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) Roger H. Reeves, Ph.D.
Click to edit Master subtitle style Institute for Genetic Medicine McKusick-Nathans
Johns Hopkins University School of Medicine rreeves@jhmi.edu Dept. of Physiology

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 HoffmanLaRoche Inc. and Elan Pharmaceuticals and on the SABs of DSRTF, RDS and the Linda Crnic Foundation. Nov. 2011

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.
Click to edit Master subtitle style

Photo from:

www.dsrtf.org

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

What do we know for sure about Down Syndrome?

What do we know for sure about Down Syndrome? It occurs due to Trisomy 21. Every individual has a different subset of features that are more common in people with trisomy 21 than in the typical (euploid) population It is among the most complex genetic conditions compatible with human survival beyond term.

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Comparative mapping
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.

This brain regions is affected in DS, too.

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.

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

J Neurosci 2004

Hippocampal deficits include excessive inhibitory input in electrophysiological studies.

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 Time

Day 3 and 4: Milk Intake in Feeding Tubes

Day 5 thru 21: Drug Administration in Feeding Tubes

Test Fernandez et al., Nat Neurosci., published online Feb 25 2007 Day 14

Test Day 21

Pentylenetetrazole Treated Ts65Dn Mice Exhibit Improved Object Recognition Memory

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

Discrimination index

40 30 20 10 0 Milk *

WT Ts65Dn

PTZ

Fernandez et al., Nat Neurosci., 2007

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

170 160 150 fEPSP (%) 140 130 120 110 100 0 10 20 30 40 50 60

Dentate LTP

wt Ts65 + PTZ Ts65Dn ctrl.

Time (min)

2 weeks of treatment, test 2-3 months later

Fernandez et al., Nat Neurosci., 2007

Arizona Cognitive Test Battery

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

Lynn Nadel Jamie Edgin Stephanie Sherman Tracie Rosser Stuart Tinker Cheryl Maslen

Len Abbeduto Rene Pierpoint Roger Reeves Iser DeLeon Melissa Allman George Capone Valerie DeLeon

Targeted Brain Regions

Cerebellum

Tested core function(s): motor responsiveness, reaction times Tested core function(s): Holding of information in active or working memory to guide action selection; executive function Tested core function(s): Storage of episodic information in long-term memory

Prefrontal cortex

Hippocampus

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. 2:149164

Research Design

(U. Wisconsin, Oregon Health Science Univ., Childrens Natl. Medical Center)

J Neurodevel Disord (2010) 2:149-164

A novel alternative approach to therapy for Down syndrome

Laura Baxter Juan Troncoso Randall Roper Nidhi Saran Donna Klinedinst Phil Beachy Ann Lawler Heidi St. John Hernan Lorenzi Ishita Das Yan 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.

Cerebellar volume is reduced in DS and in Ts65Dn mice

Euploid Down syndrome

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

Normalize d cerebellar area

0 . 0 2 . 0 1 . 8 0 1 . 6 0 1 . 4 0 1 . 2 0 1 . 0 0 . 8 0 0 . 6 0 0 . 4 0 0 . 2 0 0

Eup Ts6 loid 5Dn

* *

P
Eu. 0 Ts. N=1 N=1 0 0

P
Eu. 6 Ts. N=1 N=1 0 0

P
Eu. 1 Ts. N=1 4 N=1 0 0

Eu. P Ts. N=1 2 N=1 0 04

P
Eu. 2 Ts. N= 8 N= 7 5

P
Eu. 3 Ts. N= 5 N= 5 7

EGL is the same size at P0

Stereology demonstrates an early mitotic deficit in trisomic mice

Roper, R.J. et al., 2006. Proc. Natl. Acad. Sci. 103(5):14526.

gc precursors Euploid (n=6): 951,995 Ts65Dn (n=6): T-test: 931,976 p = 0.40

P0

Mitotic cells 16,970 13,354 p = 0.05

Mitotic index 1.8% 1.4%

P6
Euploid (n=5): Ts65Dn (n=5): T-test: 7,381,391 5,269,919 p = 0.001 135,359 98,781 p = 0.04 1.8% 1.8%

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

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

7 0 6 0 5 0 4 0 3 0 2 0 1 0 0

E u T s

CPM (x 10

1 0 2 0

0 10

7.5

Shh-Np (nM)

Roper, R.J. et al., 2006. Proc. Natl. Acad. Sci. 103(5):14526.

Can the Ts65Dn/DS granule cell deficit be cured?

SAG is a small molecule (potential drug) that has the same effects as the SHH growth factor
Frank-Kamenetsky et al., J Biol. 2002 Nov 6;1(2):10.

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.

a. Granule cell precursors

10,000, 000 9,000, 000 8,000, 000 7,000, 000 6,000, 000 5,000, 000 4,000, 000 3,000, 000 2,000, 000 1,000, 000 0
ed un tre at

b. Mitotic granule cell precursors 200,

000 180, 000 160, 000 140, 000 120, 000 100, 000 80, 000 60, 000 40, 000 20, 000 0
un tre at ed

T SA s G E ve u hi cle E SA u G

E un u tre at ed

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.

E un u at tre ed

T SA s G E ve u hi cle E SA u G

Ts

Ts

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
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Trisomy for orthologs of Hsa21 genes in mice has related effects on developmental pathways and functions 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. The Arizona Cognitive Test Battery provides a focused instrument for assessing possible improvements and represents a substantial part of the current Clinical Trial evaluation. Additional approaches in the pipeline may eliminate some congenital problems of Down syndrome altogether

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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.

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

Reeves lab

Cognition

Cerebellum project: Laura Baxter Randall Roper Nidhi Saran Juan Troncoso Ishita Das and Phil Beachy, Stanford University Current lab members: Donna Klinedinst Huiqing Li Meifang Xiao Sarah McGuire Duane Currier Sarah Edie Renita Polk Jennifer Poitras Tara Howard Ben Devenney

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

Joan T. Richtsmeier