Development and Longevity Weiqing Li Department of Biological Structure University of Washington, Seattle Conj 542, 2/1/11

Developmental Biology Stem Cell Research Mechanisms of Aging

Development Adulthood ? 1. Shared signaling cascades? 2. Antagonistic pleiotropy? 3. More intrinsic connections?

Dwarf mice live longer (4 years and 12 days old — roughly the equivalent of a 136-year-old human) R. Miller, Michigan

Tatar & Antebi, 2003

Each C. elegans can produce about 500 progeny

C. elegans as a genetic model organism short generation time (<3 days) transparent strains can be kept at -80°C simple transgenic techniques (takes < 3 days to generate a transgenic line) - gene knockdown by feeding RNAi - genetic screen and mutant mapping - gene knockout consortiums short lifespan (15-25 days) genetics: invariant linage

C. elegans organs / tissues Pancreas: sensory neurons, intestine Liver: intestine Heart: pharynx, body-wall muscle Blood: fluid in pseudocoelom Kidney: excretory canals Skeleton and bone: cuticle Steroidogenic glands: XXX neurons, skin, intestine, somatic gonad Adipose tissue: intestine, pseudocoelom Lung: pseudocoelom “Immune system”: intestine Macrophages: coelomocytes

embryo L1L2L3L4adult normal environment growth arrest increased fat extended lifespan dauer adverse environment L3 dauer Adverse environmental cues, or down- regulation of insulin signaling, induce dauer arrest in C. elegans Insulin receptor mutant

The down-regulation of the insulin pathway results in extended lifespan in C. elegans hormone longevity Insulin receptor PI3 kinase

Mice live longer when insulin receptor function is depleted in the fat tissue Khan et al., Science 299: 572, 2003 FIRKO mice Wild-type mice

Longo, 2009 Sch9 : an akt homolog

development aging & etc. metabolism insulin-like ligands: DAF-28 and INS1-37 IST-1 AAP-1 PDK-1 kinases insulin receptor-like PTEN lipid phosphatase FOXO transcription factor IRS-1 P55 PI3K PI’s PI3P’s on off A conserved insulin signaling pathway in C. elegans

Baumeister et al., 2006

ASI ASJ ciliated endings nerve ring ASI ASJ Pdaf-28 (insulin)::GFP Ciliated sensory neurons are exposed to sense the environment amphid

grow fast and reproduce delay / limit growth delay reproduction conserve energy turn on stress responses live long good environments: live normally adverse environments: protect your body (somatic maintenance) Growth vs. Survival

dauer arrest cGMP signaling insulin signaling TGF-  signaling steroid signaling TOR signaling ? The Activation of Five Signaling Pathways is Required to Prevent Dauer Arrest

Baumeister et al., 2006 pha-4

The diseases of affluence diatetes, hypertention, heart disease, stroke. Obesity Diabetes blindness, kidney failure, heart diseases, stroke, nerve problems, premature death.

Food abundant Famine diabetic survivors?

Development Adulthood ? 1. Shared signaling cascades? 2. Antagonistic pleiotropy? 3. More intrinsic connections?

development aging & etc. metabolism IRS-1 P55 PI’s PI3P’s insulins Genetics revealed the insulin/IGF-1 pathway in C. elegans forward genetics functional genomics (RNAi knockdown) classical genetics deletion knockouts identify gene functions reverse genetics mechanisms of biological processes suppressor screens: DAF-2 AGE-1 DAF-28 AKT-1 AKT-2 INS-11 AKT-1 P55 INS-7 INS-18 DAF-18/PTEN DAF-16/FOXO PDK-1 (gf) AKT-1 (gf) reporter-based screens:

Known/predicted ORFs (~19,000) E. coli expressing individual RNAi clones Score phenotypes C. elegans chromosome B A B C D E F ACDEF Add worms Eat bacteria & take up specific dsRNA Functional genomics ( RNAi screen) Andy Fraser, Ravi Kamath, Julie Ahringer, MRC/Wellcome, UK

Longevity Determined by Developmental Arrest Genes in Caenorhabditis elegans Chen et al., larval arrest genes 24 longevity regulators RNAi during adulthood Mitochondrial genes, Genes affecting translation, transcription

Lifespan Regulation by Evolutionarily Conserved Genes Essential for Viability Curran and Ruvkun, ,700 essential genes 64 longevity regulators RNAi during adulthood Insulin and metabolic pathways; Translation, RNA and chromatin factors.

skn-1/Nrf1, 2 pha-4/FoxA

Blastomere Identity in C. elegans progeny make pharynx and epidermis due to induction from MS makes mesoderm (muscle, pharynx) makes gut

1994

2007 eat-2: nicotinic acetylcholine receptor daf-16: FoxO, mediates insulin signaling

Phase II detoxification enzymes, including gcs-1 ROS: reactive oxygen species Nrf target genes: Hyung An and Blackwell, 2008

skn-1-dependent GCS-1::GFP expression in the intestine and ASI neurons Stressor: heat (or paraquat)

Stress-induced nuclear accumulation of SKN-1::GFP

dauer arrest cGMP signaling insulin signaling TGF-  signaling steroid signaling TOR signaling ? The Activation of Five Signaling Pathways is Required to Prevent Dauer Arrest

Tatar & Antebi, 2003

Each C. elegans can produce about 500 progeny

Xie, 2008

LAG-2(delta)::GFP GLP-1(notch)::RFP Germline development, notch signaling and stem cell model in C. elegans

Z1Z4 Z2 Z3 glp-1/notch (-): defective germ-line, extended lifespan Ablation of Z2, Z3: extended lifespan Ablation of Z1,Z2, Z3, Z4: normal lifespan Ablation of Z2, Z3 In daf-16/FOXO(-): normal lifespan DTC Depletion of germ-line stem cells extends lifespan Kenyon lab (UCSF)

glp-1/ notch(-) RNAi screen (LGI) for normal lifespan Identification of kri-1 (ankyrin repeats) glp-1/ notch(-) RNAi of known steroid pathway genes Involvement of steroid signaling (lifespan, DAF-16::GFP)

A functional Pkri-1::KRI-1::GFP indicates that kri-1 is expressed in the intestine

kri-1 mediates the longevity signal triggered by loss of germline specifically

RNAi of kri-1 or steroid-pathway genes blocked nuclear DAF-16::GFP kri-1 daf-16/FOXO daf-9/cyp daf-12/nhr loss of germline stem cells

The steroid signal(s) triggered by loss of germline may be generated in both the somatic gonad and intestine daf-36(oxygenase)::gfp Rottiers et al., 2006

Xie, 2008

Wang et al., 2008 glp-1 animals store less fat when daf-16 and kri-1 functions are normal

A triglyceride lipase (K04A8.5) regulates fat storage and longevity in response to loss of GCS signal K04A8.5(RNAi) Wang et al., 2008 K04A8.5 Q-PCR (WT vs. glp-1) K04A8.5 RNAi knock-down K04A8.5 overexpression

Inhibition of GCS proliferation triggers the signal

K04A8.5 Q-PCR

C. elegans development aging? metabolism? XXX cells sensory neurons food environment pituitary gland mouse hypothalamus development aging metabolism food metabolic cues Drosophila development aging metabolism? brain food environment ring gland corpora allata development and physiology external or internal signals central neuroendocrine systemic hormones 1º 2º

Kleemann and Murphy, 2008

Genetic mosaic analyses daf-2(-) daf-2(+) daf-2(-) daf-2(+) free duplication or extrachromosomal array daf-2(+) daf-2(-) daf-2(+) daf-2(-) daf-2 transgene driven by a tissue-specific promoter (Apfeld & Kenyon, 1998; Wolkow et al.,2000; Libina et al., 2003)