Dirk Isbrandt / Maria A Rüger / Michael Schroeter / Igor Jakovcevski - C 8

Novel roles for HCN/h- and Kv7/M-currents in the development of the cerebral cortex

Summary

The aim of the Institute for Molecular and Behavioral Neuroscience is to study molecular mechanisms underlying disease-associated changes in cellular excitability using transgenic mouse models. We studied the function of HCN channels in early forebrain development by creating a mouse line with ablated h-current, and by examining the effect of HCN inhibitor on neural stem cells. Our results indicate that HCN channel function in early embryonic period is essential for normal cortical development. 

Introduction 

Developmental and evolutionary expansion of the cerebral cortex relies on the controlled division of neural stem and progenitor cells, to which the membrane potential is suggested to contribute. HCN channels, which mediate I(h), a voltage-dependent non-selective cation current, depolarize the membrane potential towards the action potential threshold and, thus, importantly contribute to the dynamic control of the resting membrane potential in neurons. 

Embryonic loss of HCN/h-channel function in mouse forebrain results in impaired neural progenitor proliferation and microcephaly

We recognized the previously unknown role of HCN channels in regulating the proliferation of neural progenitors during forebrain development. We demonstrate expression of HCN subtypes, particularly HCN3 and HCN4, in human, mouse, and rat neural progenitors of the cerebral cortex. Mice with early embryonic ablation of HCN function restricted to the forebrain under the control of the EMX1 promoter had a pronounced microcephaly phenotype associated with a decrease in proliferation and premature expression of glial markers, which was absent when HCN-channel function was ablated in newborn post-mitotic cortical neurons using the Nex promoter. Pharmacological blockade of I(h) in neural stem cell cultures from rat cortices (NSC) by ZD7288 revealed a dose-dependent proliferation impairment. In addition, single-cell transcriptome analysis of ZD7288-treated and untreated rat NSCs revealed an accumulation of treated cells in the G1 phase of the cell cycle. These findings suggest that impaired I(h)-mediated membrane potential dynamics resulted in G1-phase lengthening, which is associated with decreased proliferation and premature differentiation of neuronal progenitors. 

Perspectives 

Our current data indicate that proper HCN channel function is important to forebrain development, and that mutations in HCN-channel genes could lead to brain malformations. We now plan to study the databases with patients that have brain malformations coupled with HCN subunit gene mutations. We intend to show, after we provided proof-of-principle that HCN channel function is essential for proliferation of neural progenitors, that our data are clinically relevant. In another line of study we use calcium imaging and voltage-sensitive dyes to demonstrate mechanistic coupling between the h-current inhibition and cellular proliferation. We also have data indicating that HCN inhibition plays a role in microglia proliferation and determination of microglial phenotype. Finally, we conduct the experiments to show the effects of M-current blockade on neural stem cells and forebrain morphology.

Selected publications

Schlusche, A.K., Vay, S.U., Sandke, S., Campos-Martin, R., Kleinenkuhnen, N., Florio, M., Huttner, W., Tresch, A., Roeper, J., Stockebrand, M., Rueger, M.A., Jakovcevski, I., Isbrandt, D. (2018). Embryonic loss of HCN/h-channel function in mouse forebrain results in impaired neural progenitor proliferation and microcephaly. FENS abstract F18-3491. 


Prof. Dr. Dirk Isbrandt

Inst. for Molecular and Behavioral Neuroscience / RG location - LFI Building

Prof. Dr. Dirk Isbrandt

Principal Investigator C 8

dirk.isbrandt@uni-koeln.de

Work +49 221 478 32732

Institute for Molecular and Behavioral Neuroscience
Kerpener Str. 62
50937 Cologne

Publications - Dirk Isbrandt

Link to PubMed


PD Dr. Maria A Rüger

Dept. of Neurology / RG location - LFI Building

PD Dr. Maria A Rüger

Co-Principal Investigator C 8

adele.rueger@uk-koeln.de

Work +49 221 478 87803

Dept. of Neurology
Kerpener Str. 62
50924 Cologne

Publications - Maria A Rüger

Link to PubMed


Prof. Dr. Michael Schroeter

Dept. of Neurology / RG location - LFI Building

Prof. Dr. Michael Schroeter

Co-Principal Investigator C 8

michael.schroeter@uk-koeln.de

Work +49 221 478 87239

Dept. of Neurology
Kerpener Str. 62
50924 Cologne

Publications - Michael Schroeter

Link to PubMed


Dr. Igor Jakovcevski

Inst. for Molecular and Behavioral Neuroscience / RG location - LFI Building

Dr. Igor Jakovcevski

Co-Principal Investigator C 8

igor.jakovcevski@uk-koeln.de

Work +49 221 478 32734

Institute for Molecular and Behavioral Neuroscience
Kerpener Str. 62
50937 Cologne

Publications - Igor Jakovcevski

Link to PubMed

Group Members

Anna K. Schlusche (PostDoc)
Malte Stockebrand (PostDoc)
Sabine U. Vay (PostDoc)

Figure 1

CMMC Research Isbrandt
The generation and morphology of EMX-HCN-DN mutant mice. (A) The expression of dominant negative (DN) HCN mutation is driven by the EMX1-promoter resulting in the expression of the cre recombinase, which excise the stop sequence in the Rosa26 locus leading to the expression of the tetracycline transactivator (tTA). In absence of doxycycline the tTA binds to the responsive element (Ptet), a bidirectional promoter, inducing the expression of the human dominant negative HCN subunit (hHCN-DN) and EGFP. (B) At postnatal day (P) 0 the bodyweight is decreased in mutant mice (mut) compared to control (con) or transgenic animals treated with doxycycline until P0 (mut + dox). This decrease in body size and weight is more pronounced in mutant animals at P21 compared to control or mut + dox. (C) Not only body weight, but also brain size is decreased in mut animals compared with con and dox at both P0 and P21.