Humanin-Like Peptides and the Expanding role of the Mitochondria Genomics and pharmacogenomics of growth disorders - October 27th, 2011 Pinchas Cohen Professor and Vice Chair for Research, Mattel Children’s Hospital, UCLA

Outline: . Recap of Mitochondrial Biology . Discovery and Nature of Humanin . Humanin Receptors and signaling . Cytoprotective Actions of Humanin . Metabolo-Protection by Humanin . Humanin Levels in Disease States . Mitochines: Retrograde Signals

A Primer on Mitochondrial Biology • Originally prokaryotic – regulates: • Cellular respiration • Integrator of apoptotic signals • ROS production/oxidative stress • Mitochondrial function declines with age as a result of accumulated mutations in the mitochondrial DNA • Mitochondrial dysfunction is common in multiple diseases including diabetes and neurodegeneration • Mitochondrial Dysfunction is recognized to be a contributing factor in malignancy • Transition to glycolytic metabolism • Resistance to mitochondrial apoptosis • Accumulation of mitochondrial mutations • Higher mitochondrial mRNA transcripts • The MT-DNA encodes for 13 proteins, 2 rRNA and 22 tRNAs • Multiple signals and hundreds of proteins are sent to the mitochondria – BUT – • Mitochondrial-Cellular communication remains elusive New BP3 partners: ! LTBP-1, LRP-1 (TGFßRV) BP3BP3DR receptor Partners RXR, Transferrin, Collagen, E7, Caveolin

Transferrin Novel Genes: Humanin!

Cloned by two other groups at the same time: AD survival factor (Nishimoto) & BAX partner (Reed)! Humanin: The First Mitochondrial Peptide

24 amino acid peptide transcribed from an ORF within the region of the mtDNA at the 16S ribosomal RNA locus (Nature 2003) (PNAS 2003) NMR structure

Conserved Sequence Hydrophobic Core Human: MAPRGFSCLLLLTSEIDLPVKRRA

Humanin • Produced as an mRNA, capped and polyadenylated • Gene within a gene. Expressed and secreted • Present in Brain, Testes, Prostate, Seminal Plasma, CSF, and Plasma • Cytoprotective / metaboloprotective factor Protective Actions of Humanin in vivo studies in rodents Neuroprotective/Cytoprotective • Prolongs Survival and function in ALS and Alzheimer mouse models • Protects against experimental

Metaboloprotective/Anti-inflammatory • Treats and prevents in NOD mice • Improves blood sugar in Zucker diabetic rats

Cardioprotective/Vasoprotective • Decreases myocardial infarct size • Delays atherosclerosis in an Apo-E KO mice HN rescues dying neurons Ikonen et al 2003 PNAS

Primary cortical neurons treated with Aβ and +/- HN

Humanin

10 pM 100 pM 1 nM 10 nM

Aβ

•A βHN + also protects against -induced & FAD-induced apoptosis •BP-3 HN effective in inhibiting non-neuronal cell death in various cells HN Both Treats and Prevents Type-1 Diabetes in NOD (Non-Obese Diabetic) Mice

Glucose Percent (mg/dL) survival Saline HN 100 400 90 P = 0.04 80 300 70 60 50 200 ** ** 40 ** 30 n=10 Saline 100 ** ** 20 n=10 HN 2 mg/kg/day IP 10 0 0 0 15 30 45 60 75 90 0 5 10 15 20 25 Time (minutes) Time (weeks)

Hoang et al, 2009 Metabolism HN Antagonizes Apoptosis in beta cells

Apoptosis

1.2

1.0

0.8 ** 0.6 ** ** 0.4

0.2

0 0 1000 HN (nM) HN Inhibits Inflammation and Insulitis in the Islets of Type-1 Diabetic Mice

Control Humanin

Hoang et al, 2009 Metabolism HN Inhibits Inflammation and Insulitis in the Islets of Type-1 Diabetic Mice Decreased severity of insulitis in HN treated NOD mice 100 G3 - more than 50%

75 infiltration G2 - less than 50% infiltration 50 G1 - periinsulitis

25 G0 - normal Infiltrated Islets Infiltrated (%)

0 Saline HN Hoang et al, 2009 Metabolism Humanin analogues protects against atherosclerosis and improves endothelial function in ApoE-KO mice

%Relaxation

0 * * * 0.12 Plaque size (mm) -20 * 0.10 Scrambled peptide ** ** -40 ** 0.08 Aortic Arch 0.06 % Relaxation % -60 Contorl ApoE HC S 0.04 HNGF6A -80 ApoE HC HN Control HN 0.02

-100 0.00 9 8 7 6 5 Scr HNG Acetyicholine (-log M/L) Log relaxant concentration 0.4 mg/kg/day HNGF6A for 16 weeks (Oh et al; Atherosclerosis 2011) Humanin analogue HNGF6A infusion lowers blood sugar in Zucker Diabetic Rats

400

300

200 * * * * * * * 100 *

Glucose (mg/dL) Glucose Saline HNGF6A

0 0 60 120 180 240 Minutes After Infusion Recent support for the MDP concept

(& editorial by Shadel, Cell 2011) (Dillin & colleagues, Cell 2011)

MDPs = mitokines? Mitochines? MDPs - a novel family of mitochondrial-derived peptides - are unique mediators of mitochondrial function in health and disease

• Humanin is clearly expressed and has functional significance as a cyto-protective, anti-inflammatory, and metabolo-protective factor • Regulate cell gene expression and mitochondrial function • Therapeutic potential in DM, atherosclerosis, AD • Developmentally & Hormonally regulated / reduced in aging • Serve as retrograde signals from the mitochondria • Mitokines? Or Mitochines? • Additional MDPs are produced • May be important in multiple disease states Thanks to Mito-collaborators: Laura Cobb (UCLA) Cohen lab Radhika Muzumdar (Einstein) Ed Hosono Christina Wang (LABiomed) David Lee Nir Barzilai (Einstein) Brian Miyazaki Kuk-Wha Lee (UCLA) Carmel Ashur Andy Bartke (SIU-SM) Hemal Mehta Sree Nair (Mayo) David Hwang Valter Longo (USC) Hayoung Park Andy Hoffman (Stanford) Jyotsna Keni Amir Lerman (Mayo) Hiromi Nakamura Chris Glass (UCSD) Qinglei Gao Shuo Lin (UCLA) Yung-Ping Chin Julian Whitelegge (UCLA) Junxiang Wan

Supported in part by the following grants to Pinchas Cohen: P30DK063491 (DERC), RO1ES020812, R01AG034430 (EUREKA), and a Transformative-R01: GM090311