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.
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NAD+ was first discovered by Arthur Harden and William Young 1906, during research on fermentative enzymes and was part of the 1929 Nobel Prize attributed to Harden and Hans von Euler-Chelpin (White and Schenk, 2012). It has long been known that NAD+ is a coenzyme used in multiple redox reactions and, consisting of two covalently bound mononucleotides – nicotinamide mononucleotide (NMN) and AMP (Zamporlini et al., 2014). It exists in its oxidised form NAD+, and the reduced form, NADH which provide oxidoreductive power, perhaps most notably utilised within the mitochondria to produce ATP (White and Schenk, 2012), with some of the ATP in turn being used to activate the NAMPT protein for further production of NAD+ (Hara et al., 2011). NAD+ can be synthesised either synthesised de novo from tryptophan (Zhou et al., 2015) or from salvage pathways using nicotinic acid or nicotinamide, with a third pathway – using nicotinamide riboside precursors – having recently been discovered (Billington et al., 2008).
The mitochondrial matrix is not the only cellular compartment containing an NAD+ pool, with the others being the cytosol and the nucleus as shown in figure 1 (White and Schenk, 2012). Whilst the nuclear membrane is porous to NAD+ to allow diffusion, mitochondrial membranes are not (White and Schenk, 2012). To transport NAD+ across mitochondrial membranes, biochemical shuttles are required; the four shuttles that have been found and are as follows: glycerol 3-phosphate, malate-aspartate, lactate and NADH/cytochrome c electron transport (White and Schenk, 2012). These shuttles, as well as the conversion of pyruvate to lactate replenish NAD+ levels via oxidation of the reduced form – NADH (White and Schenk, 2012). In addition to these mechanisms NADH is also oxidised into NAD+ within mitochondrial matrices by the electron transport chain during respiration (White and Schenk, 2012).
In recent years NAD+ has been accepted to be a signalling molecule, constituting integral parts of large and dynamic regulatory pathways and integrated networks (White and Schenk, 2012), including sirtuins.
Our projects ultimate goal, to increase NAD+ levels within human cells, could have been achieved a variety of ways. Before the main body of research is presented we will briefly discuss the reasoning as to why each method was discarded and how we arrived at our final decision.
One possible way would be to directly manipulate people’s genomes, but it comes with an array of risks such as lack of uniformity of genome modification – not every cell may be altered. Unintended complications may arise, such as immune response, organ failure and ultimately, death. These ethical considerations also lead on to the idea of eugenics if gene therapy became commonplace. This approach is also beyond our team’s current capabilities and conflict with iGEM regulations. Aside from the obvious ethical implications of human genetic modifications, studies have shown that in vivo upregulation of relevant genes pertaining to the NAD+ synthesis pathways, did not significantly affect the NAD+/NADH redox ratio (Frederick et al., 2015). Interestingly though, germ line knock-down of genes coding for NAD+ consuming enzymes has shown to perturb the NAD+/NADH redox ratio of cells, in favour of NAD+ (Frederick et al., 2015). This gene treatment was insufficient and was therefore inadequate to stimulate oxidative metabolism within skeletal muscle cells (Frederick et al., 2015), which is one of our project goals.
We briefly considered trying to transport the rate limiting enzyme in de novo NAD synthesis, NAMPT (Koltai et al., 2010; Hara et al., 2011; Liang et al., 2013; Canto, Menzies and Auwerx, 2015), into the viscera with the target being the cytosol of cells. We discarded this as we realised that the final product produced by the enzyme was smaller and more stable than the enzyme. Enzymes are large, require specificity when being transported – requiring human modifications to existing transport mechanisms – and can require numerous other criteria for efficacy, specific temperature, pH, and post-translational modifications.
There are non-synthetic biology alternatives, for example NAMPT can be chemically activated by P7C3 (Zhao et al., 2015), but more research is needed to confirm its safety.
Many of the papers looked at indicated that a rise in intracellular NAD+ was achieved through supplementation of NAD+ intermediates such as NMN, NR or Acipimox (see figure 2) (Zamporlini et al., 2014; van der Weijer et al., 2015; Felici et al., 2015). Whilst we could have targeted genes to upregulate NMN synthesis (see figure 2) such as Namnt within the E. coli, we decided to synthesise the end-product. We did this because with age the components maintaining the circadian rhythms decline, reducing the amount of NAMPT (Zamporlini et al., 2014). Whilst upregulating this may seem the simplest way to achieve our goal we were worried that the activity of NAMPT may be reduced due to lower ATP levels, which is required for NAMPT activation (Hara et al., 2011), due to the mitochondrial dysfunction. Later we will discuss the direct effect of NAD+ on cell biochemistry.
We were interested in the diversity of applications NAD+, conferred likely due to its role in integrated networks (White and Schenk, 2012) and as such were looking into its neurological effects on humans.
Sirtuins are a family of proteins that act as deacylases and depend on NAD+ to be biologically active (Koltai et al., 2010; Zamporlini et al., 2014). Mammals such as humans have seven sirtuins (SIRT1-7), with early work being carried out on the yeast Sir2 homologue (Ying, 2007; Koltai et al., 2010). Evidence confirms that SIRT1 promotes mitochondrial function, shown by the lack of upregulation of OXPHOS genes in both the nucleus and mitochondria when SIRT1 iKO mammals were subjected to fasting and calorie restriction (CR), leading to deacreased mitochondrial functionality (Gomes et al., 2013). Fasting and CR activate SIRT1, as does DNA damage from ionising radiation sources such as UV radiation sunlight (Zamporlini et al., 2014). It was speculated by Gomes and his team that the induced activity of increased mitochondrial oxidative metabolism – and increased transcription of mitochondrial OXPHOS genes due to these conditions – was via SIRT1 deacetylating PGC-1α (Gomes et al., 2013). However this was disproved as SIRT1 continued to function in KO PGC-1α/β mice (Gomes et al., 2013), suggesting an as of yet undiscovered mechanism.
We decided to use the NAD+ molecule for our project as we have seen evidence that it has neuroprotective effect in humans, as when NAD+ starvation occurs a misfolded amyloidogenic protein stimulates the activation of autophagic mechanisms and neurodegeneration (Zhou et al., 2015). Mitochondrial dysfunction is present in a multitude of neurodegenerative diseases including Parkinson’s (Zhou et al., 2015) and Alzheimer’s (Long et al., 2015; Zhou et al., 2015); with NAD+ loss being a contributory factor. Administration of the precursor NMN was shown to restore the respiratory capacity of ailing mitochondria, as well as reducing mitochondrial fission and reducing fragmentation – which might explain the reduction of mitochondrial bioenergetics defects – after this they identified that NAD+ may be a limiting factor when the oxygen consumption rate (OCR) deficiencies normalised upon administration (Long et al., 2015). This neuroprotective action can be mainly attributed to SIRT1 which deacetylases PGC-1α, which when activated, stimulates mitochondrial biogenesis (Long et al., 2015).
Recent research has indicated that these neurodegenerative diseases are prion based, with affected neurones having decreased ATP productions as well as reduced NAD+ levels (Zhou et al., 2015). This can be a destructive cycle as ATP is required by the rate limiting enzyme NAMPT for de novo synthesis of NAD+ which in turn is required for metabolic production of ATP, leading to a gradual decline of both.
The NAMPT-NAD+ axis has been shown to have both neuroprotective and neuroregenerative effect via the combined actions of SIRT1, SIRT2 and SIRT6 (Ido et al., 2001). These proneurogenesis effects have been corroborated by the reduced activity of SIRT1, SIRT2 and SIRT6 when these deacetylases were knocked down (Zhao et al., 2015).
There are many causes of neuronal cell death, one of which is ischemia. This is the restriction of blood flow from to areas of the body by narrowing of arteries or spasm; when this occurs this can cause a stroke, called an ischemic stroke. NAD+ has been shown to protect against ischemic cell death, as ischemic injury depletes the supply of neurological NAD+ (Zhao et al., 2015). This indicates that continued neuronal viability is dependent on the activities of NAD+, possibly due to the activity of NAD+ consuming enzymes such as the aforementioned sirtuins. However neuronal death can be caused by an array of diseases as well – Parkinson’s, Alzheimer’s and multiple sclerosis.
We did hope that the excess in NAD+ consumption by Sirtuins and other NAD+ consuming enzymes would result in a far larger pool of NMN, an NAD+ intermediate that has been consistently shown to increase NAD+ levels (Gomes et al., 2013; Frederick et al., 2015; Zhao et al., 2015). However even with sensitive instruments and assays, NMN has not been found within blood plasma of mice (Hara et al., 2011). Interestingly, however, NAD+ has been shown to be present in the blood stream at nanomolar concentrations, possibly due to the use of NAD(P) by ectoenzymes (Billington et al., 2008). We were unable to determine through literary research as to whether NAD+ is able to traverse the blood brain barrier (BBB), but it has been shown that NMN can cross the BBB to modulate intracellular NAD levels (Zhao et al., 2015). NMN is safe for long-term use for clinical applications as it is a native endogenous substrate (Wang et al., 2015). This leads us to speculate that either NMN is contained within cells by membranes and is therefore not subject to high enough diffusion gradients between cell interiors and exteriors or that the majority of NMN is used in NAD+ synthetic pathways. This could also indicate that cells are completely impermeable to NMN, however this is unlikely due to the droves of evidence that NMN supplements can increase the NAD+ pool, meaning that NMN can traverse membranes even if it is only unidirectional, which if the case will require further development of our GMO to address. Assays need to be carried out to detect whether increased NAD+ can perturb and therefore increase the NMN pool. By effectively reducing the activity of NAD+ synthesis and salvage pathways’ activities and thereby reducing NMN consumption, we hope that NMN may enter the bloodstream to create an extracellular NMN pool. Concurrently we believe that NMN levels may be actively produced from exogenous NAD+ by the proteins coded for by the NadD and NudC genes (Zhou et al., 2011) as shown in figure 4.
NMN has been shown to have a positive effect on the insulin levels secreted in culture and in vivo, due to the stimulation of SIRT1 in pancreatic β cells (Zhao et al., 2015).
Studies have revealed that inhibition of other NAD+ consuming enzymes such as PARP (1 & 2) enzymes can convey long-term neuroprotection (Ying, 2007). As a future consideration for the development of the neurological aspect of our project, this inhibition of PARPs could potentially yield a more layered and therefore more refined approach.
Our project relies on the capacity of mammalian cells to uptake NAD+ for use in various biochemical reactions. Experiments have proven that NAD+ is taken up in two stages, initially there is a rapid transport followed by a period of uptake lasting an hour or so (Billington et al., 2008), with transport rate shown in figure 5.
NAMPT has been shown to significantly increase the number of mitochondria within cells, and NAD+ increases myocyte mitochondria biogenesis (Kim, Yoon and Park, 2014). This indicates that NAMPT increases the NMN levels, which in turn, increase the NAD+ levels to increase mitochondria concentration. As previously mentioned NMN increases insulin levels – evidence shows that SIRT1 has been linked to increased insulin sensitivity (Kim, Yoon and Park, 2014). These increased insulin levels coupled with increased biogenesis may have a positive effect on bodily energy levels, reducing age-related fatigue. The site of action of this biogenesis may be due to the modulation of hypoxia inducing factor-1 alpha (HIF-1α), which is known to increase with age to combat the decline of appropriate redox conditions – especially within skeletal muscle (Koltai et al., 2010; Gomes et al., 2013). Once again returning to sirtuins, SIRT1 increases the transcriptional activity of HIF-1α – which seems counter intuitive to its other areas of effect – but SIRT3 destabilises the HIF-1α protein structure, with SIRT6 acting as a corepressor (Gomes et al., 2013). These data imply that the benefits of increasing the activity of sirtuins thus far outweighs the cost.
Nuclear activity of SIRT1 and the level of acylation of proteins show an inverse correlation (Koltai et al., 2010) indicating that with increasing age target proteins are subjected to a lesser degree of acylation, possibly contributing to the detrimental proximate effects of aging. Exercise has been shown to increase the activity of NAMPT, the rate limiting enzyme in NAD+ synthesis, which thereby increases the NAD+ pool, increasing the SIRT1 deacetylase which is dependent upon NAD+ to function (Koltai et al., 2010). SIRT6, in conjunction with SIRT1 regulate DNA repair mechanisms (Liang et al., 2013) as previously mentioned, indicating that stimulation may enable partial protection from DNA damage and increase replicative lifespan of cells as shown in figure 6.
As NAD+ is required by E. coli for glucose oxidation (Liang et al., 2013), we predict that an increase in NAD+ synthesis will confer a selective advantage over other gut fauna, promoting better growth rates as overexpression of the pncB genes has been shown to confer a significant increase in the mass of the cell (Liang et al., 2013). Experiments were done on a strain of E. coli (BA002) that could not anaerobically use glucose as an energy source due to the lack of NAD+ regeneration; the decreased NAD(H)/NAD+ ratio due to increase in the NAD(H) pool, improved glucose uptake and growth of cell – substantiated by the increase of dry mass of cells – also conferring succinic acid production (Liang et al., 2013). However the excess NAD+ may also increase the prevalence of other gut fauna and a full analysis of bacterial interaction will need to be completed before any clinical considerations as NAD+ may confer different behaviour to pathogenic organisms.
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