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HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection Du

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HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection Du Published OnlineFirst July 14, 2020; DOI: 10.1158/1541-7786.MCR-20-0292 MOLECULAR CANCER RESEARCH | NEW HORIZONS IN CANCER BIOLOGY HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment A C Helena Johard1,2,3, Anna Omelyanenko1, Gao Fei1,4, Misha Zilberter5, Zankruti Dave2, Randa Abu-Youssef1, Linnea Schmidt2, Aditya Harisankar6, C. Theresa Vincent2,7, Julian Walfridsson6, Sven Nelander2, Tibor Harkany8,9, Klas Blomgren10,11, and Michael Andang€ 1,2,3 ABSTRACT ◥ Children suffering from neurologic cancers undergoing chemo- Hyperpolarizing pharmacologic inhibition of HCN channels during therapy and radiotherapy are at high risk of reduced neurocognitive exposure to ionizing radiation protects NSCs cells in neurogenic abilities likely via damage to proliferating neural stem cells (NSC). brain regions of young mice. In contrast, brain tumor–initiating Therefore, strategies to protect NSCs are needed. We argue that cells, which also express HCN channels, remain proliferative during induced cell-cycle arrest/quiescence in NSCs during cancer treat- HCN inhibition. ment can represent such a strategy. Here, we show that hyperpo- larization-activated cyclic nucleotide-gated (HCN) ion channels are Implications: Our finding that NSCs can be selectively rescued dynamically expressed over the cell cycle in NSCs, depolarize the while cancer cells remain sensitive to the treatment, provide a membrane potential, underlie spontaneous calcium oscillations and foundation for reduction of cognitive impairment in children with are required to maintain NSCs in the actively proliferating pool. neurologic cancers. Introduction and in the subventricular zone along the lateral ventricles (SVZ; refs. 5–7). To develop new therapeutic strategies protecting against Even though neurologic childhood cancers have increased over the damage during cancer treatments a deeper understanding of the past 20 years, the survival rates have also greatly improved (1). regulatory mechanisms controlling NSC proliferation is needed. So Unfortunately, both chemotherapy and radiotherapy pose well- far, strategies to rescue neurogenesis have focused on reactivating and known risks of inducing neurocognitive damage with severe impact increasing NSC proliferation after radiotherapy (8–10). Another on these young children's quality of life and requiring extensive strategy is to selectively induce quiescence while maintaining cancer posttreatment cognitive rehabilitation therapy (2). It is likely that cells in a proliferative state. Such a strategy has not been reported yet these side effects on cognitive development stem from damage to the and requires deeper understanding of control mechanisms that diverge proliferating neural stem cell (NSC) pool, as postnatal neurogenesis in normal and transformed cells. regulates critical functions such as learning and memory (3, 4). The Proliferation of NSCs as well as cancer cells is maintained by a production of neurons by the NSCs continues at a reduced rate variety of extracellular signals in the microenvironment (11–13) throughout life in the subgranular zone of the hippocampus (SGZ) including signaling via neurotransmitters that control the membrane potential. For example, chloride-permeable GABAA receptor channels 1Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, 2 limit proliferation via hyperpolarization, while glutamate receptors Sweden. Department of Immunology, Genetics and Pathology, Uppsala activate proliferation via depolarization (14). Interestingly, highly University, Rudbeck Laboratory, Uppsala, Sweden. 3Central European Institute of Technology, Masaryk University, Brno, Czech Republic. 4College of Veterinary proliferating cancer cells are thought to have a more depolarized Medicine, Jilin University, Changchun, Jilin, China. 5Gladstone Institute of Neu- membrane potential conductive toward sustained proliferation in rological Disease, San Francisco, California. 6Department of Medicine, Karolinska comparison with nonmalignant cells (15). This suggests a divergence Institutet, Huddinge, Sweden. 7Department of Microbiology, New York Univer- in maintenance of membrane potential in malignant cells likely via sity School of Medicine, New York, New York. 8Department of Neuroscience, 9 altered expression of ion channels. Karolinska Institutet, Solna, Sweden. Department of Molecular Neurosciences, Here we explore HCN ion channels as putative target for prolifer- Center for Brain Research, Medical University of Vienna, Vienna, Austria. 10Department of Women's and Children's Health, Karolinska Institutet, Stock- ative control in NSCs. HCN channels have neither previously been holm, Sweden. 11Pediatric Oncology, Karolinska University Hospital, Stockholm, investigated in NSCs nor in cancer cells, although HCN expression has Sweden. been linked to proliferation in embryonic stem cells (16, 17). The HCN – Note: Supplementary data for this article are available at Molecular Cancer channel family consists of 4 isoforms (HCN1 4) and conduct potas- Research Online (http://mcr.aacrjournals.org/). sium and sodium, with HCN2 being also slightly permeable to H. Johard, A. Omelyanenko, and G. Fei contributed equally to this article. calcium (18). In response to membrane hyperpolarization in excitable cells, HCN channels conduct a depolarizing current, referred to as € Corresponding Author: Michael Andang, Uppsala University, Rudbeck Labora- h-current (I ),contributing toward an electric feedback loop essential tory, Uppsala 751 85, Sweden. Phone: 467-0331-4682; E-mail: h [email protected] for pace-making cyclic behavior in, for example, cardiac pacemaker cells of the heart (19) and neuronal oscillations (20), including Mol Cancer Res 2020;XX:XX–XX circadian rhythmicity in the suprachiasmatic nucleus (21, 22). doi: 10.1158/1541-7786.MCR-20-0292 We show that HCN channel activity is critical for maintaining NSCs Ó2020 American Association for Cancer Research. in the actively proliferating pool. Hyperpolarization by pharmacologic AACRJournals.org | OF1 Downloaded from mcr.aacrjournals.org on September 29, 2021. © 2020 American Association for Cancer Research. Published OnlineFirst July 14, 2020; DOI: 10.1158/1541-7786.MCR-20-0292 Johard et al. inhibition of HCN channels induces quiescence in NSCs, while leaving Flow cytometry brain tumor cells unaffected. This selective effect protects NSCs from For cell-cycle profile analyses, DNA profiles were acquired with genotoxic insult in vivo and thereby constitutes a novel approach for BD CellQuest Pro software, and percentages of G1,S,andG2 phases neuroprotection during cancer treatment by directly reducing damage were calculated and graphically presented with MODFIT software. to NSCs. For cell-cycle sorting, adherent NSCs were incubated in darkness for 30 minutes in the presence of Hoechst 33258 (10 mg/mL, Sigma-Aldrich). Cells were trypsinized and resuspended in 37C Materials and Methods conditioned Hoechst media and sorted for DNA content on a Animals FACSVantage SE (Becton Dickinson) at 4C and cell-cycle fractions Mouse strains used to generate in vitro cultures were GFAP::GFP corresponding to G1–G0,S,andG2–M were collected in conditioned reporter mice (23). In vivo drug action experiments were performed on media on ice. wild-type C57/B6 animals. Ethical permissions were obtained for all Cell cycle sorting of cells for electrophysiological recordings was animal work (permit number N397/12, N 248-13, N 163-15). Work also done by transfecting cells with Gem-AG (26) plasmid obtained was carried out in accordance with rules and regulations governing from the RIKEN BioResource Research Center DNA Bank. animal research in Sweden. Assays used for viable cell counting Neurosphere derivation and generation of NSC lines ATP-based cell number assays were used after treatment as Neurosphere cultures were prepared as described previously (24). described previously (27). The following chemical compounds used NSCs isolated from sphere cultures were further cultured in monolayer in cell-based assays: ZD7288 (Tocris Bioscience), Zatebradine (Tocris as described previously (25). Bioscience), CsCl (Sigma), and SR9009 (Calbiochem). Electrophysiology Gene expression analysis and statistics Whole-cell patch clamp recordings were performed on selected cells RNA was isolated using the RNeasy Micro Kit (Qiagen). For qRT- in voltage-clamp mode, recorded, and digitized at 10 kHz with EPC10 PCR, 1 mg of RNA was reverse transcribed using the High Capacity amplifier and digitizer (HEKA). The composition of the intracellular cDNA Reverse Transcription Kit (Applied Biosystems). Transcript fi solution was 140 mmol/L CsMeSO4, 10 mmol/L HEPES, 2 mmol/L abundance was assayed with qRT-PCR using gene-speci c primer MgCl2, 0.6 mmol/L EGTA, 2 mmol/L ATPNa, 0.3 mmol/L GTPNa, set pairs and SYBR reagents (Invitrogen) with a 7500 Fast Real-Time PCR to pH 7.2–7.3 with CsOH, osmolarity 270 –280 mOsm. The compo- machine (Applied Biosystems). sition of the extracellular solution ACSF was 124 mmol/L NaCl, Primer sequences 30 mmol/L NaHCO3, 10 mmol/L glucose, 1.25 mmol/L NaH2PO4, 3.5 mmol/L KCl, 1.5 mmol/L MgCl2, 1.5 mmol/L CaCl2. HCN positivity was ascertained as a presence of a slow-activating inward Hcn1 Forward: 50CGC CTT TCA AGG TTA ATC AGA 30 current at a 5-second step to -110 mV. Resting membrane potential Hcn1 Reverse: 50 GGC GAG GTC ATA GGT CAT GT 30 – – 0 0 (Em) was calculated from voltage current relationships
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