigated in nineteen healthy donors, including four women, five men, five girls, and five boys. With the exception of two male adult donors, none of the tested conditions gave rise to significant differences in alpha and beta diversities between individuals. Conclusion: The composition of the urinary microbiota was found to be highly resilient to changes introduced by storage temperature and duration. In addition, we did not observe any intrapersonal daily or day-to-day variations in microbiota composition in women, girls or boys. Together our study supports flexibility in study design, when conducting urinary microbiota studies. Author summaryThe discovery of bacteria native to the urinary tract in healthy people, a location previously believed to be sterile, has prompted research into the clinical potential of these bacteria. However, methodological weaknesses can significantly influence such studies, and thus development of robust techniques for investigating these bacteria are needed. In the present study, we investigated whether differences in storage following collection, could affect the bacterial composition of urine samples. Next, we investigated if this composition exhibited daily or day-to-day variations. Firstly, we found, that the bacterial composition of urine could be maintained by storage at -80 {degrees}C, -20 {degrees}C, or refrigerated at 4 {degrees}C. Secondly, the bacterial composition of urine remained stable over time. Overall, the results of this study provide information important to study design in future investigations into the clinical implications of urinary bacteria.

microbiology↗

bioRxiv2019

Prolonged Ocular Exposure Leads to the Formation of Retinal Lesions in Mice

The observation of retinal lesions in the posterior pole of laboratory mice has been found to occur for many reasons, some of which are due to native, developmental abnormalities and those that are influenced by environmental or experimental conditions. Herein, we investigated the rate and extent of retinal lesions as a result of prolonged ocular exposure following general anesthesia. Mice were housed under standard animal care conditions and transported to the laboratory for experimental preparation induction procedures (EPIP) involving general anesthesia, mydriasis/cycloplegia, and topical anesthesia to the cornea. Following EPIP, two ocular recovery conditions (protected and unprotected) were tested within two different animal recovery chambers (open or closed). During anesthesia recovery, and extending up to 2.5 months thereafter, the anterior and posterior poles were evaluated using digital color photography, scanning laser ophthalmoscopy, and spectral-domain optical coherence tomography to document the effects of eye protection and chamber recovery type on the development of retinal lesions. In some mice, electroretinograms and histological evaluations were performed to assess functional and structural changes that accompanied the retinal lesions detected by in vivo imaging. We found that the anterior segments of mice recovered in the open chamber with unprotected eyes showed substantial acute changes. At 1-hour post-EPIP, the anterior chamber exhibited corneal thinning, severe media opacities, a reduction in anterior chamber depth, and ocular lens prolapse. These changes largely resolved upon recovery. At 3- and 14-days post-EPIP, inspection of the posterior pole by fundus imaging revealed prominent lesions in the outer retina in a significant proportion of mice recovered in the open chamber. ERG testing conducted at 1-month post-EPIP revealed compromised functional responses in the eyes of affected vs. unaffected mice. Imaging at 14-days post-EPIP revealed that the outer retina lesions in affected mice almost wholly resolve over time to nearly insignificant levels. However, data collected at 80-days post-EPIP demonstrates that some lingering effects persist long-term and appear to be confined to the retinal pigment epithelium. In comparison, mice recovered in the closed chamber with unprotected eyes experienced only mild lens opacities at 1-hr post EPIP that cleared following a full recovery from the effects of sedation. Furthermore, protected eyes of mice recovered in either the open or closed chamber were completely devoid of any anterior or posterior pole complications. In sum, prolonged ocular surface exposure to circulating ambient room air leads to significant anterior and posterior segment ocular complications. We interpret these changes to be caused by dehydration and desiccation of the corneal surface of the eye. The most abundant, semi-reversible complication observed was the development of lesions in the outer retina, which had a 90% probability of occurring after 45 minutes of exposure. The lesions largely absolved short-term but some imaging evidence suggests that they may persist months after their initial appearance. DisclosuresB.A. Bell, none; V.L. Bonilha, none; S.A. Hagstrom, none; B.Anand-Apte, none; 14 J.G. Hollyfield, none; I.S. Samuels, none. Grant InformationResearch reported in this publication was supported by the National Eye Institute of the National Institutes of Health under award numbers P30EY025585, R01EY016490, RO1EY026181, RO1EY027083, R01EY014240 and R01EY027750, US Dept. of Veterans Affairs Biomedical Laboratory Research and Development Service VA Merit Award I01BX002754, an unrestricted grant from the Research to Prevent Blindness to the Cleveland Clinic Lerner College of Medicine of Case Western Reserve University, Foundation for Fighting Blindness Research Center Grant, The Wolf Family Foundation, the Llura and Gordon Gund Foundation and the Cleveland Clinic. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the US Dept. of Veterans Affairs.

pathology↗