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

Development of a cell-based assay for measuring the NAD levels in polyphenol treated cells

Glaucoma is one of the leading causes of vision loss in the world and it is characterized by the dysfunction of retinal ganglion cells (RGCs). An early pathomechanism of glaucoma is the degeneration of the axons of RGCs and finding new treatments that could prevent axon degeneration is of great interest. Increasing the concentration of coenzyme nicotinamide adenine dinucleotide (NAD) has been shown to be axon protective in a number of neurodegenerative systems. Increasing NAD could potentially be achieved by increasing the catalytic properties of the terminal enzyme involved in the cytosolic production of neuronal NAD, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2). NMNAT2 is thus an ideal therapeutic target. Polyphenol A (PA), which is a polyphenol that will not be disclosed, has been demonstrated to be NAD-boosting through positive modulation of NMNAT2. The aim was to develop a cell-based assay for screening PA and 12 novel analogues of PA for their NAD-boosting effects in brain cortex, retinal and liver cells isolated from C57BL/6J mice. The protocol involved using a bioluminescence assay for which optimization of variables such as cell concentration, substrate (nicotinamide) concentration, PA concentration and incubation time was performed. The method development resulted in a one-day protocol for testing PA and its analogues in cortical cells. PA and several of its analogues exhibited NAD-boosting effects. This protocol along with the results from the screening can be further used for the development of novel drugs that could prevent glaucoma and other axon and neurodegenerations

Development of a cell-based assay for measuring the NAD levels in polyphenol treated cells

Glaucoma is one of the leading causes of vision loss in the world and it is characterized by the dysfunction of retinal ganglion cells (RGCs). An early pathomechanism of glaucoma is the degeneration of the axons of RGCs and finding new treatments that could prevent axon degeneration is of great interest. Increasing the concentration of coenzyme nicotinamide adenine dinucleotide (NAD) has been shown to be axon protective in a number of neurodegenerative systems. Increasing NAD could potentially be achieved by increasing the catalytic properties of the terminal enzyme involved in the cytosolic production of neuronal NAD, nicotinamide mononucleotide adenylyltransferase 2 (NMNAT2). NMNAT2 is thus an ideal therapeutic target. Polyphenol A (PA), which is a polyphenol that will not be disclosed, has been demonstrated to be NAD-boosting through positive modulation of NMNAT2. The aim was to develop a cell-based assay for screening PA and 12 novel analogues of PA for their NAD-boosting effects in brain cortex, retinal and liver cells isolated from C57BL/6J mice. The protocol involved using a bioluminescence assay for which optimization of variables such as cell concentration, substrate (nicotinamide) concentration, PA concentration and incubation time was performed. The method development resulted in a one-day protocol for testing PA and its analogues in cortical cells. PA and several of its analogues exhibited NAD-boosting effects. This protocol along with the results from the screening can be further used for the development of novel drugs that could prevent glaucoma and other axon and neurodegenerations

A rheological study of hyaluronan and sodium hydroxide at different concentrations

This thesis examines how the rheological properties change depending on the composition of hyaluronan, HA and sodium hydroxide, NaOH. This was performed to see if there was any relationship between the rheological properties of a sample depending on different compositions of HA and NaOH. Moreover, the fluidity of the samples was studied by investigating . Five concentrations of HA (11, 18, 20, 25, 33 wt%) were investigated with six concentrations of NaOH (0, 1, 2, 4, 6, 8 wt%). Rheology was used to determine rheological properties of the composition and the rheometric data was obtained from three different measurements: time sweep, frequency sweep and amplitude sweep. G', G'' andwere investigated but no clear correlation was found. However, some patterns were detected for frequency sweep and amplitude sweep. The graphs generally followed the same shape and the compositions with 11% HA generally had the lowest G' and G'' values. Additionally, the majority of the samples, that could be measured, could be defined as fluids, due to  being higher than 1

A rheological study of hyaluronan and sodium hydroxide at different concentrations

This thesis examines how the rheological properties change depending on the composition of hyaluronan, HA and sodium hydroxide, NaOH. This was performed to see if there was any relationship between the rheological properties of a sample depending on different compositions of HA and NaOH. Moreover, the fluidity of the samples was studied by investigating . Five concentrations of HA (11, 18, 20, 25, 33 wt%) were investigated with six concentrations of NaOH (0, 1, 2, 4, 6, 8 wt%). Rheology was used to determine rheological properties of the composition and the rheometric data was obtained from three different measurements: time sweep, frequency sweep and amplitude sweep. G', G'' andwere investigated but no clear correlation was found. However, some patterns were detected for frequency sweep and amplitude sweep. The graphs generally followed the same shape and the compositions with 11% HA generally had the lowest G' and G'' values. Additionally, the majority of the samples, that could be measured, could be defined as fluids, due to  being higher than 1

Pyrroloquinoline quinone drives ATP synthesis in vitro and in vivo and provides retinal ganglion cell neuroprotection

Abstract Retinal ganglion cells are highly metabolically active requiring strictly regulated metabolism and functional mitochondria to keep ATP levels in physiological range. Imbalances in metabolism and mitochondrial mechanisms can be sufficient to induce a depletion of ATP, thus altering retinal ganglion cell viability and increasing cell susceptibility to death under stress. Altered metabolism and mitochondrial abnormalities have been demonstrated early in many optic neuropathies, including glaucoma, autosomal dominant optic atrophy, and Leber hereditary optic neuropathy. Pyrroloquinoline quinone (PQQ) is a quinone cofactor and is reported to have numerous effects on cellular and mitochondrial metabolism. However, the reported effects are highly context-dependent, indicating the need to study the mechanism of PQQ in specific systems. We investigated whether PQQ had a neuroprotective effect under different retinal ganglion cell stresses and assessed the effect of PQQ on metabolic and mitochondrial processes in cortical neuron and retinal ganglion cell specific contexts. We demonstrated that PQQ is neuroprotective in two models of retinal ganglion cell degeneration. We identified an increased ATP content in healthy retinal ganglion cell-related contexts both in in vitro and in vivo models. Although PQQ administration resulted in a moderate effect on mitochondrial biogenesis and content, a metabolic variation in non-diseased retinal ganglion cell-related tissues was identified after PQQ treatment. These results suggest the potential of PQQ as a novel neuroprotectant against retinal ganglion cell death

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