Roger H. Reeves, Ph.D.Dept. of PhysiologyMcKusick-Nathans Institute for Genetic MedicineJohns Hopkins University School of Medicinerreeves@jhmi.edu

Changing the future for people with Down syndrome

A discussion for general audiences of recent research advances that have led to an historic clinical trial to improve cognition in Down syndrome,

and which hold the promise of further advances. Adapted from a presentation at the Linda Crnic Institute for Down Syndrome, Denver CO

(2011)

This work was supported by NICHD and NCI under PHS award HD38384. NHLBI supports identification of genetic modifiers of Congenital Heart Disease in Down syndrome under award HL83330. The Down Syndrome Research and Treatment Foundation (DSRTF) and Research Down Syndrome (RDS) support assessment of the ameliorative effects of SAG on trisomic brain development. The Anna and John Sie Foundation supported establishment of the testing network for the DS Cognitive test battery. Dr. Reeves is a consultant for Hoffman-LaRoche Inc. and Elan Pharmaceuticals and on the SABs of DSRTF, RDS and the Linda Crnic Foundation. Nov. 2011

www.dsrtf.org

400,000 Americans have Down syndrome (DS). 6000 children are born with DS every year in the US alone.

It is the most complex genetic condition that is compatible with survival past term.

Because the root cause – over-representation of ~500 genes – is so complex and the effects of the extra chromosome are present

in all cells from conception onward, animal models are an essential tool in efforts to study and ameliorate effects of trisomy

21.

Photo from:

DS affects every cell, tissue and organ in the body from

conception onward.Trisomy 21 has many effects, and

key among them is a cognitive deficit.

A small increase in cognitive ability could greatly expand the

opportunities of tens of thousands of people with DS.

• Mouse models with three copies of the same genes present in Down syndrome develop features like people with DS.

• These models make it possible to identify

the basis for cognitive deficits, and to test drugs that diminish these deficits.

• Clinical safety trials in people with DS using some of these drugs started in

2011. (Watch http://clinicaltrials.gov/)

What do we know for sure about Down Syndrome?

What do we know for sure about Down Syndrome?

1. It occurs due to Trisomy 21.

2. Every individual has a different subset of features that are more common in people with trisomy 21 than in the “typical” (euploid) population

3. It is among the most complex genetic conditions compatible with human survival beyond term.

Comparativemapping

Genes on human Chr 21 are found on Chr

16, 17 and 10 in mouse

Ts65Dn mouse (karyotype by Gail Stetten, Sarah South)

Muriel Davisson

Can a mouse have Down syndrome?

A.Short stature*Flat facies *Flat nasal bridge *Protruding tongue *Mental retardation, “DS type” * Small cerebellum * Highly arched palate (*)

{

Yes! Trisomic mice develop a number of the same features as do trisomic people.

4. Mental retardation, “DS type”

Ts65Dn mice are significantly deficient in functions that require the hippocampus,

like the Morris water maze test.

Reeves, R.H., N.G. Irving, T. Moran, A. Wohn, C. Kitt, S. Sisodia, C. Schmidt, R.T. Bronson and M.T. Davisson. 1995. A mouse model for Down Syndrome exhibits learning and behavior deficits. Nature Genetics 11:177-184.

This brain regions is affected in DS, too.

The Down syndrome brain: functional outcomes

• Hippocampal function is robustly affected in mouse DS

models (Ts65Dn, Ts1Cje) Reeves et al., Nat. Gen. 1995; Escorihuela, Neuro Lett. 1995; Holtzman et al., PNAS 1996

•

Hippocampal deficits especially affect declarative learning and memory, affecting speech, language and short-term memory.

• J Neurosci 2004

The Down syndrome brain: functional outcomes

Hippocampal deficits include excessive inhibitory input in electrophysiological studies.

J Neurosci 2004

Fernandez et al., Nat Neurosci., published online Feb 25 2007

Proposed Mechanism for Decreased Synaptic Plasticity in Ts65Dn, Fernandez et al. 2007

Ts65Dn mice show:

Reduced spatial memory and its physiological correlate, LTP

Greater Inhibition in the Hippocampus via GABAA neurons in Ts65Dn mice (Kleschevnikov et al., 2004)

Ts65Dn shows an increase in inhibitory (GABA-ergic) inputs relative to excitatory inputs to (Hanson et al., J Phys 2007)

Approach: Restore this balance with drugs that reduce GABAA receptor activity and measure tests of hippocampal function

Experimental Protocol: Assessing the Cognitive Effects of Pentylenetetrazole in Ts65Dn Mice

Day 1 and 2: Home Cage Feeding

Day 3 and 4: Milk Intake in

Feeding Tubes

Day 5 thru 21: Drug

Administration in Feeding Tubes

Time

Test

Day 14

Test

Day 21Fernandez et al., Nat Neurosci., published online Feb 25 2007

Pentylenetetrazole Treated Ts65Dn Mice Exhibit Improved Object Recognition Memory

Milk PTZ0

10

20

30

40 WTTs65Dn

*

Dis

crim

inat

ion

inde

x

Fernandez et al., Nat Neurosci., 2007

Short term evaluation at the end of 2 wks. of treatment

fEP

SP

(%)

Time (min)

0 10 20 30 40 50 60

100

110

120

130

140

150

160

170 Dentate LTP

Pentylenetetrazole Causes a Lasting Improvement in Circuit Functionality (2-3 months)

2 weeks of treatment, test 2-3 months later

wt

Ts65 + PTZ

Ts65Dnctrl.

Fernandez et al., Nat Neurosci., 2007

Arizona Cognitive Test Battery

Lynn NadelJamie Edgin

Stephanie Sherman

Tracie Rosser Stuart Tinker

Cheryl Maslen

Len AbbedutoRene Pierpoint

Roger ReevesIser DeLeonMelissa AllmanGeorge CaponeValerie DeLeon

A battery of tests focused specifically on brain regions that are highly affected in Down syndrome

Targeted Brain Regions

• Cerebellum– Tested core function(s): motor responsiveness, reaction

times

• Prefrontal cortex– Tested core function(s): Holding of information in active or

working memory to guide action selection; executive function

• Hippocampus– Tested core function(s): Storage of episodic information in

long-term memory

Selection of Tests

• Performance not at floor or ceiling• Mostly nonverbal:

– Avoids potential confound of well-known verbal STM and language deficits (e.g. syntax, articulation) in DS

– Analogues or variants exist or can be developed for animal models of DS

• Global measures of cognitive function & measures sensitive to regions of structural and functional abnormalities in DS

• Validated (e.g., neuroimaging or lesion data)

Edgin et al., J. Neurodevelop. Disord. 2:149–164

Research Design

(U. Wisconsin, Oregon Health Science Univ., Children’s Nat’l. Medical Center)

J Neurodevel Disord (2010) 2:149-164

A novel alternative approach to therapy for Down syndrome

Laura BaxterJuan TroncosoRandall RoperNidhi SaranDonna KlinedinstPhil BeachyAnn LawlerHeidi St. JohnHernan Lorenzi Ishita DasYan Xiang

Baxter, L.L. et al. 2000. Human Mol. Genet. 9:195-202.Roper, R.J. et al., 2006. Proc. Natl. Acad. Sci. 103(5):1452-6.

Euploid Down syndrome

Cerebellar volume is reduced in DS and in Ts65Dn mice

From Kesslak et al., Neurology, 1994, 44: 1039-1045.

Ts65Dn

Baxter, L.L. et al. 2000. Human Mol. Genet. 9:195-202.

Cerebellar volume and cell density are reduced in Ts65Dn mice

Reduced granule cell density in the Ts65Dn cerebellum correctly predicted the same phenotype in DS

Baxter, L.L. et al. 2000. Human Mol. Genet. 9:195-202.

When in development do the trisomic and euploid

cerebellums begin to diverge?

Ts65Dn cerebellum is overtly normal at P0, but significantly smaller by P6

0.00

0.02

0.04

0.06

0.08

0.10

0.12

0.14

0.16

0.18

0.20

1P0 P6 P14 P24

EuploidTs65Dn

**

P28 P35

*

*

Eu. N=10Ts. N=10

Eu. N=10Ts. N=10

Eu. N=10Ts. N=10

Eu. N=10Ts. N=10

Eu. N=7Ts. N=5

Eu. N=5Ts. N=7

Normalized

cerebellar area

EGL is the same size at P0

Euploid (n=6): 951,995 16,970 1.8%

Ts65Dn (n=6): 931,976 13,354 1.4%

T-test: p = 0.40 p = 0.05

Euploid (n=5): 7,381,391 135,359 1.8%

Ts65Dn (n=5): 5,269,919 98,781 1.8%

T-test: p = 0.001 p = 0.04

P0

P6

gc precursors Mitotic cells Mitotic index

Stereology demonstrates an early mitotic deficit in trisomic mice

Roper, R.J. et al., 2006. Proc. Natl. Acad. Sci. 103(5):1452-6.

Is the growth deficit in trisomic mice related to the role of SHH in gcp

proliferation?

-20

-10

0

10

20

30

40

50

60

70

Shh-Np (nM)

CP

M (

x 10

3)

Eu

Ts

0 2 5 7.5 10

Isolated granule cell precursors from Ts65Dn mice do not respond to Sonic hedgehog to the same degree as

euploid gcp

Roper, R.J. et al., 2006. Proc. Natl. Acad. Sci. 103(5):1452-6.

Can the Ts65Dn/DS granule cell deficit be “cured”?

Frank-Kamenetsky et al., J Biol. 2002 Nov 6;1(2):10.

SAG is a small molecule (potential drug) that has the same effects as the SHH growth factor

A single dose of SAG on the day of birth restores Ts65Dn cerebellar structure at P6, ~ 1/3 of the way though cerebellar

development. Because the number of dividing cells is also normal after SAG treatment, the cerebellum may be “cured” in

adult Ts65Dn mice.

Randall J. Roper, Laura L. Baxter, Nidhi G. Saran, Donna K. Klinedinst, Philip A. Beachy & Roger H. Reeves. 2006. Defective cerebellar response to mitogenic Hedgehog signalling in Down syndrome mice. Proc. Natl. Acad. Sci. 103: 1452-1456.

0

1,000,000

2,000,000

3,000,000

4,000,000

5,000,000

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9,000,000

10,000,000

Ts u

ntre

ated

Ts S

AG

Eu un

treat

ed

Eu ve

hicle

Eu SAG

a. Granule cell precursors b. Mitotic granule cell precursors

0

20,000

40,000

60,000

80,000

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Ts u

ntre

ated

Ts S

AG

Eu un

treat

ed

Eu ve

hicle

Eu SAG

Does the SAG effect last beyond early development?

What aspects of the Ts65Dn phenotype are affected?

Basic research has changed the game for people with Down syndrome.

A number of the key findings that made this possible were supported by private Foundations,

including the Down Syndrome Research and Treatment Foundation, the John and Anna Sie Foundation and Research Down Syndrome.*

*Dr. Reeves is a member of the Science Advisory Boards of the Linda Crnic Institute, which is affiliated with the Sie Foundation, and of DSRTF and RDS. He is a grantee of DSRTF and RDS, non-profit foundations dedicated to

amelioration of cognitive deficits in DS.

Conclusions

1. Trisomy for orthologs of Hsa21 genes in mice has related effects on developmental pathways and functions

2. Trisomy affects the function of the hippocampus in similar ways in mice and people. Down-regulation of GABA-A interneurons hold promise as therapeutic targets – A Clinical trial to test this has started.

3. The Arizona Cognitive Test Battery provides a focused instrument for assessing possible improvements and represents a substantial part of the current Clinical Trial evaluation.

4. Additional approaches in the pipeline may eliminate some congenital problems of Down syndrome altogether

http://www.roche-trials.com/

The first clinical trial by a major pharmaceutical company to test a drug that may improve learning and memory in Down syndrome started in the fall of 2011.

Cerebellum project:Laura BaxterRandall RoperNidhi SaranJuan TroncosoIshita Das

and Phil Beachy, Stanford University

Current lab members:Donna Klinedinst Huiqing LiMeifang XiaoSarah McGuireDuane CurrierSarah EdieRenita PolkJennifer PoitrasTara HowardBen Devenney

Craniofacial Joan T. Richtsmeier

Reeves lab

Jamie Edgin Lynn Nadel Stephanie Sherman George Capone Julie Hoover-Fong Iser DeLeon Len Abbeduto Cheryl Maslen Valerie DeLeon

Cognition