Spotlight on Vanderbilt University

Spotlight on VanderbiltFriday, November 4

8:00-9:00am

Chair: Daniel Claassen, MDVanderbilt University

Welcome to Nashville!

HSG 2016: DISCOVERING OUR FUTURE

Vanderbilt Presenters


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


• 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


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


*

** 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


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


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


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


Bypassing the Mn deficit in mouse striatal model normalizes ATM-P53 responsiveness


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


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


Hypothesis


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


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


(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


• 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


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


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


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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