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Extra Credit Due 5 pm, February 26

Exam Questions need to be: • Multiple choice (a-e) • Factually correct (no conceptual errors)

• Test understanding (how things work) rather than memorization; no fill in the blank, no definitions • Understandable (no long essays, clear writing) • Must cover a topic discussed in this class (no exam questions from other classes) • Must be original (no plagiarism)

Submit 2 questions only, not on the same topic

Last Time

I am not advocating Anorexia!!!

(1) It is true that the average American has a caloric intake that is too high

(2) It is true that the recommended caloric intake itself is on the high side

(3) BUT, a caloric reduction of 30% from the recommendation for a given height is fairly drastic

(4) There would be a cost to reproduction (shunting of resources away from reproduction and toward maintenance and longevity—ie you’d become sterile)

Caloric Restriction

Caloric restriction is one of the few reliable ways to promote somatic maintenance and increase lifespan

(1) The key is to figure out what physiological pathways are induced by caloric restriction

(2) There are compounds that mimic caloric restriction without the cost to reproduction, such as resveratrol

Caloric Restriction

•  The physiological responses and patterns of gene expression following caloric restriction are not completely understood

• But there are two general models, in response to moderate and severe caloric restriction

Upregulate AMPK and downregulate TOR genes

Activate Sirtuins Upregulate AMPK and downregulate TOR genes

Activate Sirtuins

• Compounds such as resveratrol mimic DR, but not clear what it is doing • There is a debate as to whether resveratrol induces the response of moderate DR or severe DR •  The more popular view is that it induces the pathway of moderate DR

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•  It is known that reducing IGF/insulin signaling pathway increases longevity

•  Caloric restriction appears to reduce IGF/insulin signaling pathway

•  The link between caloric restriction and the IGF/insulin signaling pathway is not clear

•  Not clear why reducing the IGF/insulin signaling pathway promotes longevity

•  The paper that came out yesterday

•  A population of short Ecuadorians have a mutation in a growth hormone receptor

•  So that they cannot take up the growth hormone, and have a deficiency (making them short)

•  This hormone makes IGF. IGF/insulin signaling goes down

•  Not clear why reducing the IGF/insulin signaling pathway promotes longevity

Molecular Regulation of Circadian Rhythm

Example of genes, proteins, and their regulation in a system

Molecular Regulation of Circadian Rhythm

Earth Rotation: Photoperiod

Photoperiod: Period from sunrise to sunset

Circadian Clock

• Definition: an endogenous time-keeping mechanism, on a roughly 24-h cycle, that coordinates physiological processes

• The circadian period is generated at the level of the cell

• Organisms of all phyla show circadian rhythms in gene expression and physiological processes

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Circadian Clock

Free running clock: internal biological oscillator

Entrainment by external environmental cues (light, temperature, food)

•  Definition: The angular distance between a point on the Earth’s surface and the equator

Latitude Parallel

•  Values increase North/South of Equator

•  Degrees/Minutes/Seconds. Maximum value: 90o

Seasonal variation in photoperiod increases with greater distance from the equator

The mammalian pacemaker is located in the suprachiasmatic nucleus (SCN) of the hypothalamus

Pacemaker cells in the suprachiasmatic nucleus in the hypothalamus

Regulatory Control of Circadian Rhythm

•  At midday, clock (CLK) and cycle (CYC) proteins induce the expression of period (PER) and timeless (TIM)

•  However, the PER protein is rendered unstable by doubletime (DBT), through phosphorylation

Wager-Smith and Kay. 2000 Nature

Mechanism of circadian clock regulation for the fly, Drosophila melanogaster

CLK and CYC are transcription factors (induce transcription)

doubletime (DBT) regulates PER stability through phosphorylation

Wager-Smith and Kay. 2000 Nature

In Humans stability of PER sets the clock speed

Increased period stability speeds up the clock

Increased phosphorylation of period by doubletime or CK1 (in humans) destabilizes period and slows down the clock, as period cannot accumulate, and the clock cannot proceed as quickly

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•  At the same time, the CLK:CYC complex represses the CLK promoter

•  Once night falls, TIM can accumulate and interfere with the action of DBT and allow PER to accumulate

Wager-Smith and Kay. 2000 Nature

• In the middle of the night, PER:TIM dimers translocate into the nucleus (oval) and impede the functions of CLK:CYC (negative feedback)

• This brings about a cessation of PER and TIM mRNA production, and a simultaneous increase in Clk expression

Wager-Smith and Kay. 2000 Nature

•  The perception of light at dawn by cryptochrome (CRY) leads to degradation of timeless (TIM)

•  As the PER:TIM complex declines in the nucleus and no longer impedes the activity of the CLK:CYC complex

•  The increase in CLK:CYC then induces the transcription (expression) of PER and TIM and the cycle starts all over

•  Period: Per1, 2, 3

•  Cryptochrome: Cry1, 2

•  BMAL: Bmal 1 = Brain and Muscle Aryl hydrocarbon receptor nuclear translocator (ARNT)-Like 1 (analogous to Cycle in Drosophila)

•  Clock: Clock

•  casein kinase 1 delta/epsilon: CK1δ/ε (analogous to Doubletime in Drosophila)

•  Timeless has been identified in mammals, but function is unknown

In humans, there are 10 “clock genes”

• Given the mechanisms shown on how circadian rhythm is regulated, what are some of the ways in which circadian rhythm could evolve?

• Genetic disorders (or genetic variation) can offer some examples

• Such “disorders” represent genetic variation upon which natural selection could act

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“Early to bed and early to rise makes a man healthy, wealthy, and wise.”�

Benjamin Franklin

The perception above fails to account for genetic variation incircadian rhythm in human and other populations

•  Delayed Sleep Phase Syndrome (DSPS; “owls”)

•  Advanced Sleep Phase Syndrome (ASPS; “early birds”)

•  Non-24-h sleep–wake syndrome (N-24)

Genetic Variation in Circadian Rhythm within Populations

Advanced Sleep Phase Syndrome (ASPS) “Early Bird Syndrome”

•  Pacemaker runs too fast, but is still modulated by light cycle

•  Free-running clock is less than 24 hours

•  0.2% of the population go to sleep ~3 h earlier than the mean (9:30 or earlier)

•  Higher rate of depression

Delayed Sleep Phase Syndrome (DSPS) “Night-Owl Syndrome”

•  Estimated ~4.5% of the population prefer go to sleep ~3h or later than the mean (3:30 am or later)

•  Ontogenetic shift: In humans, 7% of adolescents are thought to have DSPS

•  High incidence of depression, heart disease, is noted when individuals are forced to comply with a diurnal schedule

•  Chronic fatigue in adolescents that are forced to comply with a diurnal schedule

•  Night owls can remain alert longer because of a longer running clock

Examples of Mutational Targets that would alter Circadian Rhythm

Mutations that destabilize (phosphorylate) Period will lengthen the circadian clock (Period takes longer to accumulate, and the circadian clock slows down)

•  Mutations in period at phosphorylating site (where CK1 binds)

•  Mutations in CK1 (doubletime in Drosophila) at the site that phosphorylates period

•  Mutations in timeless that prevent it from stabilizing period

•  Mutations that alter transcription of timeless or period Ebisawa, 2007. Journal of Pharmacological Science. 103:150-154

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•  Many of the mutations affect the phosphorylation status of the Period proteins

Advanced Sleep Phase Syndrome “Early Bird Syndrome”

•  Mutation in the PER2 gene –  Mutation in PER2 gene, at the phosphorylation site within the

casein kinase I (CKI)-binding domain

Serine --> Glycine at the phosphorylation site (Toh et al. Science 2001)

•  Mutation in the Casein Kinase I epsilon (CK1δ-T44A) gene: –  This enzyme is similar to Drosophila doubletime and phosphorylates

PER. The mutation within CKI decreases enzymatic activity--> more stable period

•  These mutations shorten the circadian cycle

Examples of mutations in humans:

Delayed Sleep Phase Syndrome “Night-Owl Syndrome”

•  Length variation in the PER3 gene: 75% of individuals DSPS carry two copies of the “short Per3” gene

•  Allelic Variation (Amino Acid substitutions): a particular allele (H4 haplotype) of the Per3 gene may confer susceptibility to DSPS in ~15% of affected people

•  Many other mutations found (see Ebisawa), such as in CK1

Examples of mutations in humans:

Ebisawa 2007

Christina Schmidt et al. 2009. Homeostatic sleep pressure and sustained attention in the suprachiasmatic area. Science. 324:5

http://www.sciencemag.org/cgi/content/full/324/5926/516

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•  Basic conclusion: Night owls remain alert longer (longer clock) •  Higher performance in Night owls 10.5 hrs after awakening

Selection potential in Populations

•  Long term fitness of a species relies on variation upon which selection can act, leading to adaptations to environmental change

•  Genetic variation in populations must exist for photoperiodic response to evolve

•  Polymorphism in circadian rhythm could allow populations to evolve in their photoperiodic response (nocturnal vs diurnal, or seasonal)

Genetic Variation among Populations!

•  There is known genetic variation in circadian rhythm within human populations (night-owls, early birds, etc.)

•  Anecdotal observations suggest variation among human populations: those closer to the equator tend to have higher % of night owls, and higher % early birds toward the poles

•  However, a shift in allelic frequency with latitude has not been confirmed in human populations

•  A latitudinal shift has been found in Drosophila (fruit flies)

•  Drosophila populations toward the poles (higher latitude) tend to have greater mid-day activity

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Planet X is larger than earth and has a longer daily photoperiod. Which type of mutation would LEAST LIKELY lead to a shift to accommodate the photoperiod on this planet?

(a) Increase activity of CK1 (in humans) or Doubletime (in Drosophila)

(b) Increase phosphorylation of Period (c) Increased stability of Period (d) Decreased accumulation of Timeless

Which of the following is MOST LIKELY to lead to Advance Sleep Phase Syndrome (ASPS)?

(a) A mutation in CK1 (b) A mutation in Period (c) A mutation in Timeless (d) It depends on where the mutation is located and how it

affects the conformation of the proteins above

Which of the following is LEAST LIKELY to lead to Advance Sleep Phase Syndrome (ASPS)?

(a) Period is effectively phosphorylated (b) Period is allowed to accumulate (c) Timeless accumulates more quickly (d) Reduced activity of CK1

You are a manager for a radio station, and people are complaining that your late night DJs are falling asleep on the job. Which of the following might help alleviate your problem?

(a) Hire a DJ who has a mutation in the hPER2 gene, at the phosphorylation site within the Casein Kinase I (CK1) binding domain

(b) Hire a DJ who has a mutation which causes increased gene expression of PER and TIM

(c) Hire a DJ with a mutation in TIM that increases stabilization of PER

(d) Hire a DJ with a mutation in the Casein Kinase I epsilon (CKIδ-T44A) gene