The great Breakthrough in your life comes when you realize it that you can learn anything

you need to learn to accomplish any goal that you set for yourself. This means there are no

limits on what you can be, have or do.

Albert Einstein

To my Family

List of articles

I Bjurstöm H, Wang J, Ericsson I, Bengtsson M, Liu Y, Kumar-Mendu S, Issazadeh-Navikas S, Birnir B. GABA, a natural immunomodulator of T lymphocytes. J Neuroimmunol. 2008 Dec 15;205(1-2):44-50. Epub 2008 Oct 26.

II Mendu SK, Akesson L, Jin Z, Edlund A, Cilio C, Lernmark A,

Birnir B. Increased GABA(A) channel subunits expression in CD8(+) but not in CD4(+) T cells in BB rats developing diabetes compared to their congenic littermates. Mol Immunol. 2011 Jan;48(4):399-407. Epub 2010 Nov 26.

III Suresh K Mendu, Amol Bhandage, Zhe Jin, Bryndis Birnir.

Different subtypes of GABA-A receptors are expressed in hu-man, mouse and rat T lymphocytes resulting in different sensi-tivity to drugs. PLoS One (under revision).

IV Henrik Ring, Suresh K Mendu, Shahrzad Shirazi-Fard, Bryn-

dis Birnir and Finn Hallböök. GABA maintains the prolifera-tion of progenitors in the developing chick ciliary marginal zone and non-pigmented ciliary epithelium. PLoS One (in press).

Reprints were made with permission from publishers

Articles not included in thesis

V Jin Z, Jin Y#, Kumar-Mendu S#, Degerman E, Groop L, Birnir B. Insulin reduces neuronal excitability by turning on GABA(A) channels that generate tonic current. PLoS One. 2011 Jan 14;6(1):e16188.

VI Romanico Arrighi, Jonas M Fuks, Suresh K Mendu, Zhe Jin,

Bryndis Birnir, Antonio Barragan. GABAergic signaling is linked to a hypermigratory phenotype in dendritic cells infected by Toxoplasma gondii. PLoS Pathogens( under revision).

# equal contribution

Table of Contents

Introduction ................................................................................................... 11 1. The Discovery of GABA ...................................................................... 11 2. GABA Channels ................................................................................... 12

2.1. GABAA Receptors ........................................................................ 12 2.2. GABAB Receptors ........................................................................ 12

3. Ligand-Gated Ion Channels .................................................................. 13 4. The GABAA Channel Structure ............................................................ 13 5. Distribution of GABAA Channels ........................................................ 15 6. The GABAA Channel Function ............................................................ 16 7. Two modes of GABA Generated Currents ......................................... 16

7.1.a. Phasic inhibitory currents .......................................................... 16 7.1.b. Tonic inhibitory currents ........................................................... 17 7.2 Sources of extracellular GABA ................................................... 17 7.3 The Extrasynaptic GABAA channel composition .......................... 18

8. Pharmacology of GABAA Channels ..................................................... 19 8.1. The GABA binding site ................................................................ 19 8.2. Benzodiazepines ........................................................................... 19 8.3 Neurosteriods ................................................................................. 20 8.4 Other allosteric ligands .................................................................. 20

9. Biosynthesis, Release and Uptake of GABA ....................................... 20 10. GABA and GABAA channels outside the brain- focus on the pancreatic islets ........................................................................................ 22 11. Immunology of T cells and autoimmunity ......................................... 23 12. Retinal Stem cells ............................................................................... 25

12.1. GABAergic signalling in Retinal progenitor cells ...................... 26 13. Autoimmune diseases ......................................................................... 27

13. 1. Multiple Sclerosis (MS) ............................................................. 27 13. 2. Type 1 Diabetes (T1D) .............................................................. 28

14. Hypothesis of GABA modulation of T cells ...................................... 29 15. Cross-talk between the nervous system and the immune system ....... 30 16. GABAergic components in immune cells .......................................... 31

Aims of this Thesis ....................................................................................... 33

Experimental strategy ................................................................................... 34 1. Experimental Animals and Ethics ................................................... 34

2. Real-time PCR ................................................................................ 34 3. Proliferation assay ........................................................................... 36 4. Western blot ..................................................................................... 37 5. Immunocytochemistry ..................................................................... 37 6. Patch-clamp Recordings .................................................................. 38 7. Port-a-Patch ..................................................................................... 39 8. Statistics ........................................................................................... 40

Results and Discussion ................................................................................. 41 Paper I ...................................................................................................... 41

Results ................................................................................................. 41 Discussion ............................................................................................ 42

Paper II ..................................................................................................... 43 Results ................................................................................................. 43 Discussion ............................................................................................ 43

Paper III .................................................................................................... 44 Results ................................................................................................. 44 Discussion ............................................................................................ 46

Paper IV ................................................................................................... 46 Results ................................................................................................. 46 Discussion ............................................................................................ 47

Conclusions ................................................................................................... 48

Acknowledgements ....................................................................................... 49

References ..................................................................................................... 52

Abbreviations

APC Antigen Presenting Cells

HSC Hematopoietic Stem Cells

SSADH Succinic semialdehyde dehydrogenase

[3H]-Thymidine Tritiated Thymidine

5-HT 5-hydroxytryptamine

BB Bio Breeding rats

BBB Blood-Brain-Barrier

BZ Benzodiazepines

CA1 Cornu Ammonis 1

CMZ Ciliary Marginal Zone

CNS Central Nervous System

CRAC Ca2+ release-activated Ca2+ channels

CSF Cerebrospinal fluid

CT Cycle Threshold

CTL Cytotoxic T lymphocyte

DR++ Diabetic Resistant

DRlyp/lyp Diabetic Prone

EAE Experimental Autoimmune Encephalomyelitis

EGF Epidermal growth factor

ER Endoplasmic Reticulum

FGF2 Fibroblast growth factor

GABA Gamma-aminobutyric acid

GABAA Gamma-aminobutyric acid receptor type A

GABAB Gamma-aminobutyric acid receptor type B

GABARAP Gamma-aminobutyric acid receptor-associated protein

GABA-T GABA Transaminase

GAD 65/67 Glutamate decarboxylase 65/67

GAT 1 -4 GABA transporter 1-4

GluCl Glutamate-gated chloride channel

GLUT2 Glucose transporter 2

GODZ Golgi-specific zinc finger protein

HAP-1 Huntington-associated protein 1

IL-4 and IL-5 Interleukin 4 and 5

IPSC Inhibitory Post Synaptic Currents

KCC2 potassium chloride co-transporter 2

LGIC Ligand-gated ion channels

MBP Myelin Basic Protein

MHC I & II Major Histocompatibility Complex MLN Mesenteric Lymph Nodes MOD-1 Modulation of locomotion defective

MOG Myelin Oligodendrocyte glycoprotein

MS Multiple Sclerosis

nAChR nicotinic acetylcholine

NK cells Natural killer cells

NKCC1 Na-K-Cl Co-transporter

NOD Non-Obese Diabetic mice

NPE Non-pigmented epithelium

PCR Polymerase Chain Reaction

PLN Pancreatic Lymph Nodes

PLP Proteolipid protein

PP-MS primary progressive-MS

RPE Pigmented epithelium

RR-MS relapsing- remitting-MS

RT Reverse Transcription

SLMV Synaptic-like micro vesicles

T & B lymphocytes Thymus and Bone marrow derived lymphocytes

T1D Type 1 Diabetes

TCR T CELL Receptors

TH1, TH2 , TH17 helper T cells type 1, 2 and 17

THDOC 5a-pregnan-3a-21-diol-20-one

TLR Toll-like receptor

TM (1-4) Trans Membrane (1-4)

TNF Tumor necrosis factor

TREG Regulatory T cells

ZAC Zinc-activated ion Channel

ZnT8 Zinc transporter 8

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Introduction

Membranes are essential for organisms. They compartmentalise by defining a boundary where specific ways are needed to cross the barrier. The plasma membrane contains proteins such as ion channels, pumps and transporters that have evolved to transport ions across the membrane. Ion channels are found virtually in all cells and are essential for physiology. Ion channels are pores where ions travel through down their electrochemical gradient. Ion pumps on the other hand, use the energy of ATP and transport ions against the electrochemical gradient. In coupled transporters, movement of ions against its electrochemical gradient is powered by the downhill movement of another ion species (Ashcroft 2006). These transport proteins participate in diverse processes such as nerve and muscle excitation, hormone secretion, cell proliferation, sensory transduction, learning and memory, regulation of blood pressure, salt and water balance, lymphocyte proliferation, fertilization and cell death (Ashcroft 2000). An example of plasma membrane with pro-teins, phospholipids and cholesterol looks like a fluid-mosaic (Figure 1).

Figure 1. The fluid-mosaic model of the plasma membrane contains proteins (ion channels, ion pumps and ion transporters), phospholipids, carbohydrates and choles-terol

1. The Discovery of GABA Amino acids are not only primordial units of protein structure but also oper-ate many intricate biochemical pathways that are important for the living organism. The amino acid, γ−aminobutyric acid (GABA) is readily derived from glutamate and acts as a chemical signal in protozoans, bacteria, meta-

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zoans, plants and vertebrates (Krnjevic 2010). In 1950 a key discovery illus-trated that the brain contains large amounts of an amino acid called GABA of unknown function (Awapara, Landua et al. 1950). Later in 1954, Florey’s experimental work from the crayfish extracts of cortex and spinal cord found unknown inhibitory agent (Factor I) that suppressed the firing of neurons (Florey 1954). Afterwards, Florey in collaboration with Elliott concluded that the purified Factor I contained GABA and fully accounted for the in-hibitory activity (Bazemore, Elliott et al. 1956). After various types of ex-perimental approaches such as iontophoresis, intracellular recordings and pharmacological tests, it was concluded that GABA is the principal inhibi-tory transmitter in the mammalian central nervous system (CNS) (Florey 1954).

2. GABA Channels Basic activity of nerves is determined by the balanced activity of two sys-tems, the excitatory glutamate system and the inhibitory GABA system in the CNS. The inhibitory neurotransmitter GABA exerts its action through GABA receptors. Based on the pharmacology, GABA receptors are classi-fied into GABAA and GABAB Receptors.

2.1. GABAA Receptors GABAA receptors are ion channels that are opened by GABA and a selective agonist muscimol. The competitive antagonists bicuculline and SR-95531 binding to the GABA binding site results in decreased GABAA channel cur-rents. Picrotoxin is a non-competitive blocker, blocks the GABAA channel pore. Barbiturates and benzodiazepines enhance the GABAA channel activity (Macdonald and Olsen 1994; Olsen and Sieghart 2008). There is an excep-tion in the pharmacology of GABAA channels containing the ρ(1-3) sub-units. They have different pharmacological sensitivity. These are not blocked by bicuculline nor modulated by benzodiazepines or barbiturates. They are sensitive to picrotoxin and neurosteriods. The ρ (1-3) subunits can form homomeric and also heteromeric channels (Johnston 2005). GABAA chan-nels modulatory drugs are commonly used to treat anxiety disorders, insom-nia, epilepsy, aggressive behaviours, and are used to induce anaesthesia.

2.2. GABAB Receptors GABAB receptors are G-protein coupled receptors activated by GABA and a selective agonist is baclofen. These are coupled to effector mechanisms such as adenylate cyclase system, Ca2+ and K+ ion channels by G proteins (e.g. Gi/o (Bowery, Bettler et al. 2002)). GABAB receptors exist as a heterodimer

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with two dissimilar seven transmembrane subunits comprising the functional receptor (Bowery and Smart 2006).

3. Ligand-Gated Ion Channels The super family of ligand-gated ion channels (LGIC) contains the Cys-loop receptors such as GABAA channels and glycine channels, that are selective for negative ions and are inhibitory, and the nicotinic acetylcholine (nAChR), 5-hydroxytryptamine (5-HT R) and Zinc-activated ion channels (ZAC), that are selective for positive ions and are excitatory in vertebrates. The Cys-loop superfamily of ion channels are also found in invertebrates for example, glutamate-gated chloride channel (GluCl) and MOD-1 that are permeate to negative ions whereas EXP-1 is permeate to positive ions (Les-ter, Dibas et al. 2004; Miller and Smart 2010). Each subunit has N-terminus extracellular domain containing the ligand binding site and four putative transmembrane regions (TM1- TM4) (Ortells and Lunt 1995). Family mem-bers have high degree of similarity of amino acid sequences (30%) and have highly characteristic sequence motifs at the N-terminus domain such as a 15-residue Cys-loop formed by two Cys residues (Cockcroft, Osguthorpe et al. 1990).

4. The GABAA Channel Structure The asymmetric GABAA channel is formed by co-assembly of five subunits (Figure 2). Each subunit has a large extracellular hydrophilic N-terminus, followed by four transmembrane regions (TM1-4) with a large intracellular loop between TM3-TM4 and ends with a short extracellular C-terminal. TM 2 forms the lining of the channel pore. The large intracellular loop between TM3 and TM4 contains phosphorylation sites and consensus sequences for interactions with a variety of proteins (Figure 3). Intracellular and trans-membrane proteins that interact with the intracellular domains can affect the fate, function and pharmacology of the GABAA channels (Birnir and Korpi 2007). For example, GABARAP interacts with the GABAA channel γ and β2 subunit that helps in the regulation of delivery of the GABAA channel to the surface after activity (Jacob, Bogdanov et al. 2005). Gephyrin regulates the clustering of γ2 containing (Essrich, Lorez et al. 1998) or α2 containing (Tretter, Jacob et al. 2008) GABAA channel at synapses.

GABA binds at the α/β subunits interface in the N-terminal domains (Figure 4) (Korpi, Grunder et al. 2002; Birnir and Korpi 2007). A number of positively charged residues (Arg and Lys) are clustered around the extracel-lular domain of TM1 and TM2 segment. These residues play a role in ion selectivity and concentrate ions within the channel vestibule (Kash, Trudell

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et al. 2004). To date, nineteen GABAA channel subunit genes have been cloned. These are α1−6, β1−3, γ1−3, δ, ε, θ, π, ρ1−3 of eight different classes (Olsen and Sieghart 2008). There is an exception in birds, chicken additionally expresses β4 and γ4, but lacks θ and ε. The number of GABAA

channel subunit genes that can form the channel complex is the largest one of any among the mammalian ion channel receptors families. In humans, the GABAA channel subunit genes are distributed among five chromosomes in clusters. The gene sequence of θ is closely related to β and ε to γ. Therefore, the θ and ε subunits are considered as β-like and γ-like. The rat and mouse GABAA channel subunit genes are similarly clustered as the human genes (Simon, Wakimoto et al. 2004; Uusi-Oukari and Korpi 2010).

In theory, it is possible to form many GABAA channel combinations from the 19 subunits along with multiple splice variants of some of the subunits. But the current knowledge eliminates many combinations and restricts the number of combinations (Olsen and Sieghart 2008). GABAA channel sub-types are formed from two copies of α, two copies of β, and often one copy of another subunit for example, γ, δ, or ε (Sieghart and Sperk 2002). The diversity of the subunits combinations is determined by GABAA channel subunits that assemble from their component subunits in the endoplasmic reticulum (ER). The selective oligomerisation of GABAA channel subunits with the N-terminus controls particular combinations (Kittler, McAinsh et al. 2002). The other subunits that are not used are retained in the ER and de-graded by ubiquitylation. The GABAA channel subunits molecular weight ranges from 43-66 kDa and the estimated molecular weight of GABAA channel complex is approximately 300 kDa (Michels and Moss 2007).

Figure 2. GABAA channel complex. An example of the pentameric GABAA chan-nel formed from 2α :2β : one γ. GABAA channel has two GABA binding sites at the interface of α/β , a binding site for benzodiazepines at the interface of α/γ2. Neuros-teroids bind to site in the membrane spanning transmembrane regions of the sub-units.

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Figure 3. Each GABAA subunit consists of four transmembrane domains (TM1-TM4). TM2 is believed to line the pore of the channel. There is a large extracellular NH2-terminal and a large intracellular loop between TM3-TM4, with phosphoryla-tion sites and protein interactions motifs.

Figure 4. The binding sites for GABA and the benzodiazepines (BZ) are shown. GABA binds between α/β whereas the BZs bind between α and γ2 subunits.

5. Distribution of GABAA Channels The mRNA of each of the GABAA receptor subunits is distinctively distrib-uted. Some of the subunits are ubiquitously expressed throughout brain whereas others are confined to only specific regions (Laurie, Seeburg et al. 1992). For example, the GABAA α1 subunit is almost ubiquitously present in the brain, the α5 is highly expressed in the hippocampus whereas the α6 is restricted to cerebellar granule cells (Wisden, Laurie et al. 1992). The expression of the β2 subunit strongly correlates with the expression of the α1 subunit, the γ2 is also expressed throughout brain (Wisden, Laurie et al. 1992). The ε and θ mRNA are strongly concentrated in the monoaminergic neurons (Sinkkonen, Hanna et al. 2000) and π mRNA is not identified in brain but present in peripheral organs such as uterus (Hedblom and Kirkness 1997). It was confirmed by using subunit-specific antibodies that α1β2γ2 subunits containing GABAA channels are the most abundant subtype of GABAA channel in brain, similar but less abundant subtypes are α2βγ2, α3βγ2, α4βγ2, α5βγ2, α6βγ2 and α6βδ GABAA channels (Olsen and Sieghart 2009).

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6. The GABAA Channel Function GABAA channels are anion-selective channels permeable to Cl- ions. In mature neurons, opening of GABAA channels generally results in inhibition as the equilibrium potential is for Cl- near the neuronal resting membrane potential (Martin and Olsen 2000). GABAA channels also conduct HCO3- with variable permeability (Kaila, Lamsa et al. 1997). Immature neurons contain higher intracellular [Cl-] that leads to excitation whereas in mature neurons, intracellular [Cl-] is gradually reduced resulting in neuronal inhibi-tion (Ben-Ari, Gaiarsa et al. 2007). The intracellular [Cl-] is strongly regu-lated by two cation chloride co-transporters, the NKCC1 transporter accu-mulates chloride whereas the KCC2 exports chloride. The NKCC1 takes up chloride by internalisation of one Na+, one K+ in exchange for two Cl- in electroneutral coupled fashion (Payne, Rivera et al. 2003). In immature neu-rons, the NKCC1 isoform is highly expressed and plays the main role in maintaining high intracellular chloride. On the other hand, KCC2 extrudes K+ and Cl- using energy in the electrochemical gradient of K+ and is ex-pressed in mature neurons. It maintains the low intracellular Cl– concentra-tion (Delpire 2000).

7. Two modes of GABA Generated Currents The GABA inhibitory mechanism is essential for information processing in the brain and it can be classified into two types 1. phasic inhibition and 2. tonic inhibition. GABAA channels can be located at synapses are called syn-aptic channels or outside of synapses where they are then termed extrasynap-tic channels. Phasic inhibition is mediated by synaptic GABAA channels whereas tonic inhibition is mediated by extra- and perisynaptic GABAA

channels (Semyanov, Walker et al. 2004; Birnir and Korpi 2007; Glykys and Mody 2007).

7.1.a. Phasic inhibitory currents The inhibitory information passes in a control manner across synapses from one neuron to another in a neuronal network. The phasic inhibition is the fundamental inhibitory mechanism that synchronizes neuronal network and maintains synaptic integrity (Semyanov, Walker et al. 2004). The synaptic GABAA channels are low affinity channels and activated by millimolar con-centrations of GABA. The GABA concentrations in the synaptic cleft is around 3 mM (Mozrzymas, Barberis et al. 1999). The GABA released from the presynaptic terminals of a neuron acts on GABAA channels that are lo-cated in the post-synaptic terminal and generates inhibitory post synaptic current (IPSC) within milliseconds, the phasic inhibition (Birnir and Korpi 2007; Brickley and Mody 2012) Figure 5.

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7.1.b. Tonic inhibitory currents Tonic inhibitory mechanism was identified and characterised in hippocampal dentate gyrus granule cells (Birnir, Everitt et al. 1994) and cerebellum (Kaneda, Farrant et al. 1995). Later it was confirmed that it is present throughout the brain but in a cell-type specific manner. Tonic currents were recorded e.g. in cerebellar granule cells, when application of GABAA recep-tors antagonist reduced the resting membrane conductance and produced an inwards shift in the baseline current (Semyanov, Walker et al. 2004). Tonic currents are activated by high-affinity GABAA channels with picomolar to nanomolar concentration of GABA (Lindquist and Birnir 2006; Jin, Jin et al. 2011). Tonic currents in neurons are dependent on the extracellular concen-tration of GABA and the affinity of the GABAA channels that are present in the neuron (Birnir, Everitt et al. 1994). These extrasynpatic GABAA channels activate slowly and generate the tonic currents with a latency from GABA exposure (Mody 2001; Birnir and Korpi 2007; Brickley and Mody 2012). The tonic current modulates basic excitability of neuronal networks and the extrasynaptic GABAA channels are also sites for many drugs important in a wide range of neurological disorders (Semyanov, Walker et al. 2004; Birnir and Korpi 2007; Jin, Jin et al. 2011).

Figure 5. A diagrammatic representation of various components of the GABA signaling system.

7.2 Sources of extracellular GABA Tonic currents are due to low ambient GABA concentration that is present in the extracellular space. The extracellular concentration of GABA in the brain ranges from 10 pM to 2.5 μM (Lerma, Herranz et al. 1986; Tossman and Ungerstedt 1986; Jin, Jin et al. 2011). The extracellular GABA concen-tration is maintained by several processes. 1. Diffusion of GABA out of syn-

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apses to extrasynaptic locations (Semyanov, Walker et al. 2003; de Groote and Linthorst 2007) 2. GABA can be released by astrocytes (Liu, Schaffner et al. 2000) 3. Decrease the GABA transaminase activity increase the GABA concentration (Overstreet and Westbrook 2001) 4. Vesicular release such as reverse transport by GABA transporters (Brickley and Mody 2012) 5. Non-vesicular GABA release for example, GABA passes through bestrophin channels (Lee, Yoon et al. 2010). In GABA transporter 1 (GAT-1) knockout mice, the tonic current is increased whereas the synaptic current is decreased (Jensen, Chiu et al. 2003). The extracellular concentration of GABA in hip-pocampus can also be altered with increased stress or exposed to novel envi-ronment (de Groote and Linthorst 2007).

7.3 The Extrasynaptic GABAA channel composition Tonic currents are mediated by a variety of GABAA channel subtypes in specific neuronal populations. In cerebellar granule cells the GABAA chan-nels containing the α6 and δ subunits generate tonic conductance (Semyanov, Walker et al. 2004). GABAA channel containing δ subunit exhibits smaller single channel conductance and much longer open time and slow rate of desensitization on exposure to GABA for prolong time (Saxena and Macdonald 1994). It was observed that δ subunit containing GABAA channels are highly sensitive to endogenous neurosteriods (Lambert, Belelli et al. 2003). GABAA channels containing α5βγ2 subunits generate tonic conductance in CA1 region of the hippocampus (Caraiscos, Elliott et al. 2004; Jin, Jin et al. 2011). Interestingly, it was recently shown that insulin can also induce high-affinity extrasynaptic GABAA channel in the CA1 region of hippocampus (Jin, Jin et al. 2011). With studies of single channel conductance, it was shown that tonic currents were mediated by high-affinity GABAΑ channel containing α1βγ2 or α4βγ2 (Lindquist and Birnir 2006) in hippocampal dentate gyrus granule cells. Tonic current is also mediated by extrasynaptic α4βδ subunit containing GABAΑ channel in dentate gyrus granule cells (Stell, Brickley et al. 2003). From the above studies, it is con-cluded that tonic inhibition is often mediated by α4, α5, α6, δ and γ2 sub-units containing GABAA channels but even α1 can sometime contribute to the tonic currents.

Extrasynaptic GABAA channel are targets for many drugs such as anaes-thetics, sleep-promoting drugs, neurosteriods and alcohol. Since these extra-synaptic GABAA channels do modify basic neuronal excitability, they are potential therapeutic targets in diseases like schizophrenia, epilepsy, stress and pregnancy related mood disorders and Parkinson’s disease.

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8. Pharmacology of GABAA Channels Many drugs targeting GABAA channels have been used to treat CNS dis-eases such as generalized anxiety disorders, sleep disorders, muscle spasms and seizures. Their main role is to enhance the neuronal inhibition by GABA. Currently benzodiazepines, barbiturates and anaesthetics are used in the clinics but also experimentally to identify native GABAA channel sub-types by their pharmacology.

8.1. The GABA binding site The initial GABAA channel recombinant studies in Xenopus oocytes showed that both the α and the β subunits were obligatory for GABAA channel acti-vation (Schofield, Darlison et al. 1987). Further experiments identified a specific residue in the α subunits (F65) as a critical determinant of GABA binding (Smith and Olsen 1994). It was also demonstrated that tyrosine resi-dues (Y157 and Y205) in the β subunits were involved in the binding of GABA (Amin and Weiss 1993). It appears that the GABAA channel has two GABA binding sites located at the interface between the α and the β sub-units (Kash, Trudell et al. 2004; Olsen and Sieghart 2009). Many of the ex-trasynaptic GABAA channels such as the α5βγ2, α4βγ and α6βδ have high-affinity for GABA and contribute to the tonic inhibition in neurons (Olsen and Sieghart 2008). Competitive antagonists of GABA at GABAA channel are SR-95531 (gabazine) and bicuculline (Hamann, Desarmenien et al. 1988; Lindquist, Laver et al. 2005; Sedelnikova, Erkkila et al. 2006). A non-competitive antagonist that can block the GABAA channel is picrotoxin (Ehrenberger, Benkoe et al. 1982; Sedelnikova, Erkkila et al. 2006). In this study GABA, SR-95531 and picrotoxin were used to assess the GABAA channel activity.

8.2. Benzodiazepines The benzodiazepines (BZ) bind at the interface of the α and γ2 subunits. Although the β subunit is needed to form the GABAΑ channel, it does not greatly affect the sensitivity of the BZ site-ligand (Hadingham, Wingrove et al. 1993). The benzodiazepine sensitivity is higher towards γ2 containing GABAΑ channel than those containing γ1 subunit. BZs do not bind to chan-nels containing the α4 or α6 subunits. BZ do increase frequency of opening, mean open time and conductance of channel gated by GABA. In saturating concentration of GABA, the Cl- conductance is not increased further (Eghbali, Curmi et al. 1997). The GABAA channels cannot be opened with BZ alone but need GABA first to open the channel that it is then modulated by the BZ (Rudolph and Knoflach 2011). Both genetic and medicinal ap-proaches have been used to dissect the pharmacological characteristics of

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GABAA channel subunits. Gene-knockout experiments have revealed some important information and also uncovered some limitations. Much of the current knowledge about GABAA channels was obtained from point muta-tion studies. A point mutation in α1, α2, α3 or α5 subunits from histidine-to-arginine abolish the binding of diazepam but preserve the GABA binding (Rudolph, Crestani et al. 1999). These point mutation experiments demon-strated that α1 mediated the sedative, anteriograde amnestic actions; α2 mediated anxiolytic-like and myorelaxant actions; α3 mediated anxiolytic action; α5 mediated enhance performance and memory (Wingrove, Thomp-son et al. 1997; Rudolph, Crestani et al. 2001; Dias, Sheppard et al. 2005; Rudolph and Mohler 2006; Rudolph and Knoflach 2011). However, knock-out mutation of α6 caused upregulation of K+ channels and down regulation of other GABAA subunits, and β3 and γ2 knockouts result in death of neo-nates (Rudolph, Crestani et al. 2001).

8.3 Neurosteriods Endogenous steroid hormones and their synthesised metabolite such as 5α-pregnan-3α-ol-20-one (5α3α-THPROG) and 5α-pregnan-3α-21-diol-20-one (5α-THDOC) are potent positive allosteric modulators of the GABA chan-nels (Barker, Harrison et al. 1987; Lambert, Cooper et al. 2009). It was dem-onstrated that in the presence of GABA, low concentrations of steroids in-crease the GABAA channel mean-open time. However, high concentration of steroids alone is enough to activate GABAA channels (Callachan, Cottrell et al. 1987; Lambert, Cooper et al. 2009). The extrasynaptic GABAA channels containing α4βδ and α6βδ are highly sensitivity to THDOC (Wohlfarth, Bianchi et al. 2002). In paper II, THDOC was used to study the extrasynap-tic-like GABAA channel in T lymphocytes.

8.4 Other allosteric ligands In addition to the benzodiazepines and neurosteroids, other allosteric modu-lators for the GABAA channels such as the general anaesthetics, like barbitu-rates, etomidate, propofol are used for the identification of specific sub-types of GABAA channels (Olsen and Sieghart 2009). Drugs that target the GABAA channels are used to treat disorders like Angelaman's syndrome, anxiety disorders, autism, depression, mania, premenstrual syndrome, schizophrenia and sleep disorders (Korpi and Sinkkonen 2006).

9. Biosynthesis, Release and Uptake of GABA The GABA shunt is a process to produce and conserve the supply of GABA (Figure 6). The principal source of GABA is glucose and to some extent

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pyruvate and other amino acids act as precursors. The first step in the pro-duction of GABA is the transamination of α-ketoglutarate, a Krebs cycle intermediate to L-glutamic acid by the enzyme GABA transaminase (GABA-T). Glutamic acid decarboxylase (GAD) catalyses the conversion of L-glutamic acid to GABA by decarboxylation. GABA can then be metabo-lised to succinic semialdehyde by GABA-T. Succinic semialdehyde can be oxidised to succinic acid by succinic semialdehyde dehydrogenase (SSADH) and then re-enters the Krebs cycle (Bernard W Agranoff et al 1999).

GABA is released into the synapses from the presynaptic terminal of a neuron by depolarisation in a Ca2+-dependent manner. GABA is transported from the cytosol into vesicles and these vesicles are docked at the plasma membrane. When the presynaptic terminal is depolarised by the action po-tentials, voltage-gated Ca2+ channels open and result in increase of the intra-cellular Ca2+ levels. The influx of Ca2+ leads to the fusion of GABA contain-ing vesicles with the plasma membrane and release GABA into the synaptic cleft (C. Tanaka 1996). There is some evidence for non-vesicular release of GABA at extrasynaptic sites (Mody 2001). GABAergic drugs usually exert their action by increasing the apparent affinity for GABA resulting in chan-nel activity at lower GABA concentration. The GABA concentrations in the synaptic cleft is around 3 mM (Mozrzymas, Barberis et al. 1999). GABA re-uptake from the synapses is mediated by GABA transporters. They require extracellular Na+ ions and Cl- ions and are capable of bidirectional transport. Four GABA transporters have been cloned and are named GAT1, GAT2, GAT3 and GAT4. These transporters are important in relation to fine-tuning of GABAergic neurotransmission and to avoid desensitization of the GABAA channels (Schousboe and Waagepetersen 2007).

Figure 6. The metabolic pathway of the synthesis and the degradation of GABA. Enzymes are GABA-T (GABA transaminase), GAD (Glutamic acid decarboxylase), SSADH (succinic semialdehyde dehydrogenase).

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10. GABA and GABAA channels outside the brain- focus on the pancreatic islets In contrast to the central and peripheral nervous system, GABA and GABAA channels are also present in several non-neuronal tissues such as the pancreatic islets, oviduct, liver, kidney, skin, spermatozoon and blood cells where they may participate in a wide range of physiological processes. All these tissues do contain components of GABA signalling system (Erdo and Wolff 1990).

Figure 7. The GABA signalling system in the human pancreatic islets: GABA is produced and co-released from pancreatic β cells with insulin whereas both the alpha and β cells have GABAA channels

Outside the brain, high concentration of GABA can be found in the pan-creatic islet β cells that are present in the small clusters of islets of Langerhans. Each islet consists of four different kinds of endocrine cells. The most abundant cell types in human pancreas are the insulin producing β cells (54%) followed by glucagon producing alpha cells (35%) then the somatostatin producing delta cells (11%) (Brissova, Fowler et al. 2005) and finally low number of pancreatic polypeptide-producing cells (PP cells) are present. The islets are abundantly vascularised (Bonner-Weir and Orci 1982). GAD is present in human pancreatic islets β and delta cells (Gladkevich, Korf et al. 2006) (Figure 7). Pancreatic β cells release GABA by vesicular and non-vesicular process (Braun, Ramracheya et al. 2010). GABA causes inhibitory action on glucagon and somatostatin secretion by activation of GABAA channel on α-cells and δ cells (Rorsman, Berggren et al. 1989; Pizarro-Delgado, Braun et al. 2010). A recent study claims that GABA acts on β cells in autocrine fashion activat-

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ing cell growth and survival signalling pathway (Soltani, Qiu et al. 2011). Furthermore, GABA prevents and reverses the diseases by enhancing β cell growth and survival in T1D models (Soltani, Qiu et al. 2011). GABA exerts immunomodulatory effect by decreasing cell proliferation, inflammatory cytokines and increasing regulatory T cells (Bjurstom, Wang et al. 2008; Jin, Mendu et al. 2011; Mendu, Akesson et al. 2011; Soltani, Qiu et al. 2011). Therefore, GABA may have protective function for beta cell when invaded by immune cells.

11. Immunology of T cells and autoimmunity Pluripotent hematopoietic stem cells (HSC) are divided into: common lym-phoid progenitor and common myeloid progenitor. Lymphoid progenitor gives rise to white blood cells or leukocytes which includes natural killer cells (NK cells) and the T and B lymphocytes. Myeloid progenitor cells give rise to remaining leukocytes like monocytes, dendritic cells, neutrophils, eosinophils and basophils, and erythrocytes and platelets. Monocytes enter tissues where they differentiate into macrophages (Murphy, Travers et al. 2007).

Common lymphoid progenitors migrate to the thymus through blood and mature there (Figure 8). They are then called thymus-dependent lympho-cytes or T cells. The thymus is located in the upper anterior thorax above the heart. It has three regions; the outer cortex, the inner medulla and a unique microenvironment for the T cell development called thymic stroma. The progenitors receive a signal from the stroma through Notch signalling that determines whether the cell becomes a B cell or a T cell and defines other choices like α:β versus γ:δ type and whether to develop into the CD4+ or CD8+ cell type. About 50 million thymocytes are generated each day but only 1-3 percent leaves the thymus each day as fully mature T cells. Early thymocytes do not have any CD3, CD4 or CD8 surface markers and these are called double negative thymocytes (Takahama 2006).

Thymocytes give rise to two cell lineages; the minority γ:δ and the major-ity α:β CD3. The development of α:β CD3 proceeds through several stages and forms both CD4+ and CD8+ T cells. These are called double positive cells, can divide and form the population of small double-positive cells which initially express low levels of T cell receptors (TCR). Most of the TCR cannot recognize self-peptide: self MHC molecules on thymic stroma and will fail positive selection. As a result, the cells will die and then macro-phages in the thymus will clear away the dead cells. Elimination of these T cells in the thymus prevents autoreactivity. On the other hand, double posi-tive cells that can recognize self peptide: self MHC molecules develop into single positive CD4 or CD8 or regulatory thymocytes (Takahama 2006; Murphy, Travers et al. 2007).

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Figure 8. T cell development

The mature T cells that express CD4 have receptors that recognise peptide bound to self-MHC class II molecule and are programmed to become cyto-kine-secreting cells. In contrast, cells that recognise peptide bound to self-MHC class I molecule and are programmed to become CD8 cytotoxic effec-tor cells. These are exported to peripheral lymphoid organs through the blood and then recirculate between blood and lymphoid tissue. A population of these mature T cells that have not encountered their specific antigen are known as naïve T cells. These naïve cells are continuously in recirculation surveying the peripheral lymphoid organs. When a naïve CD8+ T cell recog-nises an antigen: MHC I complex on a cell, it is activated and causes the death of the cell, hence they are called cytotoxic T cells (CTL). In contrast, CD4+ T cells are differentiated into TH1, TH2 , TH17 and TREG. The TH1 cell recognises the antigen: MHC II complex on macrophages and releases in-flammatory cytokines including interferon-γ and TNF which are associated with autoimmune diseases. The TH2 cell recognises the antigen: MHC II complex on the naïve B-lymphocytes and induces IgE class switching by cytokines like IL-4 and IL-5 (Murphy, Travers et al. 2007; Zhu and Paul 2008). Autoimmune responses develop against self-antigens (autoantigens) and give rise to autoreactive effector cells and autoantibodies that lead to a variety of chronic syndromes called autoimmune disease. There are at least three dif-ferent mechanisms to discriminate between self and non-self. The first is an immature lymphocyte recognises antigen that distinguishes between self and non-self, this leads to a negative signal that causes lymphocytes death or inactivation. This is an important mechanism of inducing self-tolerance in the thymus and the bone marrow developing lymphocytes is called as central tolerance. The second is based on high and constant antigen concentration that is expressed by every cell in the body and provides a strong signal to

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lymphocytes resulting in tolerance to the antigen. The third mechanism for differentiating between self and non-self is based on innate immunity that provides signals which are crucial for adaptive immune response. In this situation, the naïve lymphocytes encounter self-antigen, when the antigen presenting cells (APC) do not express co-stimulatory molecules. This leads to a negative, inactivation signal. It is an important mechanism for antigens that are encountered outside the thymus and the bone marrow. Peripheral tolerance is induced once the mature cell leaves the central lymphoid organs. Breakdown of natural tolerance mechanisms can be due to genetic suscepti-bility or environmental triggers such as infections resulting in the condition termed autoimmunity (Kamradt and Mitchison 2001; Murphy, Travers et al. 2007).

12. Retinal Stem cells The retina develops from an evagination of the ventral diencephalon. A bi-layered cup-shaped optic vesicle is formed by the evagination. The optic vesicle folds inwards to form a bi-layered cup, the optic cup. The outer layer of the cup becomes the retinal pigmented epithelium (RPE) and the inner layer becomes the retina. In the chicken, the majority of the cells within the retina are generated within the first 10 days of embryonic development. This proc-ess starts at embryonic day 2 and completed at embryonic day 12 (Prada, Puga et al. 1991). As the development continues, progenitor cells within the retina proliferate and differentiate into neurons in a highly ordered manner. Neuronal differentiation begins in central region of the retina and proceeds to the peripheral regions (Fischer and Reh 2003). At the peripheral edge of the retina, new neurons are generated by a population of stem cells and this region of the eye is called the ciliary marginal zone (CMZ) (Fischer 2005). Adjacent to the CMZ, is a distinct anatomical structure known as the ciliary body. It is derived from the optic cup. The ciliary body contains two layers, the outer one is a pigmented epithelium and inner layer is a non-pigmented epithelium (NPE). The ciliary body is subdivided into two regions pars plana and the pars plicata. In the chicken, cells at the retinal margin and the ciliary body express retinal progenitor markers (Reh and Fischer 2001). The pro-genitors in the CMZ are capable of producing new neurons in the presence of growth factors (Reh and Fischer 2001). NPE cell proliferation is normally quiescent but the proliferation can be stimulated by the growth factors such as insulin, EGF and FGF2 (Fischer and Reh 2003). Some cells in the NPE of the ciliary body are capable of generating neurons in vivo (Reh and Fischer 2001). Thus, NPE cells may be a source of neural stem cells that could be used to regenerate the retina. In paper IV, NPE cells were used to study the role of GABAA channels.

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Figure 9. A diagram of embryonic eye and mature eye CMZ (ciliary marginal zone), retinal pigmented epithelium (RPE). Adapated from Thomas A. Reh’s Labo-ratory, University of Washington.

12.1. GABAergic signalling in Retinal progenitor cells Retinal progenitors are multipotent cells and give rise to the different neu-ronal cell types of the retina. The retinal development is perfectly regulated throughout proliferation and cell-cycle exit. The retinal development is regu-lated by environmental factors, growth factors or neurotransmitters (Martins and Pearson 2008). There is some evidence for that the proliferation of retina progenitor cells is modulated by neurotransmitter such as glutamate (Mar-tins, Linden et al. 2006). Another neurotransmitter GABA, has been shown to regulate cell proliferation of various stem and progenitors cells. Chicken neurons abundantly express the GABA synthesising enzyme, GAD and they are already present between embryonic day 6 (E6) and E9 (Calaza, Gardino et al. 2006). At E6, GABA positive neuroblast-like cells are found in the chick developing retina (Hokoc, Ventura et al. 1990). A number of different GABAA channels are expressed in the developing retina (Martins and Pearson 2008). In the chick retina at E3, GABAA channels evoke changes in intracellular Ca2+ levels (Yamashita and Fukuda 1993). Activa-tion of the GABAA channels in chicken neuronal precursors results in de-creased cell proliferation (Salazar, Velasco-Velazquez et al. 2008). Further-more, it was demonstrated that GABAA channel signalling through histone variant H2AX controls negatively in mice ES and peripheral neural crest stem cell proliferation (Andang, Hjerling-Leffler et al. 2008). Therefore, GABA has an important role in neural development by controlling cell divi-sion and proliferation.

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13. Autoimmune diseases 13. 1. Multiple Sclerosis (MS) MS is the prototype of immune-mediated demyelinating diseases of the cen-tral nervous system affecting 0.05-0.15% of Caucasians (Noseworthy, Luc-chinetti et al. 2000; Trapp and Nave 2008). The prevalence of the disease varies and is at least, in part, based on the genetic background. Its preva-lence is highest in Caucasians but is less common in Asian or African popu-lations (Fox, Jenkins et al. 2000). MS usually starts between 20 and 40 of age affecting more women than men. There are two forms of the disease, relapsing-remitting-MS (RR-MS) and primary progressive-MS (PP-MS) (Sospedra and Martin 2005).

There are many brain proteins that can induce the disease. One of the proteins is Myelin Basic Protein (MBP). It has five isoforms, from differen-tial splicing which can be found in peripheral lymphoid organs. Experimen-tal Autoimmune Encephalomyelitis (EAE) can be induced with MBP in mouse and rat strains, and non-human primates. Another protein is the Pro-teolipid protein (PLP). It is the most abundant protein in myelin comprising more than 50% of CNS myelin protein. It has two main transcripts. The third protein is myelin oligodendrocyte glycoprotein (MOG). It is a less abundant myelin protein and is a transmembrane glycoprotein of the immunoglobulin superfamily (Wekerle, Kojima et al. 1994). These three proteins form the myelination around the axons and help in the conduction of action potential impulses. During disease progression, the myelin sheath is damaged by the inflammatory immune cells. As a result, axons no longer conduct impulses all the way down to the synapse (Neumann 2003).

MS is an autoimmune disease mainly mediated by CD4+ TH1 cells (Haf-ler 2004). This view stems from the infiltrated cellular composition that is observed in the brain and cerebrospinal fluid (CSF) of MS patients and in the EAE animal models (Sospedra and Martin 2005). In the EAE models, im-munisation with myelin antigens of susceptible animals leads to a CD4+T-mediated autoimmune diseases that is similar to MS (Hemmer, Archelos et al. 2002; Hafler 2004). Similarly, adoptive transfer of isolated immune cells from sick animal to naïve animal results in the disease again by CD4+ T cells (Hemmer, Archelos et al. 2002). It was thought that the brain was an im-mune privileged site. Recent studies in infection and autoimmune diseases show that systemic inflammation and tissue damage leads in microglia acti-vation. As a result, release of inflammatory mediators and upregulation of MHC molecules on CNS cells. Neuronal antigens are released into the lymph nodes via blood where these are processed and presented to T cells or B cells by the antigen presenting cells (APC) such as dendritic cells. The T or B cells now proliferate and are released from the lymph nodes. The primed cells migrate throughout the body and cross the blood-brain-barrier

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(BBB) at places where these cells re-encounter their priming antigen. These reactivated cells are recruited to the brain and now cause tissue damage, demyelination and degeneration (Hemmer, Archelos et al. 2002). In addition, infectious agents may induce MS by at least two mechanisms. (1) molecular mimicry in which the autoreactive T and B cells get activated by cross react-ing between self-antigens and foreign antigens during infection and migrat-ing across the blood-brain-barrier (2) bystander activation, autoreactive cells are activated by inflammatory cytokines, super antigens and molecular pat-tern recognition, e.g. Toll-like receptor activation (TLR) (Sospedra and Mar-tin 2005). It is known that MS patients have low levels of GABA and GAD in blood (Demakova, Korobov et al. 2003). It was demonstrated that antigen presenting cells express function GABAA channels and immune cells have machinery for GABA synthesis and degradation. The manipulation of GABA signalling in MS can ameliorate the Experimental Autoimmune En-cephalomyelitis (EAE) by inhibition of inflammation (Bhat, Axtell et al. 2010).

13. 2. Type 1 Diabetes (T1D) Type 1 diabetes develops as a consequence of combinations of genetic and unknown environmental factors. It is often called juvenile-onset diabetes as T1D usually starts at an early age (van Belle, Coppieters et al. 2011). It is an autoimmune organ-specific destruction of the insulin producing β cells in the islets of Langerhans within the pancreas (Anderson and Bluestone 2005; Bluestone, Herold et al. 2010). The β cells are glucose sensing bodies that release insulin to maintain physiological normal glucose levels. Once the β cells destruction happens, the control of blood glucose is lost which results in critical complications such as ketoacidosis, kidney failure, heart disease and blindness (Bluestone, Herold et al. 2010).

Two key model animals of T1D are the Bio Breeding rats (BB) (Schranz and Lernmark 1998) and the Non-Obese Diabetic mice (NOD) (Makino, Kunimoto et al. 1980). These models have been used to study effects of ge-netics, pathophysiology and environment factors on the incidence of T1D. Studies on NOD mice demonstrated that the disease may occur as a result of immune disregulation and consequences such as proliferation of autoreactive CD4+ and CD8+ T cells, autoantibody producing B lymphocytes and activa-tion of the innate immune system that collaborate to destroy the insulin se-creting β-cells. There is evidence indicating that autoreactive T cells have a major role in the initiation of disease and progression in NOD mice and the human disease. T cells are presumably activated in the pancreatic -draining - lymph nodes (PLN) because of high turnover of β cells in the islets leading to antigen presentation. Further damage of islets causes the release of self-antigens, proliferation of autoreactive T cells and eventually infiltrating T cells that are present at the time of the disease. The major auto-antigens are

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the insulin, GAD and the Zinc transporter 8 (ZnT8) (Stadinski, Kappler et al.).

The discovery of GAD autoantibodies in the pancreatic islets has raised the question of the role this protein has in autoimmune type 1 diabetes. The destruction of the insulin producing β cells can be mediated by GAD65-reactive cytotoxic T lymphocytes (Yoon, Yoon et al. 1999). Two forms of GAD (GAD65 and GAD67) are present. GAD65 is mainly membrane asso-ciated and present to the cytoplasmic membrane of the synaptic-like micro vesicles (SLMV) whereas GAD67 is mostly distributed throughout the cyto-plasm (Chessler, Simonson et al. 2002). Mice islets express mainly GAD67 while human islets and rat islets predominantly express GAD65 (Yoon, Yoon et al. 1999; Chessler and Lernmark 2000). GAD and GABA antibodies are present in the sera of type 1 diabetes patients (Solimena, Folli et al. 1990) and autoantibodies and T cells reactive to GAD65 have been detected in early diagnosed diabetes patients (Ludvigsson 2009).

14. Hypothesis of GABA modulation of T cells Lymphocyte activation causes an increase in intracellular Ca2+ through a cascade of events from internal stores and thereby induce cytokine secretion and cell proliferation (Figure 10) (Smith-Garvin, Koretzky et al. 2009). It is well known that T lymphocytes have a variety of K+ channels and Ca2+ re-lease-activated Ca2+ (CRAC) channels (Panyi, Varga et al. 2004). Calcium released from internal stores triggers the activation of the CRAC channel that is in the plasma membrane. Calcium enters the cell through this channel in a process called store-operated Ca2+ release-activated Ca2+ entry (SOCE) (Oh-hora and Rao 2008). Calcium accumulation is dependent on the chemi-cal and electrical gradient across the cell membrane. An increase in influx of intracellular calcium is believed to happen when K+

Ca2+ channels open as the K+

v channel cause hyperpolarisation of the membrane and thus increase of the electrical driving force on Ca2+ entering the cell (Chandy, Wulff et al. 2004). On the other hand, GABAA channels opening by GABA will cause depolarisation of cell due to high concentration of (30-40 mM) intracellular Cl- (Pilas and Durack 1997; Bjurstom, Wang et al. 2008) and thereby de-crease the influx of calcium (Tian, Chau et al. 1999; Tian, Lu et al. 2004). The intracellular Ca2+ concentration does therefore not increase as much in the presence of active GABAA channels and this result in decreased cytokine secretion and cell proliferation. Therefore, GABAA channels on autoreactive T lymphocytes could possibly regulate inflammation and T1D disease ag-gression.

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Figure 10. An illustration showing the T cell with GABAA channel and other ion channels. Calcium is the key player in the T cell proliferation and cytokine secretion. Activation of GABAA channel in presence of GABA, regulates negatively the influx of calcium and decrease the T cell proliferation. CRAC- Ca2+ release-activated Ca2+

channel, Kv - voltage-gated potassium channel, KCa+2 - Ca+2 –activated potassium

channel.

15. Cross-talk between the nervous system and the immune system The immune system and the nervous system communicate through messen-gers including the neurotransmitters. These two systems are connected ana-tomically and physiologically, including 'hardwiring' of sympathetic and parasympathetic nerves to the main sites of immune system in the liver, spleen, bone marrow, thymus, lymph nodes, skin and gastrointestinal system (Steinman 2004). Therefore, immune cells may be exposed to a variety of signals secreted from nerve terminals. There are many studies that show various neurotransmitters receptors are expressed on T lymphocytes (Levite 2012). Activation of these receptors can lead to a variety of responses in-cluding cytokine secretion, chemotactic migration and suppression of infec-tious diseases, proliferation, cytotoxicity, ion currents and increased intracel-lular Ca2+ concentration (Levite 2008). The T cells themselves even some-times can secrete neurotransmitters spontaneously or when induced by ex-ternal stimuli (Levite 2008). Indeed, whether the neurotransmitters action results in activation or suppression of the T lymphocytes is related to the type of neurotransmitter (Levite, Chowers et al. 2001). It appears that the GABA is the main inhibitory neurotransmitter in the nervous system and GABA is an immunomodulatory molecule in the immune system. In both cases GABA appears to the systems to maintain balance activity. T cell re-ceptor activation, cytokines and the neurotransmitter itself can all regulate the expression of the neurotransmitter signal system in T cells (Levite, Chowers et al. 2001).

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16. GABAergic components in immune cells Immune cells have machinery for the GABA synthesizing and degrading enzymes and they do express GABAA channel subunits and the GABA transporters (Jin, Mendu et al. 2011). But, there are relatively few groups that have studied the GABA signalling system in immune cells and in par-ticular, for CD4+ and CD8+ T cells there are very few studies. I will briefly summarize what is known. GABA and its synthesising enzyme have been detected in dendritic cells and macrophages (Bhat, Axtell et al. 2010) and GAD67 in the human peripheral lymphocytes (Dionisio, Jose De Rosa et al. 2011). The GABA degrading enzyme, GABA-T is found in macrophages, T cells (Bhat, Axtell et al. 2010) and human peripheral lymphocytes (Dion-isio, Jose De Rosa et al. 2011). GABAA channel subunits have been de-tected in the immune cells. In NOD mice α1, α2, β1, β2, γ3 and δ GABA subunits were identified (Tian, Lu et al. 2004) and in EAE mice α1,β1,γ2 and e subunits were identified in T cells (Bhat, Axtell et al. 2010). In macrophages α1, α2, β3 and δ subunits were detected (Reyes-Garcia, Hernandez-Hernandez et al. 2007). My own results show GABAA subunits in human, mice, rats and autoimmune diseases models (Bjurstom, Wang et al. 2008; Mendu, Akesson et al. 2011). In human PBMC expression of α1, α3, α4, β2, β3, γ2, δ and ε mRNA was detected (Alam, Laughton et al. 2006). In neurons, GABA co-transporters (GAT1-4) are important for uptake of GABA from synapses. In mice, GAT-1 was detected and knockout of GAT-1 increases EAE symptoms in T cells. Furthermore, it was demon-strated that GAT-1 negatively regulates T cell activation through PKC-dependent signalling pathway (Wang, Feng et al. 2008; Wang, Luo et al. 2009). GAT-2 mRNA and protein were detected in CD4+ T cells and macrophages (Bhat, Axtell et al. 2010). Both of the GAT 1 and GAT 2 were identified in human peripheral lymphocytes (Dionisio, Jose De Rosa et al. 2011). Only two studies have shown GABA-activated currents in immune cells apart our group. GABA application results in decrease cell proliferation and cytokine secretion (Tian, Chau et al. 1999; Tian, Lu et al. 2004; Bjurstom, Wang et al. 2008; Mendu, Akesson et al. 2011). T regulatory cells (Treg) downregulate the immune system and it was shown that GABA in-creases the regulatory T cells number in diabetic mice (Soltani, Qiu et al. 2011). It was demonstrated that GABA decreases the influx of calcium in activated human PBMC (Tian, Lu et al. 2004; Alam, Laughton et al. 2006). Recently it was shown that oral administration of GABA downregulated inflammatory responses in rheumatoid arthritis and obesity (Tian, Dang et al. 2011; Tian, Yong et al. 2011). GABA treatment delayed the incidence of type-1 disease in NOD/scid mice and multiple sclerosis (Tian, Lu et al. 2004; Bhat, Axtell et al. 2010).

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From these recent studies it is clear that the GABA signalling system is ac-tive in immune cells and has a function in regulating T cells proliferation and inflammatory cytokine secretion.

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Aims of this Thesis

The long-term objective of the project was to discover the role of GABA and GABAA channels in T lymphocytes and retina stem cells. The specific aims were:

I To examine if GABAA channel subunits are expressed in auto anti-gen-specific EAE cell line from multiple sclerosis model mice and if functional GABAA channels are formed. Determine if physio-logical concentrations of GABA modulate cell proliferation.

II To examine if CD4+ and CD8+ T cells from type 1 diabetes model

rats, DRlyp/lyp and DR+/+, express GABAA channel subunits and form functional GABAA channels. Determine if there was any dif-ference between the cell-types in terms of GABAA channel sub-types expressed and if physiological concentrations of GABA modulated cell proliferation.

III To investigate if there were any differences recorded in the

GABAA channels subunit isoforms expressed in CD4+ and CD8+ T cells from humans, mice, rats and human leukaemia T cell lym-phoblast (Jurkat cell line).

IV To investigate the effects of GABA and GABAA channels on the

proliferation of chicken NPE cells.

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Experimental strategy

1. Experimental Animals and Ethics Paper I: B10.RIII mice were used in this study. They are susceptible con-genic strains that developed experimental autoimmune encephalomyelitis (EAE) after immunization with myelin basic protein (MBP). Lymph nodes were collected from the immunized mice, T cells isolated and used for fur-ther experiments.

Paper II: Bio Breeding congenic rats of DR++ and DRlyp/lyp strains were used in this study. DR++ strains do not develop diabetes whereas DRlyp/lyp strains develop diabetes after 53 days in our animal centre. Mesenteric lymph nodes (MLN) were collected from prediabetic and diabetic DRlyp/lyp that were age matched DR++, T cells isolated and used further experiments.

Paper III: Adult Wistar rats and C57Bl/6J mice were used in this study and mesenteric lymph nodes were collected. For Human pancreatic lymph nodes: adipose tissue containing pancreatic lymph nodes (PLN) from cadaver donor were provided by the Nordic Islet Transplantation program (www.nordicislets.org) by the courtesy of prof. Olle Korsgren, Uppsala Uni-versity. T cells were isolated and used further experiments.

Paper IV: Embryonic day 12 chicken eyes were obtained from White Leg-horn chicken. Non-pigmented cells (NPE) were dissected from retina and used for further experiments.

Ethics: All the tissue collection protocols were approved by Malmö-Lund, Uppsala region animal ethics committees.

2. Real-time PCR Reverse Transcription (RT) followed by Polymerase Chain Reaction (PCR) has proven a powerful tool for the detection and quantification of mRNA. Real Time RT-PCR has been widely and increasingly used because of its sensitivity, good reproducibility and many applications (Bustin 2000). Real Time PCR means the method of collecting data throughout the PCR process as it happens, thus combining amplification and detection in a single step. It

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is the most sensitive method for detection and quantification of gene expres-sion levels of low abundance or low concentration of mRNA in limited sam-ple (Pfaffl, Horgan et al. 2002). In general, two chemistries have been used for the quantification of the amplicon 1. Gene-specific fluorescent probes, e.g. TaqMan Probes, enable the detec-tion of a specific PCR product as it accumulates during the PCR process. 2. Specific double stranded DNA binding agents, e.g. SYBR Green I, detect PCR products by binding to the double stranded DNA amplified during PCR. When SYBR Green I containing reaction mix is added to a sample, it is available with low fluorescence (Figure 11). During PCR, the target gets amplified in the presence of DNA polymerase. The SYBR Green I dye then binds to each new copy of dsDNA resulting in an increase in the fluores-cence intensity proportional to the amount of double stranded PCR product produced. For SYBR Green I assays, appropriate designed primers should be used to avoid false positive results. An ideal primer should span between exon-exon junction to avoid amplification of target gene in genomic DNA. However, it is not possible if the gene of interest has no intron. In that case, it is necessary to run RT minus control (Applied Biosystems 2009).

Figure 11. SYBR principle: A pre-made reaction mixture contains SYBR Green I, cDNA, primers, enzyme. After PCR reaction fluorescent signals attain in proportion to the PCR product in the form of cycle threshold (CT).

In this thesis, RT-PCR was used to detect the mRNA expression of GABAA channel subunits from various sources such as brain, CD4+ and CD8+ T cells of mesenteric lymph nodes from rats and mice and pancreatic lymph nodes from humans. Total RNA was isolated and complimentary DNA (cDNA) was synthesised. It is essential to measure the RNA qualitatively and quanti-tatively prior to RT. Random hexamers and Oligo(dT) were used to obtain a faithful cDNA. This cDNA was used as a template for amplification of dif-ferent genes of interest with mRNA specific primers. An important point here is to consider the variation between samples, pipette errors, RNA isola-tion, cDNA synthesis. To overcome all these problems, real time PCR data is

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usually normalized against a control gene often called the reference gene. The ideal reference gene should be expressed identically in all experimental conditions, in different tissues or cell types and unaffected by treatment. The most common reference genes are GAPDH, albumins, actins, tubulins, cyclophilin, microglobulin, 18S rRNA. The endpoint quantification for Real time PCR is the threshold cycle (CT). The CT is defined as the PCR cycle at which fluorescent signal crosses the threshold. The CT, a numerical value is inversely related to the amount of desired amplicon product in the reaction. Depending on the assay methods such as absolute or relative quantification the Real Time PCR data can be reported in copy number or change in ex-pression of the target gene. Here in this thesis, relative expression has been used to represent the data. When presenting RT-PCR data from gene expres-sion studies, it should be presented as 2-∆Ct rather than raw CT values (Ap-plied Biosystems 2009). ∆CT comes from the normalization process where ∆CT=CT (gene of interest) - CT (internal control OR reference gene) (Schmittgen and Livak 2008). Another method of presenting quantitative real-time PCR data is the comparative CT method (2-∆∆CT). The advantage of the comparative CT method is ability to present the data as fold change in expression (Livak and Schmittgen 2001). Fold Change = 2-∆∆CT

2-∆∆ct = [(CT gene of interest- CT reference gene) sample A - [(CT gene of interest- CT reference gene) sample B

In the study, I used the SYBR Green I method to quantify the GABAA recep-tor subunit's mRNA and the primers were designed using NCBI/ Primer-BLAST or from published articles. The primers pairs are appended at the end of each article. The data presented in this thesis as either 2-∆CT or 2-∆∆CT

3. Proliferation assay It is a technique that can be used to assess the ability of lymphocytes to pro-liferate in presence of various stimuli. It can be achieved by measuring the cell number present in the culture before and after the addition of antigen, antibodies or mitogens that stimulate proliferation. This method can be used to assess of the cell biological reactions or effects of drugs on the prolifera-tion in presence of stimuli. The most commonly used method to estimate increase in cell number is based on incorporation of [3H] thymidine into DNA, a process which is closely related to the underlying changes in cell number (Muul, Silvin et al. 2008). In this thesis, proliferation assay was used to determine the effects of GABA on the proliferating T lymphocytes. In Paper I, stimulation was initiated with Myelin Basic Protein (MBP(89-101)) or

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purified protein derivate whereas in Paper II stimulation was initiated with anti-rat CD3 mAbs. In Paper IV, effects of several drugs on the proliferating NPE cells were examined.

4. Western blot It is a method used to identify specific proteins from cell lysate by using a protein specific antibody. It has three major steps: 1. separation of the pro-teins based on the protein size from cell lysate, this is done using gel electro-phoresis, 2. transfer of the protein to a membrane, 3. detection of the protein by a specific antibody. Once detected, the target protein can be visualized as a band. In this thesis, Western blot was used to identify proteins from T lymphocytes in Paper III. T lymphocytes, Jurkat cells and brain protein lys-ate were extracted and subjected to separation using 10% polyacrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% fat-free milk for 1 h and incubated with primary antibody (dilutions listed in Table 1) overnight at 4oC. After washing, the secondary antibody was added and protein bands were identified.

Table 1. Primary antibodies used in Western Blot

Primary antibody

Specificity Molecular

weight (kDa) Host

Primary antibody dilution

Company

GABAA R α(1−6)

α1 (100%), α2 (80%), α3 (71%), α4 (76%), α5 (71%), α6 (55%)

51-62 Rabbit 1: 200 Santa Cruz

Biotechnology

GABAA R α1 100% 52 Rabbit 1:1000 Synaptic Systems

GABAA R β3 100% 54 Goat 1:500 Santa Cruz

Biotechnology

GABAA R γ2 100% 54 Rabbit 1:500 Synaptic Systems

5. Immunocytochemistry It is a method for localization of antigens (proteins) in cells using a specific antibody binding to the antigen thereby allowing visualization and examina-tion with a microscope. In 1955, Coons studied for the first time the cellular protein distribution with fluorescent markers (Goldstein and Watkins 2008). In this thesis, immunocytochemistry was used in Paper III to locate presence of GABAA channels. In brief, cells were fixed with 2% paraformaldehyde and block with 5% BSA for 30 min on ice. Then the cells were incubated with a primary antibody overnight at 4oC (dilutions according to the Table

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2). After washing, fluorophore conjugated secondary antibody was added and the cells incubated for 1 h at 4oC. Finally, the cell nucleus was stained with DAPI. The cells were examined with a confocal microscopy ( LSM, Carl Zeiss, Germany). Negative controls where cells that had not been ex-posed to the primary antibody. Table 2. Primary antibodies used in immunocytochemistry

Primary Antibody Host Dilution Company

GABAAR α1 Rabbit 1:50 Novus Biologicals

GABAAR α2 Rabbit 1:500 Synaptic Systems

GABAA R β3 Mouse 1:500 Synaptic Systems

GABAA R γ2 Rabbit 1:500 Synaptic Systems

6. Patch-clamp Recordings The Patch-clamp technique allows the study of ion channels in cells such as neurons, pancreatic islets, immune cells and cell lines. In the late 1970, the patch-clamp technique was developed by Erwin Neher and Bert Sakmann. They received Noble Prize in Physiology or medicine in 1991 for the dis-covery. The Patch-clamp technique can be used to measure either current, voltage or membrane capacitance of a cell. In the current-clamp method, voltage changes are measured by injecting of currents into the cell. The volt-age-clamp is used to measure ion currents changes across the membrane at a set potential also called the holding potential. In this thesis, the voltage-clamp method was used to measure GABAA currents in T lymphocytes in Paper I, II and III and NPE cells in Paper IV.

There are four patch-configurations used to record currents: 1. Cell-attached (ca) 2. Inside-out (io) 3. Whole-cell (wc) and 4. outside-out (oo) (Figure 12).

Cell-attached configuration: It is a basic configuration for patch-clamp

experiments. A glass micropipette with a sharp tip with a diameter less than 1 µm is used to seal on to the cell membrane. When a positive pressure is released or a gentle suction is applied, a tight giga-ohm (GΩ) seal forms. This is the optimal method to measure single-channel currents, as the cell interior is intact. The pipette solution can contain the drug that will be used to study the channels. The disadvantage of the cell-attached configuration is that is not able to exchange solutions in the pipette easily.

Inside-Out configuration: After forming the cell-attached configuration,

withdraw the glass micropipette from the cell. This results in ripped-off patch containing the membrane. The channel in the cell membrane is ex-posed to the external solution at the intracellular phase of the membrane.

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This configuration is useful for studying the internal milieu of the channel or applies lipophilic drugs like benzodiazepines that can cross the membrane.

Figure 12. Figures showing different forms of patch-clamp configurations.

Whole-cell configuration: When one has attained the cell-attached configura-tion, gently apply suction to the pipette interior. This results in the rupture of the cell membrane. As a result, the solution in the glass electrode interior has now access to the cell internal milieu. Thus, the ionic composition can be exchanged with the pipette solution. This configuration is useful to measure total channel activity in the cell and is hence called whole-cell configuration.

Outside-out configuration: After forming the whole-cell configuration, if the glass micropipette is withdrawn slowly, the cell membrane can reform at the end of tip. So that outside of the cell membrane became outside of the patch membrane at the tip of the pipette. This method is sometimes used to measure single-channel activity.

In this thesis, I have used the cell-attached, the inside-out and the whole-cell configuration to measure single-channel GABAA currents and whole-cell GABAA currents in Paper I, II and IV.

7. Port-a-Patch Port-a-Patch is a miniaturized patch-clamp setup where a cell is automati-cally captured and sealed by suction using a computer-controlled pump. This simplified protocol to form the whole-cell configuration. First, a sus-pension of cells is placed on the chip when applying gentle suction, a single cell is captured to aperture. Like in conventional patch-clamp, application of negative pressure to the aperture initially forms tight giga seal and then for-mation of whole-cell configuration by rupture of cell membrane. All this procedure can be done by pre-defined protocol using the software or, of

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course, it can be done manually. It combines an improved throughput with high quality data (Farre, Haythornthwaite et al. 2009; Nanion and Technolo-gies 2009). In paper III, Port-a-Patch instrument was used to measure whole-cell GABAA currents in rat T lymphocytes and Jurkat cell lines.

8. Statistics Paper I: The changes in the proliferation were calculated using Student’s t-test. Paper II: The fold change of RT-qPCR data was presented as mean ± SEM. The comparisons were assessed with one-way ANOVA and Student’s t-test. Paper III: The mRNA expressions were presented as mean ± SEM. The comparison between were made using Kruskal-Wallis one-way ANOVA on ranks, post-hoc Tukey test. Paper IV: One-way ANOVA, Tukey multiple-comparison post-hoc was used.

In all cases, P-value < 0.05 was considered as significant.

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Results and Discussion

Paper I Results It is well known that GABAA receptors are abundantly expressed in the brain (Birnir and Korpi 2007; Olsen and Sieghart 2008). Here we examined if the encephalitogenic CD4+ T cells from Experimental Autoimmune Encephalo-myelitis (EAE) mice express GABAA receptor subunits. We used RT-PCR to examine expression of GABAA channel subunits in the T cells. We de-tected α1, α4, β2, β3, γ1 and the δ GABAA channel subunits. Further, we examined intracellular proteins expressed in CD4+ T cells. It is known that specific intracellular proteins may affect the fate, function and pharmacology of the GABAA channels in neurons (Everitt, Luu et al. 2004; Birnir and Korpi 2007). The PCR result showed that the encephalitogenic CD4+ T cells expressed mRNA for intracellular proteins such as GABARAP, gephyrin, HAP-1 and GODZ. We further examined the mRNA expression of GABAA channels and intracellular proteins in antigen-specific activated T cells and naïve T cells. Interestingly, activated CD4+ T cells did not express mRNA for the GABAA β2 and γ1 subunits whereas the remaining subunits and in-tracellular proteins were unaltered as compared to naïve T cells. Next, we examined if the GABAA channels were activated by low physiological con-centrations of GABA (1 µM) in the resting and activated encephalitogenic CD4+ T cells. The whole-cell current recordings showed that GABA-activated currents were generated and were blocked with a GABA competi-tive antagonist (SR-95531) in both activated and naïve T cells (holding po-tential -50 mV). The GABAA currents in T cells behaved as currents gener-ated by extrasynaptic neuronal GABA-activated channels that usually open after a delay and have high-affinity for GABA (Lindquist and Birnir 2006). Then single-channel currents were recorded in an intact cell using cell-attached configuration. The results showed that extrasynaptic-like channels were activated after a latency of 1.9 min and had the average maximal-conductance of 34 pS. Finally, we examined, if proliferation of encephalito-genic CD4+ T cells were modulated by the low physiological concentration of GABA. The plasma GABA level is about 100 nM in healthy individuals (Bjork, Moeller et al. 2001). The T cells were stimulated in vitro with the MBP(89-101) peptide in presence 0.1, 1 and 1000 µM concentrations of

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GABA. Controls cells were not exposed to the MBP(89-101) peptide. The pro-liferation of the T cell stimulated by the MBP(89-101) peptide was significantly decreased by all the three concentrations of GABA.

Discussion This study shows that GABAA channel subunits are expressed in CD4+ T cells of EAE mice. These subunits can form functional GABAA channels and the channels are activated by low physiological concentrations of GABA. The proliferation of antigen stimulated CD4+ T cells was inhibited by GABA. The role of GABA and GABA channels are well studied in the CNS (Olsen and Sieghart 2008). GABA is present in the synapses at higher con-centration whereas the extracellular GABA concentrations ranges from about low pM to 1 µM (Lindquist and Birnir 2006; Jin, Jin et al. 2011). Only a few studies have examined the expression of GABAA channels in immune cells (Jin, Mendu et al. 2011). The subunits expression in CD4+ T cells is enough to allow formation of functional channels. Interestingly in our study, differential expression of GABAA channel subunits was seen in activated and resting cells. This is an important information and is of relevance when designing therapeutic drugs that should target only encephalitogenic CD4+ T cells and not resting T cells. The expressed channels responded to low physiological concentrations of GABA. The expressed α1, β1 and δ subunits forming GABAA channels have high-affinity for GABA and are of impor-tant for tonic conductance (Belelli, Casula et al. 2002; Lindquist and Birnir 2006). The in vitro stimulated CD4+ T cells proliferation was inhibited by GABA. The low physiological GABA concentrations that are present in the blood can potentially inhibit the proliferation of encephalitogenic CD4+ T cells that are encountered in the blood or lymph nodes during the disease condition. Even the low extracellular GABA in the brain will reduce the proliferation of these cells (Bhat, Axtell et al. 2010). The effect of GABA on the proliferation of T cells may result from decrease in the Ca2+ influx. A model describing the hypothesis is shown in Figure 10. It shows the mecha-nism of Ca2+ influx/efflux during activation of GABAA channels. From our results in Paper I, we concluded that low concentrations of GABA do acti-vate extrasynaptic-like GABAA channels in T cell and decrease activated T cell proliferation. Thus, GABA or GABA concentration enhancing drugs or modulatory drugs of GABAA channels may potentially be used to delay or hinder EAE and even multiple sclerosis disease progression.

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Paper II Results The aim of the study was to examine: 1. if CD4+ and CD8+ T cells from prediabetic and diabetic DRlyp/lyp (T1D prone) and age matched DR+/+ (T1D resistant) rats express GABAA channel subunits. The cells were isolated from the mesenteric lymph nodes (MLN). 2. If these subunits can form functional GABAA channel and be activated by GABA. 3. Finally, we inves-tigated the effect of GABA on proliferation ability of the T cells. Our results showed that BB rats express α1, α2, α3, α4, α6, β3, γ1, δ, ρ1 and ρ2 GABAA channel subunits in CD4+ and CD8+ T cells. We also examined if the expression levels differed based on the genotype (DR++ and DRlyp/lyp), the age (45 and 55 days) and the cell type (CD4+ and CD8+ T cells). Interest-ingly, the subunit expression in CD8+ T cells from DRlyp/lyp were signifi-cantly upregulated compared to the expression levels in DRlyp/lyp and DR+/+

CD4+ T cells and DR+/+ CD8+ T cells. In contrast, in diabetic CD8+ T cells from DRlyp/lyp a few GABAA subunits were significantly downregulated compared to its prediabetic stage. However, there was no difference found between diabetic and prediabetic in DRlyp/lyp CD4+ T cells. Next, we exam-ined the intracellular proteins, as they are important for channel function and pharmacology. Our results showed that the specific intracellular proteins important for the GABA signalling system were expressed in T lympho-cytes. We further examined if we could activate the GABAA channel with 1 µM GABA in T lymphocytes of DR+/+ rats. Single-channel currents were recorded with 1 µM GABA plus 100 nM THDOC. THDOC is a neurosteroid hormone that has high-affinity towards extrasynaptic-like GABAA channels (Lambert, Belelli et al. 2003). In T lymphocytes, GABAA channels were activated after a delay and increased the conductance with time. This is a characteristic feature of extrasynaptic-like GABAA channels (Lindquist and Birnir 2006). Next, we wanted to verify if physiological concentrations of GABA are high enough to modulate the proliferation of the T lymphocytes. In this study the T lymphocytes were stimulated with 0.5 µg/ml of anti-CD3 antibodies and grown in presence of 100 nM or 100 µM GABA. The results showed that T lymphocytes proliferation was inhibited with 100 nM GABA. However, the higher concentration of GABA does not increase the inhibitory activity indicating that 100 nM GABA is probably already saturating. Hence we concluded that GABA has a role as an immunomodulator affecting T lymphocytes proliferation.

Discussion From our results, it is clear that both rat CD4+ T and CD8+ T cells express many different GABAA subunits and that can mix in different combinations

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and forms of GABAA channels of varying function and pharmacology. Drug specific modulation of GABAA channels is one of the methods used to ad-dress the channel composition. It is known that GABAA channels containing the γ2 subunit are generally present in synapses. This subunit is absent in rat T lymphocytes. On the other hand, GABAA channels containing the δ subunit are confined to extrasynaptic sites and are highly sensitive to the THDOC (Lambert, Belelli et al. 2003). GABAA channels containing α4 and α6 subunits are also located extrasynaptically on the neurons (Birnir and Korpi 2007; Olsen and Sieghart 2008). Therefore, αβ3δ, αβγ1 channels are possible to form in the T lymphocytes. Intracellular associated protein are important for channel clustering, intracellular transport and recycling of GABAA subunits (Birnir and Korpi 2007; Jacob, Moss et al. 2008). These intracellular proteins were expressed in all, apart from HAP-1 that was not expressed in CD8+ T DRlyp/lyp. Our results show that α4 was not expressed in prediabetic CD8+ T DRlyp/lyp and CD4+ T DRlyp/lyp. It is known that α4 con-taining GABAA channels have high-affinity for the GABA and HAP1 in-creases the surface expression of GABAA subunits (Birnir and Korpi 2007). It is possible that lack of α4 and HAP1 in younger animals contributes to pre-disposition of the animals for diabetes. CD8+ T cells had the highest expression of GABAA subunits. It is well known that CD8+ T lymphocytes are cytotoxic T lymphocytes and participate in destruction of the pancreatic islets in type-1 diabetes (Bluestone, Herold et al. 2010). It can be speculated that the high level of GABAA subunits in CD8+ T DRlyp/lyp helps to oppose the T lymphocytes activation. GABA is secreted from pancreatic β cells and is present in the blood. From our results, we hypothesised that the role of GABA is to decrease the activation of T cells and thus GABA may poten-tially be used as an immnunomodulatory molecule to delay or decrease the progression of type-1 diabetes.

Paper III Results The objective of this paper was to examine the GABAA subunits present in CD4+ T and CD8+ T cells from humans, mice and rats as well as the Jurkat cell line. The RT-qPCR results demonstrated that there were 5, 8 and 13 different GABAA subunits expressed in human, mouse and rat CD4+ and CD8+ T cells (Table 3). In the Jurkat cells 9 different GABAA subunits were expressed. Interestingly, only mice T lymphocytes express the γ2 GABAA subunit. GABAA subunits expression was identical in CD4+ T and CD8+ T cells in each of the three species. From the RT- qPCR, it is evident that the necessary building blocks are present to form GABAA channels. Next, we wanted to confirm the GABAA subunit proteins were made in the humans,

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mice and rats CD4+ and CD8+T cells. In Western blots alpha GABAA sub-units were detected using an α(1−6) antibody, the α1 and β3 were detected using a subtype-specific antibodies in rat CD4+ and CD8+ T cells respectively as well as in Jurkat cells. The mRNA of GABAA γ2 subunit was detected only in the mouse T cells. Western blot results confirmed the γ2 GABAA protein in the mouse T cells. We further examined the localization of GABAA subunit proteins by immunocytochemistry. It revealed that α1, α2 and β3 GABAA subunit proteins were localized in the cytoplasm and in the plasma membrane of rat T cells. Similarly α2 and γ2 GABAA subunit pro-teins were localized in the mouse T cells, and α1 GABAA subunit proteins in human T cells and in the Jurkat cells. The α1 and β3 GABAA subunit pro-teins were observed in diffused manner throughout the cytoplasm in the Jur-kat cells. Next, we examined if the GABAA channels were functional and if we could detect GABA activated currents in T cells by using the patch-clamp method. Whole-cell GABA-activated (1 µM or 1 mM GABA) cur-rents were measured in rat CD4+ and CD8+ T cells and the Jurkat cell (hold-ing potential -80 or +40 mV). We also measured GABA-activated tonic like currents with 1 µM or 1 mM GABA in rat CD4+ T cell. Together the results confirmed that T cells form functional GABAA channels.

Table 3. GABAA subunits detected with RT-PCR and selected subunits were tested with Western blot and ICC

Subunits Methods Humans Mice Rats Jurkat cell

α(1−6)

RT-PCR α1, α5 α2, α3, α5 α1, α2,α3, α4,α6 α1,α3,α5,α6, Western

blotnot tested not tested α(1−6) α1

ICC α1 α2 α1, α2 α1

β(1−3)

RT-PCR β1 β2, β3 β2, β3 β(1−3) Western

blot not tested not tested β3 β3 ICC not tested not tested β3 β3

γ(1−3)

RT-PCR not tested γ1, γ2 γ1 not tested

Western blot not tested γ2 not tested not tested

ICC not tested γ2 not tested not tested

δ, ε, θ, π, ρ(1−3)

RT-PCR π, ρ2 δ θ, π, ρ1, ρ2, ρ3 π, ρ2 Western

blotnot tested not tested not tested not tested

ICC not tested not tested not tested not tested

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Discussion The results showed that human, mouse and rat CD4+ and CD8+ T cells ex-press GABAA subunits mRNA and proteins that form function GABAA channels in the plasma membrane. 5, 8 and 13 and of the 19 tested GABAA

channel subunits were detected in T cells from humans, C57BL/6J mice and Wistar rats respectively. It is known that the subunit composition determines the GABAA channel pharmacology (Olsen and Sieghart 2008). All the spe-cies have αβ subunits and have binding sites for GABA (Birnir and Korpi 2007). Different subtype of GABAA channels can form and will have differ-ent pharmacology and sensitivity to GABAA channels-targeted drugs. Inter-estingly, the GABAA γ2 subunit is only expressed in mouse T cells. It is not expressed in humans and rats T cells. Benzodiazepines bind to the γ2 subunit containing GABAA channels (Rudolph and Knoflach 2011). Care should be taken when selecting animal model for the study of GABAA channel phar-macology in immune cells. The GABAA channels were activated by 1 µM or 1 mM GABA in T cell. GABAA channels activation results in changes of the membrane potential that affects many physiological processes that take place in the cells. From our results, it is clear that humans, mice and rats have dif-ferent subtypes of GABAA channels in CD4+ and CD8+ T lymphocytes. The channels will thus vary in their pharmacological sensitivity although the way they affect the physiological process may be the same.

Paper IV Results The aim of this study was to examine how the retinal non-pigmented ciliary epithelial (NPE) cells (chicken retinal stem cell) regulate proliferation. In particular, we examined the role of the neurotransmitter GABA on the NPE cells proliferation. It has been shown that some neurotransmitters influence cell proliferation by altering the membrane potential (Martins and Pearson 2008) and GABA has been shown to regulate proliferation of embryonic stem cells (Andang, Hjerling-Leffler et al. 2008). Our RT-qPCR results demonstrated that the retina stem cells express 17 GABAA subunits as well as GAD65/67, the GABA synthesizing enzyme. Clearly, the stem cells have the necessary subunits to form GABAA channels. Next, we examined whether GABAA subunits could form functional channels in NPE cells using the whole-cell patch-clamp method. We recorded GABA-activated currents that were inhibited by SR-95531, a competitive antagonist of GABA. The GABAA channels behave like extrasynaptic neuronal channels. They were activated after a delay with low concentration of GABA. The average tonic currents in the NPE were -4.5 ± 1.4 pA (n=5) at -90 mV holding potential. Finally, the proliferation of the NPE cells was assessed in presence of

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GABA and the GABAA antagonist. The results showed that in the presence of GABAA antagonists, the proliferation of NPE cells was decreased com-pared with the GABA treated cells. However, the GABAA agonist in pres-ence of GABA did not further increase the cell proliferation but it maintains the proliferation by depolarizing the membrane potential through increase of intracellular Ca+2 and thus the proliferation.

Discussion The results show that chicken NPE cells express mRNAs transcripts encod-ing the GABAA subunits which form GABAA channels that could be acti-vated by GABA. It is known that GABA signalling system is involved in many processes including cell proliferation and migration (Ben-Ari 2002). The high number of GABAA subunits expressed indicates that multiple sub-types of GABAA channels can be formed in the cells and from the whole-cell recording it is evident that GABAA channels generate tonic currents, which are the characteristic feature of extrasynaptic GABAA channels (Lindquist and Birnir 2006; Bjurstom, Wang et al. 2008). GABAA antago-nists decreased NPE cell proliferation. Hence, we concluded that GABA and GABAA channels are required for optimal NPE cell proliferation.

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Conclusions

In this thesis, I have shown that GABA is an important signal molecule in peripheral tissues and is an important immunomodulator molecule. My re-sults are important for normal physiology but also for a number of diseases and in particular autoimmune diseases like type-1 diabetes and multiple scle-rosis.

1. GABA or GABAA channels enhancing drugs may potentially be used to treat or delay onset of multiple sclerosis due to the presence of the GABAA channels in pathogenic CD4+ T cells.

2. The activation of GABAA channels by GABA in T lymphocytes can potentially decrease the pancreatic islets inflammation by inhibiting autoreactive T cells. Hence, GABA may delay or hinder the progres-sion to type-1 diabetes.

3. The presence of distinct subtypes of GABAA channels in humans, mice and rats is an important observation. The pharmacology of the channels will differ putting a restriction on model animals used in drug discovery projects. The discovery of the subunit composition of the GABAA channel in human T cells has direct implications for drugs to be and not to use in e.g. intensive care units.

4. NPE cells express extrasynaptic-like GABAA channels that maintain the cell proliferation by regulating the membrane potential.

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Acknowledgements

This thesis would not been possible without the guidance and the help of several persons who have contributed, shared and assisted from the starting to completion of this study. First and foremost, my utmost gratitude to Professor Bryndis Birnir for believing in me and giving me an opportunity to do an excellent research project. Under your guidance and teachings, I have grown as a researcher. Without her enthusiasm, encouragement, discussions and support, this study would have hardly progressed. Every time when I meet you I learnt a new thing. It has been a pleasure working with you and I have enjoyed the nice lab-lunches and the different lab-activities every semester. It gives me a great pleasure acknowledging Dr. Zhe Jin for his valuable comments, discussions and help in many aspects. Your immense wisdom and critic always guided me in the right direction. You have been very sup-portive, dedicated and patient in everything all these years and you are also a nice person. You will always be an inspiration to me. I am deeply grateful to Professor Finn Hallböök for your help in the retinal stem cell paper. I owe my deepest gratitude to Professor Åke Lernmark and Dr. Lina Åkesson for helping with the revisions of the BB manuscript and also providing the BB rats. I want to express my gratitude to Professor Shohreh Issazadeh-Navikas and her lab members for the collaboration resulting in the EAE paper and also Helena Bjurstöm who helped me ini-tially in our lab. I would also like to thank Professor Lena Eliasson for welcoming me and introducing me to Bryndis Birnir in CRC, Lund Univer-sity, Malmö. I would like to acknowledge Associate Professor Cilio Cor-rado for teaching me how to collect lymph nodes from animals. I am indebted to my many colleagues who significantly contributed to the work. A special thanks to Amol Bhandage for his dedication in the experi-ments on western blots and immunocytochemistry a hardworking guy, Hen-rik Ring-for the enjoyable collaboration resulting in the retina paper, Anna Edlund- thanks for your help in patch-clamp experiments, Karin Nygren- I am thankful for all the help you gave me, nice discussions about Sweden, Swedish lunch and culture etc,. Yang Jin - we have had many discussions, chats and good jokes. You are very talented and straight forward guy. I have learnt many things from you, from electrophysiology to be a practicable man, thanks. Sergiy Korol- a very helpful person, open hearted and a very patient guy, thank you. Omar Babateen a very good colleague, all the best.

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Hanna Taylor- Thanks is too small to say, you make everything possible and you are a nice person. Yifan- a hardworking girl, all the best for your PhD. Many thank to Jerker Nyberg for fixing all the computer and software problems. A big thanks to Emma for all the help with the administration things. Big thanks to all the students who have passed through our lab especially Marima Mehic, Einar, Jacob, Florence, Olga. Not to forget the people in the neighbouring groups for creating the pleasant environment, people that were nice talking to, gathering every week to share a cake, I made sure to make time for the cake-time. Thanks to each of you for all the fun we had throughout these years John Sedin, Hanna Olsén Josefin Dahlbom, Olof Nylander, Markus Sjöblom, Anna Sommansson, Frank Lee, Karin Nordström, Svante Winberg, Per-ove, Salman, Gunno, Gunilla and Gunnar Flemström. I would also like to thank Pro-fessor Erik Renström and his group and Lena's group in LUDC, Malmö I should express my deepest gratitude to my beloved Parents Naana (Venkata Satyanarayana Mendu) and Amma (Siva Laxmi Mendu) for their love, encouragement and support. You always inspire me. Without your help it had not been possible for me to come to Sweden. Your dream finally comes true. To my elder brother Prasad Mendu and his wife Madhavi and younger brother Prasanna Mendu and his wife Kavitha for their concern and affection. Also I should thank my wife’s parents Gopalam Srimanna-rayana and Vilasa and my brother-in-law Vijaykrishna and grandfather RangaRao. To my cousins and Mendu family members Ravi, Gopi, Suresh and Sunil and babai & pinni. I thank my friends in Sweden for the fun we had and nice chats, uninter-rupted discussion, nice jokes, watching movies and visiting many places in Sweden and Europe. Ramesh Tati- Naidu, very loving and caring person, making atmosphere resemble India, Vini nagaraj- my loving sister, for all the nice big laughs you made, Sugata Manna- my bhaiya, Karuna -your jokes and satires I enjoy -Many thanks to all of you. I should be grateful to my friends Bekkam Govardhan & Parvathi for showing affection and their prayers, thanks for inviting me to your home in USA and I enjoyed spending time with Ashish. A very cool, enthusiastic friend Pramod Rompikuntal & his wife Deepthi, My colleague in India and friend in Sweden Kiran Yanamandra (Annaa), you are always guiding me when I ask you and never ending chatting & his wife Sireesha and their kids Anish, Souresh , nice trip to Paris with family a very pleasing moment. Thanks for hosting me when I came initially to Umeå. I also thank Azharurddin sajid for his help when I visited Malmö and for sharing his room in Malmö. I will never forget that. I also thank my friends in India Abhram, Shivasankar, Imtiaz, Pasha, Sunil, Ajay.

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My neighbors in Uppsala Suresh Surisetti & Swarna, Shyam & Reshma and Shyamraj & Sunitha for their concern, the get together dinners ,playing cards and watching movies. Finally I would like to express my loving thanks to my dear wife Amaravani, my very good friend. She helped me concentrate on my work and supported me very well all these years making this thesis possible. Without your help I cannot move forward. You are always in my heart.

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