Potassium channel expression in adult murine neural progenitor cells

Neuroscience 180 (2011) 19 –29

POTASSIUM CHANNEL EXPRESSION IN ADULT MURINE NEURAL PROGENITOR CELLS H. PRÜSS,a,b1* M. DEWES,a1 C. DERST,c F. FERNÁNDEZ-KLETT,a,d R. W. VEHc AND J. PRILLERa,d,e*

Adult neural progenitor cells (NPCs) are promising tools for the treatment of neurodegenerative diseases, brain and spinal cord injury, multiple sclerosis, or stroke (e.g Pluchino et al., 2003; Hofstetter et al., 2005). Beside self renewal, these cells are endowed with the ability to generate neuronal and glial cells, and most of the NPCs proliferate in the subventricular zone (SVZ) of the adult mammalian forebrain (Doetsch et al., 1999; Garcia et al., 2004). NPCs are commonly cultured in the presence of epidermal and fibroblast growth factor (EGF/FGF2) leading to clonal expansion of neurospheres (Reynolds and Weiss, 1992). In order to increase the yield in neuronal cells for brain repair, it is essential to identify the molecules and signal cascades that regulate NPC growth, migration and differentiation. Potassium (K⫹) channels are a group of promising molecule candidates for NPC regulation as they are important components of the signal transduction machinery in almost all cells of the mammalian body. The channels form highly regulated pores in cell membranes and can be divided into three structural classes: voltage-gated K⫹ channels (Kv), inwardly rectifying K⫹ channels (Kir) and two-pore ‘background’ (K2P) channels (Coetzee et al., 1999). At least 75 different K⫹ channel genes have been identified to date (Caterall et al., 2002). Distinct temporal and spatial expression, multiple splice variants and the ability to form hetero-oligomers, underscore the impressive diversity of K⫹ channels, which may permit the complex regulation needed to control cell proliferation and differentiation. The molecular diversity of K⫹ channels allows for the development of highly specific drugs, which target selective cell types or functional systems. Interestingly, spadin was recently identified as the first K2P channel inhibitor and natural anti-depressant peptide, which enhances hippocampal neurogenesis (Mazella et al., 2010). There is increasing evidence that K⫹ channels influence cell growth, maturation and neurogenesis. Changes in K⫹ channel activity are required for proliferation at critical cell cycle checkpoints (DeCoursey et al., 1984; Gallo et al., 1996; Wonderlin and Strobl, 1996; MacFarlane and Sontheimer, 2000; Pardo, 2004; Vautier et al., 2004). Pharmacological channel modulation revealed that K⫹ current changes may be not only concurrent with, but necessary for progression through the cell cycle (Pappas et al., 1994; MacFarlane and Sontheimer, 2000; Chittajallu et al., 2002; Liebau et al., 2006; Yasuda et al., 2008). It has long been known that K⫹ channels are expressed at neural induction (Ribera, 1990). Moreover, lack of certain functional Kv channels results in megencephaly mice with increased proliferation, neurogenesis and enhanced hippocampal cell survival (Almgren et al., 2007). Recently,

a Department of Neuropsychiatry and Laboratory of Molecular Psychiatry, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin b

Department of Experimental Neurology, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin

c

Institute for Integrative Neuroanatomy, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin

d

BCRT, Augustenburger Platz 1, D-13353 Berlin, Germany

e

NeuroCure, Charité-Universitätsmedizin Berlin, Charitéplatz 1, D-10117 Berlin

Abstract—Neural progenitor cells (NPCs) are a source of new neurons and glia in the adult brain. Most NPCs reside in the forebrain subventricular zone (SVZ) and in the subgranular zone of the dentate gyrus, where they contribute to plasticity in the adult brain. To use their potential for repair, it is essential to identify the molecules that regulate their growth, migration and differentiation. Potassium (Kⴙ) channels are promising molecule candidates for NPC regulation as they are important components of signal transduction and their diversity is ideal to cover the complex functions required for cell proliferation and differentiation. There is increasing evidence that Kⴙ channels influence cell growth and neurogenesis, however, very little is known regarding Kⴙ channel distribution in NPCs. We therefore explored the expression of a variety of voltage-gated (Kv), inwardly rectifying (Kir) and two-pore (K2P) Kⴙ channels in the SVZ of adult mice and in neurosphere cultures of NPCs during growth and differentiation. Immunocytochemical analysis revealed a differential expression pattern of Kⴙ channels in nestinⴙ SVZ precursor cells, early SVZ doublecortinⴙ neurons and (sub)ependymal cells. These findings were confirmed in neurosphere cultures at the protein and mRNA levels. The expression of some Kⴙ channel proteins, such as Kir4.1, Kir6.1, TREK1 or TASK1, suggests a role of Kⴙ channels in the complex regulation of NPC proliferation, maturation and differentiation. © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. Key words: potassium channel, neural progenitor cells, subventricular zone, rostral migratory stream, doublecortin, neurospheres. 1 HP and MD contributed equally to the study. *Correspondence to: H. Prüss or J. Priller, Department of Neuropsychiatry, Charité-Universitätsmedizin Berlin, Charitéplatz 1, 10117 Berlin, Germany. Tel: ⫹49-30-450-517209; fax: ⫹49-30-450-517962. E-mail address: [email protected] (H. Prüss) or josef.priller@ charite.de (J. Priller). Abbreviations: BMPs, bone morphogenetic proteins; DCX, doublecortin; EGF, epidermal growth factor; FGF2, fibroblast growth factor; GFAP, glial fibrillary acidic protein; Kir, inwardly rectifying K⫹ channels; Kv, voltage-gated K⫹ channels; K2P, two-pore K⫹ channels, K⫹, potassium; NPCs, neural progenitor cells; RMS, rostral migratory stream; SVZ, subventricular zone.

0306-4522/11 $ - see front matter © 2011 IBRO. Published by Elsevier Ltd. All rights reserved. doi:10.1016/j.neuroscience.2011.02.021

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H. Prüss et al. / Neuroscience 180 (2011) 19 –29

Yasuda et al. (2008) demonstrated that the differentiation of NPCs into neuroblasts in the rodent SVZ is accompanied by changes in the resting membrane potential and down-regulation of Kir channel expression. However, a detailed analysis of K⫹ channel expression and function in NPCs does not exist. We therefore explored the expression of a variety of voltage-gated, inwardly rectifying and two-pore K⫹ channels in the adult murine SVZ and in neurosphere cultures using immunocytochemistry, Western blotting, gene expression profiling by microarray and real-time PCR, as well as transient transfection experiments. A differential expression pattern of K⫹ channels was observed during growth and differentiation of NPCs, which may contribute to the complex regulation of NPC behavior and lineage determination. Some channels, such as Kir4.1, Kir6.1, TREK1 or TASK1, might selectively participate in SVZ neurogenesis and K⫹ channel variability during the cell cycle.

EXPERIMENTAL PROCEDURES NPC culture—neurospheres Adult mouse neurospheres were derived from the SVZ using a standard experimental protocol (Galli et al., 2008). All animal procedures were performed according to the local guidelines for animal research. Briefly, SVZ of C57Bl/6 mice (⬃8 weeks old) were isolated, diced and enzymatically dissociated with 0.05% trypsin–EDTA for 7 min at 37 °C, washed, and triturated into a single cell suspension. Cells were plated in serum-free NBMA medium supplemented with retinoic acid-free B27, L-glutamine, 20 ng/ml EGF and 10 ng/ml basic FGF2 (all substances from Invitrogen, San Diego, CA, USA). Primary SVZ cells were plated at a density of 3.5⫻103 cells/cm2 in lowattachment culture flasks (Nunc, Roskilde, Denmark), incubated for 7 days at 37 °C and 5% CO2 to allow neurosphere formation. The secondary neurospheres or subcultures hereof (passage 2) were dissociated into single cells and used for differentiation assays, or immunochemical studies. Differentiation was induced by removal of growth factors (EGF, FGF2).

Immunochemistry Mice were deeply anesthetized and perfused transcardially with PBS followed by 4% paraformaldehyde in PBS. Brains were dissected, fixed in 4% paraformaldehyde at 4 °C overnight, cryoprotected with 30% sucrose, shock-frozen in hexane at ⫺70 °C, and stored at ⫺80 °C. Frozen tissues were cut into 20 ␮m sections on a cryostat and washed in PBS. Sections were preincubated in 10% normal goat/donkey serum in PBS containing 0.3% Triton X-100. Sections were washed three times in PBS and incubated at 4 °C for 36 h in primary antibody solutions (10% normal goat serum, 0.3% Triton X-100). K⫹ channel proteins were detected using the following primary antibodies: rabbit Kv1.1 [1:1000] and Kv1.4 [1:100] (Veh et al., 1995); Kv1.5 [1:150] (generously provided by Prof. H.G. Knaus; Koschak et al., 1998); mouse Kv1.6 [1:500] (NeuroMab, Davis, CA, USA); rabbit Kv2.1 [1:100] (Sigma, Munich, Germany); Kv4.2 [1:500] (NeuroMab); Kv4.3 [1:500] (NeuroMab); Kir1.1 [1:100] (Alomone, Jerusalem, Israel); Kir2.1 [1:250], Kir2.2 [1:100], Kir2.3 [1:100] and Kir2.4 [1:1000] (Prüss et al., 2005); Kir3.1 [1:500] and Kir3.2 [1:200] (Eulitz et al., 2007); Kir4.1 [1:1000] (Chemicon, Billerica, MA, USA); Kir4.2 [1:50] (Alomone); Kir5.1 [1:100] (provided by Dr. Veh); Kir6.1 [1:700] and Kir6.2 [1:100] (Thomzig et al., 2005); Kir7. 1 [1:2000] (Derst et al., 2001); TASK1 [1:100] (Alomone); goat TASK3 [1:100] (Santa Cruz Biotechnology, Santa Cruz, CA, USA); TWIK1 [1:200] (Santa Cruz); rabbit TWIK2 [1:100] (Alomone); goat TREK1 [1:1000] (Santa Cruz); TREK2 [1:500] (Santa Cruz); TRAAK

[1:250] (Santa Cruz). Further antibodies: goat/rabbit doublecortin (DCX) [1:500] (Santa Cruz); mouse class III beta tubulin (TuJ-1) [1:500] (Covance, Princeton, NJ, USA); mouse nestin [1:200] (Chemicon); rabbit glial fibrillary acidic protein (GFAP) [1:200] (Dako, Hamburg, Germany); mouse RIP [1:5000] (Chemicon). After two washes, sections were incubated in PBS-A (2 mg bovine serum albumin in 1 ml PBS) for 1 h and subsequently treated with the secondary antibody (biotinylated IgG, 1:2000 in PBS-A; Vector Laboratories, Burlingame, CA, USA) at room temperature for 16 h. Two further washes were followed by avidinbiotin complex (Elite ABC, 1:1000 in PBS-A; Vector Laboratories) at room temperature for 6 h. After another three washes and preincubation in 3,3=-diaminobenzidine solution (Sigma, Munich, Germany) containing 0.5 mg 3,3=-diaminobenzidine in 1 ml 50 mmol/L Tris– buffer, pH 7.6, 10 mmol/L imidazole and 0.3% ammonium nickel sulfate. Peroxidase activity was visualized for 3 min after addition of H2O2 (0.015% final concentration). Omission of primary antibodies served as negative controls. For immunofluorescence staining, brain sections or coverslips with cultured cells were treated with the primary antibodies as described. After 16 h incubation with secondary antibodies at 4 °C (Alexa 488-/594-IgG 1:500; Invitrogen, Darmstadt, Germany), sections or coverslips were mounted with Mowiol 4-88 (Hoechst, Paris, France) and examined using a fluorescence or confocal microscope (Leica TCS SPE, Leica Microsystems, Wetzlar, Germany).

Western blot analysis Neurospheres were homogenized on ice in a dounce-homogenizer (Wheaton, Millville, NJ, USA) in homogenization buffer (4 mmol/L HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), pH 7.4, 250 mmol/L sucrose, 1 mmol/L EDTA, 5 mmol/L sodium azide, 2 mg/ml aprotinin, 1 mg/ml pepstatin A, 1 mg/ml leupeptin and 0.5 mmol/L phenylmethylsulfonylfluoride). The homogenate was centrifuged at 2500 g for 10 min, followed by centrifugation of the supernatant at 100,000 g. The pellet was resuspended in 50 mmol/L Tris–HCl, pH 7.5, and protein concentration was determined with bicinchoninic acid assay (Pierce, Rockford, IL, USA). Homogenates were heated at 90 °C in sodium dodecyl sulfate sample buffer for 3 min. 40 ␮g of protein per lane was loaded on 8% sodium dodecyl sulfate-polyacrylamide gels followed by electrophoresis. After separation, proteins were electroblotted onto nitrocellulose (Schleicher & Schuell, Dassel, Germany). The membranes were blocked with 5% low-fat milk powder in PBS containing 0.5% Tween-20 for 1 h, incubated over night at 4 °C with anti-K⫹ channel antibodies, secondary horseradish peroxidase-linked antibody (1:1000; Sigma, St Louis, MO, USA), and visualized with enhanced chemoluminescence (Pierce) using ImageMaster VDS-CL (Amersham, Freiburg, Germany).

Gene expression profiling At 0 days (spheres) and 7 days of NPC differentiation of identical subculture, cells were harvested, pelleted and total RNA was prepared from tissue specimens using Trizol (Gibco BRL, Gaithersburg, MD, USA) according to the manufacturer’s protocol. RNA obtained from three independent experiments was pooled for each time point. 5 ␮g of RNA was used for analysis with GeneChip Mouse Genome 430A 2.0 oligonucleotide microarray (Affymetrix, Santa Clara, CA, USA). Labeling of RNA targets, hybridization and calculation of gene expression levels were performed according to the manufacturer’s protocol in the Charité Laboratory of Functional Genome Research.

Quantitative real-time polymerase chain reaction At 0, 1, 2, 3, 4, 7 and 9 days of NPC differentiation, RNA was prepared as described above. Total RNA was reverse-transcribed using random hexamers and moloney murine leukemia virus re-

H. Prüss et al. / Neuroscience 180 (2011) 19 –29 verse transcriptase (Promega, Mannheim, Germany). Expression of mRNA in each sample was normalized for RNA preparation and reverse transcriptase reaction on the basis of its glyceraldehyde 3-phosphate dehydrogenase (GAPDH) mRNA content. Amplification products in reverse transcriptase PCR were detected using the LightCycler-FastStart DNA-Master SYBR-Green-I Kit (RocheMolecular Biochemicals, Penzberg, Germany) and amplified and detected using the LightCycler Relative Quantification Software (Roche Molecular Biochemicals). Quality of amplification products was confirmed after each real-time PCR by checking the melting curve and visualization on agarose gel. All samples were amplified in duplicate from the same RNA preparation and the mean values of the respective crossing points (Cp) were considered. For determination of PCR efficiencies (E), we analyzed a serial dilution of cDNA and calculated E⫽10[⫺1/slope]. The relative expression of each gene part compared with GAPDH mRNA expression was calculated using the respective Cp according to the equation E(gene)⫺Cp(gene)/E(GAPDH)⫺Cp(GAPDH). Thermal cycling was carried out at 95 °C for 15 s, 68 °C for 10 s, 72 °C for 15 s. The sequence-specific primers (Tib Molbiol, Berlin, Germany) are as follows: DCX: forward 5=-cagcaagtctccagctgactc-3=, reverse 5=-cgctgtcattggatgactctg-3=; GFAP: forward 5=-gagggacaactttgcacaggac-3=, reverse 5=-gaatcgctggaggaggagatc-3=; Nestin: forward 5=-gaggacccaaggcatttcg-3=, reverse 5=-gatctatctttgccttcacactttcc-3=; BMP7: forward 5=-cagatcacagtctatcaggtg-3=, reverse 5=-caatcaggcctgccaacttg-3=; BMP6: forward 5=-caagtcttgcaggagcatcag-3=, reverse 5=-gtcaccacactcagctggag-3=; ␤-actin: forward 5=-acccacactgtgcccatcta-3=, reverse 5=-gccacaggattccataccca-3=. For statistical analysis, data were calculated as means⫾ standard deviation and compared using one-way analysis of variance (ANOVA) followed by t-tests. Results were considered significant at a probability value ⬍0.05.

Transient transfection of NPCs Plasmids were generated by cloning Kir2.1 and Kir2.3 cDNAs into the pEGFP-C1 vector (Clontech, Hamburg, Germany). Vectors (4 –5 ␮g) were introduced into a single-cell suspension of 3– 4⫻106 cultured NPCs by nucleofection (program A-33, Mouse NSC Nucleofector Kit, Amaxa, Walkersville, MD, USA). Cells were then plated in growth medium containing EGF and FGF2 and used for proliferation or differentiation assays.

RESULTS Neurospheres Culture conditions promoted the formation of neurospheres without relevant morphological differentiation into neurons or glial cells during the first week (Fig. 1A). In line with previous reports (Gritti et al., 1996; Yasuda et al., 2008), the majority of neurosphere cells were nestin-positive (Fig. 1B) and many nestin-positive cells were also GFAP-positive (Fig. 1B=), although spheres also contain lineage-restricted precursors, post-mitotic neurons and glia as well as dead cells. Thus, cultures were enriched for astrocytic precursors, which show immunoreactivity for both nestin and GFAP in vivo (Doetsch et al., 1999; Garcia et al., 2004). Neurospheres differentiated into cells of neuronal and glial fate. The ratio of developing neurons (⬍2%, DCX-positive, Fig. 1C), oligodendrocytes (⬍1%, RIP-positive, Fig. 1E) and astrocytes (⬎95%, GFAP-positive, Fig. 1D) after 4 days of growth factor removal was comparable to the published literature.

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As outlined above, it is likely that the proliferation and differentiation of neural stem and precursor cells depend on a complex cell cycle control that is influenced by K⫹ channel activity. We therefore analyzed whether NPC maturation is associated with changes in K⫹ channel expression at the mRNA and protein levels. Analysis of Kⴙ channel gene expression using microarrays In order to identify the potassium channel genes expressed by NPCs, a gene-array screening was used with mRNA probes derived from undifferentiated neurospheres (growth phase) and differentiated NPCs after 7 days of growth factor withdrawal. It is important to note that cell populations were not homogeneous, but also contained lineage-restricted progenitors and post-mitotic cells. The microarray included many, but not all K⫹ channel genes (e.g. Kir2.4, Kir7.1 or TASK3 were missing). Some members of the three K⫹ channel families (Kir, Kv and K2P channels) showed high transcript levels, in particular Kir4.1, KCa2.2, TREK1, Kv4.2 and Kv3.1 (Fig. 1F). These findings are in good agreement with previous mRNA data from the literature in different neural stem and progenitor cell populations. For example, Kv3.1 mRNA expression was found in murine mesencephalic NPCs and embryonic rat neural tube NPCs (Cai et al., 2004; Liebau et al., 2006). Kv4.2, Kv1.6 and Kv3.1 mRNAs were identified in human NPCs derived from aborted fetal brain tissue (Schaarschmidt et al., 2009), and Kir4.1 transcripts were detected in NPCs from the adult mouse SVZ (Liu et al., 2006; Yasuda et al., 2008). The quality and reproducibility of our neurosphere cultures were confirmed using quantitative real-time PCR detection of lineage marker gene expression during the first days of differentiation (Fig. 1G). Profound and rapid upregulation was detected for GFAP and DCX mRNAs (P⬍0.05 between days 0 and 7), while changes in nestin mRNA expression were not significant (Fig. 1G). Bone morphogenetic proteins (BMPs) are relevant for neurosphere proliferation and lineage progression. We found induction of BMP-6 mRNA expression only after 7 days of NPC differentiation, paralleled by termination of BMP-7 mRNA expression (Fig. 1G, P⬍0.05 between days 0 and 7). These temporal changes correspond to published data (Bonnert et al., 2006; Deleyrolle et al., 2006). Analysis of Kⴙ channel protein expression using immunohistochemistry Selection of K⫹ channel proteins for further analysis was determined by the availability of highly specific antibodies. Apart from some commercial antibodies, the affinity-purified specific antibodies used in this study were previously characterized by competitive ELISA, Western blotting and immunohistochemical preabsorption experiments (Veh et al., 1995; Derst et al., 2001; Prüss et al., 2003, 2005; Thomzig et al., 2005; Eulitz et al., 2007). K⫹ channel protein expression was analyzed in parallel in the SVZ of adult mice and in neurosphere cultures (Table 1). Of particular interest were the neuronal progen-

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H. Prüss et al. / Neuroscience 180 (2011) 19 –29

Fig. 1. SVZ-derived neurospheres (A) contain numerous nestin-positive cells (B), many of which also express GFAP (B=). After growth factor withdrawal, they mainly differentiate into DCX-positive immature neurons (C) and GFAP-positive astrocytes (D), to a lesser degree into RIP-positive oligodendrocytes (E). A gene-array screening (F) reveals that many K⫹ channel genes are expressed in growing neurospheres (0 d), and after NPC differentiation (7 d). The signal values of the x/y-axis assign a relative measure of abundance to the particular transcripts (blue dots⫽absent, red dots⫽present, gene expression levels according to the manufacturer’s protocol). Representative real-time PCR results of lineage marker expression during the first days of differentiation (n⫽3 per timepoint, normalized to the housekeeping gene GAPDH) are used to characterize the neurosphere cultures (G). Values are expressed as arbitrary units (AU). The asterisks mark significant changes between day 0 and day 7. Scale bars represent 50 ␮m in (A), 20 ␮m in (B, B=) and 10 ␮m in (C–E). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

itors that migrate on a rostro-caudal axis to the olfactory bulb via the rostral migratory stream (Fig. 2A) and that originate in the walls of the lateral ventricles (Fig. 2B, C). Two-pore potassium channels. Of the K2P channels, the most interesting expression pattern was observed for the TASK1 channel protein, which was detected mainly in cells along the rostral migratory stream (RMS) (Fig. 2A-1 to A-4). Strongest immunoreactivity was detected in the neuroblast zone (Fig. 2D), however, high-resolution confocal imaging excluded co-localization with DCX⫹ cells (Fig. 2A-2). Instead TASK1 signal was confined to astrocytes directly surrounding

the DCX⫹ cells, which migrate along the RMS into the olfactory bulb (Fig. 2A-1 to A-4) and which are GFAP-positive (Fig. 2E). In the SVZ, TASK1⫹ cells co-expressed nestin (Fig. 2F), which is used as a marker for progenitor cells. In neurosphere cultures, TASK1 was expressed in GFAP⫹ astroglial cells after 4 days of differentiation (Fig. 2G) and other cell types (arrow in G). TASK1 expression started in a few nestin⫹ cells on day 0, whose number increased during the first week (Fig. 2H). TASK3 was expressed in the SVZ and in fibers of the neuroblast zone (Fig. 2I), but not in DCX⫹ neural progenitors (Fig. 2J). Instead, TASK3 immunoreactivity was de-

H. Prüss et al. / Neuroscience 180 (2011) 19 –29 Table 1. Immunohistochemical analysis of several members of the three major K⫹ channel classes in the adult murine SVZ and in neurosphere cultures during differentiation (for details see text) Neurosphere culture

Subventricular zone

(Sub)ependymal cells DCX⫹ progenitors GFAP⫹ DCX⫹ Kv1.1 Kv1.4 Kv1.5 Kv1.6 Kv2.1 Kv4.2 Kv4.3 Kir1.1 Kir2.1 Kir2.2 Kir2.3 Kir2.4 Kir3.1 Kir3.2 Kir4.1 Kir4.2 Kir5.1 Kir6.1 Kir6.2 Kir7.1 TASK1 TASK3 TWIK1 TWIK2 TREK1 TREK2 TRAAK

⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫺ ⫹ ⫹ ⫹ ⫹ ⫹ ⫹ ⴙ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺ ⫹ ⫺ ⫹ ⫹ ⫹ ⫺ ⫺ ⫹ ⫺ ⫺

⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫺ ⫹ ⫺ ⫺ ⫺ ⫺ ⫺

tected in nestin⫹ SVZ cells (Fig. 2K). In neurosphere cultures, TASK3 was also expressed in nestin⫹ progenitors after 3 days (Fig. 2L), and in both differentiating astrocytes and immature neurons after 7 days (Fig. 2M). Importantly, TASK3 immunoreactivity was even observed in early proliferating undifferentiated neurospheres (Fig. 2N). TWIK2 (Fig. 2O), TREK1 (Fig. 2P), TWIK1 (Fig. 2U) and TREK2 proteins (Fig. 2V) were all detected in ependymal cells of the ventricle wall, but not in DCX⫹ neural progenitors. No TRAAK immunoreactivity was detected in the SVZ (data not shown). In neurosphere cultures, TREK1 expression was observed in early nestin⫹ cells (Fig. 2T) and in differentiating GFAP⫹ astrocytes (Fig. 2Q–S). The strong immunoreactivity for TREK1 and the presence of the other K2P channels are in agreement with previous mRNA expression data in mice (Aller and Wisden, 2008). Voltage-gated potassium channels. Despite gene expression of several Kv channels in neurosphere cultures (Fig. 1F), only very few Kv channel proteins were detected by immunochemistry in the adult SVZ (Table 1). Kv4.2 immunoreactivity was observed in ependymal cells of the SVZ, but not in DCX⫹ cells (Fig. 3A–C). No Kv4.3 immunoreactivity was observed in the SVZ (Fig. 3D), while Kv4.3-expressing cells were detected in the raphe (Fig. 3E) and in cortical areas (Fig. 3F) of the same brain sections,

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underscoring antibody sensitivity. Kv1.6 immunoreactivity was observed in DCX⫹ cells of the dorsal SVZ (Fig. 3G) and in the very proximal part of the RMS (Fig. 3H), but not along the RMS (Fig. 3I–K). Kv1.6-immunoreactive cells did not express the progenitor marker nestin. Kv2.1 immunoreactivity was absent in the SVZ (Fig. 3L), although Kv2.1 was strongly expressed in cortical neurons of the same brain sections (Fig. 3M). The distribution of Kv1.6 in cells adjacent to the SVZ and the prominent expression of Kv2.1, Kv4.2 and Kv4.3 in cortical neurons are in line with the pattern of Kv channel expression in the adult brain as reported previously (Rhodes et al., 1997; Trimmer and Rhodes, 2004). We did not detect Kv1.1, Kv1.4 and Kv1.5 immunoreactivities in the SVZ or in neurosphere cultures (Table 1). Inwardly-rectifying potassium channels. Several members of the Kir family were detected in the SVZ and in neurosphere cultures. Extensive positive controls were applied, when no protein signal was detected, as shown exemplarily for the Kir2.1 and Kir2.3 members (Fig. 4A– E=). Both channel proteins were absent in DAB stainings of the SVZ (Fig. 4A, C) and in fluorescent stainings of neurosphere cultures (data not shown), but could be clearly detected in neurospheres after transient transfection with plasmids for overexpression of Kir2.1 or Kir2.3 fusion proteins with the green fluorescent protein (GFP) (Fig. 4B, E, E=). Moreover, Kir channel expression in other brain regions of the same section was used as positive control for antibody sensitivity, for example, Kir2.3 immunoreactivity was detected in cortical neurons (Fig. 4D). Kir2.4 was expressed in differentiating neurospheres and in SVZ cells. Strongest Kir2.4 immunoreactivity was observed in nestin⫹ SVZ cells (data not shown) and to a much lesser degree in DCX⫹ cells (Fig. 4F). In neurosphere cultures, Kir2.4 was predominantly expressed in astrocytic cells (Fig. 4G–I, lower panel), with lower levels in DCX⫹ neuronal progenitors after 4 days (Fig. 4G–I, upper panel). Expression was already present in nestin⫹ progenitors at the beginning of differentiation (Fig. 4J). Western blots of cultured neurospheres confirmed the presence of Kir2.4 protein (Fig. 4K). The distribution of Kir2.4 immunoreactivity in adjacent brain regions, for example, in striatal neurons (arrows in Fig. 4F), matched the results of previous studies (Prüss et al., 2005). Kir3.2 immunoreactivity was restricted to the periventricular cell layer (Fig. 4L), and Kir4.1 was expressed on nestin⫹ (data not shown) and GFAP⫹ (Fig. 4M), but not on DCX⫹ cells in the SVZ (Fig. 4N). A similar staining pattern was observed in cultured NPCs with strong Kir4.1 immunoreactivity on astrocytes (Fig. 4Q) and co-localization with nestin (Fig. 4P), but no expression on early DCX⫹ neurons after 7 days (Fig. 4O). Kir5.1 was expressed in the ependymal layer around the lateral ventricle, but not in DCX⫹ neuronal precursors and in the RMS (Fig. 4W). The expression patterns of the Kir4.1 and Kir5.1 channel proteins we observed here are in line with previous studies (Yasuda et al., 2008). Kir6.1 immunoreactivity was detected on GFAP⫹ cells (Fig. 4R), but not on DCX⫹ neuronal precursors in the SVZ (Fig. 4T) or on DCX⫹ cells in neurosphere cultures (Fig.

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Fig. 2. K2P channel protein expression in the SVZ and in neurosphere cultures. DCX-positive neural progenitor cells replenish interneurons in the olfactory bulb, where they migrate along the rostral migratory stream (A), originating in the walls of the lateral ventricles (B, C). TASK1 immunoreactivity was detected mainly in cells along the RMS, but it did not co-localize with DCX (A-1 to A-4, positions indicated in A). Strongest TASK1 expression was detected in the neuroblast zone and the SVZ (D). Here, DCX⫹ cells of the neuronal lineage neighbour nestin⫹ progenitor cells. The TASK1 signal was confined to GFAP-expressing cells (E) and/or nestin-expressing cells (F). In neurosphere cultures, TASK1 was expressed in GFAP⫹ cells (G; box is enlarged in G=/G⬙ for single fluorescent images) and other cell types (arrow in G), including nestin⫹ progenitors (H),

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Fig. 3. Kv channel protein expression in the SVZ. Kv4.2 protein was detected in ependymal cells of the SVZ, but not in DCX⫹ cells (A–C). Kv4.3 immunoreactivity was absent in the SVZ (D), while Kv4.3-positive cells were detected in the raphe (E) and the cortex (F) of the same brain sections, serving as positive control. Kv1.6 protein expression was detected in DCX⫹ cells of the dorsal SVZ (G) and in the very proximal part of the RMS (H), but not along the RMS (I–K). Kv2.1 immunoreactivity was absent in the SVZ (L), although Kv2.1 was strongly expressed in cortical neurons (M). Bars represent 50 ␮m in (A, B, D, I–L) and 20 ␮m in (C, G, H, M). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

4S), but instead on nestin⫹ cells and in the astrocytic lineage during NPC differentiation (Fig. 4U, V), and on a subpopulation of nestin⫹ cells in the SVZ (data not shown). A similar staining pattern for Kir6.1 was seen in the ventricle walls of the rat brain (Prüss et al., 2008). Kir7.1 immunoreactivity was present on epithelial cells of the choroid plexus (Döring et al., 1998), but absent on DCX⫹ cells of the SVZ (Fig. 4X). In neurosphere cultures, Kir7.1 immunoreactivity was clearly detectable on day 4 using immunocytochemistry and Western blot analysis (Fig. 4X, inset), and reduced on day 7 (data not shown). No immunoreactivites were detected for Kir1.1, Kir2.2, Kir3.1, Kir4.2 and Kir6.2 channels in the SVZ and in neurosphere cultures (Table 1).

DISCUSSION Kⴙ channel protein and mRNA expression by adult murine NPCs The comprehensive immunohistochemical analysis of several members of the three major K⫹ channel classes revealed a differential expression pattern in cells of the SVZ

and in neurosphere cultures during differentiation (Table 1). Based on the channel protein distribution, different groups of cells could be distinguished. First, many K⫹ channels were expressed on nestin⫹ SVZ precursor cells, in particular most K2P and Kir channels, including TREK1 and TASK1. Second, only few channels were expressed on early SVZ DCX⫹ neuronal precursors, that is, Kir2.4. Third, some channels were expressed in close proximity to the subventricular region, such as Kir7.1 in epithelial cells of the choroid plexus and TASK1 in astrocytes surrounding the RMS. A larger number of studies examined K⫹ channel mRNA expression in NPCs from different species. Transcripts of several K⫹ channels were identified, broadly overlapping with the data from our gene-array screening. Similar to our data, high mRNA expression was detected for Kv3.1 in murine mesencephalic neural progenitors (Liebau et al., 2006), for Kv3.1 and Kv3.3 in embryonic rat neural tube NPCs (Cai et al., 2004), for Kv4.2 and Kv1.6 in proliferating human NPCs derived from aborted fetal brain tissue (Schaarschmidt et al., 2009), and for Kir4.1, Kir5.1 and Kv3.1 in murine neurosphere-derived precursors (Ya-

underscoring the astrocytic predominance seen in the SVZ. TASK3 was expressed in the SVZ and in fibers of the neuroblast zone (I), but not in DCX⫹ neural progenitors (J). The expression was most prominent in nestin⫹ SVZ cells (K) and in nestin⫹ progenitors in neurosphere cultures (L), and to a lesser degree in TuJ1⫹ early neurons (M). TASK3 expression already started in early proliferating neurospheres (N). TWIK2 (O, box enlarged in O=/O⬙), TREK1 (P), TWIK1 (U) and TREK2 (V) immunoreactivities were all detected in ependymal cells of the ventricle walls, but not in DCX⫹ neural progenitors. TREK1 immunoreactivity was detected in neurosphere cultures with co-labelling of early nestin⫹ cells (T) and differentiating astrocytes (Q–S). OB, olfactory bulb; RMS, rostral migratory stream; LV, lateral ventricle; cc, corpus callosum. Bars represent 50 ␮m in (A-1 to A-3, D, F, I, O), 20 ␮m in (A-4, E, G, H, J, K, M, P–V) and 10 ␮m in (L). For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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H. Prüss et al. / Neuroscience 180 (2011) 19 –29

Fig. 4. Kir channel protein expression in the SVZ and in neurosphere cultures. Kir2.1 (A) and Kir2.3 (C) proteins were absent in the adult murine SVZ. In neurospheres, Kir2.1 (B) and Kir2.3 (E, E=) proteins were only detectable after transient transfection of these channels. The characteristic protein expression in other brain areas (e.g. cortical expression of Kir2.3 in the same section, D) served as additional positive control. Kir2.4 showed low expression in DCX⫹ neuronal precursors in the SVZ (F; arrows point to striatal neurons) and neurosphere cultures (G–I, upper panel), but was broadly expressed in GFAP⫹ astrocytic cells (G–I, lower panel), starting already in early nestin⫹ progenitors (J). Western blots of cultured neurospheres confirmed the presence of Kir2.4 protein (K, arrow). Kir3.2 protein expression was limited to the periventricular cell layer (L). Kir4.1 expression was also seen in astrocytes (M) with lack of immunoreactivity in DCX⫹ SVZ cells (N) and DCX⫹ cultured NPCs (O). Expression on

H. Prüss et al. / Neuroscience 180 (2011) 19 –29

suda et al., 2008). The expression of lineage and differentiation markers, such as DCX, nestin, GFAP and BMPs, during the first days of NPC differentiation was similar in our study compared with previous gene expression data (Bonnert et al., 2006; Deleyrolle et al., 2006). Only few studies analyzed the protein distribution of K⫹ channels in adult murine SVZ-derived neurospheres. The present findings support and extend those data. For example, the presence of Kir4.1 protein in some, but not all nestin⫹ cells of the SVZ and neurosphere cultures is in agreement with previous work (Liu et al., 2006; Yasuda et al., 2008). The astrocytic expression may be indicative of the known role of Kir4.1 in setting the glial resting membrane potential (Butt and Kalsi, 2006). In line with our results, Yasuda et al. (2008) found expression of Kir5.1 protein in the adult mouse SVZ and in neurosphere cultures, as well as disappearance of Kir4.1 and Kir5.1 immunoreactivity in the RMS. We did not detect Kv1.3 expression in the SVZ and in NPC cultures, which is in line with one study of SVZ-derived neurospheres (Yasuda et al., 2008), but in contrast to another using murine mesencephalic neural progenitors (Liebau et al., 2006). Moreover, Kir2.1 immunoreactivity was absent in our NPC cultures and in one previous study (Yasuda et al., 2008), but detected in another (Liu et al., 2006). Correlation between data from NPC cultures and brain SVZ sections The present mRNA data from differentiated murine NPCs were obtained from a heterogeneous population with a majority of glial and only about 1% neuronal cells. Therefore, neuron-specific appearance of channel transcripts may not be detected. Moreover, it is well known and biologically plausible that not all transcripts eventually lead to protein expression. We therefore combined the mRNA screening with protein detection at a cell-specific level. Affinity-purified specific antibodies were used, most of which were extensively characterized using competitive ELISA, Western blotting and immunohistochemical preabsorption experiments (Veh et al., 1995; Derst et al., 2001; Prüss et al., 2003, 2005; Thomzig et al., 2005; Eulitz et al., 2007). In the case of antibodies that did not result in immunostaining, several positive controls were performed, including simultaneous application to brain sections and cultures, Western analysis of brain and NPC protein homogenates and staining of transfected cells that overexpress the pertinent protein. The comparison between K⫹ channel protein expression in the SVZ and in neurosphere cultures in our study suggests that the expression pattern of K⫹ channels is largely preserved in vitro. For example, the expression of Kir4.1, Kir2.4, Kir6.1, Kir7.1, TASK3 and TASK1 and the

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absence of many Kv and Kir channels is identical in vivo and in cultured cells. A similar conclusion was drawn based on the electrophysiological properties of these cell populations (Yasuda et al., 2008). Thus, data from cultured NPCs may help to model in vivo functions in NPC behavior and lineage progression. Factors affecting the Kⴙ channel repertoire The K⫹ channel expression pattern found in the present study may not be generalized to other stem and progenitor cells. Recent literature suggests that K⫹ channels and currents are not only region-, but also species-specific. For example, while Kv3.1 protein was present in embryonic NPCs from rat midbrain (Liebau et al., 2006), it was absent in embryonic NPCs from mouse SVZ (Smith et al., 2008). Neurogenesis of dentate gyrus progenitor cells may be increased by the activation of Kv4.1 channels (Shi et al., 2007), while the Kv4.1 transcript was not identified in proliferating human NPCs from aborted fetal brain (Schaarschmidt et al., 2009). Kir currents were identified in rabbit and mouse MSCs, but not in human and rat MSCs (Tao et al., 2007). K⫹ currents are also likely to have different functions in neuronal and glial progenitors. While voltage-activated Atype currents (predominantly Kv4.2) were proposed to play a key role in the proliferation of human NPCs (Schaarschmidt et al., 2009), several delayed-rectifier channels (Kv1 family) seem to be important for the proliferation of oligodendrocyte progenitor cells (Schmidt et al., 1999; Vautier et al., 2004). Moreover, different culture conditions may account for the discrepancies, as published protocols vary with regard to the addition of growth factors (e.g. EGF) and supplements (e.g. retinoic acid-free B27). Finally, adult and embryonic NPCs respond differentially to K⫹ channel blockers. For example, tetra-ethyl-ammonium increased cell proliferation of embryonic NPCs (Liebau et al., 2006), but reduced proliferation of adult NPCs (Yasuda et al., 2008). Putative roles of Kⴙ channels There is an increasing body of evidence that K⫹ channels participate in the proliferation, maturation and differentiation of neuronal and glial cells (Ribera, 1990; Liebau et al., 2006; Pappas et al., 1994; Shi et al., 2007; Yasuda et al., 2008). Voltage-gated K⫹ channels are expressed early during brain development with a temporal and cell typespecific pattern (Spitzer, 1991; Shibata et al., 2000; Prüss et al., 2009). It has been hypothesized that changes in K⫹ channel activity are required for proliferation at critical cell cycle checkpoints (DeCoursey et al., 1984; Gallo et al., 1996; Wonderlin and Strobl, 1996; Pardo, 2004; Vautier et al., 2004). Interestingly, absence of functional Kv1.1 ion channel results in megencephaly with increased neuro-

developing astrocytes (Q) was already detectable at the stage of nestin⫹ progenitors (P). A similar pattern was observed for the Kir6.1 protein with co-labelling of GFAP⫹ cells (R) and immunoreactivity was absent in DCX⫹ SVZ cells (T) and DCX⫹ cultured NPCs (S), but high expression in cultured astrocytes (V) and early nestin⫹ progenitors (U). Kir5.1 was expressed in the ependymal layer around the lateral ventricle, but not in DCX⫹ neuronal precursors (W). Kir7.1 immunoreactivity was absent in ependymal cells (X, arrowhead) and DCX⫹ cells of the SVZ, but limited to the choroid plexus attached to the ventricle wall (X). Kir7.1 protein expression in early neurosphere cultures was confirmed by Western blot analysis (X, inset). All bars represent 20 ␮m. For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.

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H. Prüss et al. / Neuroscience 180 (2011) 19 –29

genesis (Almgren et al., 2007). The exact mechanisms linking K⫹ currents and cell proliferation are still unclear. Possible explanations include the depolarization of the resting membrane potential as a mandatory step for the NPC cell cycle resulting in Ca2⫹ influx (Wonderlin and Strobl, 1996; Beeton et al., 2001). Alternatively, K⫹ channels may regulate the cell volume, which can be critical for cell division (Rouzaire-Dubois and Dubois, 1998). The distinct localization of some K⫹ channel proteins might reflect a particular function. For example, the limited distribution of the Kv1.6 channel protein in the dorsal SVZ and proximal RMS could correspond to the newly described progenitors generating a subtype of glutamatergic neurons in the olfactory bulb (Brill et al., 2009). Moreover, restriction of TASK1 and Kir6.1 protein expression to a subset of astrocytic progenitors suggests that both channels might participate in the functional organization of the stem cell niche in the SVZ. In particular, the close, nonoverlapping proximity between DCX⫹ neuronal precursors and TASK1⫹ glial cells from the SVZ along the RMS into the olfactory bulb may suggest functional relationships. The restricted TASK1 expression in cells surrounding the RMS makes it conceivable that TASK currents provide electrophysiological cues in the neurogenic microenvironment in vivo. A putative role for TASK1 in cell proliferation or migration seems plausible, similar to findings where TASK1 (together with TASK3) channels participated in the proliferation and cytokine production of T lymphocytes (Meuth et al., 2008). Recently, a role for the K2P channel TREK1 in the regulation of hippocampal neurogenesis has been described, making this K⫹ channel a novel target in anti-depressant drug design (Mazella et al., 2010). Taken together, we present a comprehensive expression analysis of several members of the three major K⫹ channel classes in NPCs. We also identify candidates, which might participate in progenitor cell growth and the stem cell niche microenvironment. Analysis of the distinct expression patterns in neuronal and astrocytic NPCs is a prerequisite in order to estimate the potential for specific K⫹ channel modulators to control endogenous adult neurogenesis. Of particular importance seems to be the expression of two-pore-domain K⫹ channels that have not been considered so far in the context of neural stem and progenitor cells, although they play a pivotal role in modulating neuronal excitability (Goldstein et al., 2001). Further studies are required to elucidate the relationship between K⫹ channels and neurogenesis in the SVZ. The results promise to provide new approaches for regulating NPC proliferation or migration in the brain. Acknowledgments—This work was supported by grants from the BMBF, BCRT, DFG, and NeuroCure (to JP).

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(Accepted 8 February 2011) (Available online 15 February 2011)