ampk and learning poster

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Activation of AMPK Improves Cognitive Function in C. elegans Juan C. Ramirez, Caroline Escoubas, Emina Tabakovic, William Mair Department of Genetics and Complex Diseases, Harvard TH Chan School of Public Health Introduction Due to advances in public health, the average human lifespan has increased dramatically and more people are living into older age. Yet, despite these achievements, aging comes with its own share of physiological, medical, and economic burdens. In particular, patient age represents the largest risk factor for complex diseases, including neurodegeneration, multiple cancers, cardiovascular diseases, and metabolic disorders. Hence, while lifespan has seen a remarkable increase, healthspan extension remains an issue. Current medical approaches treat individual maladies separately by targeting their discrete foundations. However, the high prevalence of comorbidity in the elderly – nearly 60% in the United States alone – prevents significant progress in extending healthspan through this approach. On the other hand, an alternative methodology follows targeting aging itself, an approach supported by experiments conducted on C. elegans and conserved across higher order organisms, including primates. These experiments demonstrated that physiological aging can be altered by single-gene mutations and simple interventions, resulting in both lifespan extension and preservation of a youthful state. Among such mediations, dietary restriction holds significant promise [2]. Dietary restriction (DR) – limitation of food intake without malnutrition –induces a “low cellular energy” state mediated by conserved energetics pathways. Under DR, AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, serves as a downstream regulator of the prompted low energy state, coordinating an increase in energy production and a decrease ATP consumption. AMPK typically stimulates hepatic fatty acid oxidation and ketogenesis, lipogenesis, triglyceride synthesis, muscle glucose uptake, among multiple other energy- producing actions [3]. However, activation of AMPK alone has been demonstrated to be implicated in aging mechanisms, supporting a constitutively active (CA) AMPK model for DR [4]. In addition, AMPK is activated both directly and indirectly by a wide array of small molecules, including drugs such as resveratrol, aspirin, and the biguanides phenformin and metformin – making it an attractive pharmacological target [2][5] Multiple pathways are recognized to be implicated by AMPK and lifespan extension. However, the effects on healthspan following activation of AMPK warrant further study. In this study, we describe the effect of CA AMPK on C. elegans’ healthspan as measured through cognitive function assessment. Here, learning, a measure of cognitive function, is studied by quantifying C. elegans’ elicited association between a negative and neutral stimulus, in this case starvation and salt (NaCl), respectively. By quantifying worms’ attraction or repulsion to salt following prolonged paired exposure to both stimuli, learning – and thus cognitive function and healthspan – can be assessed. We hypothesize that CA AMPK not only extends lifespan, but also lengthens functional cognitive ability as well. AMPK Activation and Aging • AMPK [A] is a heterotrimeric protein composed of three different subunits: α serves as the catalytic subunit and both β and γ are regulatory subunits [3] Expression of a truncated version of aak-2 — the α subunit-encoding gene — allows AMPK to be CA without the need for AMP [4] CA AMPK model has been shown to increase median lifespan [B] by 38% [4] Memory Assay Protocol in C. elegans • Worms are grown until adulthood, washed and transferred onto conditioning plates for four hours [A] • Three different conditions: Food and salt (+N+E); Food and no salt (+N-E); and neither salt nor food (-N-E) • Salt is a neutral stimulus to worms, though starvation is actively avoided [B] • Pairing of CS (salt) with US (starvation) [B] induces CR — the aversion of salt [C] Quantifying C. elegans’ Cognitive Function [1] Dana P. Goldman, David Cutler, John W. Rowe, Pierre-Carl Michaud, Jeffrey Sullivan, Desi Peneva and S. Jay Olshansky. Substantial Health And Economic Returns From Delayed Aging May Warrant A New Focus For Medical Research. Health Affairs, 32, no.10 (2013):1698-1705 [2] Burkewitz, K., Zhang, Y., and Mair, W.B. (2014). AMPK at the Nexus of Energetics and Aging. Cell Metab 20, 10–25. [3] Xiao, B., Sanders, M., Underwood, E., Heath, R., Mayer, F., Carmena, D., Jing, C., Walker, P., Eccleston, J., Haire, L., et al. (2011). Structure of mammalian AMPK and its regulation by ADP. Nature 472, 230–233. [4] Mair, W., Morantte, I., Rodrigues, A., Manning, G., Montminy, M., Shaw, R., and Dillin, A. (2011). Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB. Nature470, 404–408. [5] Onken, B., and Driscoll, M. (2010). Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1. Plos One 5, e8758. [6] Saeki, S., Yamamoto, M., and Iino, Y. (2001). Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans. J. Exp. Biol. 204, 1757–1764. Conclusions and Further Directions In addition to the previously reported lifespan expansion, CA AMPK also induces a stark increase in nematode learning capability. Results suggest well-maintained neurological function in treated animals. In order to further study this effects, we aim to: Express CA AMPK in specific tissues, singling out those responsible for lifespan extension and improved neurological function Activate AMPK via DR and pharmacological intervention and compare the treated worms’ cognitive functions against recruitment method Measure nematode memory retention against time to further assess cognitive function References Post-conditioning behavior is scored and quantified [A] Difference between chemotaxis indices in -N-E and +N-E [B] conditions constitutes Memory Index [C] [5] Quantification of attainment of CR measures learning and cognitive function across worm populations Activation of AMPK Promotes Increased Learning Ability Chemotaxis index confirms for consistent repulsion to salt in conditioned wild type [A, left] and CA AMPK [A, right] animals Memory index [B] demonstrates a significantly greater learning ability in CA AMPK animals than wild type. • Normalization [C] against wild type numbers demonstrates nearly a 2-fold increase in learning ability α β γ A A B B A A B +N+E No Preference +N-E Aversive Behavior -N-E No Preference Forgetting Assays Provide Further Insight into Cognitive Function A [A] Demonstrated forgetting assay data for wild type worms A forgetting assay quantifies nematode retention of neutral and negative stimuli association over time Comparison of different strains and/or growth conditions provides a comparative view of neurological health with time considered Worms are conditioned identically to a MA but then removed from both stimuli for tested time periods (1 and 2 hours in [A]) Can serve as model for memory loss, another hallmark of aging and healthspan Wild Type CA AMPK C

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Page 1: AMPK and Learning Poster

Activation of AMPK Improves Cognitive Function in C. elegans Juan C. Ramirez, Caroline Escoubas, Emina Tabakovic, William Mair

Department of Genetics and Complex Diseases, Harvard TH Chan School of Public Health

Introduction

Due to advances in public health, the average human lifespan has increased dramatically and more people are living into older age. Yet, despite these achievements, aging comes with its own share of physiological, medical, and economic burdens. In particular, patient age represents the largest risk factor for complex diseases, including neurodegeneration, multiple cancers, cardiovascular diseases, and metabolic disorders. Hence, while lifespan has seen a remarkable increase, healthspan extension remains an issue. Current medical approaches treat individual maladies separately by targeting their discrete foundations. However, the high prevalence of comorbidity in the elderly – nearly 60% in the United States alone – prevents significant progress in extending healthspan through this approach.

On the other hand, an alternative methodology follows targeting aging itself, an approach supported by experiments conducted on C. elegans and conserved across higher order organisms, including primates. These experiments demonstrated that physiological aging can be altered by single-gene mutations and simple interventions, resulting in both lifespan extension and preservation of a youthful state. Among such mediations, dietary restriction holds significant promise [2].

Dietary restriction (DR) – limitation of food intake without malnutrition –induces a “low cellular energy” state mediated by conserved energetics pathways. Under DR, AMP-activated protein kinase (AMPK), a highly conserved cellular energy sensor, serves as a downstream regulator of the prompted low energy state, coordinating an increase in energy production and a decrease ATP consumption. AMPK typically stimulates hepatic fatty acid oxidation and ketogenesis, lipogenesis, triglyceride synthesis, muscle glucose uptake, among multiple other energy-producing actions [3]. However, activation of AMPK alone has been demonstrated to be implicated in aging mechanisms, supporting a constitutively active (CA) AMPK model for DR [4]. In addition, AMPK is activated both directly and indirectly by a wide array of small molecules, including drugs such as resveratrol, aspirin, and the biguanides phenformin and metformin – making it an attractive pharmacological target [2][5]

Multiple pathways are recognized to be implicated by AMPK and lifespan extension. However, the effects on healthspan following activation of AMPK warrant further study. In this study, we describe the effect of CA AMPK on C. elegans’ healthspan as measured through cognitive function assessment. Here, learning, a measure of cognitive function, is studied by quantifying C. elegans’ elicited association between a negative and neutral stimulus, in this case starvation and salt (NaCl), respectively. By quantifying worms’ attraction or repulsion to salt following prolonged paired exposure to both stimuli, learning – and thus cognitive function and healthspan – can be assessed. We hypothesize that CA AMPK not only extends lifespan, but also lengthens functional cognitive ability as well.

AMPK Activation and Aging

• AMPK [A] is a heterotrimeric protein composed of three different subunits: α serves as the catalytic subunit and both β and γ are regulatory subunits [3]

• Expression of a truncated version of aak-2 — the α subunit-encoding gene — allows AMPK to be CA without the need for AMP [4]

• CA AMPK model has been shown to increase median lifespan [B] by 38% [4]

Memory Assay Protocol in C. elegans

• Worms are grown until adulthood, washed and transferred onto conditioning plates for four hours [A]

• Three different conditions: Food and salt (+N+E); Food and no salt (+N-E); and neither salt nor food (-N-E)

• Salt is a neutral stimulus to worms, though starvation is actively avoided [B] • Pairing of CS (salt) with US (starvation) [B] induces CR — the aversion of salt [C]

Quantifying C. elegans’ Cognitive Function

[1] Dana P. Goldman, David Cutler, John W. Rowe, Pierre-Carl Michaud, Jeffrey Sullivan, Desi Peneva and S. Jay Olshansky. Substantial Health And Economic Returns From Delayed Aging May Warrant A New Focus For Medical Research. Health Affairs, 32, no.10 (2013):1698-1705

[2] Burkewitz, K., Zhang, Y., and Mair, W.B. (2014). AMPK at the Nexus of Energetics and Aging. Cell Metab 20, 10–25.

[3] Xiao, B., Sanders, M., Underwood, E., Heath, R., Mayer, F., Carmena, D., Jing, C., Walker, P., Eccleston, J., Haire, L., et al. (2011). Structure of mammalian AMPK and its regulation by ADP. Nature 472, 230–233.

[4] Mair, W., Morantte, I., Rodrigues, A., Manning, G., Montminy, M., Shaw, R., and Dillin, A. (2011). Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB. Nature470, 404–408.

[5] Onken, B., and Driscoll, M. (2010). Metformin induces a dietary restriction-like state and the oxidative stress response to extend C. elegans Healthspan via AMPK, LKB1, and SKN-1. Plos One 5, e8758.

[6] Saeki, S., Yamamoto, M., and Iino, Y. (2001). Plasticity of chemotaxis revealed by paired presentation of a chemoattractant and starvation in the nematode Caenorhabditis elegans. J. Exp. Biol. 204, 1757–1764.

Conclusions and Further Directions

• In addition to the previously reported lifespan expansion, CA AMPK also induces a stark increase in nematode learning capability.

• Results suggest well-maintained neurological function in treated animals. • In order to further study this effects, we aim to:

• Express CA AMPK in specific tissues, singling out those responsible for lifespan extension and improved neurological function

• Activate AMPK via DR and pharmacological intervention and compare the treated worms’ cognitive functions against recruitment method

• Measure nematode memory retention against time to further assess cognitive function

References

• Post-conditioning behavior is scored and quantified [A] • Difference between chemotaxis indices in -N-E and +N-E [B] conditions

constitutes Memory Index [C] [5] • Quantification of attainment of CR measures learning and cognitive

function across worm populations

Activation of AMPK Promotes Increased Learning Ability

• Chemotaxis index confirms for consistent repulsion to salt in conditioned wild type [A, left] and CA AMPK [A, right] animals

• Memory index [B] demonstrates a significantly greater learning ability in CA AMPK animals than wild type.

• Normalization [C] against wild type numbers demonstrates nearly a 2-fold increase in learning ability

α

β γ

A

A

B

B

A

A

B

+N+E No Preference

+N-E Aversive Behavior

-N-E No Preference

Forgetting Assays Provide Further Insight into Cognitive Function

A

• [A] Demonstrated forgetting assay data for wild type worms • A forgetting assay quantifies nematode retention of neutral and

negative stimuli association over time • Comparison of different strains and/or growth conditions provides a

comparative view of neurological health with time considered • Worms are conditioned identically to a MA but then removed from

both stimuli for tested time periods (1 and 2 hours in [A]) • Can serve as model for memory loss, another hallmark of aging and

healthspan

Wild Type CA AMPK

C