Peripheral blood mononuclear cell respiratory function is associated with progressive glaucomatous vision loss

Glaucoma, affecting approximately 80 million people by the end of this decade, is a progressive optic neuropathy causing irreversible blindness. While high intraocular pressure (IOP) is a major risk factor and the only modifiable one, many patients experience vision loss despite IOP-lowering treatment (normal-tension glaucoma, NTG). This highlights the need to identify additional risk factors and biomarkers for disease progression. Mitochondrial dysfunction and altered bioenergetics have been implicated in glaucoma, and nicotinamide adenine dinucleotide (NAD), crucial for mitochondrial function and axonal health, shows decreased levels in glaucomatous retinas. This study aimed to determine the association between PBMC mitochondrial respiratory function (assessed by OCR), NAD levels, and the rate of visual field (VF) progression in glaucoma patients, using a treated glaucoma cohort and a separate untreated glaucoma cohort as a reference.

Several studies have linked altered mitochondrial bioenergetics to glaucoma, both its presence and resistance to treatment. Decreased levels of nicotinamide (NAM), a NAD precursor, have been observed in glaucoma patients. Population studies have also shown an association between lower niacin intake and NTG, and higher niacin intake with a lower glaucoma risk. Preclinical glaucoma models show that boosting cellular NAD+ levels might improve cellular energetics and neuronal stress responses, with clinical trials showing improvement in RGC function with high-dose NAM supplementation. The accessibility of PBMCs makes them a suitable model system for assessing systemic mitochondrial function.

This study comprised three parts: (1) Association of PBMC mitochondrial function with glaucoma diagnosis (comparing NTG, high-tension glaucoma (HTG), and controls); (2) Association of PBMC mitochondrial OCR and the rate of VF progression; and (3) Association of PBMC NAD levels with glaucoma diagnosis and OCR. PBMCs were isolated from venous blood samples. Mitochondrial respiration (OCR) was measured using the XFe24 Analyzer, assessing basal, maximal, ATP-linked OCR, and reserve capacity. Total cellular NAD levels were quantified using a luciferase assay. VF progression rate was analyzed using linear mixed models, accounting for random slopes and intercepts. A separate untreated glaucoma cohort (UKGTS placebo arm) served as a reference to assess the association between IOP and VF progression without the confounding effect of IOP-lowering treatment. Statistical analyses included ANOVA, chi-squared tests, univariable and multivariable linear regression models (using LASSO regression for variable selection), and linear mixed models for VF progression analysis. Lymphocyte and monocyte populations were assessed by flow cytometry, and gene expression levels were measured in a subset of samples using digital droplet PCR.

Patients with glaucoma showed significantly reduced mitochondrial respiration (OCR) compared to controls, with NTG patients exhibiting the lowest OCR. Multivariable analysis revealed that NTG was associated with a significantly lower basal OCR than controls. There was a strong association between lower PBMC basal OCR and faster VF progression rates (P<0.001), explaining 13% of the variance in progression rate. Older age and higher mean IOP were also associated with faster progression. In the untreated UKGTS cohort, IOP explained 16% of VF progression variance. PBMC total NAD levels were significantly lower in POAG patients compared to controls (P<0.001), and were strongly associated with basal OCR (P<0.001). The association between OCR and VF progression did not significantly change across the IOP spectrum. One standard deviation difference in basal OCR was equivalent to a 7.6 mmHg IOP difference or a 21-year age difference in terms of glaucoma progression risk.

This study demonstrates a strong association between lower PBMC mitochondrial respiratory function (OCR) and faster VF progression in glaucoma patients, even after controlling for IOP. This association was similar in both NTG and HTG cohorts, suggesting a potentially important factor across the IOP spectrum. Lower NAD levels, strongly associated with OCR, further support the role of mitochondrial dysfunction in glaucoma progression. These findings suggest that non-IOP factors, like genetic predisposition or nutritional influences, might contribute to mitochondrial dysfunction and VF loss. The observed association between lower OCR and faster progression rate, even in treated patients, adds a new dimension to the understanding of glaucoma pathogenesis and highlights the need to consider factors beyond IOP.

This study provides strong evidence for an association between reduced PBMC mitochondrial respiratory function (OCR) and NAD levels and faster visual field progression in glaucoma. These findings suggest that OCR and NAD could serve as important biomarkers for identifying patients at high risk of vision loss, informing personalized treatment strategies. Future research, including the prospective NAMinG and TGNT trials, is needed to confirm causality, investigate the underlying mechanisms, and explore the potential of mitochondria-targeted therapies.

This study's observational design limits causal inferences. The OCR and NAD measurements were made after the VF observation period, potentially introducing some temporal bias. The convenience sample might not fully represent the entire glaucoma population, and some variables, like physical activity, could not be comprehensively assessed. The assays used have moderate repeatability and might not be ideal for clinical applications. The COVID-19 pandemic impacted recruitment, resulting in a smaller sample size than originally planned.