BAB-C-P3506PCT NOVEL METHOD FIELD OF THE INVENTION The present invention relates to a method of promoting and/or increasing acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in a cell, comprising increasing the activity of AMP-activating protein kinase (AMPK) while maintaining or increasing acetyl-CoA carboxylase (ACC1) activity in the cell. Also provided are a method of rejuvenating a cell, such as to a younger metabolic state, comprising promoting and/or increasing acetyl coenzyme A (acetyl-coA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways, a pharmaceutical composition comprising a combination of an activator of AMPK and an inhibitor of ACC1 phosphorylation, and the use of said pharmaceutical composition in the treatment of age-related diseases or disorders. BACKGROUND OF THE INVENTION Chromatin modifying enzymes require metabolite cofactors or donors, and many are also inhibited by endogenous metabolites, e.g. α-ketoglutarate (αKG) is the cofactor for DNA and histone lysine demethylases (TETs and KDMs), while fumarate and 2-deoxyglutarate inhibit these enzymes. Furthermore, some chromatin modifiers accept diverse substrates creating a spectrum of epigenetic marks, e.g. acyl-CoA species compete as substrates of histone acyltransferase enzymes (HATs) forming at least ten histone lysine acylations with uncertain implications for gene regulation. Thus, relative metabolite abundance will affect chromatin state, and metabolic changes across life, across diets and across environmental exposures will profoundly impact the flux of epigenetic modifications. DNA methylation is much more stable than histone acetylation, with gradual changes associated with ageing: reproducible changes in DNA methylation occur throughout life and can be measured using epigenetic clocks to accurately infer age. Features such as bivalent chromatin and CpG islands (CGIs) are also known to be eroded during ageing, but the extent to which this is driven by metabolic change remains poorly explored. Understanding how cells control the epigenome requires us to understand the rate at which metabolites are synthesised, consumed, and distributed around the cell. For example, nucleocytoplasmic acetyl-CoA available for protein (including histone) acetylation is largely derived from dietary glucose via mitochondrial citrate export or acetate from various sources, but HATs must compete against lipid synthesis for this acetyl-CoA.
BAB-C-P3506PCT Ageing is characterised by a decline in metabolic homeostasis, and metabolic decline is accelerated by adverse dietary exposures, including overnutrition and obesity. There is therefore a great need to provide methods of combating the declining health and increased disease risk associated with ageing, such as by increasing the period of an individual’s lifespan which is free from age-related diseases or disorders (their “healthspan”), in particular metabolic ageing and age-related metabolic diseases and disorders caused by the deregulation of metabolic homeostasis, as well as a need to provide methods of affecting the epigenetic changes seen during ageing through metabolic regulation. SUMMARY OF THE INVENTION According to a first aspect of the invention, there is provided a method of promoting and/or increasing acetyl coenzyme A (acetyl-coA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in a cell, comprising increasing the activity of AMP-activating protein kinase (AMPK) while maintaining or increasing acetyl-CoA carboxylase (ACC1) activity in the cell. In certain embodiments, the flux of acetyl-CoA into the protein acetylation pathway is inhibited and/or reduced, such as wherein the use of acetyl-CoA in histone acetylation is inhibited and/or reduced. In a further embodiment, the turnover of acetyl-CoA in the lipid synthesis (lipogenesis) and fatty acid oxidation pathways is increased. According to a further aspect of the invention, there is provided a method of rejuvenating a cell, comprising promoting and/or increasing acetyl coenzyme A (acetyl-coA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in the cell according to the method described herein. In another aspect, there is provided a pharmaceutical composition comprising a combination of an activator of AMPK and an inhibitor of ACC1 phosphorylation, such as a small molecule activator of AMPK and a small molecule inhibitor of ACC1, the composition optionally further comprising one or more pharmaceutically acceptable diluents, excipients and/or carriers. In a yet further aspect of the invention, there is provided a method of treating an age-related disease or disorder in a subject in need thereof, said method comprising administering the pharmaceutical composition described herein.
BAB-C-P3506PCT According to a still further aspect, there is provided the pharmaceutical composition described herein for use in the treatment of an age-related disease or disorder in a subject in need thereof. BRIEF DESCRIPTION OF THE FIGURES Figure 1: A2A Cells Accumulate Less Tom70-GFP Ageing Damage Marker While Ageing to Wild Type Levels. (A) Median signal intensities from flow cytometry images of Tom70-GFP (senescence-associated damage marker) in log phase, 24h and 48h enriched mother cells. Gated for circularity, and biotin, ~600 cells left per sample post-filtration for Tom70-GFP focus. (B) Median signal intensities of ageing as measured by bud scar accumulation via WGA-Alexa405 staining (bud scar lifespan age marker). Mixed effect analysis and post-hoc Tukey tests were performed for age and mutation (n=7-8). Figure 2: The Age-Induced Hsp104-GFP Healthy Ageing Marker Shows that A2A Cells are More Resilient to Protein Maintenance. (A) Single cell signal intensities from flow cytometry images of Hsp104-GFP in log phase, 24h and 48h enriched mother cells. Gated for circularity, and biotin-labelling, ~1500 cells left per sample post-filtration. (B) Median single cell intensities of A. Figure 3: Global Gene Regulation in A2A Appears More Youthful than Aged Wild Type Cells. MA plots of change in abundance between log and 48 hours relative to average abundance for each mRNA, for wild type (WT; left panel) and A2A mutant cells (right panel). Positive values on y-axis indicate increased abundance with age relative to average, negative values indicate decreased abundance, zero indicates no change. Abundances are size-factor normalised log2 transformed read counts from filtered poly(A) selected sequencing libraries, slopes calculated by linear regression. Figure 4: A2A Growth Kinetics are Similar to Wild Type Cells. Growth curves for wild type and A2A yeast cells grown in YPD medium obtained by optical density at 600 nm, over a 24h time period. Figure 5: The Lifespan of the A2A Mutant is Similar to Wild Type Cells. Cell viability at 2, 24, 48 and 72 hours of ageing in YPD, measured by plating of equivalent culture volumes on YPD and counting colonies formed. Figure 6: A2A Mutant Cells Demonstrate Higher Viability and Fitness. Colony size assays measuring fitness of WT vs A2A cells aged in glucose. Cells were aged 48 hours in glucose, purified live in media then placed on YPD plates by micromanipulation. Colony diameters were measured on the micromanipulator screen after 48 hours. Only cells that formed visible colonies after a further 3 days were included in the analysis. Log phase cells were taken directly from culture and colony growth measured under the same conditions.
BAB-C-P3506PCT P-values calculated by Šídák’s multiple comparisons test. Log WT (n=37), Log A2A (n=28), 48h WT (n=23), 48h A2A (n=14). Figure 7: A2A cells demonstrate higher basal respiration rates. The oxygen consumption rate of WT and A2A cells were measured by a Seahorse FX-96 oxygraphy. p values calculated by Šídák's multiple comparisons test. Figure 8: The Healthy Ageing Phenotype of A2A Cells is Independent of Sak1 Overexpression. (A) Single cell signal intensities (upper panel) and median single cell intensities (lower panel) from flow cytometry images of Tom70-GFP (senescence-associated damage marker) in log phase, 24h and 48h enriched mother cells. Gated for single cells and biotin-labelling, ~600 cells (upper panel) or ~1500 cells (lower panel) left per sample post- filtration for Tom70-GFP focus. (B) Single cell signal intensities (upper panel) and median single cell intensities (lower panel) of ageing as measured by bud scar accumulation via WGA-Alexa405 staining (bud scar lifespan age marker). DETAILED DESCRIPTION OF THE INVENTION The present invention is based on the novel finding that alterations in the metabolic regulatory system can alter the ageing trajectory of yeast cells, and it is believed that the underlying metabolic effects seen in yeast may be translated to mammalian cells, such as humans. In particular, the inventors have shown herein that the flux of acetyl coenzyme A (acetyl-CoA) into the lipid synthesis (lipogenesis) and fatty acid oxidation (β-oxidation) pathways in a cell can be promoted and/or increased by increasing the activity of AMP-activating protein kinase (AMPK) in the cell, while maintaining or increasing the activity of acetyl-CoA carboxylase (ACC1), which is normally phosphorylated in an inhibitory manner by AMPK (in particular AMPK with promoted and/or increased activity). Thus, it is hypothesised that a youthful state, such as a youthful metabolic state as described herein, may be maintained and/or induced in the cell, in particular a more youthful metabolic state compared to a cell in which AMPK activity has not been increased (e.g. a control cell). Such maintenance or induction may be referred to as “rejuvenation” and may also or alternatively include maintaining or inducing a longer “healthspan” in the cell as defined herein. Therefore, it will be appreciated that the reversal of certain pathological effects of ageing may be achieved by the methods described herein, in particular wherein said pathological effects comprise and/or are caused by the deregulation of metabolic homeostasis. While the present findings are shown in yeast, metabolite cofactors for epigenetic modifying enzymes are highly conserved, making yeast an attractive system to dissect the impact of metabolites on epigenetic change during ageing. Thus, according to a first aspect of the invention there is provided a method of promoting and/or increasing acetyl-CoA flux into the lipid synthesis (lipogenesis) and fatty acid oxidation
BAB-C-P3506PCT (β-oxidation) pathways in a cell, comprising increasing the AMPK while maintaining or increasing ACC1 activity in the cell. Acetyl coenzyme A (acetyl-CoA) is a molecule that participates in many biochemical reactions in protein, carbohydrate and lipid metabolism. Its main function is to deliver the acetyl group to the citric acid cycle (also known as the (tricarboxylic acid (TCA) cycle) and Krebs cycle) to be oxidised for energy production. Acetyl-CoA is also critical as the basic building block from which lipids are made, and provides the metabolic cofactor for protein acetylation, a common regulatory post-translational modification involved in, among other processes, epigenetic regulation. Coenzyme A (CoA) consists of a β-mercaptoethylamine group linked to the vitamin pantothenic acid (B5) through an amide linkage and 3’-phosphorylated ADP. The acetyl group of acetyl-CoA is linked to the sulfhydryl substituent of the β-mercaptoethylamine group. This thioester linkage is a “high energy” bond, which is particularly reactive. Hydrolysis of the thioester bond is exergonic (−31.5 kJ/mol). CoA is acetylated to acetyl-CoA by the breakdown of carbohydrates through glycolysis and by the breakdown of fatty acids through β-oxidation. Acetyl-CoA then enters the citric acid cycle, where the acetyl group is oxidised to carbon dioxide and water, and the energy released is captured in the form of 11 ATP and one GTP per acetyl group. GTP is the equivalent of ATP and they can be interconverted by nucleoside- diphosphate kinase. The terms “promoting”, “increasing” and “enhancing”, as well as the past and other tenses thereof, used herein refer to the increasing or promotion of e.g. acetyl-CoA flux compared to a control, such as a cell in which the activity of AMPK has not been increased. These terms also refer to the activity of AMPK activity and/or optionally the activity of ACC1 activity in the cell compared to prior to such increase/promotion, for example compared to AMPK activity prior to modulation of the phosphorylation site of AMPK, such as prior to using/administering a small molecule activator to the cell. The converse terms “inhibiting”, “reducing” and “decreasing”, as well as the past and other tenses thereof, used herein refer to the decrease (either active/direct or passive, such as by virtue of modulating an upstream protein or process) of e.g. acetyl-CoA flux or AMPK/ACC1 activity compared to a control, such as a cell in which the activity of AMPK has not been increased. They also refer to flux/activity compared to prior to decrease/inhibition, for example compared to prior to increasing AMPK activity, such as prior to modulation of the phosphorylation of AMPK and/or prior to using/administering a small molecule activator to the cell. As such, it will be readily appreciated that “promoting”, “increasing”, “enhancing”, “inhibiting”, “reducing”, “decreasing” and the like are used herein in their normal meaning unless otherwise clear from the context. More specific examples of
BAB-C-P3506PCT “promoting”, “increasing”, “enhancing”, “inhibiting”, “reducing”, “decreasing” and the like will be clear from the uses of these terms herein. The term “flux” used herein refers to the total turnover or passing of acetyl-CoA through the specified pathway/cellular process, i.e. while the total amount being entered into the pathway may be increased, so too is the processing of intermediates in the pathway and of the final products. The terms “flux” and “turnover” may therefore be used interchangeably herein. Thus, wherein the flux of acetyl-CoA into a particular pathway is promoted and/or increased, there is not necessarily a build up of any intermediates of the pathway. It will be appreciated that the amount of final product of the pathway may, however be increased. Similarly, wherein the flux of acetyl-CoA into a pathway is reduced and/or inhibited, there is not necessarily a reduction in the amount of any intermediates of the pathway or of the final products. However, as will be appreciated from the disclosures herein, wherein flux of acetyl-CoA into the protein acetylation pathway is reduced/inhibited, it is desired that the final amount of “product” of protein acetylation be reduced. In one embodiment, the flux of acetyl-CoA into the lipid synthesis (lipogenesis) pathway is promoted and/or increased. Thus, in some embodiments the amount of acetyl-CoA entering the lipid synthesis pathway is increased, and so too is the amount of acetyl-CoA being processed by said pathway. In further embodiments, the turnover of acetyl-CoA by the lipid synthesis (lipogenesis) pathway is promoted and/or increased. Lipid synthesis (lipogenesis) is the conversion of fatty acids and glycerol into fats, or a metabolic process through which acetyl-CoA is converted to triglyceride for storage in fat. Lipogenesis encompasses both fatty acid and triglyceride synthesis, with the latter being the process by which fatty acids are esterified to glycerol before being packaged into very-low- density lipoprotein (VLDL). Fatty acids are produced in the cytoplasm of cells by repeatedly adding two-carbon units to acetyl-CoA. Triacylglycerol synthesis occurs in the endoplasmic reticulum membrane of cells by bonding three fatty acid molecules to a glycerol molecule. Both processes take place mainly in liver and adipose tissue, but also occur to some extent in other tissues such as the gut and kidney. Fatty acid synthesis is the creation of fatty acids from acetyl-CoA and NADPH through the action of enzymes called fatty acid synthases in the cytoplasm. The glycolytic pathway which produces most of the acetyl-CoA which is converted into fatty acids also provides the glycerol with which three fatty acids can combine (by means of ester bonds) to form triglycerides (also known as “triacylglycerols”), the final product of the lipogenic process. When only two fatty acids combine with glycerol and the third alcohol group is phosphorylated with a group such as phosphatidylcholine, a phospholipid is formed.
BAB-C-P3506PCT Triglycerides are synthesised by esterification of fatty acids to glycerol. Fatty acid esterification takes place in the endoplasmic reticulum of cells by metabolic pathways in which acyl groups in fatty acyl-CoAs are transferred to the hydroxyl groups of glycerol-3-phosphate and diacylglycerol. Three fatty acid chains are bonded to each glycerol molecule. Each of the three -OH groups of the glycerol react with the carboxyl end of a fatty acid chain (-COOH). Water is eliminated and the remaining carbon atoms are linked by an -O- bond through dehydration synthesis. Both the adipose tissue and the liver can synthesise triglycerides. Those produced by the liver are secreted from it in the form of VLDL directly into blood, where they function to deliver the endogenously derived lipids to peripheral tissues. Thus, in further embodiments the flux of acetyl-CoA into the fatty acid synthesis/triglyceride synthesis pathways is promoted and/or increased. In yet further embodiments, the amount of acetyl-CoA entering the fatty acid synthesis/triglyceride synthesis pathways is increased, and so too is the amount of acetyl-CoA being processed by said pathways. The amount of fatty acids synthesised and the amount of triglycerides synthesised may also thus be increased. As described herein, the flux of acetyl-CoA into the lipid synthesis pathway (lipogenesis) increases the activity of said pathway and the amounts of resulting fatty acids/triglycerides. These fatty acids/triglycerides are subsequently used in fatty acid oxidation (also known as β- oxidation) pathways. References herein to “fatty acid oxidation” and “β-oxidation” are used interchangeably. Thus, in certain embodiments the flux of acetyl-CoA into the fatty acid/β- oxidation pathway is promoted and/or increased. It will be readily appreciated by the skilled person’s common general knowledge as well as from the disclosures herein, that said flux of acetyl-CoA into the fatty acid oxidation pathway may be indirect, i.e. as a result of increased flux of acetyl-CoA into the fatty acid synthesis/triglyceride synthesis pathways and the subsequent increased flux of triglycerides into the fatty acid/β-oxidation pathway. Fatty acid oxidation (β-oxidation) is the catabolic process by which fatty acids are broken down in the cytosol in prokaryotes and in the mitochondria in eukaryotes to generate acetyl-CoA, which enters the citric acid cycle to eventually produce ATP, and NADH and FADH2, which are co-enzymes used in the electron transport chain. The name “β-oxidation” refers to the beta carbon of the fatty acid undergoing oxidation to a carbonyl group. Β-oxidation is primarily facilitated by the mitochondrial trifunctional protein, an enzyme complex associated with the inner mitochondrial membrane, although very long chain fatty acids are oxidised in peroxisomes. β-oxidation in peroxisomes is not coupled to ATP synthesis like in mitochondria. Instead, the high-potential electrons are transferred to O
2, which yields H
2O
2. The hydrogen
BAB-C-P3506PCT peroxide is then converted to water and oxygen by the enzyme catalase, found primarily in peroxisomes and in the cytosol of erythrocytes. Peroxisomal oxidation can be induced by a high-fat diet and administration of hypolipidemic drugs like clofibrate. Without being bound by any particular theory, it is hypothesised that the amount of acetyl-CoA available for protein (including histone) acetylation is largely derived from dietary glucose via mitochondrial citrate export or acetate from various sources, and that histone acyltransferase enzymes must compete with lipid synthesis pathways for this acetyl-CoA. Thus, increased flux of acetyl-CoA into the lipid synthesis and fatty acid oxidation pathways in a cell is predicted to reduce/decrease the amount of acetyl-CoA available for protein acetylation, in particular histone acetylation, and therefore reduce/decrease/inhibit the resulting amount of protein acetylation. As such, in one embodiment the flux of acetyl-CoA into the protein acetylation pathway is inhibited and/or reduced. In a further embodiment, the flux of acetyl-CoA into the histone acetylation pathway is inhibited and/or reduced. In a yet further embodiment, the use of acetyl-CoA in histone acetylation is inhibited and/or reduced. According to these embodiments, the amount of acetyl-CoA available for protein acetylation is decreased, and so too is the amount of acetyl-CoA being used for said acetylation, in particular for histone acetylation. The amount of protein acetylation, in particular histone acetylation, may also thus be decreased/reduced. Therefore, in a further aspect of the invention there is provided a method of inhibiting and/or decreasing the flux of acetyl-CoA into the protein acetylation pathway in a cell. In one embodiment, the inhibition/decrease in acetyl-CoA flux into the protein acetylation pathway is by increasing the flux of acetyl-CoA into the lipid synthesis (lipogenesis) and fatty acid/β-oxidation pathways in the cell. Thus, according to this embodiment the flux of acetyl- CoA into the lipid synthesis and fatty acid/β-oxidation pathways in the cell is promoted and/or increased, thereby inhibiting and/or decreasing the flux of acetyl-CoA into the protein acetylation pathway. In another embodiment, the inhibition/decrease in acetyl-CoA flux into the protein acetylation pathway is by promoting and/or increasing the flux of acetyl-CoA into the cholesterol synthesis pathway (or ergosterol synthesis pathway in yeast) in the cell. According to this embodiment, the flux of acetyl-CoA into the cholesterol synthesis pathway in the cell is promoted and/or increased, thereby inhibiting and/or decreasing the flux of acetyl- CoA into the protein acetylation pathway. In a further embodiment, the inhibition/decrease in acetyl-CoA flux into the protein acetylation pathway is by promoting and/or increasing the flux of acetyl-CoA into the lipid synthesis, fatty acid/β-oxidation and cholesterol synthesis pathways. In a yet further embodiment of this aspect of the invention, the flux of acetyl-CoA
BAB-C-P3506PCT into the protein acetylation pathway may be inhibited/decreased by any other method of sequestering acetyl-CoA away from said pathway. A non-limiting example of such sequestration is by preventing the transport of acetyl-CoA into the nucleus of the cell from the cytoplasm, such as through nuclear pores. It Is further hypothesised that relative metabolite abundance will affect chromatin state, and that metabolic changes across life, across diets and across environmental exposures will profoundly impact the flux of epigenetic modifications. As ageing is characterised by a decline in metabolic homeostasis – metabolic decline is accelerated by adverse dietary exposures, including overnutrition and obesity – it may be possible to slow ageing or increase the portion of life during which an individual is healthy (their “healthspan”) by metabolic interventions. Thus, by promoting/increasing the flux of acetyl-CoA into the lipid synthesis and fatty acid oxidation pathways and/or inhibiting/reducing the flux of acetyl-CoA into the protein acetylation pathway, it is believed that an increase in “healthy” ageing may be achieved. Healthy ageing may be expressed using the term “healthspan” which is the period of an individual’s life in which they are considered ‘fit and healthy’, i.e. the period of an individual’s lifespan in which they do not suffer from any age-related diseases or disorders. Thus, while the methods described herein do not significantly alter total lifespan, they are predicted to increase and/or lengthen “healthspan”. In this regard, the inventors have shown herein that while the overall lifespan and growth of yeast in which the activity of AMPK has been increased while maintaining the level of ACC1 activity is not significantly altered compared to control yeast in which AMPK activity is not increased (as evidenced by the number of bud scars and the growth kinetics after culture/growth for a period of time), the expression of other ageing markers in said yeast cells is decreased/lower or less increased compared to control yeast. Thus, in one embodiment the cell has reduced expression of ageing markers when aged. In a further embodiment, the cell has a longer healthspan compared to a cell in which AMPK activity is not increased. According to this embodiment, the cell has the same lifespan as the control cell. These ageing markers include proteins involved in protein synthesis and homeostasis (proteostasis), in particular those involved in the misfolded protein response, such as Tom70 (in yeast; TOMM70/TOMM70A in humans) which is a subunit of the translocase of outer mitochondrial membrane complex involved in the recognition, unfolding, and translocation of preproteins into the mitochondria and is a marker of cellular senescence, Hsp104 (heat shock protein 104) which is a protein-remodelling machine that uses ATP hydrolysis to drive protein disaggregation and thus is a marker of protein misfolding and aggregation, Hsp26 (heat shock protein 26) which is a chaperone protein involved in the detection and clearance of insoluble protein deposits and thus is a marker of protein aggregation, and Nop15 which is a constituent
BAB-C-P3506PCT of 66S pre-ribosomal particles and is involved in 60S ribosomal subunit biogenesis. Other ageing markers include global/overall gene expression patterns which are known to alter throughout age due to the general dysregulation of gene expression in later life, and mitochondrial respiration rates (e.g. basal respiration rates) which decrease with age/aged cells display lower mitochondrial respiratory activity. In the context of yeast, the ageing marker may be a result of a fitness assay in which the yeast are subjected to stresses or new environments and their ability to survive, grow and proliferate is determined. Overall cellular gene expression may also be determined in such fitness assays. Also in the context of yeast, the number of bud scars displayed/present on the surface of the yeast cell may also be used as an ageing marker. Such bud scars are produced when the yeast cell divides (i.e. grows/proliferates) and are thus a replicative ageing marker which may be used as a read out for age and/or active time in culture. Bud scars may also be used in a fitness assay to determine the ability of a yeast cell to grow. In some embodiments, the ageing marker is a protein or proteins involved in protein synthesis and homeostasis (proteostasis). In one embodiment, the ageing marker is the senescence marker Tom70 and the expression of Tom 70 is reduced in the cell compared to an aged cell in which the activity of AMPK and/or ACC1 has not been increased (i.e. a control cell). In a further embodiment, the ageing marker is the protein aggregation marker HSP104 and expression is decreased compared to an aged control cell. In a yet further embodiment, the ageing marker is global/overall gene expression and said gene expression is less altered compared to an aged control cell. In a further embodiment, the ageing marker is respiration rate (e.g. the basal respiration rate and/or mitochondrial respiration rate) and said rate is increased and/or less decreased than in a control cell. In one embodiment, the cell has increased respiration when aged compared to an aged cell in which the activity of AMPK and/or ACC1 has not been increased. In a further embodiment, the respiration is the mitochondrial respiration rate, which may be measured by the oxygen consumption rate of the cell. Thus, in a yet further embodiment the respiration rate (e.g. basal respiration rate) is and/or is measured by the oxygen consumption rate of the cell. In a still further embodiment, the expression of Tom70 and/or Hsp104 are reduced in the cell, and/or the global/overall gene expression is less altered in the cell compared to an aged cell in which the activity of AMPK and/or ACC1 has not been increased (i.e. a control cell). According to these embodiments, the cell may be a yeast cell and the expression of the ageing markers compared to in a control yeast cell. The terms “aged” or “old” as used herein refer to a cell, individual or tissue/organ which Is older compared to another cell or tissue/organ from earlier in the lifespan/life cycle of the
BAB-C-P3506PCT individual, or compared to a cell or individual which has been growing or ‘alive’ for a shorter period of time. Thus, notwithstanding the definition provided herein, these terms are used in their normal meaning. In the context of yeast, an “aged” or “old/older” cell is a cell which has been through more cell divisions since birth, and may have been cultured for a period of time which is greater than a non-aged or “young/younger” cell. Thus, in the context of yeast, age may be defined as replicative age, based on the number of times a given cell has divided, and chronological age based on time in culture. Age may be determined by any method known in the art, including without limitation, using an epigenetic and/or transcriptomic clock, by measuring telomere length, by determining the number of bud scars (e.g. for yeast) and/or by determining growth kinetics. Thus, in a further embodiment the cell is aged by culturing for a period of time. In a particular embodiment, wherein the cell is a yeast cell, age is determined by the number of bud scars, i.e. the number of bud scars displayed by the yeast cell (e.g. present on the surface of the cell). In a yet further embodiment, the cell has reduced expression of ageing markers when aged by culturing for a period of time. In another embodiment, the cell is taken or isolated from an old individual, tissue or organ. In one embodiment, the cell according to the method herein is a yeast cell and when aged displays a similar number of bud scars as an aged yeast cell in which the activity of AMPK and/or ACC1 has not been increased (e.g. a control yeast cell). In a further embodiment, the cell is a yeast cell and when aged displays similar growth kinetics as an aged yeast cell in which the activity of AMPK and/or ACC1 has not been increased. In a yet further embodiment, the cell is a yeast cell and when aged displays a similar number of bud scars and displays similar growth kinetics. According to these embodiments, it will be appreciated that the number of bud scars and growth kinetics indicate the age of the yeast cell and whether it is “aged” but may not be considered as ageing markers in the context of the term as used herein. In other words, the cell according to the method herein may be aged to a similar amount/for a similar period of time to a control cell in which the activity of AMPK and/or ACC1 is not increased, despite the reduced expression of other ageing markers, such as proteins involved in protein synthesis and homeostasis or reduced alterations in overall gene expression compared to a control cell. In certain embodiments, the activity of AMP-activating protein kinase (AMPK; also known as 5’ adenosine monophosphate-activated protein kinase) is increased in the cell. AMPK is an enzyme that plays a role in cellular energy homeostasis, largely to activate glucose and fatty acid uptake and fatty acid/β-oxidation when cellular energy is low. The role of AMPK in promoting longevity and healthspan is also known (Salminen & Kaarniranta (2012) Ageing Research Reviews, 11(2):230-241, doi: https://doi.org/10.1016/j.arr.2011.12.005; and Ge et al.
BAB-C-P3506PCT (2022) Biochimie, 195:100-113, doi: https://doi.org/10.1016/j.biochi.2021.11.008), and the effects of metformin in this regard are thought to act through AMPK. Its involvement in obesity and chronic diseases is also known (Canbolat & Cakiroglu (2023) Crit. Rev. Food Sci. Nutr., 63(4):449-456, doi: https://doi.org/10.1080/10408398.2022.2087595). It belongs to a highly conserved eukaryotic protein family and its orthologues are Snf1 in yeast and SnRK1 in plants (therefore AMPK, Snf1 and SnRK1 may be used interchangeably herein). It consists of three proteins (subunits) that together make a functional enzyme, conserved from yeast to humans. It is expressed in a number of tissues, including the liver, brain and skeletal muscle. In response to binding AMP and ADP, AMPK activation leads to the stimulation of hepatic fatty acid oxidation, ketogenesis, stimulation of skeletal muscle fatty acid oxidation and glucose uptake, inhibition of cholesterol synthesis, lipogenesis, triglyceride synthesis, inhibition of adipocyte lipogenesis, inhibition of adipocyte lipolysis and modulation of insulin secretion by pancreatic β-cells. Regulation of activity is both allosteric and by post-translational modification (i.e. phosphorylation). The most well-defined mechanisms of AMPK activation are phosphorylation at the activatory site, threonine 172 (T172) of the α-subunit (or threonine 174 of the α2-subunit) and by AMP and/or ADP binding to γ-subunit Phosphorylation at threonine 172 of the α-subunit is regulated by at least three kinases and three phosphatases: namely, liver kinase B1 (LKB1), which exists in a heterotrimeric complex with STRAD and MO25; calcium-/calmodulin-dependent kinase kinase 2 (CaMKK2); TGFβ-activated kinase 1 (TAK1); protein phosphatase 2A (PP2A); protein phosphatase 2C (PP2C); and Mg2+-/Mn2+- dependent protein phosphatase 1E (PPM1E; Jeon (2016) Exp. Mol. Med., 48(7):e245, doi: https://doi.org/10.1038%2Femm.2016.81). In yeast, Snf1 (i.e. AMPK) is phosphorylated by Sak1 which is reported to be a specific kinase for AMPK. Further, ATP competitively inhibits the binding of both AMP and ADP to the γ-subunit, suggesting the ability of AMPK to act as a sensor of cellular AMP/ATP or ADP/ATP ratios. In energy-replete conditions, i.e. in the presence of low AMP/ATP and ADP/ATP ratios, phosphatases can access threonine 172 of the α-subunit and keep it in the unphosphorylated state. However, when energy is depleted, high levels of AMP and ADP bind to the AMPK γ-subunit, which prevents the phosphatases from accessing threonine 172 of the α-subunit, thus increasing its phosphorylation. In addition, the binding of AMP, but not of ADP, to the γ-subunit increases intrinsic AMPK activity by inducing its allosteric activation. While stoichiometric phosphorylation of threonine 172 can cause >100-fold activation of AMPK, this effect is amplified up to 10-fold further by allosteric activation (Hardie (2013) Diabetes, 62(7):2164-2172, doi: https://doi.org/10.2337%2Fdb13- 0368). Thus, in one embodiment the activity of AMPK is increased by modulating the activatory phosphorylation site of AMPK. In certain embodiments, the phosphorylation site is in the
BAB-C-P3506PCT catalytic domain of AMPK. In other embodiments, the phosphorylation site is in a non-catalytic domain of AMPK. In a further embodiment, the activatory phosphorylation site of AMPK is threonine 172, such as threonine 172 of the α-subunit. Thus, in a yet further embodiment AMPK activity is increased by modulating the phosphorylation of threonine 172 of the α- subunit of AMPK. In another embodiment, AMPK activity is increased by modulating the phosphorylation of threonine 174 of the α2-subunit of AMPK. In a further embodiment, AMPK activity is increased by mutating the activatory phosphorylation site of AMPK to mimic phosphorylation. In one embodiment, threonine 172, such as threonine 172 of the α-subunit of AMPK, is mutated to mimic phosphorylation. In a further embodiment, threonine 174 of the α2-subunit of AMPK is mutated. In a yet further embodiment, AMPK activity is increased by mutating the catalytic domain. Such mutations in the catalytic domain include those which activate AMPK in the absence of or independently of the activity of an activating subunit, such as to remove the requirement for said activating subunit to be activated for AMPK activity. In one embodiment, Snf1 activity is increased by mutating leucine 183, such as mutating leucine 183 to isoleucine (i.e. an L183I mutation). In a still other embodiment, AMPK activity is increased by modulating the binding of ATP, AMP or ADP to the γ-subunit of AMPK, such as promoting or increasing the binding of AMP or ADP to the γ-subunit thereby preventing access of phosphatase enzymes to threonine 172 of the α-subunit. In another embodiment, AMPK activity is increased by modulating the binding of AMP to the γ-subunit thereby inducing allosteric activation of AMPK. Thus, in one embodiment AMPK activity is increased by allosteric modulation. In still further embodiments, AMPK activity is increased by increasing the activity of (e.g. by increasing the expression of) an upstream activatory kinase of AMPK. In some embodiments, AMPK activity is increased by increasing the activity and/or the expression of one or more of LKB1, CaMKK2 and TAK1 kinases. In another embodiment, AMPK activity is increased by increasing the activity and/or expression of Sak1. According to this embodiment, the cell is a yeast cell. Thus, in certain embodiments AMPK activity is increased by increasing the activity and/or expression of an AMPK-specific activatory kinase. In further embodiments, the activity of AMPK is increased using an activator. Such activators may be selected from direct and indirect activators of AMPK and include small molecule chemical activators. Small molecule activators of AMPK may be selected from one or more of: metformin, a guanidine derivative, MK-3903 (compound 42) and/or EX229 (compound 991). Guanidine and its isoprenyl derivative, galegine, are natural products from the plant Galega officinalis, and metformin and phenformin are biguanides, synthetic derivatives of guanidine. AMPK activation by metformin, phenformin and galegine requires increases in AMP and/or ADP, because all three drugs fail to activate AMPK in cells expressing an AMPK γ-subunit with a mutation (R531G) that renders the complex AMP/ADP insensitive.
BAB-C-P3506PCT Metformin in particular may act indirectly on AMPK activity (Hardie (2013)). Thus, in one embodiment the AMPK activator may be an indirect activator, such as an indirect small molecule activator of AMPK. In an alternative embodiment, the activator may be a direct AMPK activator, such as a direct small molecule activator of AMPK (i.e. a small molecule that binds to AMPK or one of its subunits directly). In one embodiment, the activator of AMPK is selected from one or more of: metformin, a guanidine derivative, MK-3903 (compound 42) and/or EX229 (compound 991). In one embodiment, the activator is metformin. In another embodiment, the activator is MK-3903 (compound 42) and/or EX229 (compound 991). As will be readily appreciated by the skilled person, any combination of activators disclosed herein may be used, as well as any other AMPK activators known in the art, either alone or in combination. Further examples of AMPK activators are reviewed and discussed in Hardie (2013), which are hereby incorporated by reference. In particular embodiments, the activity of acetyl-CoA carboxylase (ACC1) in the cell is maintained or increased. ACC is a biotin-dependent enzyme that catalyses the irreversible carboxylation of acetyl-CoA to produce malonyl-CoA through its two catalytic activities, biotin carboxylase and carboxyltransferase. ACC is a multi-subunit enzyme in most prokaryotes and in the chloroplasts of most plants and algae, whereas it is a large, multi-domain enzyme in the cytoplasm of most eukaryotes (e.g. humans, mice and yeast). ACC provides the malonyl-CoA substrate for the biosynthesis of fatty acids. ACC activity can be controlled at the transcriptional level (i.e. expression) as well as by small molecule modulators and post- translational modification (e.g. phosphorylation and/or allosteric transformation by citrate or palmitoyl-CoA). The human genome contains the genes for two different ACCs – ACACA which encodes ACC1 protein, and ACACB which encodes ACC2. ACC1 is found in the cytoplasm of all cells but is enriched in lipogenic tissue, such as adipose tissue and lactating mammary glands, where fatty acid synthesis is important (Kim et al. (1996) Biochem. And Biophys. Res. Comms., 225(2):647-653, doi:
oxidative tissues, such as the skeletal muscle and the heart, the ratio of expressed ACC2 is higher. ACC1 and ACC2 are both highly expressed in the liver where both fatty acid oxidation and synthesis are important (Barber et al. (2005) Biochimica et Biophysica Acta (BBA) – Molecular and Cell Biology of Lipids, 1733(1):1-28, doi: https://doi.org/10.1016% 2Fj.bbalip.2004.12.001). The differences in tissue distribution indicate that ACC1 maintains regulation of fatty acid synthesis whereas ACC2 mainly negatively regulates fatty acid oxidation (β-oxidation). Mammalian ACC1 and ACC2 are regulated transcriptionally by multiple promoters which mediate ACC abundance in response to nutritional status. Activation of gene expression through different promoters results in alternative splicing. The sensitivity of ACC expression and splicing to nutritional status results from the control of promoters by
BAB-C-P3506PCT transcription factors such as sterol regulatory element-binding protein 1, controlled by insulin at the transcriptional level, and ChREBP, which increases in expression with high carbohydrates diets (Field et al. (2002) The Biochemical Journal, 368(3):855-864, doi: https://doi.org/10.1042%2FBJ20020731; and Ishii et al. (2004) PNAS, 101(44):15597-1602, doi: https://doi.org/10.1073%2Fpnas.0405238101). Through a feed-forward loop, citrate allosterically activates ACC (Martin & Vagelos (1962) The Journal of Biological Chemistry, 237(6):1787-1792, doi: https://doi.org/10.1016%2FS0021-9258%2819%2973938- 6). Citrate may increase ACC polymerization to increase enzymatic activity. Other allosteric activators include glutamate and other dicarboxylic acids (Boone et al. (2000) The Journal of Biological Chemistry, 275(15):10819-10825, doi: https://doi.org/10.1074%2Fjbc.275.15. 10819). Long and short chain fatty acyl-CoAs act as negative feedback inhibitors of ACC, for example, one such negative allosteric modulator is pamitoyl-CoA (Pham et al. (2022) Genetics, 221(4), doi: https://doi.org/10.1093%2Fgenetics%2Fiyac086). The main cause of ACC phosphorylation is due to a rise in AMP levels when the energy status of the cell is low, leading to the activation of AMPK. AMPK is the main kinase regulator of ACC, able to phosphorylate a number of serine residues on both isoforms of ACC, leading to an inhibition of their activity (Park et al. (2002) Journal of Applied Physiology, 92(6):2475-2482, doi: https://doi.org/10.1152%2Fjapplphysiol.00071.2002). On ACC1, AMPK phosphorylates serine 79 (the main inhibitory phosphorylation site of mammalian ACC1; serine 1157 (S1157) in yeast Acc1), serine 1200 and serine 1215. Protein kinase A can also phosphorylate ACC, with a much greater ability to phosphorylate ACC2 than ACC1. Serine 80 and serine 1263 on ACC1 may also serve as sites of regulatory phosphorylation. When insulin binds to its receptors on the cellular membrane, it activates protein phosphatase 2A (PP2A) to dephosphorylate the enzyme; thereby removing the inhibitory effect. Furthermore, insulin induces a phosphodiesterase that lowers the level of cAMP in the cell, thus inhibiting PKA, and also inhibits AMPK directly. Thus, without being bound by any particular theory, it is hypothesised that by increasing AMPK activity, but maintaining or increasing the activity of ACC1 in a cell (i.e. preventing the ability of AMPK to inhibit ACC1), both the longevity effects of increased AMPK activity and the increased flux of acetyl-CoA into the lipid synthesis and fatty acid oxidation pathways can be achieved. A further hypothesis is that autophagy promoted by ACC1-mediated lipogenesis, which has previously been shown to be involved in the survival of a yeast chronological ageing model (Gross et al. (2019) J Biol. Chem., 294(32):12020-12039, doi: https://doi.org/10.1074/ jbc.ra118.007020), either alone or potentially in combination with AMPK-induced autophagy, may contribute to the improvements of healthspan seen herein. Thus, in certain embodiments the methods described herein comprise preventing the inhibitory effects of AMPK on ACC1
BAB-C-P3506PCT activity. In a particular (preferred) embodiment, only the inhibition/reduction of ACC1 activity by AMPK is prevented, and the ability of AMPK to phosphorylate and regulate (i.e. inhibit/reduce) the activity of ACC2 is unaffected. In a further embodiment, the activity of ACC2 is not altered in the cell, such as is not increased. Thus, according to these embodiments the turnover of acetyl-CoA and subsequent β-oxidation of lipid metabolites is enhanced/increased by virtue of increased AMPK activity with maintained/increased ACC1 activity and decreased ACC2 activity (by inhibitory phosphorylation by AMPK; ACC2 inhibits β-oxidation in mitochondria). In an alternative embodiment, the inhibition/reduction of ACC1 and ACC2 activity by AMPK may be prevented. Several previous studies have investigated the link between AMPK activation and ACC1/2 activity but have failed to observe the phenotypic effects demonstrated herein. In particular, Lally et al. (2018) Cell Metabolism, 29(1):174-182 (doi: https://doi.org/10.1016/ j.cmet.2018.08.020) investigates ACC1/2 non-phosphorylatable mutants in the presence of fructose that reduces AMPK activation, and shows these promote pathological de novo lipogenesis. However, Lally et al. does not investigate lipogenesis in ACC1 single mutant cells or disclose any break in the link between AMPK and ACC1 activity alone as described herein. Fullerton et al. (2013) Nat. Med., 19(12):1649-1654 (doi:
similarly discloses experiments performed in mutants in which both ACC1 and ACC2 phosphorylation are prevented downstream of AMPK activity, and there are no results therein demonstrating an effect of AMPK activation when ACC1 phosphorylation is prevented, i.e. there is no disclosure of breaking the link between AMPK and ACC1 activity alone as described herein. Hawley et al. (2016) Diabetes, 65(9):2784-2794 (doi: https://doi.org/ 10.2337/db16-0058) discloses a beneficial effect of ACC inhibition mediated through AMPK activity in reducing lipid levels. However, all experiments in Hawley et al. are performed using ACC1/2 double mutant cells (used therein purely to demonstrate that the effect does not occur if ACC1 and ACC2 cannot be inhibited) and no effects of AMPK activation with non- phosphorylatable ACC1 alone are disclosed. Therefore, in one embodiment of the present invention the activity of ACC1 in the cell is maintained or increased. This is despite the increased activity of AMPK in the cell which would otherwise lead to increased inhibitory phosphorylation of ACC1 and a subsequent reduction in activity. As described hereinbefore, such “maintained or increased” activity of ACC1 is by virtue of breaking the link between AMPK activity and ACC1 regulation by its phosphorylation by AMPK. Thus, in certain embodiments the activity of ACC1 is maintained or increased by modulating the phosphorylation of the inhibitory site of ACC1. In one embodiment, the inhibitory site is serine 79 (S79). In a further embodiment, ACC1 activity is maintained or
BAB-C-P3506PCT increased by modulating the phosphorylation of S79. In one embodiment, modulating the phosphorylation of the inhibitory site of ACC1 comprises dephosphorylating said inhibitory site, such as S79. In a further embodiment, modulating the phosphorylation of the S79 residue of ACC1 comprises dephosphorylating said S79 residue. In another embodiment, modulating the phosphorylation of the inhibitory site of ACC1 comprises preventing phosphorylation of said inhibitory site, such as S79, in particular preventing the phosphorylation of S79. In a still other embodiment, modulating the phosphorylation of the inhibitory site of ACC1 comprises mutating said inhibitory site such that it cannot be phosphorylated, such as cannot be phosphorylated by AMPK. Mutations which can be made to achieve these effects will be readily recognised by the skilled person and are known in the art, such a mutating S79 to a non-phosphorylatable amino acid, examples of which are known in the art. In one particular embodiment, the mutation of the S79 residue of ACC1 such that it cannot be phosphorylated is S79A. In yet further embodiments, ACC1 activity is maintained or increased by modulating the conformation of ACC1. Such modulation of ACC1 conformation may be by preventing the blocking of the active site upon S79 phosphorylation, and/or by preventing a change into an inactive conformation/forcing maintenance of the active conformation independently of phosphorylation status. The access of AMPK to S79 of ACC1 may also be prevented, thereby preventing the inhibitory phosphorylation by AMPK. Thus, in one embodiment ACC1 activity is maintained or increased by blocking AMPK access to the inhibitory site of ACC1, in particular access to S79. In still further embodiments, ACC1 activity is maintained or increased by preventing the inhibitory effects of S79 phosphorylation, i.e. such that phosphorylation of S79 by AMPK does not lead to the regulation of ACC1 activity. In certain embodiments, preventing phosphorylation of the S79 residue of ACC1 comprises an inhibitor of phosphorylation. In further embodiments, preventing phosphorylation of the inhibitory site of ACC1 comprises an inhibitor of phosphorylation. Such inhibitors may be selected from direct or indirect inhibitors of ACC1 phosphorylation, specific or non-specific inhibitors of phosphorylation, and selective inhibitors of ACC1 phosphorylation. In one embodiment, the inhibitor of ACC1 phosphorylation is a small molecule inhibitor. In a further embodiment, the inhibitor may be an indirect inhibitor, such as an indirect small molecule inhibitor of ACC1 phosphorylation. Indirect inhibitors include those which act upstream of ACC1 phosphorylation on the kinase responsible for said phosphorylation. However, it will be appreciated from the disclosures herein and predicted mechanism of enhancing/increasing the turnover of acetyl-CoA and subsequent β-oxidation of lipid metabolites by virtue of increased AMPK activity with maintained/increased ACC1 activity, that such inhibitors are not AMPK inhibitors. In an alternative embodiment, the inhibitor may be a direct inhibitor, such as a direct small molecule inhibitor of ACC1 phosphorylation (i.e. a small molecule that binds to
BAB-C-P3506PCT ACC1 of one of its subunits directly to prevent phosphorylation). In particular embodiments, the inhibitor of ACC1 phosphorylation is a selective inhibitor of ACC1 phosphorylation, i.e. it does not affect the phosphorylation of ACC2. Thus, in one embodiment the inhibitor is selective for ACC1 compared to ACC2. Such inhibitors will be readily identified by the skilled person in the art, for example using methods such as that described in Pandey et al. (2018) PNAS, 115(44):E10505-E10514 (doi: https://doi.org/10.1073/pnas.1804897115). As will be readily appreciated, the embodiments hereinbefore may also be applied to ACC2 according to embodiments wherein the inhibition/reduction of ACC1 and ACC2 activity by AMPK may be prevented, e.g. the inhibitory phosphorylation site of ACC2 may be dephosphorylated or mutated, the conformation of ACC2 may be modulated as described herein, the access of AMPK to the inhibitory site of ACC2 may be prevented, or the inhibitor of may be an inhibitor of ACC2 phosphorylation. However, it will also be appreciated from the disclosures herein and predicted mechanism of enhancing/increasing the turnover of acetyl- CoA and subsequent β-oxidation of lipid metabolites by virtue of increased AMPK activity with maintained/increased ACC1 activity and decreased ACC2 activity, that such ACC2 inhibitors will not be selective for ACC2 and may therefore also inhibit phosphorylation of ACC1. The cell may be a yeast or a mammalian cell, such as a human or mouse cell. Thus, in one embodiment the cell is a mammalian cell. In a further embodiment, the cell is a human cell. In an alternative embodiment, the cell is a mouse cell. In another embodiment, the cell is a non-human primate cell. In further embodiments, the cell is a mammalian liver cell/hepatocyte, such as a human hepatocyte, a non-human primate hepatocyte or a mouse hepatocyte. In yet further embodiments, the cell is a mammalian adipocyte (fat cell), such as a human adipocyte, a non-human primate adipocyte or a mouse adipocyte. In one embodiment, the methods described herein are performed in vitro, such as in culture. Thus, in a further embodiment the cell is in vitro. In another embodiment the methods are performed in vivo, for example when rejuvenating a cell as described herein. Thus, in a yet further embodiment the cell is in vivo, such as in vivo in a subject in need thereof. Methods of Rejuvenation Therefore, according to a further aspect of the invention there is provided a method of rejuvenating a cell, comprising promoting and/or increasing acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in the cell according to the method described herein.
BAB-C-P3506PCT As described hereinbefore, as ageing is characterised by a decline in metabolic homeostasis – metabolic decline is accelerated by adverse dietary exposures, including overnutrition and obesity – it may be possible to slow ageing or increase the portion of life during which an individual is healthy (their “healthspan”) by metabolic interventions, such as the promotion/increase of acetyl-CoA flux into the lipid synthesis and fatty acid oxidation pathways. Furthermore, by promoting/increasing said acetyl-CoA flux it is believed that the amount of acetyl-CoA available for protein acetylation will be reduced/decreased. Thus, by performing the method described herein a cell may be rejuvenated to a younger state, in particular a younger metabolic state. In one embodiment, the cell is rejuvenated to a younger state as determined using one or more ageing marker as described hereinbefore, such as wherein expression of one or more ageing marker is reduced in the rejuvenated cell. In a further embodiment, the cell is rejuvenated to a younger metabolic state. Such metabolic state may be measured by the flux of acetyl-CoA into the lipid synthesis and fatty acid oxidation pathways. In some embodiments, the rejuvenated cell has a longer healthspan compared to a non-rejuvenated cell, such as compared to a non-rejuvenated cell from the same tissue or organ from which the rejuvenated cell is derived/obtained. In further embodiments, the rejuvenated cell does not have a significantly altered total lifespan compared to a non- rejuvenated cell. In particular embodiments, the non-rejuvenated cell is a cell in which AMPK activity is not increased (e.g. the non-rejuvenated cell may be a control cell). Thus, in one aspect there is provided a method of increasing the healthspan of a cell, comprising promoting and/or increasing acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in the cell according to the methods described herein. In certain embodiments, the cell has an increased healthspan compared to a cell in which AMPK activity has not been increased. In a further aspect of the invention, there is provided a cell with a younger metabolic state obtainable or obtained by the methods described herein. In another aspect, there is provided a rejuvenated cell obtainable or obtained by the methods described herein. In a yet further aspect, there is provided a cell with a longer healthspan obtainable or obtained by the methods described herein. According to these aspects, the cell may comprise a younger metabolic state, is rejuvenated or comprises a longer healthspan compared to a cell in which AMPK activity is not increased according to the methods described herein (e.g. a control cell). Thus, in one embodiment the cell with a younger metabolic state comprises increased acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways compared to a cell in which AMPK activity is not increased according to the methods described herein. In a further embodiment, the cell has a younger metabolic state compared
BAB-C-P3506PCT to a cell in which AMPK activity is not increased. As described hereinbefore, such metabolic state may be measured by the flux of acetyl-CoA into the lipid synthesis and fatty acid oxidation pathways. In a yet further embodiment, the rejuvenated cell comprises increased acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways. In a still further embodiment, the cell is rejuvenated compared to a cell in which AMPK is not increased. In another embodiment, the cell with a longer healthspan comprises increased acetyl coenzyme A (acetyl-CoA) flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways compared to a cell in which AMPK activity is not increased according to the methods described herein. In a further embodiment, the cell comprises a longer healthspan compared to a cell in which AMPK activity is not increased. The term “healthspan” may be defined as hereinbefore. In one embodiment, the method of rejuvenation is performed in vitro, such as wherein the cell to be rejuvenated is in vitro, e.g. in culture. In a further embodiment, the cell is in vitro, such as wherein the method is performed in vitro. In another embodiment, the method of rejuvenation is performed in vivo in a subject in need thereof, such as wherein the cell is in vivo, e.g. in the subject in need thereof. In a yet further embodiment, the cell is in vivo, such as wherein the method is performed in vivo in a subject in need thereof. In some embodiments, the cell is a mammalian cell, such as a human cell, a mouse cell or a cell from a non-human primate. In further embodiments, the cell is a mammalian liver cell/hepatocyte, such as a human hepatocyte, a non-human primate hepatocyte or a mouse hepatocyte. In yet further embodiments, the cell is a mammalian adipocyte (fat cell), such as a human adipocyte, a non-human primate adipocyte or a mouse adipocyte. Pharmaceutical Compositions According to another aspect of the invention, there is provided a pharmaceutical composition comprising a combination of an activator of AMPK and an inhibitor of ACC1 phosphorylation, such as a small molecule activator of AMPK and a small molecule inhibitor of ACC1. Generally, the present pharmaceutical compositions will be utilised with pharmacologically appropriate excipients or carriers. Typically, these excipients or carriers include aqueous or alcoholic/aqueous solutions, emulsions or suspensions, including saline and/or buffered media. Parenteral vehicles include sodium chloride solution, Ringer’s dextrose, dextrose and sodium chloride and lactated Ringer’s. Suitable physiologically-acceptable adjuvants, if necessary to keep a composition comprising the targeting moiety specific for a tissue or organ as defined herein in a discrete location (e.g. within a tissue or organ of interest), may be chosen from thickeners such as carboxymethylcellulose, polyvinylpyrrolidone, gelatine and
BAB-C-P3506PCT alginates. Intravenous vehicles include fluid and nutrient replenishers and electrolyte replenishers, such as those based on Ringer’s dextrose. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents and inert gases, may also be present (Mack (1982) Remington’s Pharmaceutical Sciences, 16
th Edition). Thus, in certain embodiments, the pharmaceutical composition optionally further comprises one or more pharmaceutically acceptable diluents, excipients and/or carriers. As will be appreciated from the disclosures herein, such a pharmaceutical composition comprising a combination of an AMPK activator and ACC1 phosphorylation inhibitor will find particular utility in performing the methods of the invention by promoting and/or increasing acetyl-CoA flux into the lipid synthesis (lipogenesis) and fatty acid oxidation pathways in a cell and/or in a subject in need thereof. Administration of the pharmaceutical composition may be by any suitable route or method known in the art, for example systemically, e.g. enteral or parenteral, such as via intravenous infusion, or locally, such as directly into the tissue or organ to be treated/rejuvenated, e.g. by topical administration. In one embodiment, the activator of AMPK is a small molecule activator as described herein. In a further embodiment, the activator is selected from one or more of: metformin, a guanidine derivative, MK-3903 (compound 42) and/or EX229 (compound 991). Therapeutic Uses & Methods According to a yet further aspect of the invention, there is provided the pharmaceutical composition described herein for use in the treatment of an age-related disease or disorder in a subject in need thereof. In another aspect, there is provided the pharmaceutical composition for use in the treatment of ageing. In a still further aspect, there is provided a method of treating an age-related disease or disorder in a subject in need thereof, said method comprising administering the pharmaceutical composition described herein to the subject. In another aspect, there is provided a method of treating ageing in a subject in need thereof, said method comprising administering the pharmaceutical composition described herein. Such age-related diseases or disorders include metabolic diseases or disorders typically associated with age, such as type 2 diabetes mellitus, insulin resistance, non-alcoholic fatty liver disease, vascular diseases and chronic inflammation. A decline in metabolic homeostasis is also associated with age. Thus, in certain embodiments the age-related disease or disorder is a metabolic disease/disorder. In other embodiments, the age-related disease or disorder is
BAB-C-P3506PCT a disease/disorder of the liver. In yet other embodiments, the age-related disease or disorder is a disease/disorder of the pancreas. In still other embodiments, the age-related disease or disorder is a neurological disease/disorder, such as a neurodegenerative disease/disorder. Thus, in some embodiments the ageing is neurological ageing. In other embodiments, the ageing is metabolic ageing, such as ageing of the liver and/or pancreas. In another aspect of the invention, there is provided the use of a combination of an activator of AMPK and an inhibitor of ACC1 phosphorylation as described herein in the manufacture of a medicament, in particular in the manufacture of a medicament for the treatment of an age- related disease or disorder. In one embodiment, the medicament is for the treatment of a metabolic disease or disorder. In another embodiment, the medicament is for the treatment of a neurological disease/disorder, such as a neurodegenerative disease/disorder. In a yet further embodiment, the medicament is for the treatment of ageing as described herein. It will be appreciated that references herein to a patient or subject relate equally to animals and humans and that the invention may find particular utility in veterinary treatment of any of the above mentioned diseases, disorders and conditions which are also present in said animals. It will also be appreciated that references herein to “treatment” include such terms as “amelioration”, “prevention”, “reversal” and “suppression”. Furthermore, such references include administration of the combination of an activator of AMPK and an inhibitor of ACC1 phosphorylation described herein or the pharmaceutical composition comprising said combination prior to the onset of the disease or disorder, e.g. wherein the subject is at risk of the disease or disorder. Administration of the combination or pharmaceutical composition as defined herein may also be anticipated after the induction event of the injury, damage, disease or disorder, either before clinical presentation of said disease or disorder, or after symptoms manifest. Such references further include performing the method of promoting and/or increasing acetyl-CoA flux into the lipid synthesis (lipogenesis) and fatty acid oxidation (β- oxidation) pathways in a cell as defined herein in vivo either prior to the onset of the disease or disorder, or after the induction event of the disease or disorder. Unless defined otherwise, all technical and scientific terms used herein have the meaning commonly understood by a person skilled in the art to which this invention belongs. As used herein, the term “about” when used herein includes up to and including 10% greater and up to and including 10% lower than the value specified, suitably up to and including 5% greater
BAB-C-P3506PCT and up to and including 5% lower than the value specified, especially the value specified. The term “between” as used herein includes the values of the specified boundaries. Throughout the specification and the claims which follow, unless the context requires otherwise, the word “comprise”, and variations thereof such as “comprises” and “comprising”, will be understood to imply the inclusion of a stated integer, step, group of integers or group of steps but not to the exclusion of any other integer, step, group of integers or group of steps. In addition, as used herein and the appended claims, the singular forms “a”, “an” and “the” include plural referents unless the content clearly dictates otherwise. Thus, for example reference to “a cell in which AMPK activity has been promoted/increased” includes two or more such cells, or reference to “an activator of AMPK” or “an inhibitor of ACC1 phosphorylation” include two or more such activators or inhibitors. It will be understood that all embodiments described herein may be applied to all aspects of the invention and vice versa, and such combinations would be readily apparent from the description provided herein and to those skilled in the art. Other features and advantages of the present invention will be apparent from the description provided herein. It should be understood, however, that the description and the specific examples while indicating preferred embodiments of the invention are given by way of illustration only, since various changes and modifications will become apparent to those skilled in the art. The invention will now be described using the following, non-limiting examples: EXAMPLES Materials and Methods Detailed and updated protocols may be found at https://www.babraham.ac.uk/our- research/epigenetics/jon-houseley/protocols. The detailed methods and materials below are adapted from those in Horkai et al. (2023) bioRxiv, doi: https://doi.org/10.1101/ 2022.07.19.500645). Yeast Culture and Labelling Yeast strains were constructed by standard methods and are listed in Table 1, oligonucleotide sequences are given in Table 2. Plasmid templates were pFA6a-GFP-KanMX4, pYM-N14, pAW8-mCherry, pFA6a-HIS3 and pFA6a-URA3 (Houseley & Tollervey (2011) Nucleic Acids Res, 39(2):8778-8791, doi: https://doi.org/10.1093/nar/gkr589; Janke et al. (2004) Yeast, 21(11):947-962, doi: https://doi.org/10.1002/yea.1142; Longtine et al. (1998) Yeast, 14(10):
BAB-C-P3506PCT 953-961, doi: https://doi.org/10.1002/(sici)1097-0061(199807)14:10%3C953::aid-yea293% 3E3.0.co;2-u; and Watson et al. (2008) Gene, 407 (1-2):63-74, doi: https://doi.org/10.1016/ j.gene.2007.09.024). All cells were grown in YPD media (2% peptone, 1% yeast extract, 2% glucose) at 30°C with shaking at 200 rpm. Media components were purchased from Formedium and media was sterilised by filtration. MEP experiments were performed as described with modifications (Cruz et al. (2018) eLife, 7:e34081, doi: https://doi.org/10.7554/elife.34081): cells were inoculated in 4ml YPD and grown for 6-8 hours then diluted in 25ml YPD and grown for 16-18 hours to 0.2-0.6x10
7 cells/ml in 50ml Erlenmeyer flasks. 0.125x10
7 cells per aged culture were harvested by centrifugation (15 s at 13,000g), washed twice with 125µl PBS and re-suspended in 125µl of PBS containing ~3mg/ml Biotin-NHS (Pierce 10538723). Cells were incubated for 30 min on a wheel at room temperature, washed once with 125µl PBS and re-suspended in 125µl YPD then inoculated in 125ml YPD at 1x10
4 cells/ml in a 250ml Erlenmeyer flask (FisherBrand FB33132) sealed with Parafilm. 1µM β-estradiol (from stock of 1mM Sigma E2758 in ethanol) was added after 2h. An additional 0.125x10
7 cells were harvested from the log phase culture while biotin labelling reactions were incubating at room temperature. Cells were harvested by centrifugation for 1 min, 4600rpm, immediately fixed by resuspension in 70% ethanol and stored at -80°C. To minimise fluorophore bleaching in culture, the window of the incubator was covered with aluminium foil, lights on the laminar flow hood were not used during labelling and tubes were covered with aluminium foil during biotin incubation. Lifespan assays and competition assays in the MEP background were performed as previously described (Cruz et al. (2018); and Frenk et al. (2017) Aging Cell, 16(3):602-604, doi:
CRISPR Point Mutation Protocol The Snf1
L183I point mutation strains were generated as follows using CRISPR: Guides were generated to contain approximately 20nt of specific sequence adjacent to a PAM sequence. Since Cas9 cleaves 3-4nt away from the PAM in the guide sequence, optimal Snf1
L183I guide sites were selected using the Wyrick lab online yeast Cas9 guide site tool (Table 2): http://wyrickbioinfo2.smb.wsu.edu/crispr.html. The guide sequence was then cloned into an expression vector (pML104, from the Wyrick lab) that also expresses Cas9. The guide sequence was made by annealing two oligos designed by the tool above and cloning into pML104 digested with BclI and SwaI.
BAB-C-P3506PCT To digest the pML104 plasmid mini prep pML104 using the NEB Monarch PCR and DNA Cleanup Kit (#T1030S), grown in dam- E. coli. (as BcII is methylation sensitive). Then in a PCR machine, 1μg of plasmid was digested overnight at 25°C with 1µL SwaI in 50µL total volume of NEBuffer 3.1. Next, 1µL BclI, mix was added by pipetting and incubated for 2 hours in PCR machine at 50°C with the lid at 60°C. Products were run on an 1.2% agarose gel and gel extracted using the NEB Monarch DNA Gel Extraction Kit (#T1020S), eluting the DNA in 50µL. The repair template oligo (~100nt) contained the Snf1
L183I mutation and a silent PAM sequence (NGG or NAG) mutation in the middle. Mutating the PAM site is critical to stop Cas9 from cleaving the same site after the desired target mutation is introduced. Once plasmid is digested and purified, oligos were annealed by mixing 10μL of each guide cloning oligo (100µM), 10µL NEBuffer 2 (or 2.1), and 70µL water. They were then heated to 90°C and allowed to cool on bench before diluting 1:100. 1μL of digested pML104, 1µL 1:100 annealed oligos, and 6µL water were mixed and heated to 50°C then cooled to room temperature. Then oligos were ligated using 1µL T4 DNA ligase buffer and 1µL T4 DNA ligase at room temperature for 1 hour. Cells were transformed at 42°C for 40 min and plated on ampicillin with DH5 alpha competent cells. Four colonies were miniprepped and putative positive clones were verified by sanger sequencing from M13 rev. Next, 1μL of pML104-Snf1
L183I was added to 1µL 100μM repair oligo in MEP a Acc1-S1157A and MEP a SAK1 oe strains and cells were transformed. Cells were then plated on a galactose -uracil plate as Cas9 is on a galactose promoter. Colonies were then re-streaked after a few days to another galactose -uracil plate. Colonies were then streaked on a 5-Fluoroorotic Acid (5-FOA) plate. Positive colonies were then amplified using HH65 and HH66 oligos to generate a PCR product that covers the mutation site. Products were PCR purified using NEB Monarch PCR and DNA Cleanup Kit (#T1030S), and subsequently Sanger sequenced. Cell Purification Percoll gradients (1-2 per sample depending on harvest density) were formed by vortexing 1ml Percoll (Sigma P1644) with 110µl 10x PBS in 2ml tubes and centrifuging 15 min at 15,000g, 4°C. Ethanol fixed cells were defrosted and washed once with 1 volume of cold PBSE (PBS + 2mM EDTA) before resuspension in ~100µl cold PBSE per gradient and layering on top of the pre-formed gradients. Gradients were centrifuged for 4 min at 2,000g, 4°C, then the upper phase and brown layer of cell debris removed and discarded. 1ml PBSE was added,
BAB-C-P3506PCT mixed by inversion and centrifuged 1 min at 2,000g, 4°C to pellet the cells, which were then re-suspended in 1ml PBSE per time point (re-uniting samples where split across two gradients). 25µl Streptavidin microbeads (Miltenyi Biotech 1010007) were added and cells incubated for 5 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was loaded on a QuadroMACS magnet and equilibrated with cold PBSE in 4°C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 1ml cold PBSE and eluted with 1ml PBSE using plunger. Cells were re-loaded on the same columns after re-equilibration with ~500µl PBSE, washed and re-eluted, and this process repeated for a total of three successive purifications. After addition of Triton X-100 to 0.01% to aid pelleting, cells were split into 2 fractions in 1.5ml tubes, pelleted for 30s at 20,000g, 4°C, frozen on N2 and stored at -70°C. For live purification, used for fitness viability assays, cells were pelleted, washed twice with synthetic complete 2% glucose media, then resuspended in 2ml final volume of the same media and incubated for 5 min on a rotating wheel with 10µl MyOne streptavidin magnetic beads (Thermo), isolated using a magnet and washed five times with 1ml of the same media. For colony formation assays, cells were streaked on a YPD plate and individual cells moved to specific locations using a Singer MSM400 micromanipulator. Colony size was measured 24 hours later on the screen of the micromanipulator imaging with a 4x objective. RNA Extraction Cells were re-suspended in 50µl Lysis/Binding Buffer (from mirVANA kit, Life Technologies AM1560), and 50µl 0.5µm zirconium beads (Thistle Scientific 11079105Z) added. Cells were lysed with 5 cycles of 30s 6500 ms
-1 / 30s on ice in an MP Fastprep bead beater or for 3 min at power 12 in a Bullet Blender (ThermoFisher) in cold room, then 250µl Lysis/Binding buffer was added followed by 15µl miRNA Homogenate Additive and cells were briefly vortexed before incubating for 10 minutes on ice. 300µl acid phenol : chloroform was added, vortexed and centrifuged 5 min at 13,000g at room temperature before extraction of the upper phase. 400µl room temperature ethanol and 2µl glycogen (Sigma G1767) were added and mixture incubated for 1 hour at -30°C before centrifugation for 15 minutes at 20,000g, 4°C. The pellet was washed with cold 70% ethanol and re-suspended in 10µl water. 1µl RNA was glyoxylated and analysed on a BPTE mini-gel, and RNA was quantified using a Qubit® RNA HS Assay Kit. 150ng RNA was used to prepare libraries using the NEBNext Ultra II Directional mRNAseq kit with poly(A)+ purification module (NEB E7760, E7490) as described with modifications:
BAB-C-P3506PCT Reagent volumes for elution from poly(T) beads, reverse transcription, second strand synthesis, tailing and adaptor ligation were reduced by 50%; libraries were amplified for 13 cycles using 2µl each primer per 50µl reaction before two rounds of AMPure bead purification at 0.9x and elution in 11µl 0.1x TE prior to quality control using a Bioanalyzer HS DNA ChIP (Agilent) and quantification using a KAPA Library Quantification Kit (Roche). Sequencing and Bioinformatics Libraries were sequenced by the Babraham Institute Sequencing Facility using a NextSeq 500 instrument on 75 bp single end mode. After adapter and quality trimming using Trim Galore (v0.6.6), RNA-seq data was mapped to yeast genome R64-1-1 using HISAT2 v2.1.0 (Kim et al., 2019) by the Babraham Institute Bioinformatics Facility. Mapped data was imported into SeqMonk v1.47.0 (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) and quantified for log
2 total reads mapping to the antisense strand of annotated open reading frames (opposite strand specific libraries), excluding the mtDNA and the rDNA locus, but with probes included to each strand of the rDNA intergenic spacer regions. Read counts were adjusted by Size Factor normalisation for the full set of quantified probes (Anders and Huber, 2010). Where indicated, a quantile normalisation was additionally applied using the ‘Match Distribution’ function in Seqmonk. MA plots were generated in GraphPad Prism (v9.2.0) comparing mean and difference for each gene between two conditions. Probes with very low numbers of reads post normalisation in the control condition were filtered as change between datasets cannot be accurately quantified in this case. Slope values were determined from the same filtered datasets using the lm(difference~mean) function in R. Flow Cytometry Cell pellets were re-suspended in 240µl PBS and 9μl 10% triton X-100 containing 0.3µl of 1mg/ml Streptavidin conjugated with Alexa Fluor® 647 (Life technologies) and 0.6μl of 1mg/ml Wheat Germ Agglutinin (WGA) conjugated with CF®405S (Biotium). Cells were stained for 10 min at RT on a rotating mixer while covered with aluminium foil, washed once with 300μl PBS containing 0.01% Triton X-100, re-suspended in 30μl PBS and immediately subject to flow cytometry analysis. Flow cytometry analysis was conducted using an Amnis® ImageStream® X Mk II with the following laser power settings: 405=25mW, 488=180mW, 561=185mW, 642=5mW, SSC=0.3mW. Cell populations were gated for single cells based on Area and Aspect Ratio (>0.8) values and in-focus cells were gated based on a Gradient RMS value (>50). Further gating of streptavidin
BAB-C-P3506PCT positive (AF647) cells was also applied, all in a hierarchical manner and 1000 events acquired. Before data analysis, compensation was applied according to single-colour controls and an automatically generated compensation matrix. Total fluorescence intensity values of different parameters were extracted using the Intensity feature of the IDEAS® software, with Adaptive Erode modified mask coverage. Statistical Analysis All statistical analysis was performed in GraphPad Prism (v9.2.0). Tables Table 1. Strains Used All strains are diploid derivatives of the MEP system (Lindstrom & Gottschling (2009) Genetics, 183(2):413-422, doi: https://doi.org/10.1534/genetics.109.106229). TOM70-GFP and RPL13a-mCherry markers are heterozygous to avoid growth defects. MEP α Tom70-GFP MAT α ade2::hisG his3 leu2 met15D::ADE2 ura3D0 trp1D63 RPL13A-mCherry hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1 RPL13A-mCherry- Kan Mother Enrichment MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Program – a haploid Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX MEP a acc1-S1157A MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX acc1-S1157A MEP a SAK1 oe MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX KanMX6-Pgdp-SAK1 MEP a SAK1 oe acc1- MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- S1157A Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX KanMX6-Pgdp-SAK1 acc1-S1157A Snf1 L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- mutation Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX snf1-L183I Snf1 L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- mutation – MEP a Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- acc1-S1157A HPHMX acc1-S1157A snf1-L183I Wild type MEP diploid ade2::hisG his3 leu2 met15D::ADE2/MET15 lys2/LYS2 ura3DO (MEP diploid TOM70- trp1D63 hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 GFP RPL13A-mCherry) loxP-CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A- mCherry-Kan/+ Sak1oe MEP diploid ade2::hisG his3 leu2 met15D::ADE2 ura3D0 trp1D63 hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- CDC20-Intron-loxP-HPHMX TOM70-GFP-TRP1 RPL13A-mCherry- KanMX6 KanMX6-Pgdp-SAK1
BAB-C-P3506PCT Acc1 MEP diploid ade2::hisG his3 leu2 met15D::ADE2/+ lys2/+ ura3D0 trp1D63 (MEP Tom70-GFP hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- Rpl13a-mCherry acc1- CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A-mCherry- S1157A het.) Kan/+ acc1-S1157A/+ A2A-Tom70- ade2::hisG his3 leu2 met15D::ADE2/+ lys2/+ ura3D0 trp1D63 gfp+Rpl13a-mcherry hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- MEP diploid (MEP CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A-mCherry- Tom70-GFP Rpl13a- Kan/+ KanMX6-Pgdp-SAK1/+ acc1-S1157A/+ mCherry SAK1 oe acc1-S1157A het.) A2A-hsp104-gfp MEP ade2::hisG his3 leu2 lys2/+ met17::ADE2/+ ura3DO trp1D63 diploid (MEP SAK1 oe hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- acc1-S1157A HSP104- CDC20-Intron-loxP-HPHMX KanMX6-Pgdp-SAK1/+ acc1-S1157A/+ GFP het dip) HSP104-GFP-Kan/+ Snf-L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- MEP diploid Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX L183I Tom70-GFP-TRP1 RPL13A-mCherry-Kan snf1-L183I A2A-L183I MEP diploid MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- (Snf1 L183I CRISPR + Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- acc1-S1157A – MEP HPHMX acc1-S1157A Tom70-GFP-TRP1 RPL13A-mCherry-Kan diploid) snf1-L183I Table 2. Oligos Used M13 rev AGCGGATAACAATTTCACACAGG (SEQ ID NO: 1) gRNA for Snf1
L183I TTTCAGGCTTCAGATCTCTATGG (SEQ ID NO: 2) Snf1
L183I Oligo 1 GATCTTTCAGGCTTCAGATCTCTAGTTTTAGAGCTAG (SEQ ID NO: 3) Snf1
L183I Oligo2: CTAGCTCTAAAACTAGAGATCTGAAGCCTGAAA (SEQ ID NO: 4) S
nf1 L183I Repair oligo GTCGAGTACTGCCATAGGCACAAAATTGTTCATAGAG ATCTGAAGCCTGAAAACATTCTACTAGATGAGCATCT GAATGTAAAGATTGCCGATTT (SEQ ID NO: 5) Snf1
L183I (genotyping forward) ATGGTGCACATATCGGGAACTAC (SEQ ID NO: 6) Snf1
L183I (genotyping reverse) TCTTCTGGGTGTGGTTTCAAATCTG (SEQ ID NO: 7) SAK1 oe UP45 (pYM-14 GDP TATCCAGTCACAATTTTAAAAGCACTTCGTTAACACGT promoter insertion 201bp up TTGGTTGCGTACGCTGCAGGTCGAC (SEQ ID NO: 8) from start of SAK1) SAK1 oe DN45 (pYM-14 GDP AGGTACATTGACCTCTTCGACGTTAACTTTTTTATCAC promoter insertion 45bp up from TCCTATCCATCGATGAATTCTCTGTCG (SEQ ID NO: 9) start of SAK1 start codon ATG) SAK1 oe C (genotyping primer TGGCAACATACTAGCATATGGA (SEQ ID NO: 10) forward) SAK1 oe D (genotyping primer ATCTTCAGGTCACAAGGTATACTTC (SEQ ID NO: 11) reverse) ACC1 CORE UP451 (for TTCTCCACCTTTCCAACTGTTAAATCTAAAATGGGTAT making S1157A mutation) GAACAGGGCTGTTAGGGATAACAGGGTAATCCGCGC GTTGGCCGATTCAT (SEQ ID NO: 12) TCTTAACGGAGATGACTGACTGTTTGCAACATATGAC ACC1 CORE DN451 (for AAATCTGAAACAGTTCGTACGCTGCAGGTCGAC making S1157A mutation) (SEQ ID NO: 13) ACC1 S1157A F (genotyping) GGTATGAACAGGGCTGTAG (SEQ ID NO: 14) ACC1 S1157A R (genotyping) CATATGACAAATCTGAAACAGCT (SEQ ID NO: 15)
BAB-C-P3506PCT HSP104 UP (c terminal tag) GATGACGATAATGAGGACAGTATGGAAATTGATGATG ACCTAGATCGGATCCCCGGGTTAATTAAC (SEQ ID NO: 16) HSP104 DN (c terminal tag) TACTGATTCTTGTTCGAAAGTTTTTAAAAATCACACTA TATTAAAGAATTCGAGCTCGTTTAAAC (SEQ ID NO: 17) HSP104 C (genotyping) GAAAAGCTATTCATAAGATCGTGGA (SEQ ID NO: 18) HSP104 D (genotyping) TCAAAACCATTATTGTAGTACCCGT (SEQ ID NO: 19) TOM70 UP451 AAGATTCAAGAAACTTTAGCTAAATTACGCGAACAGG GTTTAATGTGCATGCTTATGGTGAGCAA (SEQ ID NO: 20) TOM70 DN451 TAGTTTTTGTCTTCTCCTAAAAGTTTTTAAGTTTATGTT TACTGTAAGTTATACTAGTTCGTCGACTGGAT (SEQ ID NO: 21) TOM70 C (genotyping) GATTTGGCTAGAACTATGGAAGAG (SEQ ID NO: 22) TOM70 D (genotyping) TTATACGCACTGCTAATTATTTACAG (SEQ ID NO: 23) RPL13A-mCherry UP45 AAGAGAGCTAGAGAAAAGGCTGAAGCTGAAGCTGAA AAGAAGAAACGTACGCTGCAGGTCGAC (SEQ ID NO: 24) RPL13A-mCherry DN45 ATACAAAAATTGTGGATGAAAAATTCTTTGATGAAGTT TTTAGATATCGATGAATTCGAGCTCG (SEQ ID NO: 25) RPL13A C (genotyping) CAGAACCTTGAGATTAGCCAGAT (SEQ ID NO: 26) RPL13A D (genotyping) ATCTTTCGCATCTCTTCTATGC (SEQ ID NO: 27) EXAMPLE 1: A2A Cells Accumulate Less Tom70-GFP Ageing Damage Marker While Ageing to Wild Type Levels In order to increase AMPK activity while maintaining ACC1 activity in yeast, mutants were generated in which the upstream activating kinase of AMPK, Sak1, was overexpressed and Acc1 harboured the S1157A mutation to break the link between AMPK activity and Acc1 regulation by phosphorylation of this residue. These mutants are referred to as “A2A” herein. The healthy ageing phenotype of A2A cells is visible by imaging flow cytometric analysis of replicatively aged A2A double mutants and single Acc1
S1157A and P
GPD-SAK1 mutants (Figure 1). This shows that A2A mutants have significantly lower levels of ageing pathology (characterised by the senescence-associated Tom70-GFP damage marker) compared to equivalently aged wild type cells (based on WGA bud scar age marker), a clear demonstration of healthy ageing. EXAMPLE 2: The Age-Induced Hsp104-GFP Healthy Ageing Marker Shows that A2A Cells are More Resilient to Protein Maintenance Hsp104 proteins are protein disaggregases that are often used as a reporters for misfolded/ damaged protein aggregates and to assess protein quality control in aged systems. Indeed, replicative ageing studies have identified the importance of Hsp104 to mediate protein
BAB-C-P3506PCT disaggregation during replicative ageing (Erjavec et al. (2007) Genes & Development, 21(19): 2410-2421, doi: https://doi.org/10.1101%2Fgad.439307). Therefore, Hsp104-GFP (Schneider et al. (2021) Scientific Reports, 11(1):12819, doi: https://doi.org/10.1038/s41598-021-92249-1) was used as an orthogonal ageing marker in aged A2A mutants compared to wild type aged controls. As shown in Figure 2, A2A mutant yeast accumulate more Hsp104 over the course of 48 hours, demonstrating a larger capacity to ameliorate potential maladaptive protein aggregates that are typically correlated with ageing, while wild type cells lost the majority of Hsp104-GFP expression after 48 hours. EXAMPLE 3: Global Gene Regulation in A2A Appears More Youthful than Aged Wild Type Cells RNA-Seq analysis of global transcriptional dysregulation, which serves as an orthogonal marker of the ageing process, was performed on A2A mutant yeast and equivalently ages wild type control cells. As shown in Figure 3, aged A2A yeast cells have a more youthful gene expression profile than equivalently aged wild type cells (represented as the smaller slope value of -0.14 for A2A cells compared to -0.26 for WT cells). Typically when cells age, low expressed genes become highly expressed, while highly expressed genes decrease in expression, i.e. there is a dysregulation of global/overall gene expression patterns. Indeed, this observed change in overall gene expression during ageing in wild type cells is correlated with the slowing down of the cell cycle and gene dysregulation. EXAMPLE 4: A2A Mutant Growth Kinetics are Similar to Wild Type Cells Figure 4 shows the cell growth profiles of A2A yeast cells compared to wild type, demonstrating that A2A cells have similar growth kinetics to wild type cells. Thus, the A2A mutation does not impair cell cycle time or proliferation rate during normal growth. EXAMPLE 5: The Lifespan of the A2A Mutant is Similar to Wild Type Cells Figure 5 shows that A2A cells do not differ substantially in lifespan from wild type cells, showing that the improvement of healthspan is separable from lifespan extension. EXAMPLE 6: A2A Mutant Cells Demonstrate Higher Viability and Fitness To test the viability and fitness of aged (24h) cells, WT and A2A cells were aged, purified using a live purification method and then single cells were spotted onto a YPD plate. Whereas aged wild type cells are very slow to form colonies due to age-linked loss of fitness, the A2A cells maintain fitness similar to young cells (Figure 6), showing that A2A cells must have a healthier phenotype at 48h.
BAB-C-P3506PCT EXAMPLE 7: A2A cells demonstrate higher basal respiration rates To examine mitochondrial respiration rates, oxygen consumption rate (OCR) was measured in WT and A2A cells aged (48h) in YPD. Aged cells were purified using a live purification method and OCR was measured in synthetic glucose media. Aged A2A cells were more respiratory active than aged WT cells (Figure 7), showing that A2A cells shift their metabolism to respiration during ageing. EXAMPLE 8: The Healthy Ageing Phenotype of A2A Cells is Independent of Sak1 Overexpression Activation of Snf1/AMPK requires at least two events: (1) phosphorylation of the activation loop on threonine 210 by Sak1, Tos3, or Elm1 kinases; and (2) an Snf4-dependent process. Snf4 interacts with a regulatory domain of Snf1 that otherwise acts as an autoinhibitory domain (Leech et al. (2003) Eukaryotic Cell, 2(2):265-273, doi: https://doi.org/10.1128/ec.2.2.265- 273.2003). Therefore, since the A2A mutant simulates Snf1 activation by Sak1 overexpression, it was validated that the healthy ageing phenotypes observed in the A2A mutant are a result of AMPK and Acc1 activation, rather than a result of Sak1 overexpression off-target effects, by generating an A2A mutant that directly activates Snf1/AMPK. This novel A2A CRISPR mutant was engineered with a Snf1 L183I substitution in the catalytic domain that allows Snf1 to function independently of Snf4 activation. The Snf1
L183I + Acc1
S1157A (A2A-L183I) mutant displayed similar healthy ageing Tom70-GFP and WGA phenotypes to those observed in the A2A mutant (Figure 8), suggesting that the changes seen in the A2A mutant are a result of AMPK activation, rather than any off-target effects of Sak1 overexpression. Similar to as observed for mutants harbouring only Sak1 overexpression (data not shown), Snf1
L183I mutants also did not display the surprising phenotypes observed in A2A yeast demonstrated hereinbefore. It should also be noted that these surprising effects are seen in the A2A-L183I mutant, despite it differing from the wild type control strain by only two amino acid changes in the genome.
CRISPR Point Mutation Protocol The Snf1L183I point mutation strains were generated as follows using CRISPR: Guides were generated to contain approximately 20nt of specific sequence adjacent to a PAM sequence. Since Cas9 cleaves 3-4nt away from the PAM in the guide sequence, optimal Snf1L183I guide sites were selected using the Wyrick lab online yeast Cas9 guide site tool (Table 2): http://wyrickbioinfo2.smb.wsu.edu/crispr.html. The guide sequence was then cloned into an expression vector (pML104, from the Wyrick lab) that also expresses Cas9. The guide sequence was made by annealing two oligos designed by the tool above and cloning into pML104 digested with BclI and SwaI.
BAB-C-P3506PCT To digest the pML104 plasmid mini prep pML104 using the NEB Monarch PCR and DNA Cleanup Kit (#T1030S), grown in dam- E. coli. (as BcII is methylation sensitive). Then in a PCR machine, 1μg of plasmid was digested overnight at 25°C with 1µL SwaI in 50µL total volume of NEBuffer 3.1. Next, 1µL BclI, mix was added by pipetting and incubated for 2 hours in PCR machine at 50°C with the lid at 60°C. Products were run on an 1.2% agarose gel and gel extracted using the NEB Monarch DNA Gel Extraction Kit (#T1020S), eluting the DNA in 50µL. The repair template oligo (~100nt) contained the Snf1L183I mutation and a silent PAM sequence (NGG or NAG) mutation in the middle. Mutating the PAM site is critical to stop Cas9 from cleaving the same site after the desired target mutation is introduced. Once plasmid is digested and purified, oligos were annealed by mixing 10μL of each guide cloning oligo (100µM), 10µL NEBuffer 2 (or 2.1), and 70µL water. They were then heated to 90°C and allowed to cool on bench before diluting 1:100. 1μL of digested pML104, 1µL 1:100 annealed oligos, and 6µL water were mixed and heated to 50°C then cooled to room temperature. Then oligos were ligated using 1µL T4 DNA ligase buffer and 1µL T4 DNA ligase at room temperature for 1 hour. Cells were transformed at 42°C for 40 min and plated on ampicillin with DH5 alpha competent cells. Four colonies were miniprepped and putative positive clones were verified by sanger sequencing from M13 rev. Next, 1μL of pML104-Snf1L183I was added to 1µL 100μM repair oligo in MEP a Acc1-S1157A and MEP a SAK1 oe strains and cells were transformed. Cells were then plated on a galactose -uracil plate as Cas9 is on a galactose promoter. Colonies were then re-streaked after a few days to another galactose -uracil plate. Colonies were then streaked on a 5-Fluoroorotic Acid (5-FOA) plate. Positive colonies were then amplified using HH65 and HH66 oligos to generate a PCR product that covers the mutation site. Products were PCR purified using NEB Monarch PCR and DNA Cleanup Kit (#T1030S), and subsequently Sanger sequenced. Cell Purification Percoll gradients (1-2 per sample depending on harvest density) were formed by vortexing 1ml Percoll (Sigma P1644) with 110µl 10x PBS in 2ml tubes and centrifuging 15 min at 15,000g, 4°C. Ethanol fixed cells were defrosted and washed once with 1 volume of cold PBSE (PBS + 2mM EDTA) before resuspension in ~100µl cold PBSE per gradient and layering on top of the pre-formed gradients. Gradients were centrifuged for 4 min at 2,000g, 4°C, then the upper phase and brown layer of cell debris removed and discarded. 1ml PBSE was added,
BAB-C-P3506PCT mixed by inversion and centrifuged 1 min at 2,000g, 4°C to pellet the cells, which were then re-suspended in 1ml PBSE per time point (re-uniting samples where split across two gradients). 25µl Streptavidin microbeads (Miltenyi Biotech 1010007) were added and cells incubated for 5 min on a wheel at room temperature. Meanwhile, 1 LS column per sample (Miltenyi Biotech 1050236) was loaded on a QuadroMACS magnet and equilibrated with cold PBSE in 4°C room. Cells were loaded on columns and allowed to flow through under gravity, washed with 1ml cold PBSE and eluted with 1ml PBSE using plunger. Cells were re-loaded on the same columns after re-equilibration with ~500µl PBSE, washed and re-eluted, and this process repeated for a total of three successive purifications. After addition of Triton X-100 to 0.01% to aid pelleting, cells were split into 2 fractions in 1.5ml tubes, pelleted for 30s at 20,000g, 4°C, frozen on N2 and stored at -70°C. For live purification, used for fitness viability assays, cells were pelleted, washed twice with synthetic complete 2% glucose media, then resuspended in 2ml final volume of the same media and incubated for 5 min on a rotating wheel with 10µl MyOne streptavidin magnetic beads (Thermo), isolated using a magnet and washed five times with 1ml of the same media. For colony formation assays, cells were streaked on a YPD plate and individual cells moved to specific locations using a Singer MSM400 micromanipulator. Colony size was measured 24 hours later on the screen of the micromanipulator imaging with a 4x objective. RNA Extraction Cells were re-suspended in 50µl Lysis/Binding Buffer (from mirVANA kit, Life Technologies AM1560), and 50µl 0.5µm zirconium beads (Thistle Scientific 11079105Z) added. Cells were lysed with 5 cycles of 30s 6500 ms-1 / 30s on ice in an MP Fastprep bead beater or for 3 min at power 12 in a Bullet Blender (ThermoFisher) in cold room, then 250µl Lysis/Binding buffer was added followed by 15µl miRNA Homogenate Additive and cells were briefly vortexed before incubating for 10 minutes on ice. 300µl acid phenol : chloroform was added, vortexed and centrifuged 5 min at 13,000g at room temperature before extraction of the upper phase. 400µl room temperature ethanol and 2µl glycogen (Sigma G1767) were added and mixture incubated for 1 hour at -30°C before centrifugation for 15 minutes at 20,000g, 4°C. The pellet was washed with cold 70% ethanol and re-suspended in 10µl water. 1µl RNA was glyoxylated and analysed on a BPTE mini-gel, and RNA was quantified using a Qubit® RNA HS Assay Kit. 150ng RNA was used to prepare libraries using the NEBNext Ultra II Directional mRNAseq kit with poly(A)+ purification module (NEB E7760, E7490) as described with modifications:
BAB-C-P3506PCT Reagent volumes for elution from poly(T) beads, reverse transcription, second strand synthesis, tailing and adaptor ligation were reduced by 50%; libraries were amplified for 13 cycles using 2µl each primer per 50µl reaction before two rounds of AMPure bead purification at 0.9x and elution in 11µl 0.1x TE prior to quality control using a Bioanalyzer HS DNA ChIP (Agilent) and quantification using a KAPA Library Quantification Kit (Roche). Sequencing and Bioinformatics Libraries were sequenced by the Babraham Institute Sequencing Facility using a NextSeq 500 instrument on 75 bp single end mode. After adapter and quality trimming using Trim Galore (v0.6.6), RNA-seq data was mapped to yeast genome R64-1-1 using HISAT2 v2.1.0 (Kim et al., 2019) by the Babraham Institute Bioinformatics Facility. Mapped data was imported into SeqMonk v1.47.0 (https://www.bioinformatics.babraham.ac.uk/projects/seqmonk/) and quantified for log2 total reads mapping to the antisense strand of annotated open reading frames (opposite strand specific libraries), excluding the mtDNA and the rDNA locus, but with probes included to each strand of the rDNA intergenic spacer regions. Read counts were adjusted by Size Factor normalisation for the full set of quantified probes (Anders and Huber, 2010). Where indicated, a quantile normalisation was additionally applied using the ‘Match Distribution’ function in Seqmonk. MA plots were generated in GraphPad Prism (v9.2.0) comparing mean and difference for each gene between two conditions. Probes with very low numbers of reads post normalisation in the control condition were filtered as change between datasets cannot be accurately quantified in this case. Slope values were determined from the same filtered datasets using the lm(difference~mean) function in R. Flow Cytometry Cell pellets were re-suspended in 240µl PBS and 9μl 10% triton X-100 containing 0.3µl of 1mg/ml Streptavidin conjugated with Alexa Fluor® 647 (Life technologies) and 0.6μl of 1mg/ml Wheat Germ Agglutinin (WGA) conjugated with CF®405S (Biotium). Cells were stained for 10 min at RT on a rotating mixer while covered with aluminium foil, washed once with 300μl PBS containing 0.01% Triton X-100, re-suspended in 30μl PBS and immediately subject to flow cytometry analysis. Flow cytometry analysis was conducted using an Amnis® ImageStream® X Mk II with the following laser power settings: 405=25mW, 488=180mW, 561=185mW, 642=5mW, SSC=0.3mW. Cell populations were gated for single cells based on Area and Aspect Ratio (>0.8) values and in-focus cells were gated based on a Gradient RMS value (>50). Further gating of streptavidin
BAB-C-P3506PCT positive (AF647) cells was also applied, all in a hierarchical manner and 1000 events acquired. Before data analysis, compensation was applied according to single-colour controls and an automatically generated compensation matrix. Total fluorescence intensity values of different parameters were extracted using the Intensity feature of the IDEAS® software, with Adaptive Erode modified mask coverage. Statistical Analysis All statistical analysis was performed in GraphPad Prism (v9.2.0). Tables Table 1. Strains Used All strains are diploid derivatives of the MEP system (Lindstrom & Gottschling (2009) Genetics, 183(2):413-422, doi: https://doi.org/10.1534/genetics.109.106229). TOM70-GFP and RPL13a-mCherry markers are heterozygous to avoid growth defects. MEP α Tom70-GFP MAT α ade2::hisG his3 leu2 met15D::ADE2 ura3D0 trp1D63 RPL13A-mCherry hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1 RPL13A-mCherry- Kan Mother Enrichment MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Program – a haploid Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX MEP a acc1-S1157A MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX acc1-S1157A MEP a SAK1 oe MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX KanMX6-Pgdp-SAK1 MEP a SAK1 oe acc1- MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- S1157A Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX KanMX6-Pgdp-SAK1 acc1-S1157A Snf1 L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- mutation Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX snf1-L183I Snf1 L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- mutation – MEP a Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- acc1-S1157A HPHMX acc1-S1157A snf1-L183I Wild type MEP diploid ade2::hisG his3 leu2 met15D::ADE2/MET15 lys2/LYS2 ura3DO (MEP diploid TOM70- trp1D63 hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 GFP RPL13A-mCherry) loxP-CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A- mCherry-Kan/+ Sak1oe MEP diploid ade2::hisG his3 leu2 met15D::ADE2 ura3D0 trp1D63 hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- CDC20-Intron-loxP-HPHMX TOM70-GFP-TRP1 RPL13A-mCherry- KanMX6 KanMX6-Pgdp-SAK1
BAB-C-P3506PCT Acc1 MEP diploid ade2::hisG his3 leu2 met15D::ADE2/+ lys2/+ ura3D0 trp1D63 (MEP Tom70-GFP hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- Rpl13a-mCherry acc1- CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A-mCherry- S1157A het.) Kan/+ acc1-S1157A/+ A2A-Tom70- ade2::hisG his3 leu2 met15D::ADE2/+ lys2/+ ura3D0 trp1D63 gfp+Rpl13a-mcherry hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- MEP diploid (MEP CDC20-Intron-loxP-HPHMX Tom70-GFP-TRP1/+ RPL13A-mCherry- Tom70-GFP Rpl13a- Kan/+ KanMX6-Pgdp-SAK1/+ acc1-S1157A/+ mCherry SAK1 oe acc1-S1157A het.) A2A-hsp104-gfp MEP ade2::hisG his3 leu2 lys2/+ met17::ADE2/+ ura3DO trp1D63 diploid (MEP SAK1 oe hoD::SCW11pr-Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP- acc1-S1157A HSP104- CDC20-Intron-loxP-HPHMX KanMX6-Pgdp-SAK1/+ acc1-S1157A/+ GFP het dip) HSP104-GFP-Kan/+ Snf-L183I CRISPR MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- MEP diploid Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- HPHMX L183I Tom70-GFP-TRP1 RPL13A-mCherry-Kan snf1-L183I A2A-L183I MEP diploid MAT a ade2::hisG his3 leu2 lys2 ura3DO trp1D63 hoD::SCW11pr- (Snf1 L183I CRISPR + Cre-EBD78-NatMX loxP-UBC9-loxP-LEU2 loxP-CDC20-Intron-loxP- acc1-S1157A – MEP HPHMX acc1-S1157A Tom70-GFP-TRP1 RPL13A-mCherry-Kan diploid) snf1-L183I Table 2. Oligos Used M13 rev AGCGGATAACAATTTCACACAGG (SEQ ID NO: 1) gRNA for Snf1L183I TTTCAGGCTTCAGATCTCTATGG (SEQ ID NO: 2) Snf1L183I Oligo 1 GATCTTTCAGGCTTCAGATCTCTAGTTTTAGAGCTAG (SEQ ID NO: 3) Snf1L183I Oligo2: CTAGCTCTAAAACTAGAGATCTGAAGCCTGAAA (SEQ ID NO: 4) Snf1 L183I Repair oligo GTCGAGTACTGCCATAGGCACAAAATTGTTCATAGAG ATCTGAAGCCTGAAAACATTCTACTAGATGAGCATCT GAATGTAAAGATTGCCGATTT (SEQ ID NO: 5) Snf1L183I (genotyping forward) ATGGTGCACATATCGGGAACTAC (SEQ ID NO: 6) Snf1L183I (genotyping reverse) TCTTCTGGGTGTGGTTTCAAATCTG (SEQ ID NO: 7) SAK1 oe UP45 (pYM-14 GDP TATCCAGTCACAATTTTAAAAGCACTTCGTTAACACGT promoter insertion 201bp up TTGGTTGCGTACGCTGCAGGTCGAC (SEQ ID NO: 8) from start of SAK1) SAK1 oe DN45 (pYM-14 GDP AGGTACATTGACCTCTTCGACGTTAACTTTTTTATCAC promoter insertion 45bp up from TCCTATCCATCGATGAATTCTCTGTCG (SEQ ID NO: 9) start of SAK1 start codon ATG) SAK1 oe C (genotyping primer TGGCAACATACTAGCATATGGA (SEQ ID NO: 10) forward) SAK1 oe D (genotyping primer ATCTTCAGGTCACAAGGTATACTTC (SEQ ID NO: 11) reverse) ACC1 CORE UP451 (for TTCTCCACCTTTCCAACTGTTAAATCTAAAATGGGTAT making S1157A mutation) GAACAGGGCTGTTAGGGATAACAGGGTAATCCGCGC GTTGGCCGATTCAT (SEQ ID NO: 12) TCTTAACGGAGATGACTGACTGTTTGCAACATATGAC ACC1 CORE DN451 (for AAATCTGAAACAGTTCGTACGCTGCAGGTCGAC making S1157A mutation) (SEQ ID NO: 13) ACC1 S1157A F (genotyping) GGTATGAACAGGGCTGTAG (SEQ ID NO: 14) ACC1 S1157A R (genotyping) CATATGACAAATCTGAAACAGCT (SEQ ID NO: 15)
BAB-C-P3506PCT HSP104 UP (c terminal tag) GATGACGATAATGAGGACAGTATGGAAATTGATGATG ACCTAGATCGGATCCCCGGGTTAATTAAC (SEQ ID NO: 16) HSP104 DN (c terminal tag) TACTGATTCTTGTTCGAAAGTTTTTAAAAATCACACTA TATTAAAGAATTCGAGCTCGTTTAAAC (SEQ ID NO: 17) HSP104 C (genotyping) GAAAAGCTATTCATAAGATCGTGGA (SEQ ID NO: 18) HSP104 D (genotyping) TCAAAACCATTATTGTAGTACCCGT (SEQ ID NO: 19) TOM70 UP451 AAGATTCAAGAAACTTTAGCTAAATTACGCGAACAGG GTTTAATGTGCATGCTTATGGTGAGCAA (SEQ ID NO: 20) TOM70 DN451 TAGTTTTTGTCTTCTCCTAAAAGTTTTTAAGTTTATGTT TACTGTAAGTTATACTAGTTCGTCGACTGGAT (SEQ ID NO: 21) TOM70 C (genotyping) GATTTGGCTAGAACTATGGAAGAG (SEQ ID NO: 22) TOM70 D (genotyping) TTATACGCACTGCTAATTATTTACAG (SEQ ID NO: 23) RPL13A-mCherry UP45 AAGAGAGCTAGAGAAAAGGCTGAAGCTGAAGCTGAA AAGAAGAAACGTACGCTGCAGGTCGAC (SEQ ID NO: 24) RPL13A-mCherry DN45 ATACAAAAATTGTGGATGAAAAATTCTTTGATGAAGTT TTTAGATATCGATGAATTCGAGCTCG (SEQ ID NO: 25) RPL13A C (genotyping) CAGAACCTTGAGATTAGCCAGAT (SEQ ID NO: 26) RPL13A D (genotyping) ATCTTTCGCATCTCTTCTATGC (SEQ ID NO: 27) EXAMPLE 1: A2A Cells Accumulate Less Tom70-GFP Ageing Damage Marker While Ageing to Wild Type Levels In order to increase AMPK activity while maintaining ACC1 activity in yeast, mutants were generated in which the upstream activating kinase of AMPK, Sak1, was overexpressed and Acc1 harboured the S1157A mutation to break the link between AMPK activity and Acc1 regulation by phosphorylation of this residue. These mutants are referred to as “A2A” herein. The healthy ageing phenotype of A2A cells is visible by imaging flow cytometric analysis of replicatively aged A2A double mutants and single Acc1S1157A and PGPD-SAK1 mutants (Figure 1). This shows that A2A mutants have significantly lower levels of ageing pathology (characterised by the senescence-associated Tom70-GFP damage marker) compared to equivalently aged wild type cells (based on WGA bud scar age marker), a clear demonstration of healthy ageing. EXAMPLE 2: The Age-Induced Hsp104-GFP Healthy Ageing Marker Shows that A2A Cells are More Resilient to Protein Maintenance Hsp104 proteins are protein disaggregases that are often used as a reporters for misfolded/ damaged protein aggregates and to assess protein quality control in aged systems. Indeed, replicative ageing studies have identified the importance of Hsp104 to mediate protein
BAB-C-P3506PCT disaggregation during replicative ageing (Erjavec et al. (2007) Genes & Development, 21(19): 2410-2421, doi: https://doi.org/10.1101%2Fgad.439307). Therefore, Hsp104-GFP (Schneider et al. (2021) Scientific Reports, 11(1):12819, doi: https://doi.org/10.1038/s41598-021-92249-1) was used as an orthogonal ageing marker in aged A2A mutants compared to wild type aged controls. As shown in Figure 2, A2A mutant yeast accumulate more Hsp104 over the course of 48 hours, demonstrating a larger capacity to ameliorate potential maladaptive protein aggregates that are typically correlated with ageing, while wild type cells lost the majority of Hsp104-GFP expression after 48 hours. EXAMPLE 3: Global Gene Regulation in A2A Appears More Youthful than Aged Wild Type Cells RNA-Seq analysis of global transcriptional dysregulation, which serves as an orthogonal marker of the ageing process, was performed on A2A mutant yeast and equivalently ages wild type control cells. As shown in Figure 3, aged A2A yeast cells have a more youthful gene expression profile than equivalently aged wild type cells (represented as the smaller slope value of -0.14 for A2A cells compared to -0.26 for WT cells). Typically when cells age, low expressed genes become highly expressed, while highly expressed genes decrease in expression, i.e. there is a dysregulation of global/overall gene expression patterns. Indeed, this observed change in overall gene expression during ageing in wild type cells is correlated with the slowing down of the cell cycle and gene dysregulation. EXAMPLE 4: A2A Mutant Growth Kinetics are Similar to Wild Type Cells Figure 4 shows the cell growth profiles of A2A yeast cells compared to wild type, demonstrating that A2A cells have similar growth kinetics to wild type cells. Thus, the A2A mutation does not impair cell cycle time or proliferation rate during normal growth. EXAMPLE 5: The Lifespan of the A2A Mutant is Similar to Wild Type Cells Figure 5 shows that A2A cells do not differ substantially in lifespan from wild type cells, showing that the improvement of healthspan is separable from lifespan extension. EXAMPLE 6: A2A Mutant Cells Demonstrate Higher Viability and Fitness To test the viability and fitness of aged (24h) cells, WT and A2A cells were aged, purified using a live purification method and then single cells were spotted onto a YPD plate. Whereas aged wild type cells are very slow to form colonies due to age-linked loss of fitness, the A2A cells maintain fitness similar to young cells (Figure 6), showing that A2A cells must have a healthier phenotype at 48h.
BAB-C-P3506PCT EXAMPLE 7: A2A cells demonstrate higher basal respiration rates To examine mitochondrial respiration rates, oxygen consumption rate (OCR) was measured in WT and A2A cells aged (48h) in YPD. Aged cells were purified using a live purification method and OCR was measured in synthetic glucose media. Aged A2A cells were more respiratory active than aged WT cells (Figure 7), showing that A2A cells shift their metabolism to respiration during ageing. EXAMPLE 8: The Healthy Ageing Phenotype of A2A Cells is Independent of Sak1 Overexpression Activation of Snf1/AMPK requires at least two events: (1) phosphorylation of the activation loop on threonine 210 by Sak1, Tos3, or Elm1 kinases; and (2) an Snf4-dependent process. Snf4 interacts with a regulatory domain of Snf1 that otherwise acts as an autoinhibitory domain (Leech et al. (2003) Eukaryotic Cell, 2(2):265-273, doi: https://doi.org/10.1128/ec.2.2.265- 273.2003). Therefore, since the A2A mutant simulates Snf1 activation by Sak1 overexpression, it was validated that the healthy ageing phenotypes observed in the A2A mutant are a result of AMPK and Acc1 activation, rather than a result of Sak1 overexpression off-target effects, by generating an A2A mutant that directly activates Snf1/AMPK. This novel A2A CRISPR mutant was engineered with a Snf1 L183I substitution in the catalytic domain that allows Snf1 to function independently of Snf4 activation. The Snf1L183I + Acc1S1157A (A2A-L183I) mutant displayed similar healthy ageing Tom70-GFP and WGA phenotypes to those observed in the A2A mutant (Figure 8), suggesting that the changes seen in the A2A mutant are a result of AMPK activation, rather than any off-target effects of Sak1 overexpression. Similar to as observed for mutants harbouring only Sak1 overexpression (data not shown), Snf1L183I mutants also did not display the surprising phenotypes observed in A2A yeast demonstrated hereinbefore. It should also be noted that these surprising effects are seen in the A2A-L183I mutant, despite it differing from the wild type control strain by only two amino acid changes in the genome.