The Skaggs Institute
for Chemical Biology

Scientific Report 2006

Warm Sensitivity, Pyrogenic Cytokine Signaling, Thermoregulation, and Longevity

T. Bartfai, B. Conti, I. Tabarean, C. Davis, M. Sanchez-Alavez, H. Korn, M. Rebek, A. Shivanyuk, E. Mann, J. Rebek, Jr.

Life and reproduction of mammals require a constant body temperature under widely varying external temperatures. Constant body temperature is achieved by regulating heat production and heat loss after the external temperature has been sensed by temperature-sensitive nerve endings in the skin. The signals from these endings arrive at central neurons that are also sensing the brain temperature. The molecular mechanisms of the central thermosensor are not known. We have genetically manipulated the local environment of these central warm-sensitive neurons, and we have designed and synthesized chemicals to interfere with pyrogen signaling.

“The Cool Mouse ”

It is assumed that if we could turn off the burn rate of our metabolic machinery we could live longer and have a healthier life. The metabolic rate is, however, fitted to the demand to maintain a constant temperature of 36.7°C. We wondered if mice could be generated that have different, preferably lower, body temperatures.

We generated a transgenic mouse strain (Hcrt-UCP2) in which the warm-sensitive neurons are constantly heated by energy dissipated from a mitochondrial proton gradient in an adjacent cell group of no more than 3000 hypocretin-secreting neurons. By expressing uncoupling protein 2 in this small number of neurons, a sufficient amount of heat is generated to warm the anterior hypothalamus during most of the life time of a mouse. In these transgenic mice, the anterior hypothalamic neurons are exposed to a temperature 1°C higher than the normal temperature, as confirmed by direct measurement via implanted miniature bimetallic thermosensors.

These mice have their “thermostat” effectively and constitutively fooled, a situation that resulted in a lowered core body temperature during the dark phase of the day when mice are active. Such reduction of core body temperature resulted in reduced energy expenditure and a prolonged median survival (Fig. 1).

Fig 1. Survival curves for transgenic Hcrt-UP2 (M tg) and wild-type (M wt) mice show the longevity of the transgenic animals. The growth curve was determined by weighing animals every 2 weeks. Median average life span was determined as the median of the survival curve (P < .05 by Gehan test).

These findings open many interesting medical possibilities to influence metabolic rates, disease progression, and life span. We will study which molecular and cellular adjustments are responsible for the longevity of these mice.

Warm-Sensitive Neurons

No morphologic or immunohistochemical markers are available for the visualization of warm-sensitive neurons. This lack severely limits the possibility of studying these neurons. Using electrophysiologic studies and the pyrogenic substances prostaglandin E₂ and IL-1β, we identified electrophysiologic, morphologic, and immunohistochemical markers of warm-sensitive neurons. IL-1β can directly affect the warm-sensitive neurons (Fig. 2) via the Toll signaling pathway that involves MyD88 adapters. In another project, we are synthesizing compounds that can interrupt this signaling.

Fig. 2. Effects of IL-1 on the spontaneous activity of anterior hypothalamic neurons. A, Application of IL-1 (0.3 nM) hyperpolarizes and inhibits the firing of an anterior hypothalamic neuron. B, Expanded fragments of the recording in A that show the increased inhibitory postsynaptic potential frequency during and after application of IL-1. (Note: Action potentials are truncated.)

Pyrogen Signaling

IL-1β is the most potent pyrogen known; 0.5 pmol causes full fever response in humans. We found that IL-1 type 1 receptors (IL-1R1) are present on many anterior hypothalamic neurons that affect warm sensitivity. Toll-like receptor signaling occurs via recruitment of small adaptor molecules. An adaptor molecule for IL-1R1 is the small cytosolic protein MyD88. MyD88 interacts with the receptor via a homotypic interaction between large protein domains called TIR domains. Because IL-1 signaling plays a key role in severe chronic inflammatory diseases such as rheumatic arthritis, there is a strong focus on blocking IL-1 signaling.

When the crystal structure of the first TIR domain was resolved, we reasoned that the BB-loop of 3 amino acids, phenylalanine-valine-proline, might be one of the homotypic sites of TIR-TIR interactions. We designed and synthesized a set of BB-loop MyD88 mimics, of which the best studied is AS-1, and a set of dimeric ligands like EM163 (Fig 3). We showed that AS-1 and EM163 can block IL-1 signaling in cellular systems; they can inhibit the physical interaction as signified by coimmunoprecipitation of IL-1R1 with MyD88, and, even more important. they can attenuate in vivo the IL-1–induced fever response and suppress the formation of inflammatory mediators. These compounds constitute a novel class of anti-inflammatory molecules useful in the treatment of rheumatic arthritis and psoriasis.

Fig. 3. Synthesis and crystal structure of TIR/BB-loop mimetics in Ortep presentation (30% occupation thermal ellipsoids). A, Synthesis of BB-loop mimetic based on the consensus sequence in TIR domains of Toll-like receptors, IL-1R1, and MyD88. Compound 1, commercial butoxycarbonyl-L-valine hydroxysuccinimide ester; compound 2, AS-1 (B); compound 3, EM77 (C); compound 4, EM110.

Publications

Bartfai, T., Lees, G.V. Drug Discovery: From Bedside to Wall Street. Academic Press, San Diego, 2006.

Davis, C.N., Mann, E., Behrens, M.M., Gaidarova, S., Rebek, M., Rebek, J., Jr., Bartfai, T. MyD88-dependent and -independent signaling by IL-1 in neurons probed by bifunctional Toll/IL-1 receptor domain/BB-loop mimetics. Proc. Natl. Acad. Sci. U. S. A. 103:2953, 2006.

Davis, C.N., Tabarean, I., Gaidarova, S., Behrens, M.M., Bartfai, T. IL-1β induces a MyD88-dependent and ceramide-mediated activation of Src in anterior hypothalamic neurons. J. Neurochem. 98:1379, 2006.

Sanchez-Alavez, M., Tabarean, I.V., Behrens, M.M., Bartfai, T. Ceramide mediates the rapid phase of febrile response to IL-1β. Proc. Natl. Acad. Sci. U. S. A. 103:2904, 2006.

Tabarean, I.V., Conti, B., Behrens, M., Korn, H., Bartfai, T. Electrophysiological properties and thermosensitivity of mouse preoptic and anterior hypothalamic neurons in culture. Neuroscience 135:433, 2005.

Tabarean, I.V., Korn, H., Bartfai, T. Interleukin-1β induces hyperpolarization and modulates synaptic inhibition in preoptic and anterior hypothalamic neurons. Neuroscience 41:1685, 2006.