Team:Leicester/Description
Project Explanation
For our project we wanted to look at a universal issue, something that affects every person indiscriminately. This lead us to aging as one such issue. Because the aging process is a highly dynamic and complex process intrinsic to our physiology, we knew that it was practically impossible to treat the condition, so we started looking at the symptoms. As we age our muscle strength declines, we become susceptible to a host of neurodegenerative diseases, and our overall physiological health declines. One common denominator to these conditions appears to be mitochondrial dysfunction (Gomes et al., 2013). We are hoping to address a few of the proximate aging symptoms, such as senescence of muscle tissue and neurones within the brain. One of the attributes of senescence is the gradual breakdown of the circadian clock of human physiology (Zamporlini et al., 2014).
Through research we identified the coenzyme nicotinamide adenine dinucleotide (NAD+) – widely accepted as a mediator of redox reactions – as a suitable candidate for our purposes. Literature has identified the interplay of NAD+ and SIRT1 working in concert to modulate metabolism and circadian rhythm (Zamporlini et al., 2014) which are closely linked to the mitochondria (Gomes et al., 2013). A hallmark of aging is the loss on NAD+ with age (Gomes et al., 2013; Zamporlini et al., 2014), as well as mitochondrial dysfunction; It has been shown that these two phenomenon are correlated, but a causal link is still tenuous.
Our project, simply put, is a self-sustaining integrated pharmacy for patients – a pseudo-organ in effect. Our ultimate goal for our project results would be to integrate an as yet undetermined strain Escherichia coli into the human gut microbiome. The purpose of the non-native bacteria is to produce a specified chemical compound – in this case NAD+ – into the gut. We speculate that due to the large surface area and blood flow of the microvilli, as well as the low molecular weight of NAD+ (Zamporlini et al., 2014), and proven uptake by mammalian cells (Billington et al., 2008), the NAD+ will enter the blood stream and be transported rapidly and uniformly across the entire body. We predict that this will over time, re-establish the circadian rhythms of metabolic processes, restore oxidative metabolism – especially in muscles – and possibly have a neuroprotective effect against neurodegenerative disorders such as Parkinson’s and Alzheimer’s.
For our project, we aim to develop several BioBricks with the purpose of increasing net extracellular NAD+/NAD(H) levels of the host organism – in this case it would be E.coli – the purpose being to allow transfer of NAD into the gut microbiome for absorption into the bloodstream of patients.
We believe this approach will enable a long term, and with further development, full integration of a bacterial pseudo-organ into people that suffer from deleterious conditions such as weaker muscle strength with age, as well as the neurodegenerative diseases that more common with aging. The end goal of this integration would be to ameliorate the symptoms of aging, possibly increasing longevity as a side-effect. This would have a tremendous impact as it could potentially restore the quality of life and self-sufficiency to the elderly and neurodegenerative sufferers relying on carers.
Why NAD?
Practical Components
pncB
We identified one gene called pncB which encodes nicotinic acid phosphoribosyltransferase (NAPRTase) (Liang et al., 2013), as seen in figure (from Zhou et al., 2011) is responsible for the conversion of NA into NAMN. We selected this gene as experiments have shown that cooverexpression with another gene, nadE, increased the NAD(H) by 7-fold. This was of particular use to us as the shuttling mechanisms, among others, as mentioned previously are able to oxidise the NAD(H) into NAD+ (White and Schenk, 2012). From this we can deduce that whilst the NAD(H)/NAD+ axis is perturbed in favour of NAD(H) the net concentration of NAD+ increases in vivo over time due to the oxidative actions of the shuttles and other mechanisms (Liang et al., 2013).
nadD and nadE
The second and third gene sequences we were interested in were nadD, which encodes the NAMN adenylyltransferase and nadE which codes for NAD Synthetase in the NAD(H) biosynthetic pathway responsible for the interconversion of NAD+ and NMN (Zhou et al., 2011, Zamporlini et al., 2014). This is of particular use to us as we wish, as a secondary objective, to increase NMN levels for reasons discussed previously. We believe this will be feasible due to the increase in NAD+ in cells, potentially reducing a limiting factor of NMN production (see figure 4 NadD).
Experiments have shown that individually upregulating pncB, nadE and nadD increases the NAD(H) pool (Liang et al., 2013). Despite this extremely promising theoretical increase for single genes we decided to maximise the output by using all three. We did this as we were concerned that very high concentrations of NAD+ may be required to enter cells in useful concentrations, as some may be lost to other gut fauna, the gut environment itself, or not be concentrated enough in the blood to enter cells. Further experiments would be required to ascertain the efficiency of uptake whereupon we would regulate the NAD+ output.