3. Vanderbilt Presenters
HSG 2016: DISCOVERING OUR FUTURE
Aaron Bowman, PhD
Associate Professor in the Department of Neurology and
Pediatrics/Training Program in Environmental Toxicology,
Director
Jeff Conn, PhD
Lee E. Limbird Professor of Pharmacology /Center for
Neuroscience Drug Discovery, Director
Paul Harris, PhD
Professor of Biomedical Informatics/School of
Engineering, Professor of Biomedical Engineering/ Office
of Research Informatics, Director
4. Manganese and HD
Aaron B. Bowman, PhD
Departments of Pediatrics, Neurology and Biochemistry
Vanderbilt University (VU) and VU Medical Center
Nashville, TN
5. Huntington’s disease (HD) genetics and
environment
HSG 2016: DISCOVERING OUR FUTURE
• Longer CAG repeats (polyQ tracts) correlate with earlier age-of-onset and faster
progression
• Undefined environmental factors account for majority of variability in age-of-onset
after accounting for CAG repeat length
Environmental
Factors/Age
Apparent normal
neuronal function
6. Genetic x toxicant screen of 8 cytotoxic metals revealed resistance
of HD striatal cells to Mn toxicity
HSG 2016: DISCOVERING OUR FUTURE
0
25
50
75
100
125
0 200 400 600
Mn(II) µM
PercentSurvival
*
*
*
Wild-type STHdhQ7/Q7
Mutant STHdhQ111/Q111
Mechanistic studies demonstrated a deficient Mn accumulation phenotype
…see Williams et al. JNC 2010
Mutant STHdhQ111/Q111
Wild-type STHdhQ7/Q7
0
200
400
600
800
0 40 100
Manganese chloride exposure (! M)
IntracellularMnlevels
(fmolper100cells)
*
*
* P<0.01 (n=5)
MTT Assay Atomic Absorption Spectroscopy
7. Striatum showed decreased net Mn uptake in prodromal stage (3
months) YAC128Q mouse model of HD
HSG 2016: DISCOVERING OUR FUTURE
Total regional Mn by atomic absorption spectrometry
Mn-exposed animals
13.9 mg/kg Mn
Sub-cutaneous exposure, on
days 1, 4, and 7.
Collect tissue 24 hours
after last exposure
FVB-YAC128Q
…see Williams et al. JNC 2010
8. Human-based and unbiased genomic-level data support a link
between manganese biology and HD
HSG 2016: DISCOVERING OUR FUTURE
Andrew Tidbal et al Hum Mol Genet, 2015
Human
Striatal
Progenitors
Statistic FVB +/+ FVB Tg-
YAC128Q
p < 0.0005
p < 0.001
p < 0.005
q < 0.05
Ensemble alignment
9
17
94
UCSC mm10 alignment
292
3
5
36
54
# of Mn-responsive genes by p-value or q-value
1 week subcutaneous Mn exposure
Mouse HD model
In collaboration with Dr. Michael Aschner and Nancy Parmalee
Mouse
Striatal
Progenitors
9. Hypothesis
HSG 2016: DISCOVERING OUR FUTURE
HD genotype disrupts neuronal Mn transport and
homeostatic mechanisms to impair Mn biology and
block Mn toxicity
ATM is a specific Mn-activated kinase that phosphorylates target proteins such as
p53
Chan et al 2000. JBC
Canman et al 1998. Science
10. Mn activates p53 phosphorylation, this response is impaired in HD
striatal neuroprogenitors
HSG 2016: DISCOVERING OUR FUTURE
Human
Andrew Tidball et al Hum Mol Genet, 2015
11. ATM inhibition blocks Mn-dependent phosphorylation of p53 (and
other ATM targets) in human HD ISLT1 progenitors
HSG 2016: DISCOVERING OUR FUTURE
Andrew Tidball et al Hum Mol Genet, 2015
Manganese (Mn2+) n=6
essential metal and
neurotoxicant
“In Cell” Western Blots
KU = KU55933 an ATM kinase inhibitor
12. ATM auto-phosphorylation responds to Mn exposure, and this response
is deficient in HD cells
HSG 2016: DISCOVERING OUR FUTURE
Andrew Tidball et al Hum Mol Genet, 2015
13. Bypassing the Mn deficit in mouse striatal model normalizes ATM-P53
responsiveness
HSG 2016: DISCOVERING OUR FUTURE
Andrew Tidball et al Hum Mol Genet, 2015
KB-R7943 (KB-R) blocks activity of Sodium-Calcium Exchanger (NCX1); and is
known to block Mn efflux in mouse tissues
14. Cross-talk and co-regulation of the p53/AKT/mTOR
pathways and Mn in HD pathobiology
HSG 2016: DISCOVERING OUR FUTURE
IGF/PI3K
ATM/p53
AKT/mTORmutHTT Mn
For more see poster by Miles Bryan
0 13 25 50 75
Mn µM (24hr) .
p-p53(Ser15)
p-AKT(Ser473)
p-S6(Ser235/236)
Total Protein
Wild-type STHdhQ7/Q7
15. Hypothesis
HSG 2016: DISCOVERING OUR FUTURE
HD genotype disrupts neuronal Mn transport and
homeostatic mechanisms to impair Mn biology and
block Mn toxicity
Urea and citrulline-nitric oxide cycles are disrupted in HD models and patients (e.g. increased
blood citrulline levels); arginase is a rate limiting enzyme
Arg1, Arg2 and AGMAT are Mn-dependent urea hydrolases
Arg1 is absent in prodromal YAC128Q HD mouse striatum
16. Arginase pathway related metabolites are altered in HD mouse model;
Mn-exposure ameliorates this phenotype
HSG 2016: DISCOVERING OUR FUTURE
Urea
Bichell, Wegrzynowicz and Bowman et al Unpublished Data
17. Basal arginase activity is reduced in prodromal HD striatum; yet both in vivo Mn-exposure & in vitro
Mn-activation normalize activity between wild-type and HD
HSG 2016: DISCOVERING OUR FUTURE
Bichell, Wegrzynowicz and Bowman et al Unpublished Data
WT HD WT HD
Vehicle Mn-exposed
WT HD WT HD
Vehicle Mn-exposed
Ex vivo (basal) activity
Mn NOT ADDED to the enzyme assay
Mn-activated maximal activity
Mn ADDED to the enzyme assay
0
10
20
30
40
ngUrea/ugprotein
*
****
****
0
20
40
60
80
100
ngUrea/ugprotein
**
***
FVB genetic background; n=18 vehicle, n=12 Mn-exposed (7 day subQ exposure)
p<0.05
p<0.05 p<0.05
18. HD and Mn exhibit disease by toxicant/nutrient interaction effects
impacting brain urea and citrullin-NO cycles
HSG 2016: DISCOVERING OUR FUTURE
Bichell, Wegrzynowicz and Bowman et al Unpublished Data
Citrul-
line
urea
ADC
ODC
Orni-
thine
ARG
Putre-
cine
AGM
Manganese
Metabolite
Holoenzyme
Nitric Oxide
Antizyme
NOS
Argi-
nine
Poly-
amine
Agma-
tine
Crea-
tine
Glutamine
GS
NO
19. A continuum of possible HD-Mn
pathophysiological links
HSG 2016: DISCOVERING OUR FUTURE
(1) Disruptions in neuronal Mn balance are a downstream response to HD pathobiology with only a
minimal role in disease.
(2) Disruption in neuronal Mn balance is one of several pathways impacted by HD pathobiology,
mitigation would address a subset of HD symptoms
(3) HD mutations directly impact neuronal Mn handling, which disrupts Mn-dependent processes to
cause HD pathobiology; perhaps via interactions with proteins that function in Mn transport and
homeostatic processes.
HD mutation
Decreased Mn transport
HD pathophysiology 1
Decreased Mn transportHD pathophysiology 1
HD pathophysiology 2
HD pathophysiology 3?
20. Conclusions
HSG 2016: DISCOVERING OUR FUTURE
• Human and mouse models of HD exhibit deficits in Mn-
dependent and Mn-responsive neuronal/cellular processes
• Increasing neuronal Mn levels ameliorates the Mn-deficit related
phenotypes; while HD genotype blocks at least some Mn-
dependent signals/toxicity.
• However, in published data we’ve seen Mn-exposure exacerbates
other Mn neurotoxicities (e.g. see J. Madison et al 2012 PLoS
One)
21. Future Directions and Implications
HSG 2016: DISCOVERING OUR FUTURE
1. Characterize the relationship of the P53/AKT/mTOR pathway to
regulation of neuronal Mn homeostasis
2. Utilize small molecules to dissect Mn transport
3. Distinguish which HD phenotypes are upstream or downstream of
the defect in Mn handling
4. Determine the role of wild-type HTT gene in Mn-biology
5. Does dietary manganese influence HD pathobiology
Excess systemic manganese, based on current data, may worsen some
aspects of HD pathology, however, dietary deficiency of manganese
may also exacerbate Mn-relevant pathology. FDA has an established
Reference Daily Intake (RDI) for manganese (2.3 mg for adults and
children >4)
22. Future directions: Identifying selective small molecule
modifiers of HD-Mn biology
HSG 2016: DISCOVERING OUR FUTURE
vehicle
VU
0063088
VU
0047355
VU
0025173
VU
0150155
VU
0003765
VU
0135086
VU
0004838
VU
0057971
0
250
500
750
1000
1250
1500
1750
2000
2250
2500
Q7
Q111
* *
*
*
*
*
*
*
* Sig different from vehicle
# Sig different from corresponding Q7
# #
*#
*#
*
*
*
nM Manganese extracted
Control (Q7)
HD (Q111)
Vanderbilt High-Throughput Screening Core
A screen of >40,000 small molecules identified
~41 lead chemicals that modify sub-cellular
manganese levels.
Kumar et al 2014 Nat. Sci. Reports
A subset of these alter cellular Mn levels
selectively in control cells, but not the HD cell
model (mouse striatal cells) – suggesting they
are hitting a target affected by the disease gene
23. Acknowledgements
HSG 2016: DISCOVERING OUR FUTURE
My Lab (contributors to this work)
Andrew Tidball, Terry Jo Bichell, Kevin
Kumar, Kyle Horning, Miles Bryan,
Asad Al Aboud, Bingying Han, Anna
Pfalzer, Piyush Joshi, Rachana Nitin,
Michael Uhouse, Jack Feist, Mihir Odak
Vanderbilt University & VUMC
Diana Neely, Kevin Ess, David Weaver,
Jens Meiler, Peter Hedera, Daniel
Claassen, Chaz Hong, Edward Lowe
Einstein College of Medicine
Michael Aschner, Nancy Parmalee
UNC Greensboro
Keith Erikson
Funding:
NIEHS, RO1 ES016931 and RO1 ES010563 RO1
Pilot Grant from Vanderbilt Center in Molecular Toxicology
Peterson Foundation for Parkinsons
PK Hope is Alive
Vanderbilt HTS Core (Rey Redha, Josh Bauer, Paige Vinson)
24. Selective allosteric modulators of M4
muscarinic receptors for treatment of
Huntington’s Disease
P. Jeffrey Conn
Department of Pharmacology
Vanderbilt Center for Neuroscience Drug Discovery
:
26. Increased cortico-striatal EPSCs in presymptomatic 60 day
old YAC128 HD mice precedes deficits at later ages
Hypothesis: A drug that decreases excessive cortico-striatal activity at presymptomatic
stages may reduce appearance of motor symptoms
Deficits in cortico-striatal
transmission appear at 5
months of age and
parallels appearance of
motor deficits.
Normal
(WT)
Early HD
(2 Mo YAC)
Late HD
Symptomatic
27. M4-KO
WT
Control 3mM CCh
3mM CCh
in WT
3mM CCh
in M4-KO
%inhibitionofEPSC
M4 activation inhibits glutamatergic transmission at
corticostriatal synapses in HD mice
Pancani et al , 2014, ACS Chem. Neurosci.
WT and M4 KO
YAC128 X M4
KO cross
Pancani et al , 2015, PNAS
28. Positive Allosteric Modulators of M4
receptors?
Will it be possible to develop selective drug-like molecules
that selectively amplify activity of the M4 receptor?
30. Cholinergic inhibition of glutamatergic transmission at
corticostriatal synapses is mediated by M4 mAChRs
Stim Rec
Will chronic administration of the drug candidate that
amplifies M4 signaling reduce development of HD pathology
and symptoms?
31. Chronic treatment with VU0467154 reverses loss of cortico-
striatal activity in 5 mo old HD mice
VU046154 dosed daily from 2 – 5 months
Normal
HD
32. Chronic treatment with VU0467154 reverses motor deficits in 5
mo. old YAC128 mice
#
Locomotor
activity
Rotorod
performance
Exploratory
behavior
Ongoing studies evaluating potential neuroprotective effect
of M4 PAM
33. HTS Hit-to-Lead Lead Optimization Candidate IND PhI
mGlu5 NAMs
mGlu5 PAMs
M4 PAMs
GLP-1
M4 NAM
M1 PAMs
VCNDD Programs and Pipeline
mGlu4 PAMs
mGlu3 NAM
mGlu2 NAM
mGlu1 PAM
mGlu3 PAM
Schizophrenia, Alzheimer’s
PD-LID, Refractory depression
Schizophrenia, Alzheimer’s
Parkinson’s
Huntington’s, Schizophrenia
Schizophrenia
Schizophrenia
Diabetes
Refractory depression
Refractory depression, cognition
Parkinson’s
Projected entry into
clinical studies 1Q2018
34. Vanderbilt Center for Neuroscience Drug Discovery
Supported by NIMH, NINDS, Huntington's Disease Foundation and CHDI
In Vivo
Carrie Jones
Jerri Rook
Nellie Byun
Analisa Thompson
Michael Bubser
Jonathan W. Dickerson
Rebekah L. Collier
Mol Pharm/
Colleen Niswender
Alice Rodriguez
Daryl Venable
Doug Sheffler
Joy Marlo
Ashley Brady
Meredith Noetzel
Ephys
Tristano Pancani
Dan Foster
Mark Moehle
Zixiu Xiang
Adam Walker
Ayan Ghoshal
Xiaohui Lv
Kari A. Johnson
Med Chem
Craig Lindsley
Kyle Emmitte
Michael Wood
Shaun Stauffer
Kyle Emmitte
Cody Wenthur
DMPK
Tom Bridges
Scott Daniels
Annie Blobaum
Matt Mulder
Ryan Morrison
Frank Byers
39. Security Rules
2004 Gap Assessment
Researchers were using sub-standard methods
to manage data for research studies/trials
projects (pilot, R01, PPG)
40. Visual Status Data Validation
Numerous
Field Types
+ Text
(Free)
(Number)
(Phone)
(Zip)
(Date)
+TextArea
+Select
+Radio
+File
Branching
LogicAuto-Variable Coding
Human
Readable
Labels
PDFs
40
41. Exports Raw
Data + Stats
Script Files
(Labels,
Coding
Embedded
De-
Identification
Tools
41
42. Why REDCap?
REDCap filled a critical gap
at Vanderbilt
Easy way to do
the right thing ...
Security Rules
43. Early QI Survey (N=20) – Did REDCap
improve your science?
Yes (N=20)
Improving Data
Improves
Science
Improves
Health
43
46. So … how’s it
working so far?
It’s fantastic, but
could you add
some sort of
widget to …
Every Time!
Long ago … before we had real professionals doing this work ...
47. Used systems get better …
End-users will tell you what you need to know
to improve if you listen
52. So … how’s it
working so far?
It’s fantastic, but
could you add
some sort of
widget to …
Long ago … before we had real professionals doing this work ...
418,000 END USERS, 108 Countries
55. Typical Week
Overall
Local @ VU
M T W T T S S
Available to any student, faculty, staff
member for any Vanderbilt use – no cost
55
The Research Community
And The Institution