Heterochronic parabiosis: the promise of pro- and anti-geronic factors

Joseph M. Castellano, Ph.D. Stanford University School of Medicine, Wyss-Coray Laboratory

2016 OAIC Annual Meeting, April 19, 2016

Aging is a major risk factor for disease

Young (31 y) Old (88 y)

• Normal brain aging alterations: – Cognitive – Cellular – Molecular

• Aging is major risk factor for many neurological disorders

• Aged population projected to double by 2030

Young

Systemic environment

Old

Positive factors Positive factors

Conboy et al., 2005 Sinha et al., 2014

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Salpeter et al., 2013

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Loffredo et al., 2013

Salpeter et al., 2013

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Loffredo et al., 2013

Salpeter et al., 2013

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

Baht et al., 2015

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Loffredo et al., 2013

Salpeter et al., 2013

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

Villeda et al., 2011 Villeda et al., 2014 Katsimpardi et al., 2014

Baht et al., 2015

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Loffredo et al., 2013

Salpeter et al., 2013

Conboy et al., 2005

Conboy et al., 2005 Sinha et al., 2014

Villeda et al., 2011 Villeda et al., 2014 Katsimpardi et al., 2014

Baht et al., 2015

?

Young blood regulates aging processes in diverse tissues

Castellano et al., JAMA Neurol, 2015 (for review)

Adult neurogenesis occurs in lateral ventricles and the hippocampus (DG)

Signals in the systemic environment regulate neurogenesis in dentate gyrus

Villeda et al., 2011

Pro-aging factor CCL11 increases with age and impairs learning/memory

Villeda et al., 2011

Pro-aging factor CCL11 increases with age and impairs learning/memory

Villeda et al., 2011 B2M also revealed to be anti-neurogenic, pro-aging factor Smith et al., 2015, Nat Med

Blood-borne factors that promote plasticity and/or

cognition

e.g., GDF11, many others

Exposure to young blood revitalizes the aged brain

Plasticity gene networks

Dendritic spines/LTP

Microgliosis

Rejuvenation

Neurogenesis

Villeda et al., Nat Med, 2014 and unpublished

Learning/memory

Young blood

plasma

Strategy for the identification of rejuvenating factors

Cellular/molecular changes

Cognitive assessment

Mouse Aging

Heterochronic Parabiosis

Human Donors

Strategy for the identification of rejuvenating factors

Mouse Aging

Heterochronic Parabiosis

Human Donors

Umbilical cord

~ 20 years

~ 65 years

Cognitive assessment

Cellular/molecular changes

Cytokines Chemokines

Growth factors Neurotrophins

Hormone-like proteins Acute-phase proteins Complement factors Secreted receptors Sample array w/ differential

plasma protein signals

Plasma protein factors

150 µm spot

Protein microarray to assess relative plasma protein expression

Protein microarray reveals many elevated factors in cord plasma

Low High

Cord Young Old

Secreted Plasma Factors Castellano et al., in revision

Protein microarray reveals many elevated factors in cord plasma

Low High

Cord Young Old

Secreted Plasma Factors Castellano et al., in revision

Administration of human plasma in “NSG” mice

Heterochronic Parabiosis Changes

Mouse Human

Administration of human plasma in “NSG” mice

NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ aka “NOD Scid Gamma” (NSG)

Heterochronic Parabiosis Changes

Mouse Human

Age-dependent changes in immediate early gene marker c-Fos

Castellano et al., in revision

NSG mice exhibit other age-dependent hippocampal pathology

Castellano et al., in revision

NSG mice exhibit age-dependent learning and memory deficits

Castellano et al., in revision

NSG mice exhibit age-dependent learning and memory deficits

Castellano et al., in revision

Administration of human plasma in aged NSG mice

Aged NSG

Days 0 14

Human plasma donors

Behavior

Microarray/qPCR/IHC

Human plasma treatment results in distinct gene profiles in hippocampi

Castellano et al., in revision

Human plasma treatment results in distinct gene profiles in hippocampi

Castellano et al., in revision

Ontology-based activation prediction -Long-term memory -Long-term potentiation -neuritogenesis Plasticity upregulation by qPCR -c-Fos, Egr1, junb, camk2a, etc.

Aged NSG

Cord plasma increases c-Fos+ cell number in aged but not young dentate gyrus

Castellano et al., in revision

Aged NSG Young NSG

Cord plasma increases c-Fos+ cell number in aged but not young dentate gyrus

Castellano et al., in revision

Cord PLM activates DG granule neurons (excitatory)

Castellano et al., in revision

RE

SE

Treatment with cord plasma enhances LTP in dentate gyrus

Castellano et al., in revision

LTP is unaffected following young or elderly human plasma treatment

Castellano et al., in revision

Treatment with cord plasma improves learning and memory performance

Castellano et al., in revision

Strategy for the identification of rejuvenating factors

Cellular/molecular changes

Mouse Aging

Heterochronic Parabiosis

Human Aging Umbilical cord

plasma

Cognitive assessment

Strategy for the identification of rejuvenating factors

Cellular/molecular changes

Mouse Aging

Heterochronic Parabiosis

Human Aging

Cognitive assessment

In vivo “screen” of potential rejuvenating factors in aged WT mice

0 2 4 6 10

Putative Factors or vehicle i.p.

Days

Castellano et al., in revision

Plasma TIMP2 is reduced very early in life in blood and brain

Castellano et al., in revision

Human

Mouse

Systemic supplementation with TIMP2 enhances synaptic plasticity in aged mice

0 2 4 6 8 10 12 14

rTIMP2 or vehicle i.p.

Days

Castellano et al., in revision

Systemic treatment with TIMP2 improves learning and memory in aged WT mice

Castellano et al., in revision

Systemic TIMP2 is necessary for spatial memory

Training

Novel Location

Day 1

Day 2

Castellano et al., in revision

Translational potential of blood-borne proteins • TIMP2

– 21-24 kDa; non-glycosylated – Limits tumor angiogenesis and plays role in tissue

remodeling – Broad MMP inhibitor Marimastat proceeded to Phase

III trials (discontinued)

• Ongoing plasma trials related to CNS – AMBAR (Alzheimer’s Management by Albumin

Replacement - Grifols) – PLASMA (PLasma for Alzheimer’s SyMptom

Amelioration)

Conclusions

• Blood factors can regulate aging process in diverse tissues – Developmental-stage human plasma (UC) improves neuronal and

cognitive function – Young plasma proteins (TIMP2) restore or improve function of aged

hippocampus – Use of NSG mouse model allows for identification of relevant

blood-borne proteins with possible translational implications

Acknowledgements

Wyss-Coray Lab Tony Wyss-Coray Kira I. Mosher Rachelle Abbey Daniela Berdnik Jadon Shen

Xie Lab/AfaSci Simon Xie Bende Zou

Stanford Tom Rando Martin Angst

Funding Sources -NIH/NIA -Jane Coffin Childs Postdoctoral Fellowship/Simons Foundation -Stanford Child Health Research Institute Postdoctoral Fellowship -Donor’s Cure Foundation, CCAD

Youthful systemic factors

Stimulated vascular endothelium

Disrupted BBB?

Youth-associated leukocytes?

Transport? Diffusion? Secondary signals?

Synaptic plasticity

Neurogenesis Cell-to-cell Signaling?

Oligodendrocyte activity

Castellano et al., JAMA Neurol, 2015

Young blood alters brain via unknown mechanisms/factors

Human plasma does not alter the number of neuroblasts in dentate gyrus of NSG mice

Castellano et al., in revision

rTIMP2 treatment does not appear to alter DCX+ cell number in aged DG

Castellano et al., in revision

TIMP2 expression in plasma and hippocampus declines gradually with age in mice

Castellano et al., in revision

Blood-borne factor GDF11 mediates SVZ neurogenesis rejuvenation

Katsimpardi et al., 2014