Nov 21, 2008 - 1.2.3. Spatial aspects of GABAergic signalling in pyramidal neurons ............... 2. 1.3. ...... al., 1998, Zhou and Rush, 1996). ..... I X-ray films were used for visualization ...... pathological replay of developmental mechanisms?
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Helsinki 2008
ANASTASIA LUDWIG Mechanisms of KCC2 Upregulation During Development
Recent Publications in this Series:
Mechanisms of KCC2 Upregulation During Development
ANASTASIA LUDWIG Institute of Biotechnology and Department of Biological and Environmental Sciences Faculty of Biosciences and Finnish Graduate School in Neuroscience University of Helsinki
ISSN 1795-7079 ISBN 978-952-10-5039-8
Dissertationes bioscientiarum molecularium Universitatis Helsingiensis in Viikki 43/2008
43/2008
Mechanisms of KCC2 upregulation during development ANASTASIA LUDWIG Institute of Biotechnology and Department of Biological and Environmental Sciences Faculty of Biosciences and Finnish Graduate School in Neuroscience University of Helsinki
ACADEMIC DISSERTATION
To be presented, with the permission of the Faculty of Biosciences of the University of Helsinki, for public examination in auditorium 1041 at Biocenter 2 (Viikinkaari 7, Helsinki), on 21st of November 2008, at 12 noon. Helsinki 2008
Supervised by: Docent Claudio Rivera Institute of Biotechnology University of Helsinki Finland and Professor Kai Kaila Department of Biological and Environmental Sciences Faculty of Bioscience University of Helsinki Finland and Professor Mart Saarma Institute of Biotechnology University of Helsinki Finland Reviewed by: Professor Dan Lindholm Minerva Institute for Medical Research Biomedicum Helsinki University of Helsinki Finland and Professor Hans Gerd Nothwang Carl von Ossietzky University of Oldenburg Germany Opponent: Professor Eckhard Friauf Department of Biology University of Kaiserslautern Germany ISBN 978-952-10-5039-8 (paperback) ISBN 978-952-10-5035-0 (PDF, http://ethesis.helsinki.fi) Yliopistopaino, Helsinki 2008
“A joy that’s shared is a joy made double” To Pavel
TABLE OF CONTENTS TABLE OF CONTENTS ABBREVIATIONS ORIGINAL PUBLICATIONS ABSTRACT 1. REVIEW OF THE LITERATURE......................................................................... 1 1.1. GABA: an overview ............................................................................................ 1 1.2. GABAA receptor mediated responses .................................................................. 1 1.2.1. GABAA reversal potential ......................................................................... 1 1.2.2. Shunting inhibition .................................................................................... 2 1.2.3. Spatial aspects of GABAergic signalling in pyramidal neurons ............... 2 1.3. Functional aspects of GABA signalling in the immature brain .......................... 3 1.3.1. Trophic effect of GABA on immature neurons ......................................... 3 1.3.2. GABA and spontaneous activity ............................................................... 3 1.4. The family of cation-cloride cotransporters: an overview .................................. 5 1.4.1. Family members, protein structure, function and inhibitors ..................... 5 1.4.2. Kinetic regulation and oligomerization of CCC ....................................... 6 1.4.3. CCC expression in the CNS ...................................................................... 8 1.5. NKCC1 and it’s function in the nervous system ................................................. 9 1.6. KCC2, a neuronal-specific CCC ....................................................................... 10 1.6.1. Developmental profile of KCC2 expression ........................................... 10 1.6.2. Role of KCC2 in regulation of intracellular Cl- ...................................... 11 1.6.3. Role of KCC2 in spine formation ........................................................... 13 1.6.4. Regulation of KCC2 expression and function ........................................ 14 1.6.4.1. Regulation of developmental KCC2 increase ............................ 14 1.6.4.2. KCC2 regulation in injured neurons .......................................... 16 1.6.4.3. KCC2 regulation in GABAergic synaptic plasticity .................. 16 1.7. BDNF, a member of the neurotrophin family ................................................... 17 1.7.1. Neurotrophins and their receptors ........................................................... 17 1.7.2. BDNF synthesis, processing and secretion ............................................. 18 1.7.3. Expression of BDNF and TrkB in the brain ............................................ 20 1.7.4. TrkB signalling........................................................................................ 22 1.7.5. EGR family of transcription factors ........................................................ 24 1.7.6. BDNF and the GABAergic system ......................................................... 25 1.8. Summary ........................................................................................................... 26 AIMS OF THIS STUDY............................................................................................. 27 2. MATERIALS AND METHODS ........................................................................... 28 2.1. Cell culture procedures...................................................................................... 28 2.1.1. Dissociated cell culture ........................................................................... 28 2.1.2. Organotypic slice culture ........................................................................ 28 2.1.3. Pharmacological treatments of cultured cells ......................................... 29 2.1.4. Transfections ........................................................................................... 29 2.2. In vivo studies .................................................................................................... 30 2.2.1. Intrahippocampal injections .................................................................... 30 2.2.2. Kainic acid injections and seizures ......................................................... 30 2.3. Detection techniques ......................................................................................... 31
2.3.1. Immunocytochemical procedures ........................................................... 31 2.3.2. Immunohistochemical procedures .......................................................... 31 2.3.3. Western blotting ...................................................................................... 31 2.3.4. Semi-quantitative and quantitative RT-PCR analysis. ............................ 31 2.3.5. In situ hybridization ................................................................................ 32 2.4. Image analysis ................................................................................................... 32 2.4.1. Quantification of immunostaining intensity in neuronal cell bodies ...... 32 2.4.2. Quantification of immunostaining intensity in tissue sections................ 32 2.4.3. Luciferase assay ...................................................................................... 34 2.5. Functional 86Rb flux assay ................................................................................. 34 2.6. Sequence analysis .............................................................................................. 34 3. RESULTS AND DISCUSSION ............................................................................. 35 3.1. Developmental upregulation of KCC2 in neuronal cultures in the absence of activity and synaptic transmission (I) ........................................ 35 3.1.1. Developmental increase of KCC2 in dissociated hippocampal neurons 35 3.1.2. Chronic blockage of activity and synaptic transmission does not affect KCC2 upregulation ......................................................... 36 3.2. Upregulation of KCC2 expression by transcription factor Egr4 (II) ................ 36 3.2.1. Egr4 strongly and specifically activates KCC2 promoter in N2a cells ... 37 3.2.2. Egr4 increases KCC2 promoter activity in cultured hippocampal and cortical neurons .................................................................................38 3.3. On the mechanism of developmental KCC2 upregulation (III) ........................ 39 3.3.1. BDNF increases activity of KCC2 promoter via MAPK activation and Egr4 in cultured neurons .................................................................. 39 3.3.1.1. BDNF treatment robustly increases Egr4 expression in MEK/ERK dependent manner ............................................... 39 3.3.1.2. BDNF activates KCC2 promoter via Egr4 binding site ..............40 3.3.2. BDNF and neurturin increase KCC2 expression in immature cultured neurons...................................................................... 40 3.3.3. BDNF and neurturin increase KCC2 expression when injected in neonatal rat hippocampus........................................................................ 41 3.3.4. Endogenous network activity is involved in regulation of KCC2 expression in vivo......................................................................... 42 3.3.4.1. Tetanus toxin injections...............................................................42 3.3.4.2. Kainic acid injections ................................................................. 42 3.4. A novel N-terminal isoform of KCC2 ................................................................43 3.4.1. Identification of a new upstream exon in the KCC2 gene ...................... 44 3.4.2. Characterization of KCC2a expression in nervous system ..................... 44 3.4.3. Functionality of KCC2a ...........................................................................45 3.4.4. KCC2b-deficient mice retain KCC2a mRNA expression ....................... 45 4. CONCLUSIONS ..................................................................................................... 47 5. ACKNOWLEDGEMENTS.................................................................................... 48 6. REFERENCES........................................................................................................ 50
ABBREVIATIONS AMPA APV BDNF CaMK CCC CIP CKB CMV CNS CREB DAG DN-Egr4 DRG E Egr4 ERK EST Frs-2 GAB2 GABA GAD GDNF GDP GFP Grb2 IP3 KCC LTP MAPK MEK nAChR NGF NKCC NMDA OSR P PI3K PKC PLC PNS PSP Shc SOS SPAK TetTx Trk TTX VGCC VIAAT
alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (2R)-amino-5-phosphonovaleric acid brain-derived neurotrophic factor Ca2+-calmoduline-regulated protein kinase cation chloride cotransporter cotransporter interacting protein brain-type creatin kinase cytomegalovirus central nervous system cAMP response element-binding protein diacylglycerol dominant-negative early growth response 4 dorsal root ganglia embryonic day early growth response 4 extracellular signal-regulated protein kinase expressed sequence tag fibroblast growth factor receptor substrate-2 growth factor receptor bound protein 2-associated protein 1 -aminobutyric acid glutamic acid decarboxylase glial cell line-derived neurotrophic factor giant depolarizing potential green fluorescent protein growth factor receptor-bound protein 2 inositol-3-phosphate potassium chloride cotransporter long term potentiation mitogen-activated protein kinase MAP/ERK kinase nicotinic acetylcholine receptor nerve growth factor sodium potassium chloride cotransporter N-methyl-D-aspartic acid oxidative stress response 1 postnatal day phosphatidylinositol 3-kinase protein kinase C phospholipase C pereferal nervous system post-synaptic potential src homology 2 domain containing transforming protein son of sevenless guanylnucleotide exchange factor Ste20-related praline-alanine-rich-kinase tetanus toxin tropomyosin receptor kinase tetrodotoxin voltage-gated Ca2+ channels visicular inhibitory amino acid transporter
ORIGINAL PUBLICATIONS This thesis is based on the following publications herein referred to by their Roman numerals (I-IV), and on some unpublished results: I.
Ludwig A, Li H, Saarma M, Kaila K and Rivera C. Developmental upregulation of KCC2 in the absence of GABAergic and glutamatergic transmission. Eur J Neurosci. 2003 Dec;18(12):3199-206.
II.
Uvarov P, Ludwig A, Markkanen M, Rivera C, Airaksinen MS. Upregulation of the neuron-specific K+/Cl-cotransporter expression by transcription factor early growth response 4. J Neurosci. 2006 Dec 27;26(52):13463-73.
III.
Ludwig A, Uvarov P, Shuschmann S, Thomas-Crusells J, Saarma M, Airaksinen M, and Rivera C. Egr4 mediates BDNF- and Neurturin-evoked MAPK-dependent upregulation of KCC2 in developing neurons. Manuscript is under submission
IV.
Uvarov P*, Ludwig A*, Markkanen M, Pruunsild P, Kaila K, Delpire E, Timmusk T, Rivera C, Airaksinen MS. A novel N-terminal isoform of the neuron-specific K-Cl cotransporter KCC2. J Biol Chem. 2007 Oct 19;282(42):30570-6. Epub 2007 Aug 22. *equal contribution
ABSTRACT Gamma-aminobutyric acid (GABA) acting through ionotropic GABAA receptors plays a crucial role in the activity of the central nervous system (CNS). It triggers Ca2+ rise providing trophic support in developing neurons and conducts fast inhibitory function in mature neuronal networks. There is a developmental change in the GABAA reversal potential towards more negative levels during the first two postnatal weeks in rodent hippocampus. This change provides the basis for mature GABAergic activity and is attributable to the developmental expression of the neuron-specific potassium chloride cotransporter 2 (KCC2). In this work we have studied the mechanisms responsible for the control of KCC2 developmental expression. As a model system we used hippocampal dissociated cultures plated from embryonic day (E) 17 mice embryos before the onset of KCC2 expression. We showed that KCC2 was significantly up-regulated during the first two weeks of culture development. Interestingly, the level of KCC2 upregulation was not altered by chronic pharmacological blockage of action potentials as well as GABAergic and glutamatergic synaptic transmission. By in silico analysis of the proximal KCC2 promoter region we identified 10 candidate transcription factor binding sites that are highly conserved in mammalian KCC2 genes. One of these transcription factors, namely early growth response factor 4 (Egr4), had similar developmental profile as KCC2 and considerably increased the activity of mouse KCC2 gene in neuronal cells. Next we investigated the involvement of neurotrophic factors in regulation of Egr4 and KCC2 expression. We found that in immature hippocampal cultures Egr4 and KCC2 levels were strongly up-regulated by brain derived neurotrophic factor (BDNF) and neurturin. The effect of neurotrophic factors was dependent on the activation of a mitogen activated protein kinase (MAPK) signal transduction pathway. Intact Egr4binding site in proximal KCC2 promoter was required for BDNF-induced KCC2 transcription. In vitro data were confirmed by several in vivo experiments where we detected an upregulation of KCC2 protein levels after intrahippocampal administration of BDNF or neurturin. Importantly, a MAPK-dependent rise in Egr4 and KCC2 expression levels was also observed after a period of kainic acid-induced seizure activity in neonatal rats suggesting that neuronal activity might be involved in Egr4-mediated regulation of KCC2 expression. Finally we demonstrated that the mammalian KCC2 gene (alias Slc12a5) generated two neuron-specific isoforms by using alternative promoters and first exons. A novel isoform of KCC2, termed KCC2a, differed from the previously known KCC2b isoform by 40 unique N-terminal amino acid residues. KCC2a expression was restricted to CNS, remained relatively constant during postnatal development, and contributed 20–50% of total KCC2 mRNA expression in the neonatal mouse brainstem and spinal cord. In summary, our data provide insight into the complex regulation of KCC2 expression during early postnatal development. Although basal KCC2 expression seems to be intrinsically regulated, it can be further augmented by neurotrophic factors or by enhanced activity triggering MAPK phosphorylation and Egr4 induction. Additional KCC2a isoform, regulated by another promoter, provides basal KCC2 level in neonatal brainstem and spinal cord required for survival of KCC2b knockout mice.
1. REVIEW OF THE LITERATURE 1.1. GABA: an overview GABA is the principal inhibitory neurotransmitter in the central nervous system. Postsynaptic inhibition mediated by GABA was discovered 50 years ago in electrophysiological experiments on crayfish neurons and muscle fibers (Boistel and Fatt, 1958, Kuffler and Edwards, 1958) and a decade later in mammalian CNS (Krnjevic and Schwartz, 1967), for review see (Kaila, 1994, Krnjević, 2004). The understanding of GABA function in the brain was later supplemented by the finding that GABA can induce depolarization and trophic support of immature neurons (reviewed in Ben-Ari et al., 2007 and Farrant and Kaila, 2007). GABA binds to specific transmembrane receptors causing the opening of ion channels. There are two classes of GABA receptors: GABAA and GABAB. GABAA are ionotropic receptors permeable to Cl- and HCO3- . GABAB are metabotropic receptors indirectly promoting the activation of postsynaptic K+ channels and deactivation of presynaptic Ca2+ channels at the synaptic terminal. 1.2. GABAA receptor mediated responses Functionally, neurotransmiter-mediated responses can be inhibitory (reducing probability of postsynaptic action potential generation) or excitatory (increasing this probability). The functional outcome of GABAA receptors activation strongly depends on the properties of a post-synaptic cell such as GABAA reversal potential with respect to the resting membrane potential and shunting effect of GABAA receptors opening, which affects the spatial and temporal summation of excitatory and inhibitory inputs.
1.2.1. GABAA reversal potential
GABAA receptors are selectively permeable to Cl- and to lesser extent to HCO3-. Thus GABAA reversal potential is determined by the distribution of Cl- and HCO3- across the plasma membrane (Kaila, 1994). In immature neurons [Cl-]i is high because of active Cl- accumulation. When measured with gramicidin perforated patch recordings, GABAA reversal potential in these neurons is significantly more depolarized than resting membrane potential and under certain conditions it is sufficient to cause neuronal excitation and even spiking (Ben-Ari, 2002, Ben-Ari, 2007, Gao and van den Pol, 2001, Serafini et al., 1995). The hallmark of the GABAergic system development is a shift of GABAA mediated responses from depolarizing to hyperpolarizing. This is caused by a change of intracellular Cl- concentration from high to low due to the maturation of Cl- extrusion (Rivera et al., 1999, Rivera et al., 2005). Accordantly GABAA reversal potential is lower in adult neurons than in young ones. In mature hippocampal pyramidal cells the estimated GABAA reversal potential is close to the resting membrane potential. The developmental shift in [Cl-]i and GABAA reversal potential was confirmed in many CNS areas such as hippocampus, neocortex, cerebellum, hypothalamus and spinal cord (Ben-Ari et al., 2007). The reversal potential of Cl- permeable glycine receptors shows a similar age-dependent shift towards hyperpolarization in auditory brainstem (Ehrlich et al., 1999). The developmental shift in [Cl-]i seems to be a very general phenomenon with only few exceptions known so far: photoreceptors (Vu et al., 2000), horizontal retinal cells (Kamermans and Werblin, 1992, Perlman and Normann, 1990), olfactory sensory 1
neurons (Kaneko et al., 2004), and dorsal suprachiasmatic nucleus (Albus et al., 2005). The current view on the mechanisms of [Cl-]i regulation in development will be discussed in section 1.6. HCO3- (bicarbonate) reversal potential is set at approximately -10mV and thus bicarbonate currents are depolarizing (Kaila and Voipio, 1987). HCO3- efflux through GABAA receptor channels is efficiently compensated by a generation of bicarbonate from CO2 and H2O by carbonic anhydrase (Pasternack et al., 1993). HCO3--mediated GABAergic responses appear quite late in development due to a delayed expression of carbonic anhydrase (Ruusuvuori et al., 2004). In immature neurons the contribution of HCO3component to a total GABAA current is so small that it can be neglected. Bicarbonate currents make a significant contribution in mature CA1 pyramidal neurons when GABAA receptors are massively activated. Strong stimulation results in a characteristic biphasic GABAergic response consisting of a fast initial hyperpolarization, followed by a long strong depolarization (Kaila and Voipio, 1997, Smirnov et al., 1999). 1.2.2. Shunting inhibition
Additionally to a depolarization or hyperpolarization of a membrane, simultaneous activation of many GABAA receptor channels lead to a rapid decrease in membrane resistance. This effect occurs at the peak of a GABAergic response and it is called “shunting inhibition”. Regardless of the direction of membrane potential shift, the shunting effect negatively attenuates simultaneous excitatory inputs. Depolarizing glutamatergic responses can be shunted during the peak and potentiated during the decay phase of depolarizing GABA-induce responses (Chen et al., 1996). 2
1.2.3. Spatial aspects of GABAergic signalling in pyramidal neurons
There are spatial aspects of GABAergic signalling that have to be taken into account when speaking of GABAA receptormediated responses (Andersen et al., 1980). Firstly, the majority of GABAergic synapses are found in somatic and proximal dendritic areas of mature pyramidal neurons in contrast to glutamatergic synapses that are formed mainly on dendrites. Although at early developmental stages GABAergic and glutamatergic synapses are evenly distributed, later they are directed to a separate areas of pyramidal cells in a process called “synaptic segregation” (Benson and Cohen, 1996, Marty et al., 2002). Secondly, there is a differential distribution of GABAergic synapses when different types of interneurons project on a pyramidal cell. Some interneuronal subtypes project to the soma area whereas others make synapses on the initial segment of a pyramidal cell axon (Freund and Buzsaki, 1996, Somogyi, 1977). Finally, recent works have shown that there is a gradient of intracellular Cl- concentration along the axo-soma-dendritic axes with the highest Cl- level in the axon initial segment and the lowest in proximal dendrites (Khirug et al., 2008, Szabadics et al., 2006). This gradient allows a depolarizing GABA response in the axon initial segment and hyperpolarizing in somato-dendritic area of the same pyramidal neuron. Fluctuation of GABAA reversal and resting membrane potentials, shunting inhibition, as well as spatial aspects of GABAergic signalling imply that the effect of GABA is largely dependent on a target. As discussed in the next section, the dual aspects of GABAergic signalling (inhibitory vs. excitatory) are especially interesting in relation to immature brain where, apart from being an inhibitory neurotransmitter, GABA mediates
depolarizing responses, promotes neuronal differentiation and tunes spontaneous activity of developing neuronal network. 1.3. Functional aspects of GABA signalling in the immature brain 1.3.1. Trophic effect of GABA on immature neurons
GABA depolarization has an important trophic influence on immature neurons (Owens and Kriegstein, 2002, Represa and Ben-Ari, 2005). Components of the GABAergic system are functional already at neuronal precursor stage (LoTurco et al., 1995) . GABAA receptors are activated by tonic GABA release prior to formation of functional synapses (Demarque et al., 2002, Owens et al., 1999). GABAergic synapses are the first to become functional and provide most of excitatory drive to developing neurons (Ben-Ari et al., 2004, Ben-Ari, 2002, Gozlan and Ben-Ari, 2003, Hennou et al., 2002, Tyzio et al., 1999). In immature neurons activation of GABAA receptors leads to action potential generation, opening of voltage-gated calcium channels and NMDA receptor activation (Eilers et al., 2001, Gao and van den Pol, 2001, Leinekugel et al., 1995, Obrietan and van den Pol, 1995, Serafini et al., 1995, Yuste and Katz, 1991). GABA-mediated [Ca2+]i increase activates intracellular cascades that are important in mediating various trophic effects from cell proliferation to synaptogenesis and circuit formation (Fiszman and Schousboe, 2004). In particular depolarizing GABA stimulates BDNF synthesis (Berninger et al., 1995, Marty et al., 1996) via MAPK- and CREB-dependent mechanism (Obrietan et al., 2002). Despite the large body of in vitro evidence showing that GABA depolarization is an important trophic factor in developing brain, there are only
few in vivo studies in this area. Surprisingly, there are no structural alterations in early brain development of transgenic mice where GABA synthesis, vesicular transport or vesicular release is eliminated (Ji et al., 1999, Varoqueaux et al., 2002, Verhage et al., 2000, Wojcik et al., 2006). However, in a recent in vivo study (Cancedda et al., 2007), where GABAergic excitation was eliminated by premature KCC2 expression, morphological maturation of KCC2 expressing neurons was altered while neuronal migration was unaffected. A selective enhancement of GABAergic but not glutamatergic synapses is observed when GABAergic depolarization is prematurely terminated in cultured neurons (Chudotvorova et al., 2005). The significance of GABA as a regulator of multiple developmental processes is further supported by the exciting observations that immature properties of GABAergic signalling are recapitulated during adult neurogenesis, when newly-born neurons must grow and integrate into already established network (Ge et al., 2007) 1.3.2. GABA and spontaneous activity
Another important function of depolarizing GABA is its role in synchronized spontaneous activity of immature neuronal network. Synchronized activity, generated independently of sensory input, is a prominent feature of many immature networks (Ben-Ari, 2002, Sipila et al., 2005). The original intracellular recordings of such activity were made in slice preparations taken from neonatal rats and termed “giant depolarizing potentials” (GDP; Ben-Ari et al., 1989). Despite the ongoing dispute on the mechanisms of GDP generation and propagation, it is commonly accepted that it is based on the interplay between GABA and glutamate acting on GABAA, NMDA, and AMPA 3
receptors (Ben-Ari et al., 2007, Sipilä and Kaila, 2008). Depolarizing GABA input plays an important role in GDPs formation as the main component of intracellulary recorded GDP is blocked by GABAA receptor antagonist (Ben-Ari et al., 1989). Similarly, the sodium potassium chloride cotransporter (NKCC1) inhibitor bumetanide abolishes depolarizing driving force for GABA and blocks GDP (Sipilä et al., 2006) .
During development, GABAergic responses undergo a maturation process that is reflected by a shift in EGABA from depolarizing towards more negative values. It coincides with the cease of GABAinduced Ca2+ rise and GDP termination (Rivera et al., 2005). The shift in EGABA is caused by a developmental change in the system controlling intracellular Clconcentration, more precisely the change in the functionality of two cation-chloride cotransporters: NKCC1 and KCC2 (Fig. 2).
NCC
NCC
Figure 1. Phylogenetic tree and proposed topologies for the members of CCC family. Numbers indicate degree of identity. Modified from Gamba (2005). 4
1.4. The family of cation-chloride cotransporters: an overview Ion transporters are integral membrane proteins that control cellular uptake and efflux of inorganic ions. The function of the Na-K ATPase, a primary active transporter is to generate inward Na+ and outward K+ concentration gradient using the energy of ATP-hydrolysis. Cation-chloride cotransporters (CCC) are secondary active transporters that utilize the Na+ or K+ concentration gradients to move Cl- across the membrane. CCCs were identified as important regulators of cellular Cl- concentration (Gamba, 2005). They are directly or indirectly involved in several crucial physiological processes such as cellular pH and volume regulation, control of the cell cycle and determining the polarity of neuronal responses to inhibitory neurotransmitters GABA and glycine (Payne et al., 2003). 1.4.1. Family members, protein structure, function and inhibitors
The CCC family consists of 9 members, 7 of which are well-characterized and include one gene encoding a Na+-Clcotransporter, two genes encoding Na+-K+2Cl-cotransporters and four genes encoding K+-Cl- cotransporters (Fig. 1; reviewed in Gamba, 2005 and Hebert et al., 2004). The functions and ion specificity of the newest known CCC family members CCC9 and CIP (cotransporter interacting protein) have not been definitively determined yet (Caron et al., 2000, Gamba, 2005). N(K)CCs, KCCs and CIP are large integral membrane proteins, possessing 12 transmembrane domains flanked by hydrophilic amino and carboxy terminal domains. The most distantly related member CCC9 possesses only 11 transmembrane domains and the carboxy terminal domain is predicted to be located outside the cell.
The molecular diversity of CCC members is further increased by alternative splicing (reviewed in Adragna et al., 2004 and Gamba, 2005). At least 6 isoforms are known for NKCC2, three of which possess truncated carboxiterminal domain generated via utilization of an alternative polyadenylation site. Two isoforms of NKCC1 differ by deletion of 21st exon. Two alternately spliced isoforms are predicted for KCC1 based on cDNA analysis. Two KCC3 isoforms are generated by utilization of alternative promoter and alternative splicing of the first exon and differ in their expression pattern. And finally, two KCC4 cDNAs are characterized; however one codes a short and probably non-functional protein. The functional significance of CCC splice variants is poorly understood. All characterized members of CCC family couple Cl- transport with cation movement (Na+ and K+) in a stoichiometry that does not generate current and changes in membrane potential. Because Na+ and K+ gradients are quickly restored by ion pumps, the principal result of CCC activation is generation of a Cl- gradient. CCCs are bidirectional transporters and can carry out ion influx or efflux depending on the concentration gradients of transported ions. Under normal physiological conditions NKCCs ensure active Cl- accumulation and KCCs – Clextrusion (Fig. 2). Several substances were found to inhibit CCCs function. The Na+Cl- cotransporter is sensitive to the diuretic thiazide (Stokes, 1984) whereas Na+-K+-2Cl- cotransporters and K+-Clcotransporters are sensitive to ‘loop’-type diuretics like furosemide and bumetanide (Gillen et al., 1996, O’Grady et al., 1987). Furosemide has about equal potency for both NKCCs and KCCs (Ki ~ 25-50 M) whereas bumetanide has a ~500-fold 5
greater affinity for NKCCs (Ki ~ 0.1 M) and at low concentrations can be used as a specific inhibitor of NKCCs (Payne et al., 2003). 1.4.2. Kinetic regulation and oligomerization of CCCs
In many biochemical studies functional characterization of CCC members was obtained by exogenous expression in HEK293 cells or Xenopus laevis oocytes and subsequent 86Rb flux assay (Gamba et al., 1994, Gillen et al., 1996, Hiki et al., 1999, Mercado et al., 2000, Mercado et al., 2005, Payne et al., 1995, Payne et al., 1996, Race et al., 1999, Song et al., 2002, Xu et al., 1994). The functional properties of the transporters, e.g. affinity constants of corresponding ions and inhibitors, activation by medium tonicity as well as the effect of kinase/phosphatase inhibitors were studied (reviewed in Gamba, 2005). One interesting conclusion can be made based on comparison of affinity constants. KCC2 has the highest affinity for K+ among all KCCs. In neurons where KCC2 is expressed, under normal physiological conditions [K+]o is low and KCC2 mediates Cl- efflux thus lowering [Cl-]i. However, under conditions of heavy neuronal activity or under pathophysiological conditions [K+]o is elevated. Rise in [K+]o will result in reverse of KCC2 transport direction and reuptake of extracellular K+ at the expense of intracellular Cl- accumulation (Jarolimek et al., 1999, Payne, 1997). This would intensify network activity in response to the K+-induced depolarization and also because fast hyperpolarizing GABAergic inhibition is based on low [Cl-]i in neurons. Cotransporters display distinct kinetic regulation. Classical studies of Na-K-2Cl cotransport in non-neuronal cells showed that it is activated by cell shrinkage, low 6
intracellular Cl-, as well as by agents that stimulate intracellular kinase activity or reduce phosphatase activity. On the contrary, K-Cl cotransport is stimulated by cell-swelling and agents that stimulate phosphatase activity or reduce kinase activity (Flatman, 2002, Lauf and Adragna, 2000). Kinetic regulation of cation-chloride cotransport is implicated in the control of cell volume. Following cell swelling, K-Cl cotransport is activated and mediates ion efflux, thus decreasing intracellular osmolarity and accomplishing regulatory cell volume decrease. Following cell shrinkage, Na-K-2Cl cotransport is activated and mediates ion influx, thus increasing intracellular osmolarity and accomplishing regulatory cell volume increase. Both NKCC and KCC activation by tonicity of the medium require phosphorylation or dephosphorylation events respectively (Jennings and Schulz, 1991, Lytle and Forbush, 1992). Further studies have revealed that [Cl-]i is involved in regulation of its own transport by affecting the cotransporters’ osmotic sensitivity (Gillen and Forbush, 1999, Lytle and McManus, 2002). From all K-Cl cotransporters only KCC2 is active under isotonic conditions whereas others are activated in hypotonic medium (Payne, 1997, Song et al., 2002; reviewed in Gamba, 2005). This implies that in contrast to others, KCC2 is most likely not involved in cell volume regulation. However, the studies of KCC2 regulation by medium tonicity were carried out in non-neuronal cells and their results cannot be extrapolated to neurons. Although it is generally accepted that phosphorylation status affects cotransporter activity, for a long time the direct phosphorylation was demonstrated only for NKCC1 (Lytle and Forbush, 1992). The exciting story of finding
residues that are phosphorylated upon NKCC1 activation is reviewed in Gamba (2005). Finally, Darman and Forbush (2002) identified three phosphorylated threonine residues in N-terminal part of the protein (residues 184, 189 and 202 in shark NKCC1 sequence). Mutation of one of them resulted in complete inhibition of the cotransporter whereas mutations of two others altered regulation of NKCC1 activity by [Cl-]i. Recently some progress was achieved in studies of KCC2 phosphorylation. There are consensus sites for both tyrosine and serine-threonine kinases in the KCC2 sequence. A canonical tyrosine kinase motif is located in the carboxy terminal domain of KCC2; the tyrosin in this motif is conserved among all KCCs. When the conserved tyrosin was substituted with aspartate, mimicking the phosphorylation event, transport activity of KCC2 under isotonic conditions and hypotonic activation were dramatically reduced (Strange et al., 2000). However, pharmacological blockade of tyrosin kinases had no effect on KCC2 activity in experiments performed in oocytes. Notably, the same reagents inhibit K-Cl cotransport in neuronal cultures (Kelsch et al., 2001). The consensus sites for serine-threonine kinases PKA and PKC are located in both carboxy and amino termini of KCC2. A recent study revealed that Ser940 is a major site for PKC-dependent phosphorylation of KCC2 (Lee et al., 2007). Importantly, PKC-dependent phosphorylation of Ser940 increases KCC2 activity and enhances KCC2 surface stability by slowing endocytosis. Similar mechanisms may be involved in the surface stability changes implicated in activity-dependent downregulation of KCC2 in hippocampal slices (Rivera et al., 2004).
Several other kinases are implicated in kinetic regulation of CCC members based on their interactions with cotransporters, namely Ste20-related proline-alanine-richkinase (SPAK), oxidative stress response 1 (OSR1), WNK family kinases and braintype creatin kinase (CKB). SPAK and OSR1 recognition motif was found in NKCC1 as well as in other members of CCC family, except for KCC2, TSC and two orphan members (reviewed in Delpire and Gagnon, 2006). The interaction of the cotransporters with SPAK and OSR1 was shown using the yeast two-hybrid system (Piechotta et al., 2002). Cotransfection of HEK293 cells with NKCC1 and dominant negative SPAK drastically reduced NKCC1 phosphorylation and inhibited its activation with hypertonic medium or low Cl- (Dowd and Forbush, 2003). Surprisingly, mutation of SPAK-OSR motif completely prevented the interaction between NKCC1 and SPAK however did not alter hypertonic or low chloride induction of NKCC1 activity (Piechotta et al., 2003). This result was not confirmed by subsequent studies from the same group, which demonstrated that SPAK binding is essential for NKCC1 activation (Gagnon et al., 2007). Investigations using SPAK binding domain as bait identified WNK kinases as possible partners of SPAK (Piechotta et al., 2003). Recently WNK kinases were shown to reciprocally regulate the activity of KCCs and NKCCs, affect cotransporter sensitivity to osmotic changes and increase NKCC1 and NKCC2 phosphorylation (reviewed in Kahle et al., 2006). WNK and SPAK expression was found in the brain areas where kinetic regulation of NKCC1 and KCC2 activity was predicted. WNK3 is expressed in the brain in suprachiasmatic nucleus where circadian [Cl-]i oscillations allow opposite 7
response to GABA during day and night time (Shimura et al., 2002, Wagner et al., 1997). SPAK expression was found in the brainstem cochlear nucleus (Piechotta et al., 2003) where existence of inactive KCC2 form was reported (Vale et al., 2003, Vale et al., 2005) CKB interaction with KCC2 was identified be the means of two-hybrid system using KCC2 C-terminus as bait and confirmed by coimmunoprecipitation studies (Inoue et al., 2004). Overexpression of CKB activates KCC2 (Inoue et al., 2006). Creatin kinases are involved in ATP regeneration, so CKB may provide ATP for other molecules interacting with KCC2. In addition to phosphorylation, the transport activity of CCC members is influenced by their quaternary structure. Formation of hetero- and homooligomers has been shown for nearly all cotransporters (Blaesse et al., 2006, Casula et al., 2001, Moore-Hoon and Turner, 2000, Simard et al., 2007, Starremans et al., 2003, de Jong et al., 2003). Interestingly, coexpression of non-functional N-terminal truncated KCC1 with functional full-length KCC1 leads to a dominant-negative effect suppressing wild-type KCC1 functionality (Casula et al., 2001). This implies that oligomerization is essential for KCC1 activation. Analysis of one of the newest members of CCC family, CIP, revealed that although this protein seems to be non-functional as ionic transporter, it is able to interact with NKCC1 and effectively suppress its activity (Caron et al., 2000). These data open the possibility of regulation of CCC functionality via oligomerization. 1.4.3. CCC expression in the CNS
In CNS, the CCC family is represented by NKCC1 and four KCCs: KCC1, KCC2, KCC3 and KCC4. In the original studies 8
reporting cloning and characterization of the cotransporters, all of them except for KCC2 were found to be expressed in several tissues, including brain (Delpire et al., 1994, Gillen et al., 1996, Mount et al., 1999). In contrast to others, KCC2 shows strictly CNS-specific expression pattern (Payne et al., 1996) and was confirmed by subsequent works (Kanaka et al., 2001, Rivera et al., 1999, Williams et al., 1999) to be expressed exclusively in neurons. The levels and patterns of cotransporters expression in immature and adult rodent brain have been carefully studied (Clayton et al., 1998, Hübner et al., 2001, Kanaka et al., 2001, Le Rouzic et al., 2006, Li et al., 2002, Lu et al., 1999, Mikawa et al., 2002, Plotkin et al., 1997a, Plotkin et al., 1997b). Expression of the cotransporters seems to be strictly regulated in time and space. For example, during embryonic development KCC2 is expressed in mature regions, KCC3 in newly differentiated regions, KCC4 and NKCC1 in undifferentiated regions (Li et al., 2002). Differential temporal and spatial expression patterns of CCC members in the brain imply distinct roles for the various cotransporters in neuronal development and function. Differences in the cotransporters’ amino acid sequences might result in a differential kinetic regulation (Mount et al., 1999) which makes them suitable for different tasks. Yet, there is no clear evidence of KCC1 and KCC4 function in the nervous system (Gamba, 2005). KCC3–/– mice displayed axon swelling and progressive neurodegeneration in the peripheral nervous system (PNS) and CNS; altered regulatory volume decrease in neurons, reduced neuronal [Cl-]i and lower seizure threshold (Boettger et al., 2003, Byun and Delpire, 2007)
The data concerning NKCC1 and KCC2 expression and function in nervous cells suggest that these two cotransporters are the major players in the regulation of neuronal [Cl-]i (Payne et al., 2003). 1.5. NKCC1 and its function in the nervous system There are a number of thorough studies addressing NKCC1 mRNA and protein expression in developing and adult rodent brain. Unfortunately the results reported in these studies are somewhat contradictory. During embryonic development profound NKCC1 mRNA and protein levels were detected in the ventricular zone of the ganglionic eminences while moderate levels were found in the ventricular zone of neocortex, suggesting that NKCC1 might be involved in neuronal proliferation (Hübner et al., 2001, Li et al., 2002). These data are contradictory to the previously published work where NKCC1 mRNA was detected in cortical plate but not in ventricular zone (Clayton et al., 1998). During postnatal development some studies report general decrease of NKCC1 mRNA and protein expression with neuronal maturation in forebrain and cerebellum (Plotkin et al., 1997b, Yamada et al., 2004); others show increase of NKCC1 mRNA and protein expression in developing rat brain in general (Clayton et al., 1998, Yan et al., 2001), and more specifically in cerebellum (Mikawa et al., 2002) and hippocampus (Wang et al., 2002). In immature and adult brain, NKCC1 expression is high in choroid plexus where the cotransporter might be involved in cerebrospinal fluid secretion and volume regulation along with KCC1, KCC3 and KCC4 (Kanaka et al., 2001, Karadsheh et al., 2004, Le Rouzic et al., 2006, Li et al., 2002, Plotkin et al., 1997a).
In mature brain moderate to low expression of NKCC1 is detected in the brainstem, olfactory bulb, thalamus, cortex, pyramidal and granular neurons of hippocampus and Purkinje and granular neurons of cerebellum, although the data on expression levels and patterns are conflicting (Clayton et al., 1998, Kanaka et al., 2001, Plotkin et al., 1997a, Wang et al., 2002). Immunohistochemistry data in the studies mentioned above were generated using two different NKCC1 antibodies: rabbit polyclonal (amino acids 938-1011; Kaplan et al., 1996) and mouse monoclonal (amino acids 902-1212; Lytle et al., 1995). Although in Western blots both antibodies recognize 140 kDa band corresponding to NKCC1, immunostainings with monoclonal antibody display non-specific pattern in ferret retina (Zhang et al., 2006) and in NKCC1 deficient mice (Sipila et al., 2007, Zhang et al., 2007). Taking into account the conflicting results gained by RNA detection methods and possible nonspecific signal in immunohistochemical detection of NKCC1 protein more studies are needed to confirm NKCC1 expression pattern in immature and adult brain. The function of NKCC1 has been studied in NKCC1 knockout animals (Delpire and Mount, 2002). Targeted disruption of NKCC1 gene revealed hearing loss, growth retardation, salivation impairment and deficient spermatogenesis (Delpire et al., 1999, Evans et al., 2000, Flagella et al., 1999, Pace et al., 2000). However, no striking neuronal phenotype was found. Nevertheless, in neurons, as well as in other cells, NKCC1 is implicated in regulation of intracellular Cl- concentration. In DRGs it is responsible for Cl- accumulation and participates in the modulation of GABA neurotransmission and sensory perception (Sung et al., 2000). In olfactory sensory 9
neurons it maintains elevated [Cl-]i, which is necessary for the responsiveness to odour stimulation (Kaneko et al., 2004). NKCC1 is also involved in Cl- uptake and depolarizing GABA responses in immature neurons of the spinal cord (Delpy et al., 2008, Rohrbough and Spitzer, 1996), hippocampus (Sipila et al., 2006) and neocortex (Achilles et al., 2007, Yamada et al., 2004). However, it seems that NKCC1 is not the only protein responsible for Claccumulation in immature neurons. In some regions Cl- accumulation is clearly independent of NKCC1 expression and function. For instance NKCC1 is not detected in lateral superior olive (LSO) region of the brainstem although at early postnatal age LSO neurons have high [Cl-]i and depolarizing GABA response (Balakrishnan et al., 2003). Similarly in immature retinal ganglion and amacrine cells, deleting or blocking NKCC1 does not affect elevated [Cl-]i (Zhang et al., 2007). Thus at least in retina and LSO the nature of inward Cl- transport system is not identified. 1.6. KCC2, a neuronal-specific CCC In mature CNS KCC2 expression is abundant and strictly neuron-specific. A wide variety of brain areas were reported to express KCC2, including cortex, hippocampus, cerebellum, olfactory bulb, retina, brainstem and spinal cord (Kanaka et al., 2001, Vu et al., 2000). KCC2 is not detected in the PNS (Kanaka et al., 2001, Rivera et al., 1999). An interesting observation is that despite broad KCC2 expression in the CNS there is a minority of central neurons where KCC2 was not detected. These include dopaminergic neurons in substantia nigra (Gulacsi et al., 2003), and a population of neurons in the nucleus reticularis thalami (Bartho et al., 2004). On subcellular level KCC2 10
is expressed in plasma membrane of neuronal soma and dendrites, including spines, but it is absent from axons (Gulyas et al., 2001, Li et al., 2007, Williams et al., 1999). 1.6.1. Developmental profile of KCC2 expression
During brain development KCC2 undergoes profound upregulation in most of the brain areas following neuronal maturation in caudal-rostral direction (Li et al., 2002, Stein et al., 2004, Wang et al., 2002). In rat, KCC2 mRNA is detected as early as E12.5 in ventral horns of spinal cord and spreads over the rest of spinal cord by E18.5 (Hubner et al., 2001, Li et al., 2002). At E14.5 KCC2 expression is observed in pons, hypothalamus, basal ganglia and olfactory bulb (Li et al., 2002, Stein et al., 2004). In a newborn rat all brain areas already express KCC2, except for cortex, hippocampus and cerebellar granule and molecular layers. During postnatal development KCC2 is strongly upregulated in the forebrain areas (Clayton et al., 1998, Mikawa et al., 2002, Rivera et al., 1999). In whole brain, from postnatal day (P) 1 to adult stage, KCC2 mRNA expression is increased 7-10 times, protein levels are increased 4 times (Lu et al., 1999). As KCC2 expression follows the development of individual neurons and brain regions, it can be used as a marker of neuronal maturation (Li et al., 2002, Mikawa et al., 2002, Rivera et al., 1999, Shimizu-Okabe et al., 2002, Stein et al., 2004). This is also reflected by the observation that steep KCC2 upregulation is correlated with synaptogenesis (Gulyas et al., 2001, Takayama and Inoue, 2006, Takayama and Inoue, 2007, Zhang et al., 2006). The functional consequence of
this upregulation is the induction of the shift in GABAergic responses towards hyperpolarization (Rivera et al., 2005). 1.6.2. Role of KCC2 in regulation of intracellular Cl-
Expressed virtually in all CNS neurons and isotonically active, KCC2 is the best candidate for the role of neuronal Clextruder (Payne et al., 1996, Payne, 1997). Many studies have revealed a connection between furosimide-sensitive K-Cl cotransport activity, KCC2 expression and changes in intracellular Cl- concentration (DeFazio et al., 2000, Ehrlich et al., 1999, Fukuda et al., 1998, Jarolimek et al., 1999, Kakazu et al., 1999, Shimizu-Okabe et al., 2002, Ueno et al., 2002, Vardi et al., 2000, Wang et al., 2005). The developmental increase in functional KCC2 expression
is considered as a main factor in the developmental [Cl-]i decline and the shift in GABAergic/glycinergic responses towards hyperpolarization (Rivera et al., 2005; Fig. 2). The first direct evidence that KCC2 is intimately involved in maturation of GABAA responses came from the study of developing hippocampus (Rivera et al., 1999). In this work KCC2 expression was knocked-down by the means of antisense oligonucleotides. As a consequence, suppression of GABAA-mediated hyperpolarization was observed. Later, studies of KCC2 deficient mice confirmed the importance of KCC2 for hypopolarizing synaptic inhibition (Hubner et al., 2001, Woo et al., 2002). Transgenic mice that are fully deprived of KCC2 die at birth due to respiratory
Figure 2. Regulation of intracellular Cl- by NKCC1 and KCC2. Under physiological conditions, in neuronal cells, Cl− uptake is mediated by NKCC1 and Cl− extrusion by KCC2. NKCC1- and KCC2- mediated Cl- transport is driven by Na+ and K+ concentration gradients generated by Na+/K+ ATPase. Modified from Payne et al (2003). 11
failure (Hubner et al., 2001). In spinal cord and brainstem of E18 homozygote knockout embryos motoneurons are strongly depolarized by GABA and glycine. This is accompanied by severe motor defects and inability to generate a respiratory rhythm (Hubner et al., 2001). Interestingly, lack of respiratory activity is also observed in the brainstem preparations from glutamic acid decarboxylase (GAD) 65/67 double knockout mice and vesicular inhibitory amino acid transporter (VIAAT) knockout mice, the two mouse models with a major deficit in GABAergic activity (Fujii et al., 2007). Another mouse model with altered KCC2 expression is KCC2 knockdown mice that retain about 5% of KCC2 levels (Woo et al., 2002). In a subsequent study these mice were shown to be specific knockout of KCC2b isoform (Uvarov et al., 2007). Homozygote KCC2b knockout pups survive after birth but are hyperexcitable and die within two postnatal weeks presumably due to repetitive seizure episodes. Electrophysiological recordings confirmed increased excitability and impaired Cl- regulation in cortical and hippocampal neurons of these animals (Woo et al., 2002, Zhu et al., 2005). KCC2 hypomorphic mice retain 15 – 20% of wild-type KCC2 levels (Tornberg et al., 2005). These animals display increased anxiety and seizure susceptibility, poor spatial learning and memory, hypersensitivity to thermal and mechanical stimuli and reduced weight, although they retain normal locomotor activity and motor coordination. The authors suggest that fast hyperpolarizing inhibition but not shunting inhibition is altered in KCC2 hypomorphic mice and that requirements for fast hyperpolarizing inhibition may differ among various functional systems of the CNS (Tornberg et al., 2005). 12
Functional expression of KCC2 depends on the mechanisms regulating KCC2 mRNA and protein synthesis, transport and membrane insertion of the protein, as well as kinetic activation of the cotransporter. In cortex and hippocampus the onset of KCC2 expression is wellcorrelated with the increase in the efficiency of Cl– extrusion (Clayton et al., 1998, Rivera et al., 1999, Shimizu-Okabe et al., 2002). When KCC2 is exogenously expressed in cortical immature neurons it induces a substantial negative shift in the GABAA reversal potential (Lee et al., 2005). This indicates that in the forebrain KCC2 gene expression is ratelimiting for the maturation of functional Cl- extrusion. However this is not the case for all brain areas. In retina, spinal cord and auditory brainstem in vivo (see below), and in developing hippocampal neurons in vitro (Khirug et al., 2005), the onset of KCC2 expression precedes its functional activation. It was suggested that in these systems regulation of KCC2 localization, phosphorylation status and oligomerization, rather than KCC2 gene expression, is rate-limiting for functional Cl- extrusion. KCC2 expression has been detected in the retina of several species (Sernagor et al., 2003, Vardi et al., 2000, Vu et al., 2000). KCC2 levels are increased gradually during development coinciding with the maturation of GABAergic responses (Vu et al., 2000). In mature retina KCC2 has a differential expression pattern in various retinal neuronal types, and it is well correlated with GABAA reversal potential of these neurons (Vardi et al., 2000). However, in retinal amacrine and ganglion cells the gradual increase in KCC2 expression cannot explain the relatively fast shift of GABAA reversal potential towards more negative values. It was proposed that mechanisms regulating
KCC2 membrane integration determine the time of the shift in these neurons (Zhang et al., 2006). Interestingly, in some retinal neurons, like rod bipolar cells or starburst amacrine cells, KCC2 is compartmentalized allowing opposite response to GABA within one cell (Gavrikov et al., 2006, Vardi et al., 2000). KCC2 functional expression has been intensively studied in the auditory brainstem. Maturation of auditory neurons is accompanied by a decrease in [Cl-]i and by a shift of glycinergic responses towards hyperpolarization during the first and second postnatal weeks (Ehrlich et al., 1999, Kandler and Friauf, 1995, Lohrke et al., 2005). Similar to forebrain regions, in the auditory brainstem the developmental decrease in [Cl-]i is attributable to the increase of active Cl- extrusion (Kakazu et al., 1999). However, most of the KCC2 upregulation in the brainstem occurs during embryonic stages (Li et al., 2002). KCC2 levels are similar in P3 and P12 superior olive complex (SOC) neurons that are respectively depolarized or hyperpolarized by glycine (Balakrishnan et al., 2003). Similarly, KCC2 levels do not change in cochlear nucleus at the period of glycinergic shift (Vale et al., 2005) Thus in the auditory brainstem the developmental shift in glycinergic responses seems to be independent of the increase in KCC2 gene expression, but coincides with a change in KCC2 phosphorylation status (Vale et al., 2005) and oligomerization of the cotransporter (Blaesse et al., 2006). In summary, the analysis of agedependent KCC2 expression has revealed that the developmental increase of KCC2 expression levels is not always directly translated into a shift of intracellular Cl- concentration. In CNS regions where early induction of KCC2 gene expression precedes the time of [Cl-]i shift,
mechanisms regulating KCC2 localization, phosphorylation and oligomerization determine when Cl- extrusion becomes functional. In neocortex and hippocampus, KCC2 gene expression is delayed and coincides with the time of [Cl-]i shift, implying that KCC2 is activated as soon as it is synthesized. It is interesting that KCC2 can still be functionally deactivated in neurons from these regions under certain conditions like in vitro culturing (Khirug et al., 2005) or intensive stimulation used for plasticity induction (Fiumelli and Woodin, 2007). 1.6.3. Role of KCC2 in spine formation
Paradoxically, KCC2 expression was detected in close proximity of excitatory synapses (Gulyas et al., 2001). Recent finding revealed a structural role of KCC2 in spine formation (Li et al., 2007). Spine maturation is altered in cortical neurons of KCC2-deficient mice: knockout neurons develop long dendritic protrusions that are branched and highly motile. Fulllength KCC2 and non-functional KCC2 mutant with N-terminal deletion, rescue the spine morphology of knockout neurons equally efficiently; it means that the morphogenic role of KCC2 does not require its ion transporter function. Rather, KCC2 affects spine formation through the interaction of its C-terminal domain with the cytoskeleton-associated protein 4.1 (Li et al., 2007). This study points to a novel role of KCC2 in neuronal development: increase in KCC2 expression is likely to stimulate both glutamatergic synaptic transmission via spine morphogenesis and GABAergic hyperpolarizing inhibition via active Cl extrusion. Thus KCC2 might act as a factor that synchronizes functional development of excitatory and inhibitory neurotransmission.
13
1.6.4. Regulation of KCC2 expression and function
Taking into account the importance of Clhomeostasis for neuronal development and mature neuronal network activity, it is not surprising that KCC2 expression and function are tightly regulated. Longterm changes in chloride homeostasis such as developmental or chronic trauma conditions involve modification of KCC2 gene expression and protein synthesis. Short-term changes in [Cl-]i may involve functional activation or deactivation of the reserve pool of KCC2 molecules. 1.6.4.1. Regulation of developmental KCC2 increase The developmental increase in KCC2 expression has attracted substantial attention in recent years. Lots of data have been gathered that can be roughly divided into three categories as discussed below. However, this division is artificial because all of the factors that were shown to affect KCC2 expression are interconnected with each other. 1.6.4.1.1. GABA auto-regulation hypothesis One of the first studies addressing the regulation of KCC2 expression in immature cells was the work done on dissociated embryonic hippocampal neurons by Ganguly et al. (2001). During in vitro development these cells undergo both the shift in GABAergic responses towards hyperpolarization and the increase in KCC2 expression observed in vivo. In cultured neurons the shift of GABAergic responses is delayed and KCC2 expression is reduced when GABAA receptors are chronically blocked by bicuculline, (Ganguly et al., 2001). The effect was dependent on [Ca2+]i but independent of neuronal spiking. This study gave rise to an attractive hypothesis that depolarizing 14
GABA auto-regulates its developmental transition towards hyperpolarisation via [Ca2+]i elevations. The GABA auto-regulation hypothesis was also tested in vivo, in developing turtle retina. Dark-rearing from hatching enhanced spontaneous activity and caused GABA to retain a depolarizing component (Sernagor et al., 2003). Chronic GABAA receptor blockade by bicuculline enhanced spontaneous activity and reduced KCC2 expression (Leitch et al., 2005). However, subsequent in vitro and in vivo studies suggest that maturation of Cl- homeostasis may also be GABAindependent. Chronic blockade of GABAA receptors in cultured neurons had no effect on KCC2 levels (Ludwig et al., 2003) or developmental change in EGABA (Titz et al., 2003). Mice deficient in VIAAT have a drastically reduced GABAergic and glycinergic synaptic transmission; however KCC2 induction and function in these animals remain intact (Wojcik et al., 2006). 1.6.4.1.2. Synaptogenesis and neuronal activity In vivo KCC2 expression shows a correlation with the level of activity input received by neurons. It follows neuronal maturation, development of innervation and synaptogenesis (Gulyas et al., 2001, Takayama and Inoue, 2006, Takayama and Inoue, 2007, Zhang et al., 2006). Transient high frequency stimulation of the inputs induces a shift of EGABA towards hyperpolarization as well as KCC2 functional upregulation in deep cerebellar nucleus (Ouardouz and Sastry, 2005). These authors provide evidence that cAMP-PKA pathway and protein synthesis are involved, although it is not clear whether KCC2 expression or function is affected.
The developmental decrease of [Cl-]i and increase of KCC2 expression is disturbed when activity input is blocked in LSO by cohlear ablation before the hearing onset (Shibata et al., 2004). Interestingly, a similar disruption of Cl- regulation was observed with either ipsilateral ablation (disturbing predominantly glutamatergic input) or contralateral ablation (disturbing GABAergic/glycinergic input). This leaves open a question whether the pattern and not the activity per se is important for the maturation of Cl- regulatory mechanisms. Afferent input is essential for KCC2 developmental upregulation in immature cat visual cortex (Kanold and Shatz, 2006). The innervation of cortical layer 4 was disrupted by means of subplate ablation in neonatal cats. One month after KCC2 expression was significantly lower in the ablated cortex compared to nonmanipulated one, although no neuronal loss was observed. Surprisingly, areas of ablated cortex with decreased KCC2 expression show abnormally high levels of BDNF. This is inconsistent with the data on activity-dependent BDNF expression (section 1.7.3), with the study of KCC2 levels in BDNF-overexpressing mice (Aguado et al., 2003; section 1.6.4.1.3) and is rather reminiscent of trauma models, where BDNF increase parallels KCC2 downregulation (Rivera et al., 2002, 2004). An interesting study was made using chick ciliary ganglion (CG) and dissociated hippocampal neurons to analyze the effect of nicotinic signalling on maturation of GABAergic responses (Liu et al., 2006). The chick embryo is a useful model to perform chronic treatments in ovo. Both chick CG neurons treated in ovo with nicotinic antagonists and hippocampal neurons from 7-nAChR knockout mice retained the excitatory GABA responses (Liu et al., 2006). In 7-nAChR knockout
hippocampal neurons NKCC1 expression was increased and KCC2 expression was decreased implying that endogenous nicotinic cholinergic activity controls maturation of GABAergic signalling. As 7-nAChR has a relatively high Ca2+ permeability, this result is consistent with the hypothesis that Ca2+ influx is important for developmental KCC2 upregulation. On the other hand, the most prominent expression of 7-nAChR in hippocampus is found in interneurons (Jones and Yakel, 1997), thus the effect of nicotinic cholinergic activity might be indirect, mediated through depolarizing GABAergic input. 1.6.4.1.3. BDNF hypothesis Neuronal activity, neurotrophin release, and development of GABAergic system are tightly interconnected in the developing brain (Huang et al., 1999, Marty et al., 1997, Zhang and Poo, 2001). Consistently, KCC2 developmental expression is enhanced in BDNF-overexpressing mice and reduced in mice deficient in expression of BDNF-receptor tropomyosin receptor kinase (Trk)B (Aguado et al., 2003, Carmona et al., 2006). Already at E18 KCC2 mRNA is profoundly expressed in BDNF-overexpressing in contrast to wild type hippocampi. Yet, the consequences of chronic BDNF-overexpression are very broad and at first place include increase of GABAergic and non-GABAergic synaptogenesis and spontaneous correlated activity (Aguado et al., 2003). Thus it is unclear whether KCC2 expression is enhanced by BDNF directly, via activitydependent pathways or as a side-effect of general acceleration of neuronal development in the transgenic mouse. Despite the vast amount of data concerning the mechanisms involved in developmental KCC2 expression, a general hypothesis that would combine all these 15
facts is apparently absent. Intracellular cascades that connect extracellular signals such as BDNF or neuronal activity with KCC2 expression still remain elusive. 1.6.4.2. KCC2 regulation in injured neurons Traumatic insults to the brain are often accompanied by glutamate release and excitotoxic neuronal damage (Lipton, 1999). Additionally, brain injury is followed by depolarizing GABA responses reminiscent of those in immature neurons (Payne et al., 2003). GABA-induced Ca2+ elevations, depolarizing EGABA shift and impaired Cl- extrusion were described in different types of neuronal trauma in vitro (Fukuda et al., 1998, Katchman et al., 1994, van den Pol et al., 1996) and in vivo (Jin et al., 2005, Nabekura et al., 2002, Price et al., 2005, Toyoda et al., 2003). The depolarizing EGABA shift is attributable to acute downregulation of KCC2 activity (Wake et al., 2007) and KCC2 gene expression (Nabekura et al., 2002, Shimizu-Okabe et al., 2007, Toyoda et al., 2003). Importantly, impaired NKCC1 and KCC2 expression was also found in surgically resected human brain specimens from patients with drug-resistant epilepsy (Huberfeld et al., 2007, Munoz et al., 2007, Palma et al., 2006). Some insights in the molecular mechanisms of trauma-induced KCC2 downregulation were provided by studies of in vitro epilepsy models. Epileptic activity increases expression of BDNF mRNA and protein (Binder et al., 2001). Kindling, interictal-like activity or exogenously applied BDNF induces TrkB dependent rapid downregulation of KCC2 expression in hippocampus (Rivera et al., 2002). BDNF-TrkB intracellular signalling is mediated by recruitment of the adaptor proteins Shc/FRS-2 and phospholipase Cγ 16
(PLCγ) (Huang and Reichardt, 2003; Fig. 4). Binding of the adaptor proteins activate well-known signalling cascades: Shc activates Ras/MAPK and phosphoinositide 3 kinase (PI3K) pathways, PLCactivates the calcium/calmodulin kinase (CaMK) pathway (see section 1.7.4). Using transgenic mice with point mutations in either Shc/FRS-2 or PLC binding sites (generated by Minichiello et al. (2002)), it was determined that activity-dependent KCC2 downregulation requires both cascades (Rivera et al., 2004). Surprisingly, activation of Shc pathway alone induces increase in KCC2 expression, suggesting a role in developmental upregulation of KCC2. At first glance, the rapid fall in KCC2 levels and consequent loss of hyperpolarizing inhibition cannot be beneficial for neurons that are already damaged by trauma-induced cytotoxicity. However, it is known that injured adult neurons activate genes that are normally expressed at immature state (Cohen et al., 2003). It has been suggested that neurons gain greater flexibility by turning back in development to the state when they are able to regrow processes, migrate over lesion area, or form new contacts. It is likely that GABA-induced depolarization, known to exert trophic effects in immature brain, promotes survival and recovery of damaged neurons. 1.6.4.3. KCC2 regulation in GABAergic synaptic plasticity It is well established that coincident firing of pre- and postsynaptic neurons induces potentiation of excitatory synapses. More recently it became obvious that the strength of inhibitory synapses is also prone to activity-induced changes (Dan and Poo, 2006, Gaiarsa et al., 2002). In the hippocampus, when pre-
and postsynaptic cells are coincidently activated the strength of GABAergic synapses is reduced (Woodin et al., 2003). Repetitive postsynaptic spiking (3 Hz) within 20 ms before or after the activation of GABAergic synapses leads to a rapid reduction of inhibitory strength mediated by a local decrease in KCC2 activity in the postsynaptic cell and consequent positive shift of EGABA. Interestingly, the effect depends on Ca2+ influx and it is largely synapse-specific (Woodin et al., 2003). Later it was shown that repetitive spiking of higher frequency (10 Hz) alone can induce the acute Ca2+dependent depolarizing shift of EGABA via downregulation of active Cl- extrusion (Fiumelli et al., 2005). The shift in EGABA happens within minutes after the stimulation and thus it is attributable to downregulation of Cl- extrusion function rather than gene expression. Evidence that Cl- extrusion is mediated by KCC2 comes from the experiments where immature neurons are normally non-sensitive to repetitive spiking stimulation, but gain the sensitivity after being transfected with KCC2. The magnitude of EGABA shift depends on activity attaining larger values with higher frequency or longer duration of stimulation. Interestingly, the effect of repetitive spiking on Cl- extrusion is also dependent on protein kinase C (PKC) activation meaning that functionality of KCC2 in this model might be reduced by PKC-dependent phosphorylation (Fiumelli et al., 2005). Clearly, PKC-mediated regulation of KCC2 functionality turns out to be a complicated issue as in the recent study, Lee et al. (2007) have shown that PKC-dependent KCC2 phosphorylation enhances cell surface stability and activity of the cotransporter rather than reduce it as it was suggested by Fiumelli et al., (2005).
BDNF regulates postsynaptic [Cl-]i at GABAergic synapses (Wardle and Poo, 2003). Acute application of BDNF induces an EGABA shift towards depolarization mediated by rapid downregulation of KCC2 activity. Thus BDNF is suggested to participate in GABAergic synaptic plasticity by reducing GABA hyperpolarizing inhibition. Remarkably, this is yet another way how BDNF affects Cl- homeostasis and GABAergic responses in addition to slow developmental increase of KCC2 gene expression and rapid downregulation of KCC2 levels in injured neurons. 1.7. BDNF, a member of the neurotrophin family 1.7.1. Neurotrophins and their receptors
BDNF, along with nerve growth factor (NGF), neurotrophin-3 (NT-3), neurotrophin-4/5 (NT-4/5), neurotrophin-6 (NT-6), and neurotrophin-7 (NT-7) belong to the family of neurotrophic factors collectively called neurotrophins (Fig. 3). Neurotrophins are structurally related, secreted signalling proteins that stimulate neuronal survival, differentiation and functionality (Bibel and Barde, 2000, Huang and Reichardt, 2001). NGF, BDNF, NT-3, and NT-4 are expressed in mammals whereas NT-6 and NT-7 are found only in fish. Two unrelated types of receptors were identified for neurotrophins: high affinity tropomyosin related receptor tyrosine kinases (Trk) and low-affinity panneurotrophin receptor p75NTR (reviewed in Huang and Reichardt, 2003; Fig. 3). Three Trk receptors bind neurotrophins with specific selectivity: NGF is a preferable ligand for TrkA, BDNF and NT-4 for TrkB, and NT-3 for TrkC. Some cross-reactivity 17
was observed between neurotrophins and their receptors as NT-3 can also bind to and activate other Trks (Barbacid, 1994, Davies et al., 1995). Upon ligand binding Trk receptors dimerize. Dimerization leads to the activation of the receptor tyrosine kinase domains, auto-phosphorylation and activation of intracellular signal transduction cascades (reviewed in Huang and Reichardt, 2003; see also section 1.7.4). Differential splicing generates several TrkB isoforms with distinct functional properties (Huang and Reichardt, 2003). Among others, truncated TrkB isoforms lacking tyrosine kinase domain were described (Klein et al., 1990, Middlemas et al., 1991). Deletion of the tyrosine kinase domain eliminates tyrosine kinase activity of the receptor and thus abolishes conventional signal transduction. The function of truncated isoforms is not
completely understood but collected evidence highlights their importance in the regulation of cellular response to BDNF. Truncated receptors were suggested to exert a dominant negative effect by inhibiting productive dimerization and activation of full length receptors (Eide et al., 1996, Ninkina et al., 1996) or by scavenging BDNF from the extracellular space (Biffo et al., 1995). p75NTR was characterized as panneurotrophin receptor that binds all of the neurotrophins with similar low affinity (Johnson et al., 1986, Radeke et al., 1987, Rodriguez-Tebar et al., 1990). p75NTR is a member of tumor necrosis factor receptor superfamily. Its intracellular carboxy terminal part contains a socalled death domain that is implicated in the transduction of cell death signals (Chapman, 1995, Feinstein et al., 1995). p75NTR does not exhibit intrinsic catalytic activity and signals through association with cytoplasmic adaptor proteins (reviewed in Roux and Barker, 2002). Neurotrophins and their receptors are expressed nearly in all neuronal populations of the CNS and PNS and have well recognized roles in neuronal survival, process outgrowth, and synaptic plasticity. 1.7.2. BDNF synthesis, processing and secretion
BDNF is a stable dimeric protein that shares about 50% sequence identity with other neurotrophins (Radziejewski et al., Figure 3. Neurotrophins and their receptors. Trk and p75NTR structural features such as cysteine clusters (C); leucine-rich repeats (LRR), immunoglobulin-like domains (Ig), and cysteine-repeat domains (CR) are depicted. Physiologically more significant interactions are shown in solid arrows, less significant – in dashed arrows. Adopted from Reichardt (2006). 18
1992). It is produced and secreted by a wide variety of neuronal and nonneuronal cells in the PNS and CNS (Lessmann et al., 2003/4). BDNF is synthesized as prepro-BDNF that is cleaved into 32 kDa precursor protein, pro-BDNF. To produce the mature 14 kDa isoform, pro-BDNF is cleaved in trans-Golgi network by protease furin or in secretory granules by pro-protein convertases (Mowla, 2001, Seidah et al., 1996). BDNF is secreted via constitutive pathway in non-neuronal cells and via both constitutive and regulated pathways in neurons (Seidah et al., 1996). In vitro, pro-BDNF is also secreted, and it accounts for a significant proportion of a total BDNF that is released extracellularly (Mowla et al., 1999, Teng et al., 2005). Interestingly, recent data suggest that in CNS neurons do not secrete pro-BDNF (Matsumoto et al., 2008). Pro-BDNF is detected in brain tissue (Lee et al., 2001) where it has a similar expression pattern as the mature form and it is localized predominantly to nerve terminals (Zhou et al., 2004). This evidence, along with the fact that the prodomains of individual neurotrophins are highly conserved among vertebrate species (Suter et al., 1991), suggests that proBDNF as well as other pro-neurotrophins may have additional biological functions. Indeed, Lee et al. (2001) demonstrated that pro-NGF binds to p75NTR receptor with 5-fold higher affinity than mature NGF and effectively induces p75NTRmediated apoptosis. Similarly, Teng et al. (2005) showed that pro-BDNF is released from cultured cortical neurons and it is ten times more active in stimulating p75NTRmediated apoptosis than mature BDNF. Thus, in contrast to mature BDNF, proBDNF interacts predominantly with p75NTR and exerts distinct biological effects. The function of pro-BDNF versus mature BDNF may be as opposite as mediating
of cell death versus cell survival (Lu et al., 2005). Pro-BDNF may be cleaved to produce mature BDNF extracellularly by several matrix metalloproteinases and serine protease plasmin (Lee et al., 2001). In this context, the regulation of intracellular and extracellular cleavage of BDNF has an important impact on its physiological functions. Indeed, plasminmediated cleavage was shown to be an essential step in establishing of long-term hippocampal plasticity (Pang et al., 2004). However, it is important to stress that the interpretation of these results needs special care, as pro-BDNF secretion may be an experimental artifact. In neurons BDNF is secreted mainly via regulated (Ca2+-dependent) pathway (reviewed in Lim et al., 2003 and Lessmann et al., 2003/4). Increase of [Ca2+]i is mandatory for triggering regulated BDNF secretion. Ca2+ may come from different sources. Depolarizationinduced Ca2+ flux from voltage-gated Ca2+ channels or activated NMDA receptors as well as Ca2+ release from endoplasmic reticulum were shown to be involved (Goodman et al., 1996, Griesbeck et al., 1999, Marini et al., 1998, Wetmore et al., 1994). CaMKII acts downstream of Ca2+ signalling in the pathway leading to regulated BDNF secretion (Kolarow et al., 2007). Interestingly, neurotrophins can regulate their own secretion via activation of corresponding Trk receptors (Canossa et al., 1997, Kruttgen et al., 1998). Neurotrophin-dependent secretion of neurotrophins requires induction of PLCγ signalling pathways and Ca2+ release from intracellular Ca2+ stores (Canossa et al., 2001). BDNF release at the synaptic level was addressed using overexpression of BDNF fused with fluorescent protein GFP (Hartmann et al., 2001, Kojima et al., 19
2001, Kolarow et al., 2007). These studies demonstrated secretion of BDNF-GFP from post-synaptic sites at glutamatergic synapses and confirmed the essential role of intracellular Ca2+ signalling in this process. Apart from post-synaptic secretion of BDNF-GFP, pre-synaptic secretion of tagged BDNF was also demonstrated. BDNF-GFP was anterogradely transported along the axon and transferred to the postsynaptic neuron in an activity- and TrkBdependent manner (Kohara et al., 2001). Experiments with electrical stimulation of neurons revealed that patterned electrical activity induces BDNF secretion more efficiently than continuous depolarization (Balkowiec and Katz, 2000, Gartner and Staiger, 2002, Lever et al., 2001). The higher frequency stimulation (up to 100 Hz) induces more release of BDNF than the lower frequency one, and the most efficient stimulation protocols are tetanic stimulation (bursts of 100 pulses at 100 Hz) as well as theta burst stimulation (trains of 25 bursts, each burst of 4 pulses at 100 Hz) (Balkowiec and Katz, 2002). Remarkably, the two most efficient stimulation protocols are associated with induction of synaptic plasticity: tetanic stimulation is often used for induction of long term potentiation (LTP) whereas theta burst stimulation is aimed to mimic the typical firing pattern of hippocampal neurons during learning. In line with the previous studies, Ca2+ influx through voltage-gated Ca2+ channels (VGCC) and Ca2+ release from intracellular Ca2+ stores are both required for the induction of BDNF release by electrical stimulation, and this induction can occur in the absence of glutamatergic synaptic activity (Balkowiec and Katz, 2002). Secretion of BDNF in an activitydependent manner plays a vital role in synaptic plasticity. The importance of 20
activity-regulated BDNF secretion was highlighted by a finding that human subjects with valine (val) to methionine (met) substitution in pro-region of BDNF gene exhibit impaired hippocampal activity and memory function whereas in vitro assay demonstrated that regulated secretion of met-BDNF was markedly reduced as compared with val-BDNF (Chen et al., 2004, Egan, 2003). 1.7.3. Expression of BDNF and TrkB in the brain
Detailed studies have been performed to identify expression pattern of BDNF and TrkB in immature and adult brain using either in situ hybridization or immunodetection (Conner et al., 1997, Ernfors et al., 1990, Ernfors et al., 1992, Friedman et al., 1991, Friedman et al., 1998, Fryer et al., 1996, Hofer et al., 1990, Ivanova and Beyer, 2001, Klein, 1990, Kokaia et al., 1993, Maisonpierre et al., 1990, Schmidt-Kastner et al., 1996, Yan et al., 1997). In rodents, BDNF and TrkB expression starts at early embryonic stages and gradually increases during development (Ivanova and Beyer, 2001, Klein, 1990, Maisonpierre et al., 1990). Expression of the full length TrkB isoform reaches adult levels around birth. Interestingly, expression of the truncated TrkB isoform lacking tyrosine-kinase domain is also developmentally increased. Truncated TrkB levels stabilize at two weeks after birth and thereafter this isoform predominates over full-length TrkB (Fryer et al., 1996). Of all neurotrophins BDNF has the most widespread expression in the developing and mature nervous system. Similarly to BDNF, its receptor TrkB has an extensive expression pattern (Klein, 1990). BDNF and TrkB are coexpressed
in the same neurons in many neuronal populations, highlighting the importance of paracrine and autocrine BDNF signalling (Kokaia et al., 1993). For example, in the PNS BDNF and TrkB are prominently expressed in a subpopulation of dorsal root ganglia (DRG) neurons (Ernfors and Persson, 1991, Ernfors et al., 1990, Mu, 1993, Wright, 1995) and BDNF was suggested to be an autocrine survival factor for these neurons (Acheson et al., 1995). BDNF is also prominently expressed in inner ear sensory epithelium and supports in a paracrine manner survival of inner ear sensory neurons (Pirvola et al., 1992). In the CNS, the highest level of BDNF is observed in hippocampus and neocortex where it is coexpressed with TrkB (Hofer et al., 1990, Kokaia et al., 1993, SchmidtKastner et al., 1996). In this area, locally released BDNF is important not only for promoting neuronal survival and differentiation but also for mediating synaptic plasticity in the hippocampal and cortical circuits (Bramham and Messaoudi, 2005). Apart from its local effect, BDNF that is synthesised in hippocampal neurons may have a target-derived trophic function, as it was shown to promote survival and differentiation of neurons, that innervate this area: basal forebrain cholinergic neurons and locus coeruleus noradrenergic neurons (Alderson et al., 1990, Arenas and Persson, 1994, Friedman et al., 1993, Knusel et al., 1991). Within hippocampus BDNF mRNA and protein levels are higher in the dentate gyrus and CA3 than in the CA1 subfield, and no BDNF mRNA is detected in stratum oriens (Conner et al., 1997). Interestingly, BDNF mRNA is detected only in pyramidal and granular cells but not found in hippocampal interneurons (Rocamora et al., 1996a, Schmidt-Kastner et al., 1996). However, interneurons
express TrkB receptors implying that although they do not synthesize BDNF, they might be regulated in paracrine manner by BDNF released from pyramidal neurons. Indeed, BDNF was shown to promote differentiation of hippocampal and cortical GABAergic neurons (Marty et al., 1997; also see section 1.7.6). BDNF protein was found not only in neuronal cell bodies but also in nerve fibers and nerve terminals (Conner et al., 1997, Friedman et al., 1998, Yan et al., 1997). Analysis of BDNF immunoreactivity provided the first evidence of anterograde BDNF transport from distal neuronal cell bodies along their axons towards axonal terminals (Altar, 1997, Conner et al., 1997, Smith, 1997). Anterograde transport of BDNF was confirmed by experiments where inhibition of axonal transport or selective afferent elimination depletes BDNF immunoreactivity in corresponding nerve terminals (Altar, 1997, Conner et al., 1997). Remarkably, DRG neurons were the first model where anterograde transport of a neurotrophin was directly observed. BDNF was shown to accumulate proximally to the ligation site of the sciatic nerve or to the crush site of the dorsal roots, implying that it is anterogradely transported from DRG neurons in both peripheral and central directions (Tonra et al., 1998, Zhou and Rush, 1996). BDNF expression is regulated by a large variety of stimuli in physiological and pathological conditions (Lindholm et al., 1994). BDNF gene is controlled by cAMP response element-binding protein (CREB) and thus BDNF expression is activated by Ca2+ influx and neuronal activity (Ghosh et al., 1994, Shieh et al., 1998, Tao et al., 1998, Timmusk et al., 1993, Zafra et al., 1992). BDNF mRNA synthesis can be induced by convulsant agents like kainic acid, muscarinic 21
receptor agonist pilocarpine, as well as GABAA receptors antagonist bicuculline (Dugichdjordjevic et al., 1992, Lindholm et al., 1994, Wetmore et al., 1994, Zafra et al., 1990, da Penha Berzaghi et al., 1993). On the contrary, GABAA receptor agonist muscimol and the positive allosteric modulator diazepam substantially reduce BDNF synthesis (Zafra et al., 1991). In contrast to mature hippocampal neurons, at early postnatal age GABA is depolarizing and it is able to induce BDNF expression via MAPK/CREB-dependent mechanism (Berninger et al., 1995, Marty et al., 1996, Obrietan et al., 2002). BDNF expression is influenced by physiological activity in vivo. Synthesis of BDNF mRNA is regulated by visual stimuli in visual cortex of both immature and adult rats (Castren et al., 1992) and increased in the barrel cortex after whisker stimulation in mice (Rocamora et al., 1996b). Surgical transection of septal cholinergic afferents result in a reduction of BDNF levels in developing and adult rat hippocampus demonstrating that endogenous cholinergic innervation tonically control BDNF synthesis in this area (Lapchak et al., 1993, Lindefors et al., 1992, da Penha Berzaghi et al., 1993). Similarly to BDNF, expression, trafficking and membrane insertion of TrkB receptors is also regulated by neuronal activity (reviewed in Nagappan and Lu, 2005). For example, LTP-inducing tetanic stimulation significantly increases TrkB mRNA levels and the number of surface TrkB receptors (Dragunow et al., 1997, Du et al., 2000). Activity-dependent modulation of TrkB receptors provides a basis for selective potentiation of active synapses by BDNF. 1.7.4. TrkB signalling
BDNF binding to TrkB triggers dimerization and autophosphorylation 22
of the receptor. Upon TrkB activation, a number of tyrosine residues are phosphorylated in its intracellular part. Some of these residues serve as docking sites for several adapter proteins and enzymes. TrkB autophosphorylation leads to the activation of the three major signalling pathways involving MAPK, PI3K and PLC (reviewed in Reichardt, 2006; Fig. 4). Phosphorylated tyrosine residue Y816 (in mouse TrkB sequence) serves as a direct docking site for PLC PLC activation mediates generation of inositol trisphosphate (IP3) and diacylglycerol (DAG), mobilization of Ca2+ stores, as well as activation of Ca2+ and DAG-dependent protein kinases, such as PKC and CaMK. Adaptor protein Shc binds to phosphorylated Y515 (in mouse TrkB sequence) of TrkB and it results in the activation of small GTPase Ras and subsequent transient induction of the extracellular signal-regulated protein kinase (Erk)1/2 cascade. In this pathway Raf is activated downstream of Ras and it leads to phosphorylation of MAP/ERK kinase (MEK)1 and/or MEK2 which in turn phosphorylate Erk1 and Erk2. Apart from Ras-dependent transient MAPK activation, Trk receptors may also induce sustained MAPK phosphorylation, mediated through a distinct pathway (Marshall, 1995; reviewed in Arevalo and Wu, 2006 and Huang and Reichardt, 2003). Sustained MAPK activation requires recruitment of adapter proteins Frs-2 and Crk, internalization of Trk receptor into endosomal compartment and activation of small GTPase Rap1 (Kouhara et al., 1997, Wu et al., 2001, York et al., 2000). Frs-2 competes with Shc for the Y515 binding on Trk receptor and this competition is likely to discriminate between proliferation (promoted by transient MAPK activation) and differentiation (promoted by sustained
MAPK activation) in PC12 cells (Meakin et al., 1999). The physiological function of PLCand Shc-mediated signalling have been tested in vivo by mutating of PLC or Shc binding site in TrkB (Minichiello et al., 1998, Minichiello et al., 2002). These transgenic mice have a rather modest phenotype in terms of BDNF-dependent neuronal survival and differentiation, especially when compared with transgenic mice lacking TrkB tyrosine kinase domain
that exhibit prominent loss of sensory neurons (Luikart et al., 2003). This implies that signaling cascades other than mediated by PLC, Frs-2 and Shc plays an important role in survival-promoting TrkB function. Analysis of mice with double mutation in both Shc and PLC binding sites revealed delayed migration of cortical neurons as well as alterations in neuronal and glia differentiation (Medina et al., 2004). Remarkably, mice with mutation of PLC binding site demonstrate
TrkB
Figure 4. TrkB signaling. MAPK, PI3K, and PLCpathways are depicted. Modified from Reichardt (2006). 23
deficiencies in induction of both the early and late phases of LTP, probably due to the strongly reduced phosphorylation of CaM kinases and CREB (Minichiello et al., 2002). Mice with mutation of Shc binding site exhibit normal LTP induction (Minichiello et al., 1998), despite the fact that MAP kinases have a well established role in LTP formation (Adams and Sweatt, 2002). Both transient and sustained MAPK activation is greatly reduced in these animals; however it is not fully inhibited even in the double Shc/PLC mutants (Medina et al., 2004). Crosstalk between PLC and Shc pathways as well as MAPK activation through distinct, unidentified mechanisms may explain LTP induction in mice with impaired Shc binding. Neurons retrogradely transport neurotrophins released by their innervation targets (DiStefano et al., 1992). The mechanism of the retrograde transport implies that Trk receptors are internalized upon ligand binding and transported together with the ligand in vesicles towards the neuronal cell body (Watson et al., 1999). Internalization and transport of Trks bring activated receptors into close proximity of nucleus where Trk signalling is required to mediate specific cellular response, such as gene transcription (reviewed in Heerssen and Segal, 2002). Endocytosis also promotes transfer of activated Trk receptors to different membrane compartments and it may control the efficiency and duration of Trk-mediated signalling, in part because many signalling components are localized to specific membrane compartments (York et al., 2000). Experimental acceleration or inhibition of ligated Trk internalization prevents normal cellular responses to these complexes (Saragovi et al., 1998, Zhang et al., 2000). Endocytosis of Trks was shown to regulate prolonged versus transient ERK1/2 phosphorylation as well 24
as specific activation of ERK5 signalling pathway (reviewed in Arevalo and Wu, 2006, Heerssen and Segal, 2002, and Reichardt, 2006). In addition to the activation of the classical signalling cascades, BDNF binding to TrkB may cause rapid (within the millisecond range) membrane depolarization of neurons from different brain regions (Kafitz et al., 1999). The authors suggested that depolarization was caused by a rapid opening of Na+ channels triggered by a direct or indirect interaction of TrkB with the channel. Subsequent study revealed that tetrodotoxin-insensitive sodium channel NaV1.9 underlies the neurotrophin-evoked excitation (Blum et al., 2002) 1.7.5. EGR family of transcription factors
Neurotrophic factors promote long term effects via activation of gene transcription. Transcription of immediate early genes is induced rapidly and transiently in response to neurotrophic factor stimulation and does not require de novo protein synthesis. Many of immediate early genes are the last step in signal transduction cascades and encode transcription factors that mediate neurotrophins-induced gene expression (reviewed in Herdegen and Leah, 1998). Activation of MAPK induces transcription of a variety of immediate early genes, such as c-fos, c-jun and early growth response genes (Segal and Greenberg, 1996). The family of early growth response (Egr) genes encode structurally related zinc finger transcription factors and includes four members: Egr1-4. At least two of Egrs, Egr1 and Egr4, are dramatically upregulated by NGF in neurons and PC12 cells and thus are alternatively termed NGF-induced transcription factors (NGFIA and NGFIC respectively). In the nervous system
expression of Ergs is increased by stress, trauma as well as by physiological and pathological neuronal activity including seizures, LTP induction and sensory stimulation (Beckmann and Wilce, 1997, O’Donovan et al., 1999). Egr1 is the best studied member of Erg family and it is implicated in neuronal plasticity, learning and memory formation (reviewed in Davis et al., 2003 ans Knapska and Kaczmarek, 2004). Expression of Egr1 and Egr2 is induced by TrkB-mediated BDNF signalling (Calella et al., 2007, Minichiello et al., 2002) and it is strongly impaired in mice with PLC docking site mutation. Egr1 expression is also regulated by MAP kinases in NGFstimulated PC12 cells and BDNF-treated cerebellar granular neurons (Harada et al., 2001, Kumahara et al., 1999). Another member of Egr family, Egr4, was cloned by Crosby et al. (1991). Unlike other Erg genes that are widely expressed outside of the nervous system, Egr4 expression is restricted to the CNS where it is increased during development (Crosby et al., 1992). Egr4 levels are strongly and transiently induced in PC-12 cells by NGF, in adult rat somatosensory cortex by whisker stimulation and in adult rat cortex and hippocampus after seizures (Crosby et al., 1992, Honkaniemi and Sharp, 1999, Mack et al., 1995). Although Egr4 expression is sensitive to NGF (Crosby et al., 1991), BDNF-induced Egr4 upregulation has not been demonstrated yet. 1.7.6. BDNF and the GABAergic system
BDNF signalling is of outstanding importance in developing and adult nervous system. In addition to classical neurotrophic survival-promoting effects, it plays an essential role in neuronal differentiation and function (Lewin and
Barde, 1996). BDNF supports development and maintenance of cortical circuitry by regulation of synapse formation, stabilization and strength (Aguado et al., 2003, Vicario-Abejon et al., 1998). It is involved in activity-dependent synaptic plasticity and LTP formation (McAllister et al., 1999). BDNF levels are upregulated following injury and it has a well recognised neuroprotective effect (Hofer and Barde, 1988). BDNF is intimately involved in the development of the GABAergic system. In the hippocampus, BDNF is synthesised and secreted in the activity-dependent manner by pyramidal neurons and affects local TrkB-expressing interneurons (see section 1.7.3). In their in vitro study, Marty et al. (1996) demonstrated BDNFmediated regulation of interneuronal phenotype in immature hippocampal slice cultures. Later, BDNF was shown to induce an enlargement of the interneuronal somata, expression of GABAA receptor subunits and GABA-synthesising enzyme GAD65/67 as well as formation of GABAergic synapses (Vicario-Abejon et al., 1998, Yamada et al., 2002). Interestingly, in immature neurons depolarizing GABA induces BDNF expression (Obrietan et al., 2002) and through BDNF-mediated mechanism affects hippocampal interneurons (Marty et al., 1996). So, early in development GABA and BDNF seems to be interconnected by a feed-back loop mechanism with GABA enhancing BDNF production and BDNF supporting the development of GABAergic neurons. Remarkably, during maturation of GABAergic hyperpolarization, GABA switches from enhancing to repressing BDNF expression (Berninger et al., 1995) and thus the BDNF/GABA feed-back loop ceases to exist in mature neurons. The model of BDNF/GABA regulation was later confirmed by in vivo 25
studies of transgenic mice expressing BDNF in the nervous system earlier in development and at higher levels than wild type mice. The transgenic mice exhibit enhanced correlated spontaneous activity as well as dramatic increase in GABAergic synaptogenesis and GAD65/67 expression (Aguado et al., 2003). Interestingly, KCC2 levels in the hippocampus of BDNF overexpressing mice were strongly increased. KCC2 upregulation ensures that the feed-back loop between depolarizing GABA and BDNF will be eventually terminated by the decrease in [Cl–]i and the shift of EGABA towards more negative values. Apart from the regulation of GABAergic synaptogenesis (Marty et al., 2000), BDNF is also implicated in the modulation of GABAergic synaptic strength. In immature neurons BDNF induces a rapid and reversible potentiation of GABAA receptor-mediated responses (Mizoguchi et al., 2003). The increase in GABAergic responses was dependent on TrkB activation, PLC-induced [Ca2+]i rise and CAMKII. However, during development BDNF effect is reversed as BDNF was shown to reduce GABAergic spontaneous activity and evoked responses in older hippocampal neurons (Tanaka et al., 1997). The GABAergic response reduction may be mediated via rapid downregulation of GABAA receptor surface expression (Brunig et al., 2001, Cheng and Yeh, 2003) and/or downregulation of
26
KCC2 expression and function (Rivera et al., 2002, Wardle and Poo, 2003) in the postsynaptic cell. BDNF-mediated maturation of GABAergic inhibition has a dramatic effect on visual cortex development. In the visual cortex of transgenic mice with genetically enhanced BDNF expression the maturation of cortical interneurons as well as GABAergic innervation and inhibition is accelerated (Huang et al., 1999). Enhanced development of cortical inhibitory circuit is accompanied by the precocious developmental decline of LTP formation in the visual cortex and premature termination of critical period for ocular dominance plasticity. 1.8. Summary The reviewed experimental data suggest that developmental KCC2 upregulation serves not only maturation of GABAergic inhibition, but it is also a hallmark of the general maturation of GABA/ BDNF/activity signaling highlighted by the appearance of hyperpolarizing GABA responses, cessation of immature spontaneous network activity and closure of critical periods for synaptic plasticity. With this in mind, we aimed to investigate the factors that regulate developmental increase of KCC2 expression, which is crucial in the transition of the hippocampal neuronal circuitry from immature to the adult stage.
AIMS OF THIS STUDY The aim of this work was to study the mechanisms controlling KCC2 upregulation during early postnatal development of rodent hippocampus. The specific aims were: 1. To test the effect of chronic activity blockade on KCC2 expression in immature cultured hippocampal neurons. 2. To determine what transcription factors might be involved in developmental KCC2 expression. 3. To investigate whether neurotrophic factors regulate KCC2 developmental expression and what intracellular cascades are involved. 4. To investigate the existence of KCC2 splice variants.
27
2. MATERIALS AND METHODS The methods used are described in detail in the “Materials and Methods” section of the respective publications. All experimental procedures using laboratory animals were performed according to ethical guidelines approved by local authorities. 2.1. Cell culture procedures For studies of KCC2 developmental expression, mouse dissociated and organotypic slice cultures were used. Both systems provide a very useful tool to study neuronal development in vitro. However there are some differences between these two models that have to be taken into consideration. Neurons in dissociated cultures provide an easy access for pharmacological treatments and visualization after immunostainings. However cells in such cultures lack some essential factors guiding their maturation. The levels of cell-cell interactions, synaptic density and spontaneous activity are lower compared to in vivo, thus development of dissociated neurons might be delayed or disturbed. Organotypic slice cultures preserve the local neuronal circuit much better, but cells in such culture require time to recover after damage caused by cutting procedure. They are less accessible for drug application and microscopic visualization. Additionally, in our hands, dissociated neurons survive better than organotypic slices in long-term cultures usually employed for developmental studies. In publications II and IV two immortalized cell lines were used in addition to primary neurons. Mouse neuroblastoma neuro-2a (N2a) cells, which express a low level of KCC2 and no Egr4 (unpublished observations), were used to study transcriptional activity of 28
KCC2 intact or mutated promoter, and for Egr4 binding studies. Human embryonic kidney HEK293 cells were used for 86Rbflux assay. Both cell lines were cultured in DMEM medium supplemented with 10% fetal calf serum, penicillin 100 units/mL and streptomycin 100 μg/mL. 2.1.1. Dissociated cell culture
Dissociated neurons were prepared from E17 embryonic hippocampi or cortex and plated either on glass coverslips or directly on plastic bottom of the tissue culture plate. The surface in both cases was pretreated with polyornithyne to improve neuronal adhesion. Only defined Neurobasal medium with serum-free B27 suppliment (Gibco) was used. No serum, antibiotics or L-glutamine was added to neuronal cultures at any stage. To improve neuronal survival, before contact with neurons, the medium was preincubated overnight on the monolayer of glia cells. Glia cell cultures were prepared from E17 mouse hippocampi (Banker and Goslin, 1998) and kept during 3-4 months in DMEM medium supplemented with 10% fetal calf serum, penicillin 100 units/mL and streptomycin 100 μg/mL. The density of neuronal cultures varied from 5×103 cells/cm2 to 5×105 cells/cm2; depending on the experiment. When lowdensity cultures were plated (publication I), coverslips with neurons were kept in dishes containing glia monolayer for extra trophic support. 2.1.2. Organotypic slice culture
Organotypic slice cultures were prepared from hippocampi of P0 mouse pups. Hippocampi were dissected; transverse 350 m slices were cut and immediately placed on sterile Millicell-CM membranes
(Millipore, Bedford, MA, USA) in 6-well culture trays with 1 ml of plating medium. The plating medium was Neurobasal medium containing B27 supplement (Gibco, Life Technologies), penicillin 100 units/ml and streptomycin 100 μg/ml. 24 hours after plating the medium was changed to the growth medium Neurobasal/B27 without antibiotics. 2.1.3. Pharmacological treatments of cultured cells
Neuronal cultures were treated with blockers of action potential generation and synaptic transmission, blockers of kinases involved in signalling cascades as well as neurotrophic factors (Table 1). Activity blockers were added daily from frozen stocks prepared at 500–1000-fold the final concentration. Before addition, drugs were diluted to the final concentration in the fresh medium preincubated for 24 h with glial culture. Neurotrophic factors and signalling cascade blockers were added once in a fresh glia-conditioned medium and no
medium change was performed until the time of analysis (5 minutes – 3 days later). If a signalling cascade blocker was used together with a neurotrophic factor, then the blocker was added 30 min before the neurotrophic factor. 2.1.4. Transfections
HEK293 cells, N2a cells and dissociated neurons were transfected with luciferase reporter constructs or KCC2a and KCC2b expression constructs using lipofectamine 2000 (Invitrogen) according to the manufacturer’s protocol. The constructs list is presented in Table 2. To avoid cytotoxicity, relatively low amounts of the expression plasmids were used (0.5 μg per 1-cm-diameter well). In case of HEK293 cells and N2a cells the antibiotic-free medium was applied before transfection. In case of dissociated neurons the medium was completely changed 4 hours after transfection to improve cell survival. Transfection efficiency was 30-50% in HEK293 cells, 15% in N2a cells and 5-10% in dissociated neurons.
Table 1. List of drugs used for cell culture treatment and in vivo administration Drug Tetrodotoxin (TTX)
NBQX Picrotoxin (PTX) BDNF
Function Blocks action potentials by binding to voltage-gated sodium channels Selective NMDA receptor antagonist AMPA receptor antagonist GABAA receptor antagonist Neurotrophic factor
Neurturine
Neurotrophic factor
APV
U0126
Blocker of mitogen-activated protein kinase kinase (MEK) K252A Blocker of tyrosine kinase receptors Kainic acid Agonist of kainate receptors SL327 MEK blocker, brain penetrant Tetanus toxin (TeTx) Blocker of synaptic release
Final concentration 10 μm 1 M 40 μm
Used in I III I
10 μm 50 μm 10-100 ng/ml; 100 ng per intrahippocampal injection 10-100 ng/ml; 1 g per intrahippocampal injection 20 M
I I III
III
200 nM
III
2 mg/kg (ip injections) 100 mg/kg (ip injections) 5ng per intrahippocampal injection
III III III
III
29
Table 2. List of constructs used for transfections Construct KCC2(–1398/+5445) KCC2(–1398/+42) KCC2(–309/+42) KCC2(–309/+42)Egr4mut KCC2(–180/+42)
Egr1 Egr4 DN-Egr4
Description A luciferase reporter that includes 1398 bp of the upstream regulatory region, exon-1, intron-1, and part of exon-2 of the mouse KCC2 gene. A luciferase reporter that includes 1398 bp of an upstream regulatory region as well as 42 bp of the 5’untranslated region (5’-UTR) of the mouse KCC2 gene. A luciferase reporter that includes 309 bp of an upstream regulatory region as well as 42 bp of the 5’-untranslated region (5’-UTR) of the mouse KCC2 gene. A luciferase reporter that is similar to KCC2(–309/+42) but two critical dG nucleotides are replaced with two dTs in the Egr4 binding consensus sequence. A luciferase reporter that includes 180 bp of an upstream regulatory region as well as 42 bp of the 5’-untranslated region (5’-UTR) of the mouse KCC2 gene. It does not contain Egr4 binding consensus sequence. Egr1 expression vector, CMV promoter. Egr4 expression vector, CMV promoter. An expression vector that contains the C-terminal part and all three zinc fingers, but not the N-terminal part of Egr4, CMV promoter.
2.2. In vivo studies 2.2.1. Intrahippocampal injections
To study the effect of neurotrophic factors and activity blockade on KCC2 expression in vivo we performed injections of the drugs in hippocampus of P5-P6 rat. Before the injection rat pups were hypothermically anesthetised and the head was fixed in a surgical mask to maintain the skull stable. The stereotactic coordinates for injection were: anteroposterior -1.8 mm (relative to the bregma); mediolateral 2 mm (relative to the bregma); dorsoventral -2 mm from the cortical surface. The injection site was located in CA1 area of dorsal hippocampus. The certain amount of BDNF, neurturin or TeTx (as indicated in table 1) was dissolved in 4l of standard ACSF solution and injected in hippocampus at ~1l/min. In case of TeTx administration both sides were injected with the drug. In case of neurotrophic factor administration, the drug was injected only in the right30
Used in II II II, III II, III II
II II II
side hippocampus and the left-side hippocampus was injected with 4 l of saline solution. After injection procedure the rat was allowed to fully recover in a warm chamber before being placed back with littermates. 2.2.2. Kainic seizures
acid
injections
and
To study the effect of enhanced neuronal activity in vivo we performed intraperitoneal injections of kainic acid (KA), the agonist of kainate receptors. KA is well-know for its ability to induce seizures in adult rats. We used a low dose of KA (2 mg/kg) which was still enough to induce seizures in P5 rats. There were virtually no lethality among injected pups during seizures and 1 day after. Within 15 minutes after KA injection all rats developed the first sings of specific seizure behavior such as scratching, rapidly followed by lost of posture control and periods of lying on one side in tonic
position. One hour after KA injection, the seizure was ceased by intraperitoneal injection of paraldehyde (0.3 l/g). Paraldehyde was also administrated to control rats. In order to study MAPK involvement in activity-dependent regulation of KCC2 transcription, MEK blocker SL327 was administrated by intraperitoneal injection 30 minutes before KA (Table 1). 2.3. Detection techniques 2.3.1. Immunocytochemical procedures
The protocol used for immunodetection of antigens in dissociated neurons is described in details in the original articles (I, II and IV). List of the antibodies is provided in Table 3. 2.3.2. Immunohistochemical procedures
The protocol used for immunodetection of antigens in fixed tissue sections is described
in details in (III). After immunostaining sections were visualized with Leica TCS SP2 AOBS confocal system. List of the antibodies is provided in Table 3. 2.3.3. Western blotting
The protocol used for western blotting protein detection is described in details in the original articles (I, II, and III). In study I X-ray films were used for visualization of the chemifluorescent signal, developed with ECL-plus (Amersham, Pharmacia Biotech). In later studies Luminescent image analyzer LAS-3000 (Fujifilm) was used for the same purpose. 2.3.4. Semi-quantitative and quantitative RT-PCR analysis.
The protocols used for total RNA isolation and RT-PCR analysis of mRNA expression are described in the original publications (II, III and IV). Primers used for RT-PCR were designed manually, and contained, when possible, intronic sequence in between (Table 4). For quantitative RT-
Table 3. Primary antibody used in immunochemistry and Western blotting WB – western blotting; ICC – immunocytochemistry; IHC – immunohistochemistry Rb – rabbit; Mo – mouse Primary antibody Host KCC2 Rb polyclonal KCC2 Rb polyclonal MAP2 synaptophysin β-tubulin (βIII) Egr4 pERK 1/2 ERK 1/2 pAKT AKT
Source/Reference Dilution (Williams et al., 1999) 1:500 (Ludwig et al., 2003) 1:5000
Mo monoclonal Chemicon Mo monoclonal Boeringer Rb polyclonal BabCO, Berkeley, CA BioSite, Sweden Rb polyclonal (Zipfel et al., 1997) Mo monoclonal Santa Cruz Biotechnology, USA Rb polyclonal Santa Cruz Biotechnology, USA Rb polyclonal Cell Signalling Technology, USA Rb polyclonal Cell Signalling Technology, USA
Used in I I, II, III
1:1000 1:1000 1:10000
Method ICC ICC IHC WB ICC ICC WB
1:300 1:500 1:1000 1:1000
WB ICC WB WB
II III
1:1000
WB
III
1:1000
WB
III
I I I, II, III
III
31
PCR the cDNA samples were amplified in triplicates using the SYBR Green PCR Master Mix (Applied Biosystems, Foster City, CA, USA) and detected via the ABI Prism 7000 Sequence Detection System (Applied Biosystems). 2.3.5. In situ hybridization
In situ hybridization on paraffin-embedded sections was done as described (Li et al., 2002). Sagittal sections (thickness 7 μm) were hybridized using 35S-labelled antisense and sense (control) cRNA probes: Ret-specific probe (nucleotides 2595–3191, X67812), GFR1 specific probe (nucleotides 294-1039, U59486) and GFR2 (full-length, AF003825) (Kokaia et al., 1999). No labeling above background was observed in the sense controls. 2.4. Image analysis 2.4.1. Quantification of immunostaining intensity in neuronal cell bodies
For KCC2 intensity measurements in studies I and II, images were obtained with wide-field fluorescent microscope (Olympus AX70). On the images of MAP2 immunostaining MAP2-positive cells were randomly chosen and neuronal cell bodies were manually outlined. Intensity of KCC2 signal was measured in corresponding neurons on grey-scale images of KCC2 immunostaining using Image-Pro Plus for Windows (version 4.1.0.0; Media Cybernetics L.P.). In wide-field fluorescent images the background level is substantial and may vary between the coverslips. To determine the background level, three neuron-free areas were manually outlined on each picture. The mean intensity of background areas was subtracted from the mean intensity of the neurons from the same coverslip. 32
2.4.2. Quantification of immunostaining intensity in tissue sections
Intensity measurements of KCC2 immunoreactivity were used to analyze KCC2 expression in BDNF- and neurturininjected hippocampi (study III). This method was chosen in place of RT- PCR or western blot because it allowed us to see the spatial distribution of KCC2 expression levels. Due to diffusion gradient of the injected neurotrophic factor and because in vitro KCC2 expression was dependent on the neurotrophic factor concentration, an uneven change in KCC2 expression levels was expected. For each injected brain a series of coronal hippocampal sections was made at P8-P9, three days after the injection. The sections in the series were numbered in succession starting from the most frontal part of hippocampus. Each section contained growth factor injected side (right) as well as control side (left). KCC2 levels were analyzed by immunostaining in sections #45, 90, 132, 177, 222 and 273. The section thickness was 7M which gives a sample interval of 315m. In each brain analyzed the injection site (defined by a scar) was found between section #60 and section #90 and located in right and left hemisphere in CA1 area of hippocampus. Confocal images of immunostained tissue were made. For each image 9 consecutive optical slices (1.3 m) were made and merged for quantification of KCC2 signal intensity. The region of interest was manually highlighted and total intensity of immunostaining in the region was divided by its area. Then the KCC2 intensity in growth factor injected hemisphere was normalized to the KCC2 intensity in corresponding area of the same section control hemisphere.
Table 4. List of primers used for RT-PCR quantification and 5’RACE Primers used in quantitative real-time PCR are marked with an asterisk Primer
Sequence
Location
F1a* (KCC2a-frw) F1a-2
KCC2 sense 5’-GCCGGCTCCCGAGGGAAG-3’ 5’-CCCGAGGGAAGACGTCAAAG-3’
exon-1a exon-1a
F1a-3 F1b* (KCC2b-frw) F1b-2
5’-CGCGCCATGAGCCGCAGGTTCAC-3’ 5’-GCCACCATGCTCAACAACCT-3’ 5’-TTCTTCCTCCGTGGAGCGGAG-3’
exon-1a exon-1b exon-1b
R1a R1a-2
KCC2 antisense 5’-CTTTGACGTCTTCCCTCGGGAG-3’ 5’-GGCAGCGAGGTGACCGTGAAC-3’
exon-1a exon-1a
R1b R1b-2 R2 R2-2* (KCC2-rev) R3 R4 R7 R26 R26-2
5’-CCGTCAGGTTGTTGAGCATGGTGGC-3’ 5’- GCGCCGGGCTCGGGAT-3’ 5’-CTTCTCCGTGTCCGTGCTGTTGATG-3’ 5’-TGAAGGGACTGCTCTCTTTGG-3’ 5’-GTAGGTTGGTGTAGTTGG-3’ 5’-GCAGAAGGACTCCATGATGCCTGCG-3’ 5’-TCAGCATGGCGGCCGCCTCCCCACTG-3’ 5’-CGCCTCGCCACCTTTATTGC-3’ 5’-GGGTCTTATGTAAACGGTGACAGCG-3’ GAPDH
exon-1b exon-1b exon-2 exon-2 exon-3 exon-4 exon-7 3’UTR 3’UTR
GAPDH-fwd* GAPDH-rev*
5’-TGCACCACCAACTGCTTAGC-3’ 5’-TGGCATGGACTGTGGTCATG-3’ Egr4 5’-TCTCTCCAAGCCCACCGAAG-3’ 5’-AACCGCCTGGATGAAGAAGC-3’ TrkB 5’-TCAAGTTGGCGAGACATT-3’ 5’-ATGTACTCAAAGACCATGATGAG-3’ BDNF 5’-GGCCCAACGAAGAAAACCAT-3’ 5’-GCACTTGACTGCTGAGCATCA-3’ Ret 5’-ATGAAAGGGTACTGACCATGG-3’ 5’-AGGACCACACATCACTTTGAG-3’ GFRα2 5’-TATTGGAGCATCCATCTGGG-3’ 5’-AGCAGTTGGGCTTCTCCTTG-3’ 5’RACE 5’-CCATCCTAATACGACTCACTATAGGGC-3’ 5’-ACTCACTATAGGGCTCGAGCGGC-3
exon-4 exon-5
Egr4-fwd* Egr4-rev* TrkB-fwd TrkB-rev BDNF-fwd* BDNF-rev* Ret-fwd Ret-rev GFRa2-fwd GFRa2-rev AP1 AP2
exon-1 exon-2 exon-12 exon-15 last exon last exon exon-14 exon-16 exon-2 exon-4 adapter adapter
33
2.4.3. Luciferase assay
Luciferase assay was used to study transcriptional activity of different KCC2 gene regulatory regions. For this purpose reporter constructs were generated bearing firefly luciferase gene under control of the promoter of interest. Cells were cotransfected with a firefly construct and a basic renila construct. Renila signal was used for normalization of transfection efficiency. Two days after transfection cells were lysed in Passive Lysis Buffer (Promega). Renila and firefly luciferase activities were measured with a Dual-Luciferase Reporter Assay System according to the manufacturer’s protocol. 2.5. Functional 86Rb flux assay Rb flux assay was used to determine the functionality of the novel KCC2a isoform in study IV. HEK 293 cells were chosen for this assay because they exhibit high transfection efficiency and support K-Cl cotransport activity of exogenously expressed KCC2 (Payne, 1997). The cells
86
34
were transiently transfected with KCC2a and KCC2b expression plasmids. The 86Rb flux assay protocol is described in details in study IV and in Payne (1997). 2 μCi/ ml of 86RbCl (Amersham Biosciences) was used. 86Rb influx in the presence or absence of 2 mM furosemide was analyzed by scintillation counting (Wallac 1450 Microbeta; PerkinElmer). 2.6. Sequence analysis Sequence analysis is described in details in original publications (II and IV). Mouse, rat, human, and chimpanzee KCC2 genomic sequences, corresponding to a 1.4 kb sequence upstream of the transcription start site as well as a proximal part of intron1, were aligned by the ClustalW program (Chenna et al., 2003). Putative TF binding sites within this region were identified by using MatInspector software. CpG islands inside the KCC2 gene were analyzed using the CpG Plot tool. Phosphorylation patterns were predicted with Scansite (Obenauer et al., 2003).
3. RESULTS AND DISCUSSION 3.1. Developmental upregulation of KCC2 in neuronal cultures in the absence of activity and synaptic transmission (I) Many lines of evidence support the idea that KCC2 expression correlates with synaptogenesis. KCC2 is not expressed in cell lines of neuronal or glia origin but it is expressed in neurons (Payne et al., 1996, Williams et al., 1999). KCC2 is absent from immature neurons devoid of synapses (Li et al., 2002). In cortex and cerebellum the developmental onset of KCC2 expression occurs when a neuron has completed migration and has settled at its final position (Li et al., 2002, ShimizuOkabe et al., 2002, Takayama and Inoue, 2006, Takayama and Inoue, 2007). The level of synaptic activity correlates with the amount of synaptic contacts received by a neuron. So we aimed to explore (1) whether in dissociated hippocampal cultures KCC2 expression correlates with synaptogenesis and (2) whether KCC2 developmental increase requires synaptic activity. 3.1.1. Developmental increase of KCC2 in dissociated hippocampal neurons
Dissociated cultures were plated from hippocampi of E17 mouse embryos. At this embryonic stage virtually no KCC2 expression is found in hippocampus (Li et al., 2002). We observed earliest KCC2 immunoreactivity after 3 days in vitro (div). As the culture developed KCC2 expression gradually increased. At early stages distal dendrites (indicated by MAP2 immunoreactivity) were devoid of KCC2. KCC2 expression started in cell bodies and spread to dendrites later (Fig. 2 in I). Upregulation of KCC2 levels in dissociated cultures coincided with abrupt
increase in synapse number as indicated by double immunostaining of developing neurons with KCC2 and synaptophysin antibody (Fig. 4 in I). The temporal pattern of KCC2 expression in dissociated neurons is in good agreement with in vivo data and confirms the relevance of this model for studying developmental expression of KCC2. However the spatial distribution of KCC2 at early stages seems to be different in vitro and in vivo. In our cultures KCC2 signal was first observed in neuronal cell bodies whereas immunogold labeling of P2 and P4 hippocampal sections revealed early KCC2 signal in plasma membrane and transport vesicles of distal dendrites (Gulyas et al., 2001). It is plausible that the regulation of KCC2 transport requires factors deficient in young cultured neurons, where posttranslational activation of KCC2 might be altered (Lee et al., 2007). Consistent with nonfunctional stage of KCC2 in immature hippocampal cultures, application of the broad-spectrum kinase inhibitor staurosporine produced a rapid KCC2 activation in cultured neurons, but not in neonatal acute slices (Khirug et al., 2005) We also analyzed KCC2 increase in organotypic slice cultures prepared from hippocampi of P0 mouse pups. In these cultures KCC2 upregulation was stronger than in dissociated neurons (in dissociated cultures: from div4 to div15 – 5 fold; in organotypic slices: from div3 to div14 – 10 fold; Fig. 3 in I). In organotypic slices neuronal connections are preserved better and consequently synaptic density is higher which might explain the stronger KCC2 upregulation. It is also possible that in organotypic slices neurons develop faster
35
and represent more mature developmental stage compared to dissociated neurons. 3.1.2. Chronic blockage of activity and synaptic transmission does not affect KCC2 upregulation
Neurons grown in vitro develop synaptic contacts and are spontaneously active (Banker and Goslin, 1998). To investigate the effect of neuronal activity on KCC2 expression, we grew dissociated and organotypic cultures during two weeks in the presence of activity blockers (table 1). The blockers were applied in various combinations daily starting from the first day after plating. Tetrodotoxin (TTX) was used to block action potentials, and picrotoxin (PTX) was used to inhibit GABAergic signalling. PTX was used with TTX because it may cause elevated activity due to insufficient inhibition. AMPA and NMDA receptor antagonists NBQX and APV were used to block glutamate receptor activity. Surprisingly, none of the combinations, including the one where all the antagonists were added together, prevented or significantly altered developmental KCC2 increase (Fig. 5 in I). Apparently, synaptic activity also does not contribute significantly to the difference between KCC2 levels in dissociated and organotypic cultures. It suggests that whatever controls the scale of age-dependent KCC2 increase in vitro, it is not synaptic activity. Our results are in striking conflict with Ganguly et al. (2001) study, where KCC2 mRNA levels were decreased by 70% when hippocampal neurons were grown for 14 days in the presence of GABAA receptor antagonists bicuculline and PTX. However, the diverse protocol used by Ganguly et al. (2001) for culturing of hippocampal neurons might lead to different properties of the culture in general and GABAergic activity in 36
particular. Whatever is the reason for this discrepancy (discussed in the section 3.3.4.2), it is clear that GABAergic activity is not an absolute requirement of developmental KCC2 increase. This was confirmed later by a study of VIAAT knockout mice displaying severe reduction of GABAergic and glycinergic activity but still normal KCC2 levels (Wojcik et al., 2006). 3.2. Upregulation of KCC2 expression by transcription factor Egr4 (II) Upregulation of KCC2 levels in developing neurons implies the activation of gene transcription and regulation of promoter activity. We aimed to study KCC2 promoter region and analyze transcription factors that might be involved in KCC2 developmental expression. Uvarov et al. (2005) have shown previously that genomic fragment bearing 1.4 kb sequence upstream of the transcription initiation site is sufficient to induce CNS- specific pattern of KCC2 expression. We analyzed 1.4 kb upstream promoter and proximal intron-1 regions of KCC2 gene from four different species and identified ten highly conserved transcription factors binding sites (Fig. 1 in II). We found that among others, KCC2 promoter bears a consensus binding site for neural-specific transcription factor Egr4 (Crosby et al., 1992). Egr proteins are induced by growth factors and neuronal activity (O’Donovan et al., 1999/4/1). Egr4 expression is increased during postnatal development of rats (Crosby et al., 1992) and mice (Fig. 2 in II) synchronously with the course of KCC2 upregulation. The predicted Egr4binding site in the KCC2 promoter and the data on Egr4 expression let us suggest
that Egr4 plays a role in regulation of developmental KCC2 expression. 3.2.1. Egr4 strongly and specifically activates KCC2 promoter in N2a cells
In order to study the regulation of KCC2 gene we have generated a series of deletion constructs in which a firefly luciferase reporter was placed under the control of different KCC2 gene regulatory regions (Fig. 3 in II; table 2). The construct KCC2(-1398/+5445) included 1.4 kb upstream promoter, exon-1, intron-2 and a part of exon-2. The constructs KCC2(1398/+42) and KCC2(-309/+42) included 1.4 kb or 0.3 kb of upstream promoter sequence respectively. The shortest KCC2(-180/+42) included only 0.18 kb of the promoter and consequently was deficient in Egr4-binding site. Human neuroblastoma N2a cells do not express Egr4 (unpublished observation). After transfection of the constructs in N2a cells, we did not see any significant difference between the basal reporters’ activities regardless of the promoter size (Fig. 3 in II). The only exception was the longest KCC2(1398/+5445) that had lower activity presumably due to the unknown repressor elements in the proximal part of intron-1 (Uvarov et al., 2005). However, when the reporters were coexpressed in N2a cells together with Egr4, there was a robust induction of luciferase activity in case of the constructs bearing Egr4-binding site. Notably, no induction was observed for KCC2(-180/+42) lacking Egr4 element. The binding consensus for Egr1 is very similar to that of Egr4 (Swirnoff and Milbrandt, 1995). In order to estimate how specific Egr4-mediated induction of KCC2 promoter is, we compared the
effect of Egr1 and Egr4 overexpression. Importantly, Egr1 failed to induce activity of the promoter bearing Egr4 binding site. The specificity of Egr4 action was further confirmed by Egr1/Egr4 competition assay (Fig. 3 in II). Electrophoretic mobility shift assay (EMSA) has demonstrated the effective interaction of Egr4 with Egr4 binding site in KCC2 promoter (Fig. 4 in II). Next, we produced two constructs with modified Egr4 binding properties. The first one was KCC2(-309/+42) construct in which Egr4 consensus was mutated by the substitution of two crucial nucleotides (KCC2(-309/+42)Egr4mut). EMSA confirmed that this mutation completely prevented Egr4 binding to the consensus (Fig. 4 in II). Egr4 induction of mutated KCC2 promoter was significantly lower than the induction of the non-mutated one (Fig. 5 in II). Interestingly, 50% of Egr4-mediated induction still occurred despite the binding failure. It indicates that Egr4 overexpression renders both direct and indirect effect on KCC2 promoter activity. The second construct was modified KCC2(-180/+42), in which two copies of Egr4-binding consensus sequence were inserted upstream of short KCC2 promoter region lacking Egr4 element. When overexpressed in N2a cells together with Egr4, this construct showed 4-fold higher luciferase activity that the one without the insertion (Fig. 5 in II). These experiments showed that the Egr4-binding site mediates the effect of Egr4 on KCC2 promoter activity in N2a cells. Our next step was to determine whether Egr4 affects KCC2 expression in neurons.
37
3.2.2. Egr4 increases KCC2 promoter activity in cultured hippocampal and cortical neurons
Both Egr4 and KCC2 are expressed in developing hippocampus in vivo (Fig. 2 in II). Similarly, in cultured neurons Egr4 and KCC2 are upregulated synchronously during first two weeks of culture development (Fig. 7 in II). As a tool to study Egr4-mediated KCC2 gene regulation we produced a dominant-negative Egr4 isoform (DNEgr4) which could bind to Egr4 consensus but could not activate gene expression. In N2a cells DN-Egr4 was able to inhibit up to 70% of Egr4-mediated increase in KCC2 promoter activity (Fig. 6 in II). If Egr4 is involved in regulation of KCC2 gene expression in neurons, disruption of Egr4 binding should affect KCC2 levels. Indeed, when DN-Egr4 was expressed in cultured neurons for 48 hours, it notably (by 25%) decreased endogenous KCC2 levels as it was detected by immunostaining with KCC2 antibody (Fig. 7 in II). DN-Egr4 as well as Egr4specific siRNA suppressed activity of KCC2(-309/+42) construct in div10 but not in div5 cultured neurons (Fig. 8 in II). This experiment revealed low regulatory activity of endogenous Egr4 in young neurons in agreement with low Egr4 expression levels at early stage of culture development (Fig. 2 in II). Accordingly, Egr4 overexpression increased KCC2(-309/+42) activity by 100% in div5 neurons but failed to change it in div10 cultures. Interestingly, in all our experiments with cultured neurons, blockade of Egr4 signalling by means of DN-Egr4 or siRNA knock-down leaded to not more than 2535% reduction of luciferase activity or endogenous KCC2 levels. It is possible that the amount of DN-Egr4 or siRNA is not enough to prevent endogenous Egr4 38
signalling completely. On the other hand, overexpression of DN-Egr or siRNA in cultured neurons could potentially affect transcription of other genes, including other transcription factors, and thus part of the effect on KCC2 promoter activity might be indirect. To evaluate the direct impact of endogenous Egr4 on KCC2 promoter, we compared the fold increase in KCC2(309/+42) and KCC2(-309/+42)Egr4mut expression in cultured neurons from div5 to div10. During this period, the activity of KCC2(-309/+42) increased 4.2-fold, while the activity of KCC2(-309/+42)Egr4mut increased only 2.7-fold. This indicates that the disruption of Egr4 binding to KCC2 promoter decreases KCC2 expression by 35%, which is in perfect agreement with DN-Egr4 and siRNA data. Egr4-deficient mice were characterized by Tourtellotte et al. (1999). Despite the mostly neuronal pattern of Egr4 expression, Egr4-deficient mice display no obvious malfunction in the CNS. On the contrary, mice that have impaired KCC2 expression exhibit severe CNS dysfunction (Hubner et al., 2001). The phenotype is milder in animals expressing higher KCC2 levels (Tornberg et al., 2005, Woo et al., 2002). The mice that retain 30-40% of normal KCC2 expression exhibit no obvious neuronal phenotype or behavioral abnormalities (J Tornberg, personal communication). Consequently, the fact that Egr4 knockout mice exhibit no pronounced alteration in CNS development can be explained by mild (2535%) impact of Egr4 on KCC2 expression. Alternatively, the compensation by other transcription factors e.g. other Egrs is also possible. In summary, we characterized the 1.4 kb mouse KCC2 promoter region and identified multiple transcription factor binding sites that potentially regulate
KCC2 expression. We found that one of these factors, Egr4 binds to a specific sequence in KCC2 promoter and increases the activity of mouse KCC2 gene in neuronal cells. 3.3. On the mechanism of developmental KCC2 upregulation (III) During recent years, extensive research has revealed several stimuli that affect KCC2 levels in immature brain and might be involved in the regulation of KCC2 expression. These are: depolarizing GABAergic input acting via [Ca2+]i increase (Ganguly et al., 2001), chronic overexpression of BDNF (Aguado et al., 2003) and activity input on developing neurons (Kanold and Shatz, 2006, Liu et al., 2006; see section 1.6.4.1). However, in the studies mentioned above, neurons were chronically exposed to the stimulation that increases the probability of indirect mechanism affecting KCC2 expression. Moreover, BDNF release, synaptic activity and depolarizing GABA are interconnected. Indeed, BDNF overexpressing mice have higher level of spontaneous activity, increased GAD65/67 expression, and enhanced GABAergic synaptogenesis (Aguado et al., 2003); depolarizing GABAergic signalling induces BDNF expression (Berninger et al., 1995, Obrietan et al., 2002); neuronal activity is known to regulate BDNF release (Lessmann et al., 2003/4). It remains unclear whether the mentioned extracellular stimuli act separately through different signal transduction pathways or they work in synergy and trigger the same intracellular cascade that eventually lead to the activation of KCC2 gene transcription. In our study we decided to look at the matter the other way round, from the gene regulation point of view. Keeping in
mind that Egr4 is involved in regulation of KCC2 gene transcription (II) we designed a series of experiments aimed to elucidate whether growth factors and neuronal activity increase KCC2 expression through Egr4 induction in immature neurons. 3.3.1. BDNF increases activity of KCC2 promoter via MAPK activation and Egr4 in cultured neurons
3.3.1.1. BDNF treatment robustly increases Egr4 expression in MEK/ERK dependent manner Using mice that carry TrkB receptor with mutated PLC or Shc binding site, Rivera et al. (2004) demonstrated that in adult hippocampus BDNF/TrkB-mediated activation of Shc/MAPK pathway induces KCC2 upregulation. This suggests that in immature neurons the mechanism of BDNF/TrkB-mediated KCC2 increase may use the same pathway. Importantly, although there is no published evidence that Egr4 is induced by BDNF, other Egr transcription factors were shown to be induced by BDNF and NGF in a MAPK/ ERK dependent manner (Calella et al., 2007, Harada et al., 2001, Roberts et al., 2006). Here we used immature (div5-6) dissociated hippocampal cultures to study the effect of BDNF on KCC2 promoter activity. Already 5 minutes after BDNF (50 ng/ml) application an abrupt increase in ERK1/2 and AKT phosphorylation was determined by Western blot (Fig. 1 in III). MEK1/2 inhibitor U0126 effectively blocked ERK1/2 but not AKT phosphorylation. Egr transcription factors are induced within 1-2 hours of NGF exposure (Crosby et al., 1991). Accordantly, a robust increase of Egr4 mRNA level was observed 1 hour after BDNF application (Fig. 1 in III). 39
Importantly, this increase was sensitive to MEK1/2 blocker indicating that Egr4 is induced via MEK/ERK rather than AKT pathway. 3.3.1.2. BDNF activates KCC2 promoter via Egr4-binding site To determine the activity of KCC2 promoter upon BDNF stimulation, we transfected neurons with KCC2(-309/+42) construct. Despite the short (0.3 kb) promoter sequence included, in N2a cells Egr4 activates KCC2(-309/+42) construct to the same extent as other constructs bearing longer KCC2 promoter sequences (Fig. 3 in II). In trasfected neurons, BDNF (50ng/ ml) increased KCC2 promoter activity by 30%, as indicated by luciferase assay (Fig. 2 in III). Lower or higher concentrations of BDNF (10ng/ml or 200ng/ml) produced smaller and more variable effect. BDNFinduced activation of KCC2 promoter was abolished by both tyrosine kinase receptor (Trk) blocker K252a and MEK1/2 inhibitor U0126. This implies that BDNF regulation of KCC2 promoter is mediated through Trk/MAPK signalling. BDNF can induce network activity (Lang et al., 2007). To rule out the possibility that BDNF-induced network activity affected KCC2 regulation we perform BDNF treatment in the presence of TTX. Pretreatment with TTX did not alter BDNF- induced activation of KCC2 promoter (Fig. 2 in III). In order to determine whether Egr4 is involved in mediating BDNF effect on KCC2, we compared BDNF-induced activation of KCC2(-309/+42) and KCC2(-309/+42)Egr4mut constructs. The later one is deficient in Egr4 binding because of two point mutations that are introduced in Egr4 consensus (Fig. 4 in
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II). Disruption of Egr4 binding completely prevented BDNF-induced activation of KCC2 promoter indicating that Egr4 is an essential player in BDNF signalling cascade (Fig. 1 in III). 3.3.2. BDNF and neurturin increase KCC2 expression in immature cultured neurons
DN-EGR4 downregulates endogenous KCC2 protein level when overexpressed during div8-div10 in immature hippocampal cultures (Fig. 7 in II). To examine whether BDNF-mediated induction of Egr4 affects endogenous KCC2 expression, we analyzed KCC2 protein level in BDNF-treated cultures two days after BDNF application. BDNF induced a substantial increase in KCC2 protein levels when it was applied on neurons at div4 or div11 (Fig. 3 in III). Older cultures failed to upregulate KCC2 in response to BDNF in agreement with the previous data that BDNF reduces KCC2 levels in mature neurons (Rivera et al., 2002, Rivera et al., 2004, Wake et al., 2007). Interestingly, no developmental decrease in TrkB expression was observed in vitro (Fig. 4 in III) and in vivo (Ivanova and Beyer, 2001). This implies that developmental change in BDNF effect was due to some other reason than receptor expression, for instance due to a change in TrkB responsiveness (Knusel et al., 1994) or a change in predominantly activated signal transduction pathway (Huang and Reichardt, 2001, Rivera et al., 2004). BDNF failed to induce Egr4 mRNA rise in the presence of MEK1/2 inhibitor U0126 (Fig. 1 in III). It emphasizes the key role of MAPK activation in regulation of KCC2 promoter activity. We tested whether another neurotrophic factor that is able to induce MAPK
phosphorylation, can upregulate KCC2 expression. Neurturin is the member of glial cell line-derived neurotrophic factor (GDNF) growth factors family, it operates through receptor tyrosine kinase RET/ GFR2 co-receptors and induces MAPK phosphorylation in cultured sympathetic neurons (Baloh et al., 2000, Kotzbauer et al., 1996, Airaksinen and Saarma, 2002). Expression of neurturin co-receptors RET and GFR2 was found in hippocampal neurons throughout culture development (Fig. 4 in III). When neurturin (50 ng/ml) was applied on cultured neurons it induced rapid increase in ERK1/2 phosphorylation that was inhibited by U0126 (Fig. 4 in III). Similarly to BDNF, 1 hour neurturin treatment stimulated significant rise in Egr4 mRNA expression. The fold induction of Egr4 levels by neurturin was much lower than the one caused by BDNF (1.6 fold versus 22 fold), however it is compatible with Egr4 induction found in other studies (Mack et al., 1995). Although acting through completely different type of tyrosine kinase receptor neurturin increased KCC2 expression essentially in the same manner as BDNF. In hippocampal neurons of different age the strongest effect (130% of non-treated control) was observed during the second week in vitro (Fig. 4 in III). In summary, we performed a series of in vitro experiments that provide us with a model of KCC2 gene regulation by extracellular stimuli in immature hippocampal neurons. According to this model, neurotrophic factors, such as BDNF or neurturin, upon binding to their receptors induce MAPK phosphorylation and EGR4 expression. In turn, Egr4 binds to KCC2 promoter and increase KCC2 levels.
3.3.3. BDNF and neurturin increase KCC2 expression when injected in neonatal rat hippocampus
We extended our studies in order to see whether the model obtained in vitro is relevant in vivo. Chronic infusions and acute injections in the brain are widely used tool for studies of in vivo signalling of BDNF (Croll et al., 1994, Croll et al., 1998, Messaoudi et al., 1998, Ying et al., 2002). BDNF administration in hippocampus induces TrkB and ERK1/2 phosphorylation (Croll et al., 1998, Ying et al., 2002). We injected 100 ng of BDNF in hippocampus of P5 rats. The controlateral hippocampus was injected with saline solution and used as a control. Three days after the injection a series of coronal sections was made and KCC2 protein levels were analyzed by immunostaining. The summarized results for different hippocampal layers are presented in Fig. 5 (study III). The Fig. reflects minimum, average and maximum KCC2 increase among all sections that were analyzed. For the details of image analysis see section 2.4.2. In BDNF- injected hippocampus KCC2 protein level was on average increased by 15% compared to controlateral saline-injected hippocampus. The maximal increase varied from 20% to 40% depending on the hippocampal area. We also studied the effect of intrahippocampal neurturin injections. In contrast to BDNF, no significant increase in KCC2 expression was detected after administration of 100 ng of neurturin. So in subsequent injections we used 1 g of neurturin. KCC2 levels were analyzed three days after the injection by immunostaining in a similar way to BDNF injection analysis. On average, KCC2 levels in neurturin-injected hippocampus were increased by about 15% compared to 41
contralateral saline- injected hippocampus. The maximal increase varied between 3080% (Fig. 5 in III). Considering that BDNF was shown to promote differentiation of GABAergic neurons in vitro and in vivo (Aguado et al., 2003, Marty et al., 1996), we expected to see stronger BDNF effect in the areas where interneurons were localized. However, there was no significant difference between neurotrophic factors effect in different layers of hippocampus. In vivo data was confirmed by in vitro experiments where similar BDNF-induced increase in KCC2 expression was observed in both GABAergic and non GABAergic cultured neurons (data not shown). In summary, BDNF and neurturin intrahippocampal administration confirmed that in immature neurons KCC2 is upregulated by growth factors and, taking into account our in vitro model, this upregulation is likely to be direct rather the mediated by enhanced neuronal maturation. 3.3.4. Endogenous network activity is involved in regulation of KCC2 expression in vivo
Egr transcription factors are induced by neuronal activity and seizures (Beckmann and Wilce, 1997, O’Donovan et al., 1999/4/1). In particular, prolonged Egr4 expression is induced in hippocampus after systemic kainic acid injection (Honkaniemi and Sharp, 1999). MAPK activation is also regulated by network activity (Adams and Sweatt, 2002). Considering that both Egr4 and MAPK are involved in KCC2 gene regulation we aimed to analyze the effect of neuronal activity on immature KCC2 expression in vivo. For this purpose we performed two series of experiments in which network activity in hippocampus
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of neonatal rats was either inhibited or enhanced. 3.3.4.1. Tetanus toxin injections Tetanus toxin prevents the release of neurotransmitters by specifically cleaving the vesicle-associated protein synaptobrevin 2 that is necessary for presynaptic vesicle exocytosis (Schiavo et al., 2000). Single injection of tetanus toxin in neonatal rat hippocampus strongly reduces the frequency of spontaneous synaptic events for several days (Groc et al., 2001, Groc et al., 2002). We performed injections of 5ng of tetanus toxin in hippocampi of P6 rats. Western blot analysis of the area around injection site revealed significant decrease in KCC2 expression 24 hours after tetanus toxin administration (Fig. 7 in III). Importantly, the administrated tetanus toxin dose was relatively low and did not alter the behaviour of the pups. Right after the injection, as well as 24 hours later, the pups injected with tetanus toxin were undistinguishable from the ones injected with saline solution. 3.3.4.2. Kainic acid injections Kainic acid (KA) is well known for its ability to enhance network activity and to induce seizures in immature and adult brain (Holmes and Thompson, 1988, Holmes, 1991). We carried out intraperitoneal (ip) injection of KA in P6 rats and studied the immediate effect of artificially enhance network activity on KCC2 expression. The dose of KA (2 mg/kg) was the lowest one that still induced visible seizure symptoms. Within 15 minutes after KA administration virtually all rats developed scratching, rapidly followed by loss of posture control and periods of tonic seizures. In rare cases rats did not develop seizures within 30 minutes after KA injection. These rats were not taken for further analysis. One
hour after the injection rats (also controls) received 0.3 l/kg of paraldehyde ip to stop the seizures. Already at this stage ERK1/2 phosphorylation was enhanced in KA injected rats (Fig. 8 in III). In some rats brain-penetrant MEK1/2 inhibitor SL327 was administrated ip 30 minutes before KA injection. Importantly, SL327 blocked ERK1/2 phosphorylation but did not alter seizure development. Realtime PCR analysis showed that BDNF, Egr4 and KCC2 mRNA levels were strongly increased 4-6 hours after KA injection. Notably, disruption of ERK1/2 phosphorylation by SL327 fully prevented the rise in Egr4 and KCC2 expression. Taken together these data demonstrate that network activity in neonatal hippocampus is able to regulate KCC2 expression. Furthermore, this process requires the activation of the MAPK cascade with subsequent induction of Egr4. Interestingly, animals injected with very low doses of KA (0.2 mg/kg) that did not have any apparent seizure behavior also showed increased KCC2 expression levels (data not shown). Our results are in line with recent work demonstrating activity-dependent regulation of KCC2 levels in immature brain in vivo (Kanold and Shatz, 2006, Liu et al., 2006, Ouardouz and Sastry, 2005). On the contrary, ours and other in vitro studies demonstrate the existence of activity- independent KCC2 expression (study I; Ganguly et al., 2001). Additionally, as was discussed in section 3.1.2, the same disagreement also emerges for the studies of the role of depolarizing GABA in KCC2 developmental increase. These discrepancies indicate that the activity- and/or depolarization-dependent regulation rides on top of an activityand depolarization-independent, intrinsic mechanism. In favour of intrinsic
mechanism speaks the fact that in all documented cases where activity or GABA depolarization was blocked, the residual level of KCC2 expression was still high (Ganguly et al., 2001, Kanold and Shatz, 2006, Leitch et al., 2005, Liu et al., 2006, Ludwig et al., 2003, Wojcik et al., 2006; see also Fig. 7 in III). The activity- and depolarization- independent KCC2 expression, apart from the intrinsic developmental program, may also be regulated by constitutive secretion of neurotrophic factors (Egan, 2003). Considering that Egr4 increases KCC2 expression in MAPK-dependent manner, it is tempting to speculate that activation of MAPKs by various stimuli will eventually result in KCC2 increase. However, this idea is clearly oversimplified. In some cases when MAPKs are known to be activated, KCC2 levels do not rise and even fall. For example, KA induced seizures increase MAPK phosphorylation (Choi et al., 2003, Crespo-Biel et al., 2007, Ferrer et al., 2002), BDNF levels (Kornblum et al., 1997, Rudge et al., 1998) and Egr4 (Honkaniemi and Sharp, 1999) in adult and neonatal rats (Fig. 8 in III). However, after KA administration, KCC2 levels are downregulated in adult rats (H. Lahtinen, unpublished observation) and upregulated in neonatals (Galanopoulou, 2007 and Fig. 8 in III). It indicates that the mechanism suppressing KCC2 expression interferes with Egr4-induced KCC2 gene activation and may work through one of the multiply additional transcription factors identified in the KCC2 promoter (Fig. 1 in II). 3.4. A novel N-terminal isoform of KCC2 The diversity of CCC family members is increased by alternative splicing. Several isoforms of NKCC1, NKCC2, KCC3 and KCC4 were identified (discussed in 43
section 1.4.1). N-terminal heterogeneity was reported in case of KCC3, including two isoforms that are generated via utilization of alternative promoters and first exons (Mercado et al., 2005). No KCC2 isoforms were identified before our study; however, an analysis of KCC2-deficient mice suggests their existence. Deletion of exon-5 (Hubner et al., 2001) or disruption of exon-4 (Tornberg et al., 2005) result in a complete lack of KCC2 protein. In contrast, disruption of exon-1 produces mice that on average retain ~5% of KCC2 protein level (Woo et al., 2002). This implies that there is a KCC2 isoform that do not include exon-1 and probably utilize alternative promoter. Since regulation of gene transcription by alternative promoters is a common phenomena (Kimura et al., 2006), we analysed the sequence upstream of exon-1 to identify possible promoter regions. 3.4.1. Identification of a new upstream exon in the KCC2 gene
CpG islands are associated with transcription start sites (Larsen et al., 1992). Using CpG islands finding strategy we identified several CpG islands within the previously characterized KCC2 promoter and one CpG island ~7kb upstream of it (Fig. 1 in IV). The latter one was the candidate for an alternative KCC2 promoter. The putative novel exon sequence downstream of the new promoter was highly conserved among several mammalian species (Fig. 2 in IV). Using the upstream CpG island sequence as a query for BLAST search, we found two human expressed sequence tag (EST) clones in which novel exon-1 (now termed exon-1a) was spliced directly to exon-2 and subsequent KCC2 exons. However, no similar mouse ESTs were present in the database. To address whether the transcript 44
of the novel isoform is expressed in mice, we analyzed adult mouse brain samples by RT-PCR. PCR with primers for exon-1a and exon-2 yielded a product of expected size; sequencing of the PCR product further supported a direct splicing of exon-1a to exon-2 (Fig. 1 in IV). Full-length cDNA for the KCC2 isoform containing exon-1a (KCC2a) was obtained by nested PCR. Sequencing of the PCR product confirmed that KCC2a differs from the previously known KCC2 (now termed KCC2b) only by a presence of exon-1a instead of exon1b; all other exons are identical. When analyzing sequence alignment, we found that amino acid residues 3-12 in the exon-1a are highly conserved among several species (Fig. 2 in IV). These residues comprise a putative SPAK-OSR1 binding site. Similar SPAK-OSR1 binding motifs are found in N-terminal parts of NKCC1, NKCC2, KCC1, KCC3a, KCC3b, and KCC4 but not KCC2b (Piechotta et al., 2002). SPAK and OSR1 kinases are implicated in kinetic regulation of cotransporters’ activity (discussed in section 1.4.2). Thus, KCC2a possess a distinct N-terminus that includes SPAKOSR1 binding motif absent from KCC2b. It implies that KCC2a and KCC2b might be differently regulated in brain areas where SPAK/OSR expression is found, e.g. brainstem. 3.4.2. Characterization of KCC2a expression in nervous system
Since the two KCC2 isoforms are driven by different promoters it is possible that KCC2a and KCC2b expression patterns are different. Previous studies used PCR primers, in situ probes, or antibodies that detect both KCC2 isoforms or KCC2b only (see section 1.6). In order to address KCC2a expression specifically, we performed RNA protection assay (RPA)
and quantitative real-time PCR (RT PCR) with KCC2a and KCC2b specific primers. We found that KCC2a, similarly to KCC2b is expressed only in neuronal tissues (figures 3 and 4 in IV). At E17 KCC2a accounted for almost half of total KCC2 levels whereas at P14 KCC2a expression was three to six times lower than KCC2b expression (Fig. 3 in IV). The data on comparative KCC2a/KCC2b expression was indirectly confirmed by the finding that KCC2a promoter is twice less active in cortical neurons than the previously studied KCC2b promoter (Fig. 5 in IV). We found that the developmental KCC2 upregulation occurs because of dramatic increase in KCC2b levels. On the contrary, KCC2a remained relatively stable during development (Fig. 4 in IV). Although the data on KCC2a expression pattern gathered in this study is quite preliminary, it implies that the novel isoform may play an important role in embryonic neurons of spinal cord and brainstem where KCC2a/ KCC2b ratio is rather high. However, more detailed analysis of KCC2a expression, including immunostaining, is required to detect whether high levels of KCC2a are present also at some other specific locations in the brain. 3.4.3. Functionality of KCC2a
Rb flux assay allows direct measurement of the cotransporter activity (Gillen and Forbush, 1999, Payne, 1997). We studied functional 86Rb uptake in HEK293 cells transiently transfected with KCC2a or KCC2b expression vectors. Upon transfection with any of two isoforms, 86Rb uptake increased about 2.5-fold compared to non-transfected, control cells (Fig. 6 in IV). K-Cl cotransporters are sensitive to “loop” diuretics (e.g., furosemide and bumetanide) and are activated by the thiol alkylating reagent N-ethylmaleimide 86
(NEM) (Gamba, 2005). In the case of KCC2a, pretreatment with furosemide (2 mM) reduced 86Rb uptake to nontransfected control level. On the contrary, pretreatment with NEM (1 mM) increased the uptake 2.2 fold (Fig. 6 in IV). Among all KCCs, KCC2 is unique in mediating constitutive K-Cl cotransport because of its distinct C-terminal domain (Mercado et al., 2006). In this study we show that two KCC2 isoforms share the same C-terminal part. Accordingly, both of them can mediate isotonic 86Rb uptake that is comparable to KCC2-mediated 86Rb influx reported in previous studies (Payne, 1997). 3.4.4. KCC2b-deficient mice retain KCC2a mRNA expression
Finally, we analyzed the expression of KCC2a isoform in transgenic mice with targeted disruption of exon-1b (KCC2b-/-; Woo et al., 2002). These mice retain intact exon-1a and 5-8% of normal KCC2 protein levels (at P10-P12). We suggested that the residual KCC2 expression and postnatal survival of KCC2b deficient mice is attributable to KCC2a isoform. Analysis by RT-PCR confirmed that no KCC2b mRNA was expressed in KCC2b-/- brain, while KCC2a mRNA was present (Fig. 7 in IV). Accordingly, the proportion of KCC2b to KCC2a mRNA in KCC2b+/- brain was 50% of that in the wild-type mouse brain. KCC2a mRNA expression remained unchanged between the phenotypes (Fig. 7 in IV). KCC2b deficient mice do not die at birth as full KCC2 knockout mice (Hubner et al., 2001, Woo et al., 2002). Our data suggest that KCC2a expression in KCC2b deficient mice is enough to maintain basic inhibitory functions required for survival. However, KCC2b deficient mice eventually die by the end 45
of second postnatal week due to repetitive spontaneous seizures. Presumably, KCC2a levels in the cortex are too low to maintain higher Cl- extrusion capacity required for hyperpolarizing inhibition in mature cortical neurons. Indeed, cortical neurons isolated from KCC2b deficient mice show impaired Cl- extrusion (Zhu et al., 2005). In summary, we have identified a novel KCC2 isoform with distinct N-terminus generated by the usage of the alternative promoter and first exon. N-terminal part of KCC2a but not of previously known KCC2b possesses putative SPAK/OSR binding motif. KCC2a encode a protein
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with isotonic K-Cl cotransport activity that is similar to the activity of KCC2b isoform. KCC2a mRNA is expressed in the brain and contributes 20-50% of total KCC2 mRNA in neonatal brainstem and spinal cord. During development KCC2a mRNA expression remains stable in contrast to KCC2b mRNA that is strongly upregulated. We also show that the mice with targeted disruption of exon-1b have intact KCC2a mRNA levels and we speculate that KCC2a ensures survival of these mice after birth in contrast to full KCC2 knockout.
4. CONCLUSIONS The main findings of this thesis are: 1. KCC2 expression is upregulated during development of hippocampal neurons in vitro. This upregulation does not require synaptic activity. 2. KCC2 promoter contains multiple putative transcription factor binding sites, one of which, Egr4, is able to enhance KCC2 transcription in cultured neurons. 3. In developing neurons in vitro, KCC2 expression is increased by BDNF- or neurturin. Growth factor -dependent KCC2 increase requires MAPK activation and Egr4 binding to KCC2 promoter. BDNF and neurturin also upregulate KCC2 protein levels in vivo, when injected in hippocampus of neonatal rats. 4. Neuronal activity affects KCC2 mRNA expression in neonatal rat hippocampus in vivo. A period of KA-induced seizures increases Egr4 and KCC2 mRNA levels in a MAPK-dependent manner. Reduction of spontaneous activity by tetanus toxin decreases KCC2 protein expression. 5. KCC2 gene produces at least two isoforms different in their promoter region and alternatively spliced first exon. Similarly to the previously known KCC2b isoform, the novel KCC2a isoform is expressed in the CNS. However, KCC2a is not upregulated during postnatal rodent brain development. KCC2a accounts for a significant part of total KCC2 levels in neonatal brainstem and spinal cord and prolongs survival of KCC2b deficient mice for two weeks in comparison to the mice lacking both KCC2 isoforms.
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ACKNOWLEDGMENTS This study was carried out at the Institute of Biotechnology and Department of Environmental and Biological Sciences, University of Helsinki, under the supervision of Dr Claudio Rivera, Professor Kai Kaila and Professor Mart Saarma, during 20002008. The study was supported by Academy of Finland, Finnish Graduate School for Neuroscience and the Center for International Mobility (CIMO), Finland. I would like to express my sincere appreciation to many people who professionally and personally participated in realization of this thesis. I am deeply grateful to Dr. Claudio Rivera, the leader of our group, for his supervision and assistance, patience and kindness, endless positive attitude and exceptionally good sense of humor. Daily exciting discussions with Claudio shaped my scientific interests and taught me to think beyond the narrow topic of current research. Especially I would like to thank Claudio for creating and maintaining the vibrant and inspirational atmosphere in our multinational group that made my PhD experience truly exceptional. I am deeply thankful to Professor Kai Kaila, my supervisor and head of Finnish Graduate School for Neuroscience, for giving me the opportunity to be his student, for scientific guidance and encouragement, fruitful discussions and substantial help in many difficult situations. I am also very grateful to Dr. Katri Wegelius, the coordinator of Finnish Graduate School for Neuroscience, for arranging many interesting courses and social events for the School students and for always being there when anyone of us needed help or advice. I express my sincere gratitude to Professor Mart Saarma, my supervisor and head of the Institute of Biotechnology, for valuable recommendations and suggestions during the course of the study, preparation of the manuscripts and work on the thesis text. In addition, excellent facilities and resources of the Institute deserve special mention and I am thankful to Mart for providing such a superb environment for research work. I would like to acknowledge Professor Kristian Donner for his valuable guidance through formalities of University bureaucracy starting from my first days as a student and for rescuing me every time I failed to remember the deadline for academic term registration. I warmly thank the official reviewers of my thesis, Professor Dan Lindholm and Professor Hans Gerd Nothwang, for giving their time to pre-examine and revise the manuscript. I also thank my coauthors: Marika Markkanen, Matti Airaksinen, Stanislav Khirug, Leonard Khiroug, Peter and Anne Blaesse, Kristiina Huttu, Juha Voipio, Chunlin Cai, Sarah Coleman, Kari Keinänen, Julia Kolikova, Ramil Afzalov, Eric Delpire, Priit Pruunsild, Tõnis Timmusk, and Sebastian Shuschmann for a very fruitful collaboration. I cordially thank my colleagues from “Neuro group”: Maria Lindahl, Pia Runenberg-Roos, Päivi Lindholm, Johan Peränen, Maili Jakobson, Urmas Arumäe, Mari Heikkinen, Satu Åkeberg and many others for stimulating working environment and for the help in many practical issues. I extend my thanks to all the personnel at the Institute of Biotechnology, with a special mention to Timo Päivärinta for his excellent computer and graphic assistance. 48
My deepest gratitude goes to the former and actual members of “Rivera’s group”, Hong Li, Judith Thomas Crusells, Hannele Lahtinen, Marjo Heikura, Miika Palviainen, Sergey Smirnov, Anastasia Shulga, Shetal Soni, Olaya Llano, Ana Cathia Magalhaes, and Tero Rosenqvist, for creating relaxed working atmosphere and for the nice time we all shared together in the lab and outside it. I would like to especially acknowledge Miika and Marjo for their exceptional technical expertise, assistance in my research and never-ending fun in the lab. At the Institute and outside it, I was lucky to meet many Russian friends, whose warm welcome and continuous support made me feel at home from my very first day in Helsinki. I would like to thank all of you, as “no road is long with a good company”. I thank Andrey Golubtsov, Natalia and Evgeny Kulesskiy, Julia Kolikova, Ramil Afzalov, Dmitri Chilov, Yulia Sidorova, Konstantin Ivanov and Olga Samuilova for our interesting and sometimes “hot” discussions, lunches in “Kartano”, and for sincere joy life that you share with me. My warmest gratitude goes to Dmitry Poteryaev, Anna Popsueva, Alexey Veselov, Maxim Bespalov, Svetlana Vasilieva, Ivan Pavlov and Elena Kushnerenko, for helping me to organize my life in Helsinki, for our “sauna” and “mökki” parties and for a long-term friendship. I express my sincere affection to my life-time friend Maria Semenova and her husband Artur for their infinite support and care. I am deeply grateful to my parents Lyudmila Popova and Vladimir Popov for their trust, help, encouragement, and love. You have always been a shining example for me. I also want to thank my children, Dmitri and Elizaveta, for their unconditional love and infinite patience, as, I believe, nobody suffered from my thesis more then them. Lately, but enormously, I want to thank my husband Pavel Uvarov for sharing every moment of my life in the lab and at home. Thank you for your ideas and advices, for our late-evening discussions, for your undivided attention to my needs and for immense help. Continuous feeling of togetherness brings the meaning to my life.
Helsinki, November 2008
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