Rossi, Francesca
(2016)
NMNAT1 and its role on ageing and age-realted diseases.
PhD thesis, University of Nottingham.
Abstract
NAD metabolism is increasingly implicated in a variety of biological functions, including regulation of gene transcription, lifespan, cell death, circadian rhythm and glucose home-ostasis. The three mammalian isoforms of the central NAD biosynthetic enzyme, nicotin-amide mononucleotide adenylyltransferases (NMNATs), have recently emerged as crucial players in neuronal maintenance and protection, in ageing-processes and in many neurodegenerative diseases, such as Alzheimer’s disease. The biological basis of ageing and its related pathologies are not fully elucidated and there is an urgent need to develop valid therapeutic strategies to minimize the impact that these conditions have on the society.
In the present study, I used two mouse models, heterozygous Nmnat1 knockout mice (Nmnat1+/-) and mice overexpressing Nmnat1 (Nmnat1 tg), in order to assess the role of the nuclear isoform NMNAT1 in ageing and age-related diseases.
First, I asked whether modulating the expression of NMNAT1 influences ageing-related mechanisms. To this aim, Nmnat1 tg and Nmnat1+/- mice were subjected to metabolic analysis and behavioral tests for measuring locomotor activity. Furthermore, NMNAT enzyme activity, NAD levels, gene expression and protein levels were analyzed with bio-chemical techniques at 6 and 24 months of age. I found that Nmnat1 tg mice had a reduced body weight and increased locomotor activity during ageing, while Nmnat1+/- weighted more at 12 months than their wild type littermates. NMNAT enzyme activity, significantly higher in Nmnat1 tg mice at 6 months, did not change during ageing. In contrast, NMNAT enzyme activity in Nmnat1+/-, which is already low in young mice, showed a trend of decrease with ageing.
Second, in order to investigate the role of the nuclear isoform NMNAT1 in age-related diseases, I analyzed the effect of modulating NMNAT1 levels on behavioral and neuro-pathological traits in a mouse line which expresses non-mutant human tau isoforms (htau mouse), representing a model of tauopathy relevant to Alzheimer’s disease.
To this aim, htau were crossed to Nmnat1 tg and Nmnat1+/- mice to produce experimental mouse groups with four genotypes that were all heterozygous for murine tau (mtau+/-): Nmnat1 tg/htau, Nmnat1+/-/htau and wild type littermates. Mice were subjected to a bat-tery of specific tests to assess potential behavioral abnormalities that correlate with dys-functions characteristic of AD. Furthermore, image analysis was performed to assess the integrity of the brain areas mainly impaired in AD. Finally, biochemical studies were conducted in order to test whether modulating NMNAT1 levels caused changes in NMNAT activity and in NAD levels in htau mice.
I found that htau mice have an early, selective deficit in food burrowing, a behavioral task used to assess activities of daily living which are impaired early in Alzheimer’s dis-ease, and that overexpression of Nmnat1 ameliorates this defect. Despite the behavioral abnormalities, htau mice did not show neurodegenerative impairments in cortex and hippocampus. Modulating NMNAT1 levels produced a corresponding effect on NMNAT enzymatic activity but it did not alter NAD levels in htau mice. My results suggest beneficial effects of NMNAT1 on the early behavioral deficits in this mouse model of tauopathy.
Finally, I asked whether modulation of Nmnat1 can influence the ischemic cell death, which is at least in part caused by an overactivation of PARP1 and NAD depletion. I hypothesized that reducing NMNAT1 levels exacerbates the ischemic brain damage, whereas increasing these levels confers protection by respectively decreasing or increas-ing NAD availability within the nucleus.
To address this question, I used a stable NSC-34 clone expressing Wlds/NMNAT1, as well as wild type NSC-34 cells. I measured NAD levels and then tested cell viability af-ter genotoxic stress induced by N-Methyl-N-nitro-N-nitrosoguanidine (MNNG), which causes DNA damage and activation of PARP1, mimicking some mechanistic aspects of ischemic cell death. I first confirmed that Wlds was expressed in all cellular fractions in Wlds/NMNAT1 cells, but not in wild type cells. Furthermore, both wild type and Wlds/NMNAT1 cells showed lower NAD levels in the nuclear fraction compared to the cytoplasmic fraction. Paradoxically, NAD levels tended to be even lower in all cellular fractions of Wlds/NMNAT1 cells respect to the wild type cells. Correspondingly, they show higher susceptibility to cell death induced by MNNG. In addition to the in vitro experiments, I also started to set-up an in vivo model of cerebral ischemia. Even though time constraints did not allow analyzing the effects of Nmnat1 modulation, my study will be useful for future investigations to test whether overexpression of Nmnat1 or its downregulation may protect or exacerbate the effect of cerebral ischemia in an in vivo model.
Taken together, my results suggest that modulating Nmnat1 correspondingly influences ageing and neurodegeneration processes and underline the utility of the Nmnat1 tg and Nmnat1+/- mice as a tool for future research in this field.
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