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Final Report Summary - REGUNMNAT1 (NAD synthesizing enzyme NMNAT1 as a fine regulator of the survival of neuron)

The high incidence of neurodegenerative diseases due to the increases in life expectancy of modern societies underpins a need to understand neuronal degeneration in order to prevent it. Adult neurons do not divide and are lost during normal ageing; therefore supporting the physiological function of the neuron could delay and attenuate neuronal dysfunction due to ageing or disease. Neurodegeneration involves many factors but one of them is of particular interest: neurons are especially energetic cells and it is increasingly evident that neuronal degeneration is an active process that may involve energetic failure. The energy motors of the cell are the mitochondria, organelles with the main function of producing energy. When mitochondria fail to function upon ageing, a cellular mechanism, mitophagy, is engaged to clear the dysfunctional organelles while new ones are generated. Mitophagy is a type of autophagy, a more general process that can be activated in stress conditions or nutrient starvation, in order to ensure a healthy functional cell. Nicotinamide adenine dinucleotide (NAD) is a central molecular component of cellular energy production and also a substrate for the enzyme SIRT1 (NAD dependent deacetylase), which is known to be a longevity factor and a neuroprotective agent, including by inducing autophagy and stimulating the production of new mitochondria. The enzymes nicotinamide mononucleotide adenylyltransferase (NMNATs) perform the last and obligate step for NAD biosynthesis in mammals, converting nicotinamide mononucleotide (NMN) to NAD. Three isoforms exist with distinct subcellular localization, NMNAT1 in the nucleus, NMNAT2 in the cytoplasm and NMNAT3 in the mitochondria. Neurons are unique cells composed of a cellular body containing the nucleus and the axon, which is responsible for information transfer to other axons determining the brain connectivity and function. This project aimed to investigate the role of NMNAT1 as a regulator of the survival of neurons via modulating NMN and NAD levels, and in maintaining the energetic reservoir of these cells by interacting with the autophagic process as a powerful mechanism to prevent neurodegeneration. We also wanted to explore if the overexpression of NMNAT1 could protect in some form of neurodegeneration which also presents energetic failure. We also extended the interest in looking for the effect of NMNAT2 in the axonal compartment again by the regulation of NMN and NAD levels, because NMNAT2 has been defined as the axonal survival factor.

*Objectives and main scientific results*
We wanted to investigate if NMNAT1 downregulation affected SIRT1 activity via modulating the level of NAD/NMN in the cell. SIRT1 co-localizes with NMNAT1 in the nucleus and lower levels of both SIRT1 and NMNAT1 are related to retinal defects (eye vision defects). Interestingly, nicotimanide (Nm), a NAD precursor, is a physiological strong inhibitor of SIRT1; in the same way NMN can accumulate upon NMNAT1 depletion and inhibit SIRT1. The deletion of the nmnat1 gene in cortical cells (of the mouse brain) was not achieved in an efficient manner. It would be interesting to be able to deplete NMNAT1 to study the changes in the genetic and metabolic profile of the cells regulated by SIRT1. No difference in NAD level of the brain and the eye, due to different expression level of NMNAT1 in transgenic mice, was found. We think we should in the future look more specifically into the nuclear compartment, where there could be an accumulation of NMN following the downregulation of NMNAT1, which inhibits SIRT1, although less efficiently than Nm, as we demonstrated in some experiments (Fig.1). Main result - NMNAT1 modulation regulates SIRT1 activity in stimulating the biogenesis of mitochondria and in exerting neuroprotective effects by inducing autophagy.

The importance of autophagy in preventing neuron degeneration and its connection with NAD metabolism are important evidences suggesting that NMNATs could directly regulate the autophagic process. In our experimental conditions, the overexpression of NMNATs in a neuronal cell line was not sufficient to protect from the drugs treatment (rotenone and CCCP) inducing mitochondrial dysfunction, which eventually led the cells to die. But, at a basal level, the overexpression of the isoform NMNAT2 (and to some degree NMNAT3) lead to the increase of LC3-II (an autophagic marker), which indicates induced autophagy (Fig.2). The analysis for the same markers of post-mortem brains and muscle (which are particularly energy dependent tissues) of transgenic mice over-expressing NMNAT1 (nmnat1 tg), depleted for NMNAT1 (nmnat1het), and the wild type (nmnat1wt), revealed that NMNAT1 does not directly regulate basal level of autophagy (Fig.3). Main result - NMNAT2 and to some degree NMNAT3 directly regulate autophagy at a basal level.

Finally, in order to establish if the overexpression of NMNAT1 could protect from neurodegeneration combined with mitochondrial dysfunction (which suggest a link with an altered NAD biosynthesis as discussed above), we mated the mouse model pcd5J (purkinje cells degeneration - neurons located in the cerebellum) with a mouse over-expressing NMNAT1 (nmnat1 tg). The pcd5J mouse presents loss of motor coordination due to neurodegeneration, energetic failure due to mitochondrial dysfunction and defect in eye vision. We analysed the cerebellum of the litters generated: wild type mice; pcd5j mice; pcd5j mice where nmnat1 is overexpressed; nmnat1 overexpressing mice. Immunohistochemistry techniques were used to detect calbindin, a protein marker of the purkinje cells, in slides of frozen cerebellum, as an indication of the degeneration of the neurons. The degeneration of the neurons was ~70% at 20 days of the mice life and complete at 60 days, but in the pcd5j mice where NMNAT1 was overexpressed the degeneration was ~60% at 20 days, indicating that NMNAT1, at least in early stages, can partially protect the degeneration of these cells (Fig.4A-B-C). Main result - The overexpression of NMNAT1 and the increased levels of NAD can counteract the purkinje cells degenerative process and potentially ameliorate mitochondrial functionality and eye vision defect.

In recent studies NMNAT2 is defined as an axonal survival factor and is degraded quickly after axons are injured (cut from the cellular body), leading to a rise in the NMN level that can trigger degeneration. Previous studies in our group showed that the introduction in the wild type mouse of the bacterial enzyme NMN deamidase, which consumes NMN, delayed axon degeneration after the axon cut compared to the wild type mouse. We wanted to establish if the scavenging of NMN protects from axonal degeneration in a mouse chemical model of Parkinson’s disease. The brains of wild type and transgenic deamidase mice (expressing the bacterial enzyme NMN deamidase) were injected with the drug 6-hydroxydopamine (6-HDA), which induces degeneration of dopaminergic neurons (typical of Parkinson’s disease). Immunohistochemistry by the detection of tyrosine hydroxylase, a protein enriched in the dopaminergic neurons, was used to identify the intact axons and therefore the degree of degeneration, but scavenging NMN could not significantly prevent the degeneration of the dopaminergic neuronal axons (Fig.5A-B). SARM1, a Toll-like receptor protein, was recently found to be essential for axonal degeneration; therefore we hypothesized a connection of this protein with NMN. In our attempts to determine the potential activation of SARM1 by NMN, no changes in SARM1 were observed after axon cut, when NMN is expected to accumulate. Main result - NMN could constitute a trigger of axon-degeneration by activating SARM1, but scavenging NMN didn’t protect neurodegeneration in a chemical model of Parkinson’s disease.

*Impact*
Unravelling the impact of NAD metabolism, as a central core of energy, in the neurons is of particular interest to understand the regulation of neuronal life and intervene with efficacy to prevent neuron degeneration. The activity of NMNATs in consuming NMN and producing NAD has an important role in regulating many processes in the neuronal cells that ensure survival keeping the cells healthy. We expect a nuclear change in the NMN/NAD level that regulates SIRT1’s broad spectrum of action, following NMNAT1 modulation, which determines the survival of the cell body. NMNAT2 can regulate the autophagic process, potentially preventing axon degeneration due to energetic failure by clearing dysfunctional mitochondria (mitophagy). Studying the eventual binding of SARM1 and NMN would help to confirm that NMNAT2 activity is necessary to deplete the NMN in excess that lead to the activation of SARM1, which commit the axon to degenerate. Since overexpressing NMNAT1 delayed the degeneration of the purkinje cells in a mouse model of neurodegeneration combined with energetic failure and defect in the retina, we expect to find improvement in the mitochondrial functionality and eye vision as well. All this would clarify the role of NMNATs in the neurodegenerative process modulating the levels of NMN/NAD in the cell, and the contribution of the regulation of the autophagic machinery to their beneficial effects. Because it is increasingly evident that mitochondrial dysfunction is an underlying cause of neurodegeneration, this would also have an important impact on preventing mitochondrial diseases.

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THE UNIVERSITY OF NOTTINGHAM
United Kingdom
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