Spotlight on VanderbiltFriday, November 4
8:00-9:00am
Chair: Daniel Claassen, MDVanderbilt University
Welcome to Nashville!
HSG 2016: DISCOVERING OUR FUTURE
Vanderbilt Presenters
HSG 2016: DISCOVERING OUR FUTURE
Aaron Bowman, PhDAssociate Professor in the Department of Neurology and Pediatrics/Training Program in Environmental Toxicology, Director
Jeff Conn, PhDLee E. Limbird Professor of Pharmacology /Center for Neuroscience Drug Discovery, Director
Paul Harris, PhDProfessor of Biomedical Informatics/School of Engineering, Professor of Biomedical Engineering/ Office of Research Informatics, Director
Manganese and HD
Aaron B. Bowman, PhDDepartments of Pediatrics, Neurology and
BiochemistryVanderbilt University (VU) and VU Medical Center
Nashville, TN
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
Genetic x toxicant screen of 8 cytotoxic metals revealed resistance of HD striatal cells
to Mn toxicity
HSG 2016: DISCOVERING OUR FUTURE
Mn(II) µM
Perc
ent
Surv
ival
*
*
*
Wild-type STHdhQ7/Q7
Mutant STHdhQ111/Q111
Mechanistic studies demonstrated a deficient Mn accumulation phenotype
…see Williams et al. JNC 2010Mutant STHdhQ111/Q111
Wild-type STHdhQ7/Q7
*
** P<0.01 (n=5)
MTT Assay Atomic Absorption Spectroscopy
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 animals13.9 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
Human-based and unbiased genomic-level data support a link between manganese
biology and HD
HSG 2016: DISCOVERING OUR FUTUREAndrew Tidbal et al Hum Mol Genet, 2015
HumanStriatal Progenitors
Statistic FVB +/+ FVB Tg-YAC128Q
p < 0.0005
p < 0.001
p < 0.005
q < 0.05
Ensemble alignment9
17
94
UCSC mm10 alignment
292
3
5
36
54
# of Mn-responsive genes by p-value or q-value
1 week subcutaneous Mn exposureMouse HD model
In collaboration with Dr. Michael Aschner and Nancy Parmalee
MouseStriatal Progenitors
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. JBCCanman et al 1998. Science
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
ATM inhibition blocks Mn-dependent phosphorylation of p53 (and other ATM targets)
in human HD ISLT1 progenitors
HSG 2016: DISCOVERING OUR FUTUREAndrew Tidball et al Hum Mol Genet, 2015
Manganese (Mn2+) n=6
essential metal andneurotoxicant
“In Cell” Western Blots
KU = KU55933 an ATM kinase inhibitor
ATM auto-phosphorylation responds to Mn exposure, and this response is deficient in HD
cells
HSG 2016: DISCOVERING OUR FUTUREAndrew Tidball et al Hum Mol Genet, 2015
Bypassing the Mn deficit in mouse striatal model normalizes ATM-P53 responsiveness
HSG 2016: DISCOVERING OUR FUTUREAndrew 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
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/mTOR
mutHTT Mn
For more see poster by Miles Bryan
0 13 25 50 75 Mn µM (24hr)
.
p-p53(Ser15)
p-AKT(Ser473)
p-S6(Ser235/23
6)
Total Protein
Wild-type STHdhQ7/Q7
Hypothesis
HSG 2016: DISCOVERING OUR FUTURE
HD genotype disrupts neuronal Mn transport and homeostatic
mechanisms to impair Mn biology and block Mn toxicityUrea and citrulline-nitric oxide cycles are disrupted in HD models
and patients (e.g. increased blood citrulline levels); arginase is a rate limiting enzymeArg1, Arg2 and AGMAT are Mn-dependent urea hydrolases
Arg1 is absent in prodromal YAC128Q HD mouse striatum
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
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 FUTUREBichell, Wegrzynowicz and Bowman et al Unpublished Data
WT HD WT HD Vehicle Mn-exposed
WT HD WT HD Vehicle Mn-exposed
Ex vivo (basal) activityMn NOT ADDED to the enzyme assay
Mn-activated maximal activityMn ADDED to the enzyme assay
FVB genetic background; n=18 vehicle, n=12 Mn-exposed (7 day subQ exposure)
p<0.05
p<0.05 p<0.05
HD and Mn exhibit disease by toxicant/nutrient interaction effects impacting brain urea and
citrullin-NO cycles
HSG 2016: DISCOVERING OUR FUTUREBichell, Wegrzynowicz and Bowman et al Unpublished Data
Citrul-line
urea
ADC
ODC
Orni-thin
e ARG
Putre-cine
AGM
Manganese
Metabolite
Holoenzyme
Nitric Oxide
Antizyme
NOS
Argi-nine
Poly-amin
e Agma-tine
Crea-
tine
Glutamine
GS
NO
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 transportHD pathophysiology 1
Decreased Mn transportHD pathophysiology 1
HD pathophysiology 2HD pathophysiology 3?
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)
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 transport3. Distinguish which HD phenotypes are upstream or
downstream of the defect in Mn handling4. Determine the role of wild-type HTT gene in Mn-biology5. 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)
Future directions: Identifying selective small molecule modifiers of
HD-Mn biology
HSG 2016: DISCOVERING OUR FUTURE
nM Manganese extractedControl (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
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 OdakVanderbilt University & VUMCDiana Neely, Kevin Ess, David Weaver, Jens Meiler, Peter Hedera, Daniel Claassen, Chaz Hong, Edward LoweEinstein College of MedicineMichael Aschner, Nancy Parmalee UNC GreensboroKeith Erikson
Funding: NIEHS, RO1 ES016931 and RO1 ES010563 RO1 Pilot Grant from Vanderbilt Center in Molecular Toxicology Peterson Foundation for Parkinsons PK Hope is AliveVanderbilt HTS Core (Rey Redha, Josh Bauer, Paige Vinson)
Selective allosteric modulators of M4 muscarinic receptors for treatment of
Huntington’s Disease
P. Jeffrey ConnDepartment of Pharmacology
Vanderbilt Center for Neuroscience Drug Discovery:
Cortex
Pre-symptomatic Huntington’sNormal
Striatum
Symptomatic Huntington’s
Cortex
Striatum
Cortex
Striatum
Htt mutations may induce excessive activity at Cortico-Striatal synapses long before appearance of HD symptoms
Hypothesis: early increases in cortico-striatal transmission contributes to cell death and behavioral deficits that appear at symptomatic stages.
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 HDSymptomatic
M4-KO
WT
Control 3mM CCh
3mM CChin WT
3mM CChin M4-KO
% in
hibi
tion
of E
PSC
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
Positive Allosteric Modulators of M4 receptors?
Will it be possible to develop selective drug-like molecules that selectively amplify activity of the M4 receptor?
160,000compounds
817 primary hits
10 µM
singlicate10 point CRC
Mol Pharm Characterization
41 confirmedPAMs
Optimization of novel M1 PAMs as research tools and drug candidates
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?
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
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
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
Vanderbilt Center for Neuroscience Drug Discovery
Supported by NIMH, NINDS, Huntington's Disease Foundation and CHDI
In VivoCarrie Jones
Jerri RookNellie Byun
Analisa ThompsonMichael Bubser
Jonathan W. DickersonRebekah L. Collier
Mol Pharm/ Colleen Niswender
Alice Rodriguez Daryl VenableDoug Sheffler
Joy MarloAshley Brady
Meredith Noetzel
EphysTristano Pancani
Dan FosterMark MoehleZixiu Xiang
Adam WalkerAyan Ghoshal
Xiaohui Lv Kari A. Johnson
Med ChemCraig LindsleyKyle Emmitte
Michael WoodShaun StaufferKyle EmmitteCody Wenthur
DMPKTom Bridges Scott Daniels
Annie BlobaumMatt Mulder
Ryan Morrison Frank Byers
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