1. MTA-SE NAP-B Molecular Psychiatry Research Group 2. MTA-ELTE NAP-B Opto-Neuropharmacology Group 3. Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of

Sciences 4. Department of Psychiatry and Psychotherapy, Semmelweis University

Pharmacological investigation of alpha7 nAChR and mGluR2 in human pluripotent

stem cell derived dentate gyrus neurons: implications for schizophrenia

Edit Hathy1, Krisztina Pesti2, Gergő Vőfély3, László Homolya3, Balázs Sarkadi3, Árpád Mike2,

Ágota Apáti3, János Réthelyi1,4

Molecular characterization of stem cell derived PROX1 neurons

Conclusions

1. We derived and characterized NPCs and mature neurons from human IPSCs, which

were differentiated according to the DG PROX1 protocol. The neurons express mature

neuronal markers and have dendritic spines.

2. Human PROX1 neurons express CHRNA7 and GRM2 at the RNA level and alpha7

nAChR and mGluR2 at the protein level. Alpha7 nAChRs are localized to neuronal

somata and processes, mGluR2 seems to be localized both pre- and postsynaptically.

3. PROX1 neurons show spontaneous, TTX-sensitive activity during visualization with

calcium-imaging.

4. Alpha7 nAChR ligands modulate calcium-transients in PROX1 neurons. Small dose

choline, a selective agonist, causes short increase in the calcium intensity of neurons,

while choline and PNU (a positive allosteric activator) have a strong effect on intensity

and frequency of calcium-signals. Interestingly, PNU alone increases calcium-signals,

suggestive of a silent agonist of alpha7 nAChR or an intrinsic metabotropic effect of

PNU. MLA inhibits the effect of choline but can’t inhibit choline + PNU.

5. mGluR2 ligands have a drastic effect on calcium-signals in PROX1 neurons. DCG-IV, a

selective agonist inhibits sponataneous activity, while APICA, a selective antagonist

causes a manifold increase in calcium intensity. The functional role of mGluR2 confirms

the molecular identity of these in vitro PROX1 granule cells.

6. Single-cell electrophysiology complemented the previous results. We found that choline

and PNU administered together activate an inward current, but not PNU or choline

alone. MLA abolished the effect of choline and PNU, but PNU administered together

with MLA inhibits the previously described inhibitory effect.

7. Our results provide proof-of-concept for using this model system for further

experiments implicating these receptor subtypes and neuronal cell lines derived from

diseased individuals.

Pharmacological characterization of PROX1 neurons using single cell

electrophysiology

Acknowledgements / Funding: The authors thank Szilvia Szalóki, Beáta Haraszti, Kornélia Szebényi, and

Nóra Varga. The IPSC line 62F, the RNASeq data and the LV-pPROX1-EGFP virus was kindly provided by Professor Fred Gage

(Salk Institute for Biological Studies, La Jolla, California). We thank János Szabadics for providing DCG-IV and APICA. The

study is funded by the Hungarian Brain Research Program (NAP) (Grant NAP-B KTIA_NAP_13-2014-0011 to JR and Grant

NAP-A KTIA_13_NAP-A-I/6 to AÁ and LH).

PROX1 neurons exhibit spontaneous

Ca-transients. Administration of

choline and PNU result is signficant

increase of Ca-signals in amplitude.

PNU alone activates neurons, while

choline alone elicity only a small

effect. MLA abolishes the effect of

choline, but is can’t inhibit PNU +

choline. DCG-IV inhibits

spontaneous calcium-transients,

while APICA causes massive rise in

calcium intensity (not shown).

Human IPSCs differentiated by means of the Prox1 protocol give rise to Nestin and Sox2

expressing neuronal progenitor cells and Map2, NeuN, Prox1 positive neurons.

Map2/Prox1/NeuN/Dapi Nestin/Sox2/Dapi

P.1.g.086

email: hathyedit@ gmail.com

Abstract

Hippocampal information processing is achieved by precise, multi-level regulation of the

entorhinal cortex-dentate gyrus–CA3-CA1 trisynaptic circuit. Alpha7 nAChR and

mGluR2 have been shown to play an important modulating role in this circuit.

Both alpha7 nAChR and mGluR2/3 have been implicated in schizophrenia, furthermore

agonists and positive allosteric modulators have been developed recently for these

receptor subtypes (Conn et al. 2009). Initial trials with alpha7 nAChR and mGluR2

agonists showed improvement in cognitive dysfunction and significant alleviation of

positive symptoms, respectively.

Following the protocol of Yu et al. (2014) we derived PROX1 expressing dentate gyrus

(DG) granule cells from human induced pluripotent stem cells (IPSCs) for further

pharmacological investigation of two receptor subtypes, alpha7 nAChR and mGluR2.

We examined the expression of neuronal acetylcholine receptor subunit alpha7

(CHRNA7) and metabotropic glutamate receptor 2 (GRM2) genes at the RNA level in

IPSCs, neuronal precursors (NPCs) and differentiated PROX1 neurons using RNASeq

data. At the protein level, we investigated expression and localization in PROX1 neurons

by means of immunocytochemistry (ICC) and bungarotoxin-staining.

We sought to characterize alpha7 nAChR and mGluR2 pharmacologically in

differentiated PROX1 neurons using calcium-imaging and single-cell patch clamp

electrophysiology.

Targeted differentiation of human pluripotent stem cells into PROX1

expressing dentate gyrus neurons

Yu et al, Stem Cell Reports, 2014

PROX1 neurons transduced

with the lentivirus LV-pPROX1-

EGFP.

Alpha7 nAChR and mGluR2 expression and localization in PROX1 neurons MAP2/mGLUR2 BTX-Alexa 488

Choline (40uM)

PNU (4uM) Choline (4 mM)

Choline (40uM)

PNU (4uM)

KCl (50 mM)

KCl (50 mM)

F/F

0

F/F

0

KCl (50 mM)

s s

F/F

0

F/F

0

Choline (4 mM)

MLA (10 uM)

PNU(4 uM) KCl (50 mM)

Pharmacological investigation of the effects of specific α7 nAChR and

mGluR2 ligands in PROX1 neurons using calcium-imaging

1. Prox1 neurons exhibit sodium

currents.

2. ABA-20 protocol. A=PNU 10uM,

B=Choline 10mM + PNU 10uM.

3. ABA-20 protocol. A=Ch 10mM,

B>Choline 10 mM+ PNU 10uM.

4. BAB-20 protocol. A=Choline 10mM+

MLA 1 uM, B=Choline 10 mM+PNU

10uM.

5. BAB-20 protocol. A=Ch oline 10 mM+ PNU

10uM+MLA 1uM, B=Choline 10mM + PNU

10uM

3. Comparison of recordings 4

and 5.

mR

NA

rea

ds

(RP

KM

)

RNASeq data from an independent differen-

tiation experiment show increasing neural

expression of CHRNA7 and GRM2.

DCG-IV (1 uM)

0,001

0,01

0,1

1

10

IPS NPC Neuron 3 w Neuron 6 w

CHRNA7

GRM2

Using bungarotoxin-Alexa 488 staining alpha7 nAChR is shown to localize

both on neuronal somata and processes. mGluR2 is predominantly localized

presynaptically and colocalizes with synaptophysin.

s

Ca-imaging was carried out after administration of the calcium-indicator dye Fluo-4 in HBSS buffer using confocal microscopy. Effects of choline, PNU 120596, methyllycaconitine (MLA), DCG-IV, APICA were examined

Single cell electrophysiology: Voltage gated Na+ currents and agonist/antagonists/postive allostreic modulator evoked currents

(choline, PNU, MLA, DCG-IV, APICA) were investigated by means of whole-cell configuration voltage clamp using the Axopatch 200B amplifier and the pClamp software.

Prox1 protocol: HUES and hIPS cells are differentiated as EBs in the presence of

SMAD inhibitors Noggin and SB431542, the aunticudalizing Wnt antagonist Dkk1 or

XAV939, and the SHH antagonist cyclopamine. After plating and rosette formation

neural progenitors (NPCs) are dissociated and expanded or further differentiated into

neurons in the presence of Wnt3A, BDNF, cAMP and ascorbic acid (AA).