Dr. H. Craig Heller, co-director of Stanford's Down Syndrome Research Center, discusses Down syndrome cognition research, treatment, sleep, and circadian rhythms.

1. Today’s Agenda
 Overview of DSRTF: Carolyn Cronin, DSRTF
Executive Director
 Sleep’s Impact on Learning and Memory: Dr.
Craig Heller, Stanford University
 How You Can Help
E-mail: dsrtf@dsrtf.org
Down Syndrome Research and
Treatment Foundation

2. Down Syndrome Research and
Treatment Foundation Mission
• Stimulate and fund cognition research to
improve learning, memory, and speech for
individuals with Down syndrome
• Translation of research to deliver treatments
to allow individuals to:
• Participate more successfully in school
• Lead more active and independent lives
• Prevent or delay early cognitive decline
Down Syndrome Research and
Treatment Foundation

3. Why Cognition Research?
• Cognitive challenges present throughout life
• Generally, mild to moderate cognitive impairment
• Significant presence of neuropathology of
Alzheimer’s disease by the age of 40
Down Syndrome Research and
Treatment Foundation

4. DSRTF Strategy
FOCUS
Pioneer in stimulating cognition research
RESOURCES
Leaders in funding and executing Ds-specific research strategy
COLLABORATION
Interdisciplinary coordination and communication
TRANSLATION
Accelerate the move from research to treatments
Down Syndrome Research and
Treatment Foundation

5. 2003 Down Syndrome Research
• No evidence of what causes impaired cognition
in people with Down syndrome
• No targets on which to focus efforts
• Minimal government funding
• Few researchers focused on DS cognition
Down Syndrome Research and
Treatment Foundation

6. Research Results
• Eight drug targets
Areas of the brain that work differently —
“mechanisms”
• Three candidate drugs — and more work in this area
• Two clinical trials — and investment in tests for
efficacy
Down Syndrome Research and
Treatment Foundation

7. Government Funding:
NIH Per Capita Investment
52X
39X
6X 27X
13X
3X
28X
2X
5X
400,000 400,000 400,000 45,000 30,000 30,000 30,000
1,500,000 1,500,000 17,500
Down Syndrome Research and
Treatment Foundation
Number of Individuals affected in the U.S.

8. Progress Towards Improving Cognition in
Individuals with Down Syndrome
Craig Heller and Craig Garner
Co-directors Down Syndrome Center
Stanford University
DSRTF Webinar, January 29, 2013

9. Down Syndrome
Clinical Assessment
• Caused by the triplication of Chromosome 21
(~250 genes).
• Common Disorder: 1/700 Births: Incidence
higher when mothers are over 35
• 350,000 afflicted in US; 500,000 Europe;
> 3 Million world wide
• Cognitive impairment, mild-severe (IQ 20-80)
• Progressive cognitive decline
• Deficits in speech and language skills
• Deficits in short- and long-term memory
• Propensity for early onset Alzheimer Disease
(~30 years of age)

10. Current Treatment Strategies for
Cognitive Impairment in DS
Drug Description Trial Outcome Adverse
Vitamin Antioxidants, folinic Numerous, including No significant benefit
supplement acid, vitamins A, C, E… placebo-controlled
and more
Vasopressin Peptide hormone One trial: short, No significant benefit N/A
placebo-controlled
Piracetam Nootropic, GABA One trial: placebo- No significant benefit Various,
derivative. Site of controlled common
action unknown.
Donepezil Acetylcholinesterase Various. DS + AD, Mixed. No clear Various,
inhibitor adults, children. significant benefit for common
Large trial ongoing non-AD.
Rivastigmine Cholinesterase 2 trials: DS + AD No benefit DS + AD, 7/11 in
inhibitor. placebo-controlled; small improvement adolescents
adolescents open adolescents
label
None have been shown to be effective

11. Developing Therapies for Cognitive
Impairment due to Down Syndrome
Our strategy
• Define what is wrong in
humans.
• Explore animal models that
reflect the problems.
• Discover the underlying
mechanisms.
• Develop rationale drug
therapies to fix these
mechanisms.

12. Neuropsychological Assessment of Learning and
Memory in Down Syndrome
• (see Lynn Nadel, Genes,Brain and Behavior 2:156 2003)
– Learning is normal in very young subjects <6 month, but declines
progressively in the first year.
– A second decline occurs in adulthood as the risk of early onset
Alzheimer disease takes it toll.
– Disproportionately affected are memory processes that involve
the hippocampus and prefrontal cortex.
– Impairments are mostly seen in declarative memory, though
procedural memory is also affected.
– Impairments affect speech, language and verbal short term
memory and IQ.

13. The hippocampus is important for
memory
Henry Molaison (HM)
1926-2008

14. The Ts65Dn Mouse Model of DS
Karyotype analysis
(visual display of the chromosomes grouped by their size,
number and shape)
TS Mouse WT or 2N Mouse

15. Synapses and synaptic plasticity in DS mice
– Brain anatomy is altered.
– Synaptic plasticity is impaired.
–Electrophysiological studies indicate that
excessive inhibition is suppressing
synaptic plasticity in neural circuits critical
for memory processing.
Inhibition is under-emphasized in models of how the brain works.

16. A working hypothesis:
Intellectual disability is due to over-inhibition in the CNS
Fabian Fernandez
Over-inhibition could impair the transfer Short Term Memory to Long Term.
Major inhibitory system in brain is GABA… (very much involved in Sleep
and Circadian Rhythms).
Can GABA antagonists restore learning and memory in TS mice?
If so, is the action through modulation of sleep and/or circadian systems?

17. A mouse model of Down syndrome shows poor
learning and memory performance

18. A mouse model of Down syndrome shows poor
learning and memory performance
Normal mice
DS mice
Fernandez & Garner, 2007

19. Study Design: Evaluation of chronic treatment
with GABA antagonists on learning and memory.
Drugs Used:
Object recognition
Picrotoxin
training
Bilobilide
Pentylenetetrazole
Flumasinil
Daily drug treatment
Day 1 Day 17
Object recognition
testing
Drugs given during the light phase
Training/testing trials
carried out at various
Fabian Fernandez, Damien Colas, Bayara times after treatment
Chuluun, Craig Heller, Craig Garner, et al.
ends.

20. Memory improvement is long-lasting after daily
pentylenetetrazole (PTZ) dose
Normal mice
DS mice
1 week post-treatment 2 months post-treatment
Fernandez & Garner, 2007

21. GABAA Receptor Antagonists Tested and Shown
to be Efficacious
• Picrotoxin:
– Pros: Potent compound (IC50 1uM), excellent bioavailability
– Cons: narrow therapeutic window
• Bilobalide:
– Pros: Potent compound (IC50 2uM), excellent bioavailability, good therapeutic
window
– Cons: currently available in plant extract only (Gingko Biloba), difficult synthesis.
• Pentylenetetrazole:
– Pros: Excellent pharmacokinetic values, oral delivery, excellent bioavailability, good
therapeutic window, long history in humans
– Cons: Currently not approved by FDA
• Alpha5 inverse agonist:
– Pros: Excellent pharmacokinetic values, oral delivery, excellent bioavailability, good
therapeutic window. Specific for a subset of hippocampal GABAA receptors.
– Cons: currently not approved by FDA
• Flumazenil:
– Pros: Excellent pharmacokinetic values, good therapeutic window, approved by
FDA for the treatment of benzodiazepine overdose
– Cons: poor oral bioavailability, acute IV administration

22. Goals of recent studies
• Preclinical development of PTZ
– Dose, safety, age, pharmacokinetics
• Investigation of mechanism of drug therapy
– Dosing strategy
– Developing New Biometrics
– Understand Mechanism

23. PTZ is effective at very low dose levels
0.03 mg/kg dose
75 0 hrs
Learning index (%)
24 hrs
50
25
2N NaCl 2N PTZ TS NaCl TS PTZ
Colas & Chuluun

24. PTZ is effective at all ages: not a
developmental effect.
75
0 hrs
24 hrs
Learning index
50
(%)
25
TS NaCl TS PTZ TS NaCl TS PTZ
8 months 12 months
Colas & Chuluun

25. Assessing Safety: Continuous EEG
monitoring before, after and during PTZ treatment
1000hr of continuous EEG monitoring:
no evidence of seizure events
PTZ regimen also does not increase seizure threshold
Damian Colas

26. GABA antagonists can rescue the learning disability of Down
Syndrome……… BUT,
These effects are circadian phase dependent.
0 hrs
80 ** 2N Ts65Dn
Learning index (%)
*
24 hrs
LI (%)
40
20
0
PTZ PTZ
Treatment during the dark phase (wake)
Colas & Chuluun

27. A model system for research on circadian rhythms and photoperiodicity.

28. Siberian hamsters have normal circadian behavior
Aschoff’s Rule Phase Response Curve
Phase Shift (h)
Time of Day (h) Circadian Time (h)
Ruby et al.

29. Normal re-entrainment to short shifts in photocycle
Reentrainment: ± 3 h
Time of Day (h)
Ruby et al.

30. But, if we try to phase shift the
hamster’s circadian rhythm by 5
or 6 hours – a disastrous result!

31. Total Arrhythmia for the rest of their lives!
Time of Day (h)
And, hamsters made arrhythmic with such a phase shift
are learning impaired! Ruby et al. 1996

32. Novel object recognition (NOR)
5 min 5 min
Time
Familiarization Phase Testing Phase

33. Spontaneous alternation (SA): spatial working memory

34. Memory deficits in SA and NOR
Discrimination Index Zeitgeber Time (h)
Ruby et al. 2008

35. Memory is rescued by the chronic treatment protocol
with the GABAA receptor antagonist PTZ
Discrimination Index
The circadian clock mechanism releases GABA.
Is a continuously active circadian clock interfering with memory formation?
Ruby et al. unpublished

36. Arrhythmia Can Also Be Induced by Ablation of
the Circadian Clock in the Brain (the SCN)
SCN Lesion (SCNx) Disruptive Phase Shift (DPS)
Ruby et al. unpublished

37. SCN ablation has no effect on SA or NOR
Ruby et al. unpublished

38. Can SCN ablation rescue memory in DPS hamsters?
DPS
SCNx
Ruby et al. unpublished

39. SCN ablation rescues memory in arrhythmic hamsters
Evidence that the SCN actively suppresses neuroplasticity at a particular
circadian phase.
Why????
Ruby et al. unpublished

40. Memory Consolidation Requires a Sleep Phase
(C57Bl6 mice)
75
training testing
Discrimination Index (%)
50
25
Dark 12 hr Dark 24 hr Light 12 hr Light 24 hr
Chuluun, Colas et al.
Does Memory Consolidation Require a Certain Quality of Sleep?

41. Using Optogenetics to Fragment Sleep Without Altering
Total Sleep Duration to see Effects on Memory
Training Testing
5 min 5 min
Optogenetic stimulation – 4 hrs
0 12 24
Time (hrs)
Mice trained early in light phase and then stimulated optogenetically at
30, 60, 120, or 240 sec intervals for 4 hrs, or sleep deprived, or left undisturbed.
Testing 24 hrs after training.
Rolls, Colas et al. 2011

42. Stimulation results in a greater number of brief episodes of
wake, but does not decrease total sleep time!
Lower delta, higher theta
Rolls, Colas, et al 2011.

43. Major Finding: Memory consolidation requires minimal
quanta of NREM sleep
Rolls, Colas, et al. 2011

44. But, still a circadian component --
Sleep fragmentation only interferes with learning when
delivered during the light phase
Animals are trained in the Dark phase and
tested 24 hrs later in the Dark phase.
Learning is normal.
Rolls, Collas, et al. 2011

45. The Converse: Enhancing SWA in Ts65Dn mice
improves their learning and memory without GABA
antagonists.
Sleep deprivation for 4 hrs
prior to training.
Normalization
Baseline
of delta power
following SD
4 hr sleep deprivation before training
85 **
75 **
NOR training NOR testing **
0H 24H 65
DI (%)
55
50%
45
35 0H
24H
25
2N BL TS BL 2N SD TS SD
Colas et al. n=8 n=11

46. A Bold Hypothesis
When short term memory is being
transferred to long term memory during
sleep, the circadian system suppresses
neuroplasticity to protect the fidelity of the
memory transcripts.
Could circadian suppression of neuroplasticity be too
great in DS?
Could the sleep related processes of memory
consolidation be impaired by high levels of GABA
activity?

47. Conclusions (tentative)
• Tonic over-inhibition via GABAergic mechanisms can
produce learning disability.
• Inhibitory tone can be reset long-term with a short-term
treatment with GABAA antagonists at the proper
circadian phase.
• PTZ is an excellent candidate for clinical trials. It’s
efficacy is not age dependent.
• Quality sleep is needed for memory consolidation and for
efficacy of GABAA antagonist treatment.
• The circadian system suppresses neural plasticity during
consolidation via GABAergic mechanisms. The
functional significance may be to stabilize memory
transcripts.

48. Acknowledgements
Garner Laboratory Heller Laboratory
Craig C. Garner H. Craig Heller
Fabian Fernandez Bayara Chuluun
Martina Blank Damien Colas
Deepti Warrier Norman Ruby
Jackie Rodriguez Grace Hagiwara
Dan Wetmore
Funding Support NSF, NIH, Stanford Spark Program, Coulter
Foundation, DSRTF, Fidelity Foundation, Stanford
Neuroscience Institute

49. Thanks!

50. Ways You Can Help
• Increase awareness of DSRTF and the promise and
progress of cognitive research
• Tell us how we can provide additional value and
information
• Participate online and invite others to join to continue to
grow the DSRTF/plus15 community
• Invite us to share our mission with other groups with
whom you are affiliated
• Increase the funding we can make available for research
Down Syndrome Research and
Treatment Foundation