Prophylactic nicotinamide treatment protects from rotenone-induced neurodegeneration by increasing mitochondrial content and volume.

Leber's hereditary optic neuropathy (LHON) is driven by mtDNA mutations affecting Complex I presenting as progressive retinal ganglion cell dysfunction usually in the absence of extra-ophthalmic symptoms. There are no long-term neuroprotective agents for LHON. Oral nicotinamide provides a robust neuroprotective effect against mitochondrial and metabolic dysfunction in other retinal injuries. We explored the potential for nicotinamide to protect mitochondria in LHON by modelling the disease in mice through intravitreal injection of the Complex I inhibitor rotenone. Using MitoV mice expressing a mitochondrial-tagged YFP in retinal ganglion cells we assessed mitochondrial morphology through super-resolution imaging and digital reconstruction. Rotenone induced Complex I inhibition resulted in retinal ganglion cell wide mitochondrial loss and fragmentation. This was prevented by oral nicotinamide treatment. Mitochondrial ultrastructure was quantified by transition electron microscopy, demonstrating a loss of cristae density following rotenone injection, which was also prevented by nicotinamide treatment. These results demonstrate that nicotinamide protects mitochondria during Complex I dysfunction. Nicotinamide has the potential to be a useful treatment strategy for LHON to limit retinal ganglion cell degeneration.

Nicotinamide provides neuroprotection in glaucoma by protecting against mitochondrial and metabolic dysfunction.

Nicotinamide adenine dinucleotide (NAD) is a REDOX cofactor and metabolite essential for neuronal survival. Glaucoma is a common neurodegenerative disease in which neuronal levels of NAD decline. We assess the effects of nicotinamide (a precursor to NAD) on retinal ganglion cells (the affected neuron in glaucoma) in normal physiological conditions and across a range of glaucoma relevant insults including mitochondrial stress and axon degenerative insults. We demonstrate retinal ganglion cell somal, axonal, and dendritic neuroprotection by nicotinamide in rodent models which represent isolated ocular hypertensive, axon degenerative, and mitochondrial degenerative insults. We performed metabolomics enriched for small molecular weight metabolites for the retina, optic nerve, and superior colliculus which demonstrates that ocular hypertension induces widespread metabolic disruption, including consistent changes to α-ketoglutaric acid, creatine/creatinine, homocysteine, and glycerophosphocholine. This metabolic disruption is prevented by nicotinamide. Nicotinamide provides further neuroprotective effects by increasing oxidative phosphorylation, buffering and preventing metabolic stress, and increasing mitochondrial size and motility whilst simultaneously dampening action potential firing frequency. These data support continued determination of the utility of long-term nicotinamide treatment as a neuroprotective therapy for human glaucoma.

Retinal Ganglion Cell Degeneration in a Rat Magnetic Bead Model of Ocular Hypertensive Glaucoma.

Purpose: Glaucoma remains a leading cause of irreversible blindness worldwide. Animal glaucoma models replicate high intraocular pressure, a risk factor for glaucoma, to induce retinal ganglion cell (RGC) degeneration. We describe an inducible, magnetic bead model in the Brown Norway rat in which we are able to determine degeneration across multiple RGC compartments at a time point that is appropriate for investigating neurodegenerative events and potential treatment effects. Methods: We induced ocular hypertension through injection of magnetic microspheres into the anterior chamber of Brown Norway rats; un-operated (naïve) rats served as controls. Intraocular pressure was recorded, and eye diameter measurements were taken before surgery and at the terminal end points. We assessed RGC degeneration and vascular changes through immunofluorescence, and axon transport to terminal brain thalami through intravitreal injection of fluorophore-conjugated cholera toxin subunit ß. Results: We observed clinically relevant features of disease accompanying RGC cell somal, axonal, and dendritic loss. RGC axonal dysfunction persisted along the trajectory of the cell into the terminal brain thalami, with clear disruption at the optic nerve head. We also observed vascular compromise consistent with human disease, as well as an expansion of global eye size with ocular hypertension. Conclusions: The magnetic bead model in the Brown Norway rat recapitulates many clinically relevant disease features of human glaucoma, including degeneration across multiple RGC compartments. Eye expansion is likely a result of rodent scleral elasticity, and we caution that this should be considered when assessing retinal density measurements. Translational Relevance: This model offers a disease-relevant platform that will allow for assessment of glaucoma-relevant therapeutics.

When Is a Control Not a Control? Reactive Microglia Occur Throughout the Control Contralateral Pathway of Retinal Ganglion Cell Projections in Experimental Glaucoma.

Purpose: Animal models show retinal ganglion cell (RGC) injuries that replicate features of glaucoma and the contralateral eye is commonly used as an internal control. There is significant crossover of RGC axons from the ipsilateral to the contralateral side at the level of the optic chiasm, which may confound findings when damage is restricted to one eye. The effect of unilateral glaucoma on neuroinflammatory damage to the contralateral pathway of RGC projections has largely been unexplored. Methods: Ocular hypertensive glaucoma was induced unilaterally or bilaterally in the rat and RGC neurodegenerative events were assessed. Neuroinflammation was quantified in the retina, optic nerve head, optic nerve, lateral geniculate nucleus, and superior colliculus by high-resolution imaging, and in the retina by flow cytometry and protein arrays. Results: After ocular hypertensive stress, peripheral monocytes enter the retina and microglia become reactive. This effect is more marked in animals with bilateral ocular hypertensive glaucoma. In rats where glaucoma was induced unilaterally, there was significant microglia activation in the contralateral (control) eye. Microglial activation extended into the optic nerve and terminal visual thalami, where it was similar across hemispheres in unilateral ocular hypertension. Conclusions: These data suggest that caution is warranted when using the contralateral eye as a control and in comparing visual thalami in unilateral models of glaucoma. Translational Relevance: The use of a contralateral eye as a control may confound the discovery of human-relevant mechanism and treatments in animal models. We also identify neuroinflammatory protein responses that warrant further investigation as potential disease-modifiable targets.