The Biophysics of Na+,K+-Atpase in Neuronal Health and Disease

kth royal institute of technology

Doctoral Thesis in Biological Physics The Biophysics of Na+,K+-ATPase in neuronal health and disease

EVGENY AKKURATOV

Stockholm, Sweden 2020 The Biophysics of Na+,K+-ATPase in neuronal health and disease

EVGENY AKKURATOV

Academic Dissertation which, with due permission of the KTH Royal Institute of Technology, is submitted for public defence for the Degree of Doctor of Philosophy on Friday the 18h December 2020, at 1:00 p.m. in Fire, Science for Life Laboratory, Tomtebodavägen 23A, Solna.

Doctoral Thesis in Biological Physics KTH Royal Institute of Technology Stockholm, Sweden 2020 © Evgeny Akkuratov

ISBN 978-91-7873-738-3 TRITA-SCI-FOU 2020:47

Printed by: Universitetsservice US-AB, Sweden 2020 Abstract

Na+,K+-ATPase is one of the most important proteins in the mammalian cell. It creates sodium and potassium gradients which are fundamental for the membrane potential and sodium-dependent secondary active transport. It has a second role in the cell as a receptor that by binding chemicals from the cardiotonic steroids family, the most knowledgeable of them is ouabain, triggers various signaling pathways in the cell which regulate gene activation, proliferation, apoptosis, etc. It has been shown that several severe neurological diseases are associated with mutations in the Na+,K+-ATPase encoding genes. Although Na+,K+-ATPase was discovered already in 1957 by the Danish scientist Jens Skou, the knowledge about the function of this enzyme is still not complete.

In the studies included in the thesis, we have learned more about the function of Na+,K+-ATPase in different aspects of health and disease. In study I we showed a mechanism of ouabain-dependent regulation of the NMDA receptor, one of the most important receptors in the nervous system, via binding with Na+,K+-ATPase. This allows us to look at the Na+,K+-ATPase as regulator via protein-protein interaction. In study II we investigated a different aspect of Na+,K+-ATPase functioning – to look at how binding of ouabain to Na+,K+-ATPase activates a number of signaling cascades by looking at the phosphoproteome status of the cells. This allows us to see the whole picture of ouabain-mediated cascades and further characterize them. In study III we focused on the role of Na+,K+-ATPase in severe epileptic encephalopathy caused by a mutation in the ATP1A1 gene. We performed a molecular and cellular study to describe how mutations affects protein structure and function and found that this mutation

iii converts the ion pump to a nonspecific leak channel. In study IV we performed a translational study of the most common mutation for rapid- onset dystonia-parkinsonism. We studied how this mutation affects the nervous system on the protein-, cellular-, and organism level and found that the complete absence of ultraslow afterhyperpolarization (usAHP) could explain gait disturbances found in patients. In the on-going study we showed that Na+,K+-ATPase can oligomerize and that this effect is triggered by ouabain binding to the Na+,K+-ATPase. In this study, we utilized a novel fluorescence labelling approach and used biophysical techniques with single molecule sensitivity to track Na+,K+-ATPase interactions.

In summary, we applied biophysical and molecular methods to study different aspects of the function of Na+,K+-ATPase, and gained insights that could be helpful not only for answering fundamental questions about Na+,K+-ATPase but also to find a treatment for patients with diseases associated with mutations in this protein.

Keywords

Na+,K+-ATPase, NMDA receptor, ouabain, phosphoproteome, oligomerization, rapid-onset dystonia-parkinsonism

Sammanfattning

Na+,K+-ATPas är ett av de viktigaste proteinerna i däggdjurscellen. Det skapar natrium- och kaliumgradienter som är grundläggande för den elektriska potentialen över cellmembranet och för natriumberoende sekundär aktiv transport. Det har dessutom en roll som receptor som genom att binda hjärtstimulerande steroider, varav den mest kända är ouabain, startar olika signalvägar i cellen som bl.a. reglerar genaktivering, prolifiering och apoptos. Det har visats att flera allvarliga neurologiska sjukdomar är kopplade till mutationer i Na+,K+-ATPas gener. Trots att Na+,K+-ATPas upptäcktes redan 1957, av Dansken Jens Skou, är vår kunskap om enzymets funktion ännu inte komplett.

I studierna i denna avhandling har vi lärt oss mer om Na+,K+-ATPas funktion inom hälsa och sjukdomar. I studie I påvisade vi en ouabain- beroende reglering av NMDA-receptorn – en grundläggande receptor i nervsystemet – via bindning till Na+,K+-ATPas. Detta visar att Na+,K+- ATPas fungerar som en regulator genom att direkt interagera med andra proteiner. I studie II undersökte vi en annan sida av Na+,K+-ATPas funktion – hur bindning av ouabain till Na+,K+-ATPas aktiverar flera signal-kaskader – genom att titta på cellens fosfoproteoms-status. Vi kunde på så sätt få en mer heltäckande bild av ouabain-styrda kaskader, och karakterisera dem. I studie III fokuserade vi på Na+,K+-ATPas roll i svårartad epileptisk encefalopati orsakad av en mutation i ATP1A1-genen. Vi utförde en molekylär och cellulär studie för att beskriva hur en mutation påverkar proteinets struktur och funktion, och fann att mutationen omvandlar jonpumpen till en ospecifik läckkanal. I studie IV genomförde vi en translationell studie för den vanligaste mutationen vid dystoni parkinsonism med snabb debut. Vi studerade hur mutationen påverkar

v nervsystemet på protein-, cell-, och organismnivå och fann att frånvaro av ultralångsam efterhyperpolarisering skulle kunna förklara patienters problem med gången. I pågående studie visade vi att Na+,K+-ATPas kan oligomerisera och att detta startas av bindning till ouabain. I denna studie utvecklade vi en fluorescensmärkning av Na+,K+-ATPas, och oligomeriseringen studerades med fluorescenstekniker med en-molekyl- känslighet.

Sammanfattningsvis har vi använt biofysikaliska och molekylära metoder för att studera olika aspekter av Na+,K+-ATPas funktion och nått insikter som kan vara till hjälp, inte bara för att beskriva grundläggande molekylära funktioner men även för att hitta botemedel mot sjukdommar kopplade till mutationer i Na+,K+-ATPas.

Sökord

Na+,K+-ATPas, NMDA-receptorn, ouabain, fosfoproteom, oligomerisering, dystoni parkinsonism med snabb debut

List of publications

This thesis is included in the following articles and manuscripts: I. Akkuratov EE*, Westin L*, Vazquez-Juarez E, de Marothy M, Melnikova AK, Blom H, Lindskog M, Brismar H, Aperia A. “Ouabain Modulates the Functional Interaction Between Na,K- ATPase and NMDA Receptor”. Mol Neurobiol. 2020 Oct;57(10):4018-4030. II. Panizza E, Zhang L, Fontana JM, Hamada K, Svensson D, Akkuratov EE, Scott L, Mikoshiba K, Brismar H, Lehtiö J, Aperia A. ”Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+,K+-ATPase control of cell adhesion, proliferation, and survival”. FASEB J. 2019 Sep;33(9):10193- 10206. III. Ygberg S*, Akkuratov EE*, Howard RJ, Taylan F, Jans DC, Mahato DR, Kinoshita PF, Nennesmo I, Lindskog M, Andersson M, Lindstrand A, Brismar B, Aperia A. ”Severe epileptic encephalopathy caused by a de novo germline Trp931Arg mutation in ATP1A1 that converts Na,K-ATPase into an ion channel” (Manuscript, submitted). IV. Akkuratov EE*, Sorrell F*, Sousa V, Paukar M, Picton L, Jans DC, Andersson M, Fritz N, Zhang X, Liebmann T, Lindskog M, Svenningsson P, Miles G, Brismar H, Aperia A. “Mechanisms by which the T613M mutation causes mobility and gait disturbances in Rapid-Onset Dystonia-Parkinsonism” (Manuscript).

* Equal contribution The author contribution to the publications and manuscripts in this thesis: I. Conception, experimental design, performed part of experiments, analyzed data, prepared figures, and wrote manuscript. II. Performed and analyzed data for sodium imaging part, prepared figure, and relevant text for this part of the study. III. Experimental design, performed part of experiments, analyzed data, prepared several figures, and contributed to the manuscript. IV. Conception, experimental design, performed almost all experiments, analyzed data, prepared figures, and wrote the manuscript.

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Ongoing project

The following project is on-going, and some preliminary results are presented in the thesis

Akkuratov EE1, Schach KL1, Heimgärtner J2, Meineke B2, Elsässer SJ2, Wennmalm S1, Sezgin E3, Brismar H1,3. ”Analysis of Na,K-ATPase oligomerization in plasma membrane”

1 – Science for Life Laboratory, Department of Applied Physics, Royal Institute of Technology, Stockholm, Sweden 2 – Science for Life Laboratory, Department of Medical Biochemistry and Biophysics, Division of Genome Biology, Karolinska Institutet, Stockholm, Sweden 3 – Science for Life Laboratory, Department of Women's and Children's Health, Karolinska Institutet, Stockholm, Sweden

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List of abbreviations

aaRS aminoacyl-tRNA synthetase ADP adenosine diphosphate AMPAR α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor AHC alternating hemiplegia of childhood ATP adenosine triphosphate Cas9 CRISPR associated protein 9 CD cytoplasmic domain CRISPR clustered regularly interspaced short palindromic repeats CTS cardiotonic steroids D-AP5 D-2-Amino-5-phosphonovaleric acid EGFR epidermal growth factor receptor ERK extracellular signal-regulated kinase FCCS fluorescence cross-correlation spectroscopy FCS fluorescence correlation spectroscopy FHM familial hemiplegic migraine FRET Förster resonance energy transfer FXYD Phe-Xaa-Tyr-Asp (FXYD) motif GABA γ-aminobutyric acid GFP green fluorescence protein GPCR G protein-coupled receptor GST glutathione S-transferase HA hemagglutinin HEK human embryonic kidney cell line

IP3R inositol trisphosphate receptor ncAA non-canonical amino acid NCX sodium-calcium exchanger

NKA Na+,K+-ATPase NMDAR N-methyl-D-aspartate receptor PCR polymerase chain reaction PDB protein data bank pHluorin pH-sensitive green fluorescent protein PI3K phosphoinositide 3-kinase PLA proximity ligation assay PylT pyrrolysine-tRNA RDP rapid-onset dystonia-parkinsonism SPIEDAC strain-promoted inverse electron-demand Diels-Alder cycloaddition STORM stochastic optical reconstruction microscopy TCOK trans-cyclooct-2-ene-L-lysine TM transmembrane domain tRNA transfer ribonucleic acid

Table of contents

ABSTRACT ...... III

SAMMANFATTNING ...... V

LIST OF PUBLICATIONS ...... VII

ONGOING PROJECT ...... VIII

LIST OF ABBREVIATIONS ...... IX

TABLE OF CONTENTS ...... XI

1 INTRODUCTION ...... 1

1.1 NA+,K+-ATPASE: STRUCTURE AND FUNCTION ...... 1

1.2 CATALYTICAL CYCLE OF NA+,K+-ATPASE ...... 3

1.3 CARDIOTONIC STEROID FAMILY ...... 5

1.4 NA+,K+-ATPASE AND SIGNALING PATHWAYS ...... 9

1.5 NA+,K+-ATPASE AND PROTEIN-PROTEIN INTERACTIONS ...... 11

1.6 NA+,K+-ATPASE, AND CELLULAR ION HOMEOSTASIS ...... 13

1.7 MUTATIONS IN NA+,K+-ATPASE’S SUBUNITS ...... 14

1.8 INVOLVEMENT OF NA+,K+-ATPASE IN THE PROGRESSION OF

NEURODEGENERATIVE AND PSYCHIATRIC DISEASES ...... 17

2 MATERIALS AND METHODS ...... 19

2.1 MODEL SELECTION ...... 19 2.1.1 Mouse model ...... 19 2.1.2 Primary culture of neurons ...... 20 2.1.3 Immortalized cell lines ...... 21

2.2 CELL BIOLOGY METHODS ...... 21 2.2.1 Transfection ...... 22

2.2.2 Cell survival assay ...... 22 2.2.3 Electrophysiology ...... 23

2.3 FLUORESCENCE MICROSCOPY STUDIES ...... 23 2.3.1 Live widefield imaging ...... 24 2.3.2 Confocal microscopy ...... 25 2.3.3 Stochastic optical reconstruction microscopy (STORM) .. 26 2.3.4 Fluorescence correlation spectroscopy ...... 27

2.4 ANIMAL STUDIES ...... 28 2.4.1 Genotyping of animals ...... 28 2.4.2 Behavioral tests ...... 29

3 SUMMARY AND DISCUSSION ...... 31

3.1 PAPER I. OUABAIN MODULATES THE FUNCTIONAL INTERACTION

BETWEEN NA+,K+-ATPASE AND NMDA RECEPTOR ...... 31

3.2 PAPER II. OUABAIN-REGULATED PHOSPHOPROTEOME REVEALS

MOLECULAR MECHANISMS FOR NA+,K+-ATPASE CONTROL OF CELL ADHESION,

PROLIFERATION, AND SURVIVAL ...... 34

3.3 PAPER III. SEVERE EPILEPTIC ENCEPHALOPATHY CAUSED BY A DE NOVO

GERMLINE TRP931ARG MUTATION IN ATP1A1 THAT CONVERTS NA,K-ATPASE

INTO AN ION CHANNEL ...... 36

3.4 PAPER IV. MECHANISMS BY WHICH THE T613M MUTATION CAUSES

MOBILITY AND GAIT DISTURBANCES IN RAPID-ONSET DYSTONIA-

PARKINSONISM ...... 39

4 ONGOING STUDY ...... 43

5 CONCLUSIONS AND FUTURE PERSPECTIVE ...... 53

5.1 NEUROLOGICAL DISEASES AND NA+,K+-ATPASE ...... 53

5.2 NA+,K+-ATPASE AS A SIGNAL TRANSDUCER ...... 55

xii

5.3 NA+,K+-ATPASE AND ITS INTERACTOME ...... 56

6 ACKNOWLEDGMENTS ...... 59

7 REFERENCES ...... 63

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1 Introduction

1.1 Na+,K+-ATPase: structure and function The Na+,K+-ATPase (NKA) or sodium pump is an integral membrane protein of all animal cells. It drives three sodium ions out of the cell and two potassium ions into the cell and uses the energy of one ATP molecule1. It creates an electrochemical gradient of sodium and potassium ions which is used to set the membrane potential and is the driving force for secondary active transport across the plasma membrane. NKA was discovered in 1957 by Jens Skou who received the Nobel prize for this discovery in 19972. The first crystal structure was published in 20073 and additional conformational states were published in the years following4–6.

A functionally active NKA protein contains 2 subunits: catalytical α- subunit and regulatory β-subunit. Usually, it contains a third subunit which belongs to the FXYD family of proteins7 (Fig 1.1A). Previously, it was shown that NKA can form a dimer or even tetramer of α-β complexes. Dimerization has been shown by crystallography using two-dimensional membrane crystals8, high-performance gel chromatography with purified protein9, and a cross-linking approach10. However, there have not been many recent papers about this topic. This dimerization might occur due to an interaction between two α subunits9 but a β-β interaction has also been proposed10.

A B CT

outside

1 2 3 4 5 6 7 8 9 10

inside

CT NT CT alpha beta FXYD NT

Figure 1.1. A) schematic representation of the α (gray), β (yellow), and FXYD (orange) subunits of NKA. B) 3D structure of NKA based on 4HQJ PDB structure. Α subunit domains A- (green), N- (cyan), and P- (red) domains highlighted.

The α-subunit weighs around 110 kDa, contains 10 transmembrane domains (TM1-TM10) and the N-terminus and C-terminus are located in the cytoplasm. The N-terminus together with cytoplasmic domain 2 (CD2) between TM2 and TM3 form an actuator domain (A-domain). The cytoplasmic domain 3 (CD3) between TM4 and TM5 forms two domains – the nucleotide-binding domain (N-domain) and the phosphorylation domain (P-domain) (Fig. 1.1B). The transmembrane domains are α-helixes and they form binding sites for the transported ions3,5. There is a binding site on the α-subunit for specific inhibitors belonging to the class of cardiotonic steroids (CTS). In mammals, there are four isoforms of NKA – α1 to α4 each of which has different affinities to sodium, potassium, ATP, and CTS. The α1-subunit is expressed by all cell types whereas the α2- subunit is mostly expressed in glia, heart, muscles, and adipocytes. The α3- subunit is primarily expressed in neurons and the α4-subunit is expressed in the testis11. The sequence identity between human α1-, α2- and α3- subunits is 87% while the α4-subunit has a sequence identity of only 78%

2 with the other subunits. The sequence identity between α-isoforms from different species is 85% or higher7.

The β-subunit is highly glycosylated extracellularly and its weight can range from 35 to 55 kDa. It contains just 1 transmembrane domain and the N-terminus is located in the cytoplasm and the C-terminus is extracellular. The β-subunit is essential for the occlusion of potassium ions12, for trafficking to the membrane13, and participates in cell-cell adhesion14,15. There are three isoforms β1-β3, which are much less identical to each other than α-isoforms – 35-47%, and between species – 50% or higher7. The β1- subunit is expressed by all cells whereas the β2-subunit was found in skeletal muscle, pineal gland, and nervous tissues, and β3-subunit was found in testis, retina, liver, and lung11.

FXYD proteins are the smallest subunit in NKA – only around 10 kDa. They are all characterized by the Phe-Xaa-Tyr-Asp (FXYD) motif and contain just 1 transmembrane domain. The N-terminus is located in the extracellular space and the C-terminus is in the cytoplasm. There are 7 known isoforms of FXYD proteins in mammals although this family is much larger in other organisms16,17. This subunit regulates the affinity of NKA to sodium and potassium18, however, the presence of FXYD proteins is not required for functional expression of NKA19.

1.2 Catalytical cycle of Na+,K+-ATPase There are two conformational states of NKA during the catalytic cycle – E1- state which has a high affinity to sodium and E2-state which has a high affinity to potassium. They were crystallized in 20135,6 and 20073 respectively and the exact positions of sodium and potassium binding sites are known. The E1-state is specific to Na+ ions and only Li+ and H+ ions can

3 partially substitute Na+6. The E2-state allows the binding of not only K+ but also other cations such as Li+, Cs+, Tl+, NH4+, and finally Rb+20, which can be used to measure NKA pump activity by Rb+ uptake. During the catalytic cycle, NKA is phosphorylated at aspartate-369 and there are two phospho- states of the NKA: E1-P and E2-P1. The total reaction can be summarized as:

ATP + 3Na+in + 2K+out => ADP + Pi + 3Na+out + 2K+in

A schematic description of the NKA catalytic cycle was proposed by Post and Alberts following some adjustments5,21 (Fig. 1.2). NKA in the E1-state has a high affinity to Na+. Na+ binding to NKA drives phosphorylation of the enzyme, converting it to the E1-P and then, via an occluded state where sodium ions are not accessible from either side, NKA advances to the E2-P state which has a low affinity for Na+ and high affinity for K+. Sodium ions are released to the extracellular space and potassium ions bind to NKA. This drives dephosphorylation eof th enzyme and it is converted to the E2 state where it can bind ATP again and, via an occluded state where potassium sodium ions are not accessible from either side, NKA progresses to the E1 state with bound ATP and the cycle is repeated. Each reaction has been reproduced in experimental studies and many laboratories have described the effect of the mutations using this approach. Small molecules from the class of cardiotonic steroids can only bind NKA in the E2 conformation and this binding blocks the catalytical cycle.

E1P-ADP E2P E2P +ADP out out out in in in

H2O

E1-ATP E2/Pi + out Na out K+ in in

Pi E1-ATP E1-ATP E2 out out out

in in in ATP

Figure 1.2. Post-Alberts scheme of the NKA catalytical cycle5.

1.3 Cardiotonic steroid family Cardiotonic steroids (also known as cardiac glycosides) are cyclopentane perhydro phenanthrene derivatives that contain an unsaturated lactone ring at C-17. There are two subclasses of cardiotonic steroids (CTS): cardenolides, which contain a 5-membered lactone ring, and bufadienolides, which contain a 6-membered lactone ring. Some of the CTS can be glycosylated at C-3 with one or more sugar residues (Fig. 1.3). Ouabain, derived from the plant Acokanthera schimperi is widely used in experimental studies due to its high-water solubility among CTS.

Ouabain binds NKA in the E2-state and enters a pocket which is formed by transmembrane domains 1 and 6 of the α-subunit, which is followed by a conformation rearrangement of the cytoplasmic part of the α-subunit22,23.

The position of the N- and P-domain in ouabain-bound NKA is almost identical to NKA without ouabain. The A-domain rotates about 10° with transmembrane domains 1 and 2 moving towards the ouabain molecule24. The crystallization of the NKA with other CTS such as digoxin and bufalin shows that the level of CTS glycosylation affects the strength of CTS binding. Substituents of the CTS steroid core affects the position of transmembrane domains 1 and 225.

Figure 1.3. Chemical structures of ouabain, digoxin (both - cardenolides), and bufalin (bufadienolide). Sugar motifs (to the left of the molecule) and lactone rings (to the right of the molecule) are highlighted.

Chemicals from the cardenolides subfamily have been isolated from plants while chemicals from the bufadienolides were found in amphibians. These chemicals have been widely used to treat a cardiac failure for several centuries26. Recently it was shown that a few of the CTS might be produced in mammals and, in particular, in humans. The first evidence of the existence of endogenous ouabain was discovered in the 1980s. Hamlyn and

6 colleagues showed by mass spectrometry the presence of an endogenous cardiotonic steroid in blood plasma which they identified as ouabain27. They also found ouabain in the adrenals of humans, cows, and rats as well as in cultures of adrenal cells. Ouabain is associated with many diseases including hypertension28, congestive heart failure29, primary aldosteronism, and essential hypertension30. Most of the studies above have been performed by using the immunoreactivity method which was described before31. An isomer of ouabain has been found in the bovine hypothalamus32 and in 2012, ouabain was found in the cerebrospinal fluid of mammals including humans33.

In addition to ouabain, other endogenous cardiotonic steroids could also be hormones. Digoxin from cardenolides has been found in urine34 and bovine adrenal glands35. There are specific antibodies against digoxin called DigiBand36 which are being tested for medical application37. Digoxin has been shown to prevent ouabain-induced hypertension38. Although the results were observed repeatedly39, the mechanism of this antagonism action is not understood.

Marinobufagenin (MBG), which belongs to another subclass of CTS (bufadienolides) has also been proposed as a hormone26. Bufalin-like immunoreactivity has been detected in human bile40, urine41, and blood plasma42. Bufadienolide compounds have been detected by mass- spectrometry in human cataractous lenses43 and placentae44. Using both mass-spectrometry and nuclear magnetic resonance, bufalin-like compounds were identified as MBG. Evaluation of MBG is associated with chronic renal failure, idiopathic hyperaldosteronism, essential hypertension, acute congestive heart failure45, and preeclampsia46. Fedorova and colleagues postulated that a neurohormone termed

“endogenous ouabain” stimulates the adrenocortical production of marinobufagenin47.

There is also research that does not support the hypothesis of endogenous ouabain48–50. Most of the studies of endogenous CTS have used an immunochemical method to measure CTS concentrations. The method is based on the binding of antibodies to a specific CTS which leads to a high risk of cross-reactivity with closely related compounds. The pathways of ouabain, digoxin, and marinobufagenin synthesis in mammals are not well understood. There is some evidence that ouabain could be synthesized in the adrenal glands51,52 or the hippocampus53 but exact enzymes are unknown. Despite these uncertainties, there is an abundance of other articles that show data about the existence of endogenous ouabain26,54–57 which is strong evidence that ouabain is produced in mammals.

The binding site of CTS consists of amino acids Q111, Q119, G120, and N122 in the first extracellular loop of the α subunit and they play an important role in the sensitivity of the enzyme to CTS. These amino acids are highly conserved in all animals but mutations in them do not affect enzymatic function58. This conservation supports the idea that the siter fo CTS binding is required for other functions of the NKA besides its enzymatic function. In animals who feed on organisms with high CTS concentrations, these amino acids have been mutated to make the α-subunit CTS-resistant but this has only happened in one isoform of the α subunit: α3 in snakes and α1 in butterflies and rodents59, while the other subunits in these animals remain sensitive to CTS. It has also been shown that ouabain is a signaling molecule, activating several signaling processes in diverse cell types7,60–62. This has led to the hypothesis that CTS is a hormone with the NKA as its receptor.

1.4 Na+,K+-ATPase and signaling pathways The first evidence that the binding of ouabain to NKA drives signaling cascades came from a study from Askari and Xie groups in which the exposure of myocytes to ouabain resulted in a calcium-dependent activation of c-fos and c-jun mRNA63. This paper started a new research era regarding NKA's role as a receptor. During the following years, the same group reported that ouabain stimulated the Ras-Raf-MEK-ERK pathway64 and activated tyrosine Src kinase and epidermal growth factor receptor65. Later it was proposed that NKA and Src can form a complex and the binding of ouabain to NKA freed the kinase domain of Src resulting in its activation66. An NKA-derived peptide Src inhibitor was developed to prevent ouabain-triggered signaling67. However, a few recent studies question the existence of an NKA/Src complex68,69 (Fig. 1.4).

In parallel to these studies, the Aperia group showed that ouabain induces regular low-frequency Ca2+i oscillations which leads to activation of Nf- kB70. These effects are realized through activation of the IP3R which is in this case InsP3 independent71. Activation of IP3R occurs due to an interaction of the N-terminus of NKA and the IP3R with ankyrin B acting as an adapter molecule72,73. Digoxin and marinobufagenin can also drive this signaling pathway and the presence of Src inhibitors abolish calcium oscillations62.

Ouabain application has also been reported to activate phosphatidylinositide 3-kinase (PI3K) and Akt74. The PI3K-Akt pathway is involved in the regulation of NKA function75 and PI3K binds to a proline- rich motif of the α-subunit of NKA76. Interestingly, the Ras-Raf-MEK-ERK signaling pathway is not PI3K-dependent74 and PI3K-Akt signaling is not Src-dependent61.

Ouabain

out NKA EGFR in AnkB

Src ER IP3R PI3K Ras

Akt Raf

2+ Ca MEK

ERK1/2 Nuclear

Figure 1.4. The three most studied ouabain-triggered signaling cascades

Interestingly, multiple groups have shown that ouabain’s signaling effect is observed not only at concentrations where it significantly blocks NKA activity but also at lower concentrations than do not block NKA activity. Ouabain has also been shown to trigger various additional pathways 77–83 which seem to be not only isoform-specific but also cell type-specific. These processes alter gene expression, cell proliferation, and anti-apoptotic processes but the whole picture of ouabain- or other CTS-driven signaling is still unknown.

1.5 Na+,K+-ATPase and protein-protein interactions NKA is a well-known partner in many different protein-protein interactions7. One of the first pieces of evidence that NKA interacts with other proteins came from signaling studies where multiple groups showed interactions with Src kinase66, IP3R72, PI3K76, and more. It has been shown that NKA is involved in protein-protein interactions with caveolin-1 which is a scaffolding protein participating in the formation of caveolae84. It is hypothesized that caveolin-bound NKA forms a signaling, non-pumping pool of NKA since its interaction with caveolin might affect its enzymatic function85. However, a separate group showed that the activity of caveolar and non-caveolar bound NKA are similar86 and that caveolin-1, annexin-2, and NKA form a large multiprotein complex.

Usually, the N-terminus of NKA is involved in protein-protein interactions. This may be because the end part of the N-terminus is not crucial for pump function and truncation of the N-terminus does not affect enzymatic function87. Moreover, crystallography studies showed that the N-terminus tail has a high degree of disorder23 and binding of nouabai leads to conformational changes of the A-domain which includes the N-terminus7. This suggests that the mechanism of ouabain-dependent regulation of protein-protein interactions might involve an N-terminus conformational change. NKA interacts with aquaporin 4 (AQP4), a water channel expressed in astrocytes, via the N-terminus as well. AQP4 interacts with both the α1 and α2 isoforms of NKA and at the same time interacts with the metabotropic glutamate receptor mGluR588. This macrocomplex might be important for the regulation of water and potassium homeostasis in astrocytes.

NKA interacts with other glutamate transporters and receptors which indicates the involvement of NKA in the functional regulation of

11 glutamatergic neurons. It has been shown that NKA co- immunoprecipitates with the glutamate transporter 1 (GLT-1 or EAAT2) and glutamate aspartate transporter (GLAST or EAAT2) which are expressed mostly in astrocytes and participate in the re-uptake of glutamate into the cell. Interestingly, in synaptosomes, ouabain showed dose-dependent inhibition of the glutamate transporter while in astrocytes, ouabain concentrations lower than 1uM increased glutamate transporter activity whereas higher concentrations lead to inhibition of glutamate transporter activity89. In neurons, NKA co-precipitates with the ionotropic glutamate receptors – NMDAR and AMPAR 90,91, and in both cases ouabain leads to a reduction in total protein levels of both the receptors. Taken together, there is evidence of deep involvement of NKA and ouabain in glutamatergic synapses. Interestingly, the gene encoding the neuron-specific α3 isoform of NKA is a neighbor of the gene encoding the kainite receptor subunit GRIK5 (5000 bp between them) which belongs to the third ionotropic glutamate receptor – kainite receptor. This may predict the co-regulation of these genes and possible functional and protein-protein interactions between them, but it has not been studied so far.

NKA interacts and colocalizes with other receptors, channels, and transporters – with dopamine receptors D1 and D292,93, with glycine transporter GlyT294, sodium-calcium exchanger NCX95, and Nax channels96. Since NKA interacts with the scaffolding protein PSD-9597 which is involved in the interaction between many different channels and receptors98, it can be predicted that NKA can form a macromolecular complex with Na-dependent and independent receptors and channels in synapses. The variety of such interactions should be studied systematically.

1.6 Na+,K+-ATPase, and cellular ion homeostasis NKA is a primary-active transport protein which means that it uses energy from ATP to make an electrochemical gradient of sodium and potassium ions. The sodium gradient is the driving-force for secondary-active transport in animals. Sodium goes from the extracellular side of the plasma membrane to the cytoplasm and other molecules can be co-transported with sodium (symport) or be transported in opposite direction (antiport). The sodium/calcium exchanger (NCX) is an example of Na+-Ca2+ counter- transport where the gradient of sodium is used to remove calcium from the cytoplasm. Interestingly, NKA and NCX can colocalize with each other which probably allows Ca2+ regulation in a local microenvironment95. This may also be regulated by endogenous ouabain in the brain99.

Another example of sodium-dependent antiport is the sodium/proton exchanger. The SLC9A1 gene encodes a Na+/H+ antiporter that is a member of the solute carrier family 9 and is involved in pH regulation100. Mutations in this gene lead to Lichtenstein-Knorr syndrome characterized by cerebellar ataxia and sensorineural hearing loss101,102. The sodium/potassium/chloride co-transporter is an example of the symport of three ions in the stoichiometry 1Na+:1K+:2Cl- which makes it electroneutral. This protein regulates ion balance and cell volume and it participates in the reabsorption of these electrolytes from urine103. Antagonists of the sodium/potassium/chloride co-transporter, encoded by the NKCC1 gene, leads to the restoration of low chloride levels in neurons which might be useful for the treatment of neurological and psychiatric disorders104. Also, the sodium gradient is used for the co-transport of many metabolites including glucose105, amino acids106, and more. NKA plays an important role in the nervous system for the re-uptake neurotransmitters107.

The formation of a gradient of potassium and sodium ions is important to maintain the resting membrane potential in cells. Due to the high permeability of the membrane for potassium, it has the most impact on setting the membrane potential108. Resting membrane potentials vary in different cell types. Astrocytes, myocytes, and neurons have the lowest resting membrane potentials109,110 of all cells. During the action potential, cation gradients are disturbed and the NKA is involved in the restoration of these gradients to the resting state levels111. NKA mediates afterhyperpolarization in neurons112 which is affects the excitability of neurons113. Taken together, it is clear that the NKA is essential for all organs in animals, especially the brain, and it is not surprising that NKA can use up to 50% of the ATP produced in the brain114.

1.7 Mutations in Na+,K+-ATPase’s subunits It is known that a homozygous knockout of the α1, α2, or α3 subunit is lethal whereas heterozygous knockouts are viable and have small abnormalities. These include higher muscle and heart contraction for α2 knockouts, a lower cardiac contraction for α1 knockouts, and a few behavior abnormalities for α2 and α3 knockouts115–117. However, humans with genetic pathogenic variations in these subunits, even in the heterozygous state, can result in several different diseases and syndromes.

Pathogenic variants in the ATP1A2 gene encoding the α2 subunit lead to familial hemiplegic migraine type 2 (FHM2) and the link between genetic variations in ATP1A2 and the disease was first found in 2003118. FHM2 is an autosomal dominant inherited form of migraine with aura119. There are 4 different types of FHM characterized by pathogenic variants in different genes. Most of the pathogenic variants in ATP1A2 are single nucleotide variants which lead to amino acid changes. Mutations causing FHM2 has

14 a scattered distribution along the protein molecule and there are no preferable positions120.

Pathogenic variants in ATP1A3 give rise to several diseases. Mutations in this gene leading to rapid-onset dystonia-parkinsonism (RDP) were first described in 2004121. RDP was described in 1993 and it is an autosomal- dominant disease characterized by the sudden onset of dystonia and parkinsonism122. Most of the RDP mutations are single nucleotide variants. They are located in transmembrane domains and cytoplasmic and extracellular loops. The most common mutation, T613M, is located in the P-domain123,124. The link between pathogenic variants in ATP1A3 and other diseases, such as alternating hemiplegia of childhood (AHC), was found in 2012125,126. AHC is a rare neurological disorder characterized by early-onset episodes of hemiplegia, dystonia, various paroxysmal symptoms, and developmental impairment. Most of the mutations causing this disease are located in transmembrane regions and the phenotype of this disease is often more severe but there are phenotypes which are in between RDP and AHC120,123,127. Most of these mutations lead to reduced NKA activity and some of them affect protein expression. There is a third disease associated with ATP1A3 which is CAPOS (cerebellar ataxia, areflexia, pes cavus, optic nerve atrophy, and sensorineural deafness) syndrome, which is characterized by one mutation, E818K128. There are several other ATP1A3- related disorders: early infantile epilepsy and encephalopathy (EIEE)129, fever-induced paroxysmal weakness, and encephalopathy (FIPWE)130, and relapsing encephalopathy with cerebellar ataxia (RECA)131.

It has been shown that the ATP1A1 gene is intolerant to missense and loss- of-function variants132. Somatic mutations in the ATP1A1 gene has been shown for adrenal aldosterone-producing adenomas133. However, recently it has been found that a mutation in ATP1A1 leads to Dominant Charcot-

Marie-Tooth Type 2 which is characterized by distal weakness and atrophy, sensory loss, and absence of reflexes132,134. Another syndrome, hereditary spastic paraplegia (HSP), was shown to be associated with a mutation in ATP1A1 as well135. Most of these mutations were located in the cytoplasmic regions of the protein. Also, mutations in ATP1A1 lead to hypermagnesemia, refractory seizures, and intellectual disability136. The phenotype of these patients was more severe, and these three mutations were located in transmembrane domains (Fig. 1.5).

Figure 1.5. Distribution of pathogenic variants in ATP1A1. Mutations labeled by blue and yellow stars are associated with Charcot-Marie-Tooth132,134. Mutations labeled by green stars are associated with hypermagnesemia, refractory seizures, and intellectual disability136. Mutation labeled by red star is associated with severe epileptic encephalopathy described in paper III from this thesis.

Taken together, pathogenic variations in the three genes encoding α1-3 lead to diseases in the nervous system which also indicates the importance of NKA for brain functioning.

1.8 Involvement of Na+,K+-ATPase in the progression of neurodegenerative and psychiatric diseases NKA is involved in the progression of multiple diseases. Parkinson’s disease belongs to a group of neurodegenerative diseases called synucleinopathies. α-synuclein is a protein that aggregates in Lewy bodies which contributes to Parkinson’s disease. It has been shown that α- synuclein interacts with the α3 subunit of NKA and this interaction leads to cluster formation and decreases stimulus-triggered efficiency of Na+ extrusion137. The authors found that two amino acids which are α3-specific, Leu878, and Asn879, in the extracellular loop between transmembrane domains 7 and 8 are responsible for this interaction. Interestingly, another group independently showed that almost the same amino acids from the α3 subunit of NKA, Asn879, and Trp880, play a key role in the interaction between NKA and β-amyloid aggregates, amylospheroids138. These aggregates are formed during the progression of a second neurodegenerative disease, Alzheimer’s disease. Also, it has been shown that the α3 subunit of NKA can interact with tau assemblies which have prion-like properties139 and with misfolded SOD1 during the progression of amyotrophic lateral sclerosis140. Interestingly, in the last case, this interaction is mediated by intracellular domains of the α3 subunit. As these studies have shown that these interactions are unique for α3, there is good evidence that the neuronal-specific α3 subunit of NKA is involved in the pathogenic progression of prion-like diseases.

The α3 subunit of NKA is expressed mostly in GABAergic interneurons in the brain141. Loss of GABAergic interneurons was found in patients with psychiatric diseases such as bipolar disorder and schizophrenia142. Interestingly, there is an ouabain-triggered model of bipolar disorder143 and there are studies that endogenous ouabain might be involved in the mechanisms underlying bipolar disorder144. Recently it was shown that the

17 protein level of the α3 subunit was lower in GABAergic neurons in the frontal cortex and hippocampus in bipolar disorder and schizophrenia as compared with the controls. However, in the temporal cortex, the level of α3 subunit expression was higher145. This data opens up a discussion about the involvement of the α3 subunit of NKA not only in prion-like neurodegenerative diseases but also in the progression of psychiatric diseases.

2 Materials and Methods

2.1 Model selection We need to select an experimental model that is appropriate for the particular research question. This is an important step in the design of a study because every model has advantages and limitations. To answer biomedical questions, the best model would be humans, but this is impossible due to ethical dilemmas. Researchers have developed several different alternative models, and in this thesis, I have used several of them.

2.1.1 Mouse model To study processes on the organism level we would like to use a model that is as close to humans as possible regarding anatomy and physiology. The model should reproduce relatively fast and be amenable to genetic modification to mimic human diseases. Mice are one of the most popular choices due to their fast reproductive cycle (3 months from birth to be sexually mature), ease of genetic modification (many well-established techniques available) and it is relatively close to humans (it belongs to the class Mammalia and suborder Euarchontoglires which includes rodents and primates). Their genome is sequenced and, using the CRISPR-Cas9 technique it is possible to get knockout or knock-in models in six months.

Using a model organism, we can study how genetic modifications and/or treatments affect animal behavior, biochemical processes in different tissues, and, using microscopy, study neuronal circuits in the central nervous system. However, the use of model organisms does have a few limitations. Firstly, for many research questions that can be addressed on a single-cell level, it is better to use cell models that require the use of much fewer animals. Also, it can be important to use a more robust model that

19 gives better reproducibility without the individual variability observed between animals.

2.1.2 Primary culture of neurons To study processes that do not require the original anatomical structure and specific connections between neurons we can use a primary culture of rat neurons. This model allows us to study processes on the single-cell level and cells maintain neuronal characteristics such as physiology, morphology, and synaptic connections. Neurons are isolated from the rat hippocampus at embryonic day 18.5 and after 2-3 weeks in vitro culture, they form dendrites and axons. Primary neuron cell culture is a very robust model for studying neuronal metabolism. It requires the use of fewer animals which is preferred according to the 3R principles. Primary neuron cell cultures can be transiently transfected and used to study how protein modifications affect neuronal metabolism. Cell cultures are beneficial as they are much easier to transfect in comparison to tissue slices. This model is commonly used for live imaging and measuring changes in intracellular sodium and calcium concentration.

Neurons from rat hippocampus were prepared from embryonic day 18.5 embryos of both sexes. The hippocampus was removed, washed in Hank’s balanced salt solution (HBSS) containing 20 mM HEPES, then incubated for 10 min at 37 °C in HBSS with 20 mM HEPES and 0.25% trypsin and dissociated in minimum essential media (MEM) by pipetting using a fire- polished Pasteur pipette. Cells were plated at a density of 4*104/cm2 for imaging or 10*104/cm2 for immunocytochemistry on glass coverslips previously coated overnight with poly-ornithine (80 µg/ml) in MEM containing 10% horse serum, 2 mM L-glutamine, and 1 mM sodium pyruvate for three hours. Then the medium was replaced with Neurobasal medium containing 2% B27, 0.5 mM L-glutamine, and 1%

20 penicillin/streptomycin. Half of the medium volume was replaced with Neurobasal medium containing 2% B27, 0.125 mM L-glutamine, and 1% penicillin/streptomycin twice per week.

Although this model is robust and requires fewer laboratory animals, sometimes we want to study individual proteins that do not have a neuronal origin. Also, the primary culture of neurons is characterized by a high level of heterogeneity and can only be used for up to 3-4 weeks. Immortalized cell lines can instead be used as a more robust model with less variability.

2.1.3 Immortalized cell lines To study protein function or protein-protein interactions, immortalized cell lines can be used. They are easy to work with, do not require any use of laboratory animals, and grow fast. There many cell lines available and in the presented studies we have used HEK293 and COS-7 cell lines which are derived from human embryonic kidney and monkey kidney tissue respectively. They were grown in DMEM supplemented with 10% FBS and 1% penicillin/streptomycin. To maintain them, the cells were passaged every 3-4 days. Cells at early passages were frozen in FBS/DMSO for further studies. This model in combination with studies in silico on structural data could shed light on protein function and be used to study protein conformational changes, activity, and membrane expression. Also, these cell lines are a general model for all mammalian cells, and we have used this concept in our phosphoproteomic study.

2.2 Cell biology methods To study different aspects of cell metabolism we applied different cell biology techniques. To express a protein of interest we transfected a

21 primary cell culture or cell line with a plasmid containing the sequence of the protein of interest. For testing, whether a mutation affects protein function, we used a survival assay. For the analysis of membrane potential, we used a whole-cell electrophysiological approach.

2.2.1 Transfection We transfected cells to make them express a protein of interest. We transfected them using lipofectamine – a commercial reagent that creates liposomes in which plasmids carrying the coding sequence of the protein of interest are transferred to the cell. These plasmids usually have a CMV (cytomegalovirus) promoter which leads to overexpression in all mammalian cells. The efficiency of transfection depends on the cell type – in primary cultures of neurons the efficiency is quite low and does not exceed 5-10% while the efficiency of transfection of a cell line can be up to 100%. Cultures were transfected 24-48 h before experiments using Lipofectamine 2000 (Thermo Fisher Scientific) or Lipofectamine LTX (Thermo Fisher Scientific) for neurons and cell lines respectively according to the manufacturer's protocol. Transfected cells were identified by the presence of a fluorescent protein-tagged to the protein of interest or the presence of a separate fluorescent protein on the different plasmid. This approach is used in all studies in this thesis.

2.2.2 Cell survival assay NKA maintains the sodium and potassium gradient across the plasma membrane and a cell will not survive if the NKA is not functional. This fact makes cell lines a good model for screening whether mutations in NKA affect its function. To test this, we first needed to eliminate the endogenous NKA function which we did use ouabain-selection. HEK293 and COS-7 cells only express the α1 subunit of NKA which, in primates, is ouabain-

22 sensitive with a dissociation constant (Kd) of around 10 nM. We transfected a cell line with a plasmid carrying the α1 coding sequence containing mutations that lead to ouabain-resistance. After transfection, 10 µM ouabain was added to the cell media for 24-48 hours to block endogenous NKA activity without affecting exogenous NKA activity. This approach allowed us to specifically select transfected cells that express ouabain- resistant NKA. By comparing wild-type and mutant NKA, we could test whether the mutation affects NKA function and thereby cell viability.

2.2.3 Electrophysiology To measure the resting membrane potential and the rate of spontaneous activity of primary neurons in cell culture, we used a whole-cell patch- clamp setup. Using a micropipette of 3-7 megaohm resistance filled with an intracellular mimicking solution (120 mM potassium gluconate, 24 mM

KCl, 4 mM NaCl, 4 mM MgCl2, 0.16 mM EGTA, 10 mM HEPES, 4 mM K2- ATP, pH 7.2 adjusted with KOH) attached to neurons. We broke the membrane and connected the solution in the pipette with the cell cytosol. A second electrode was located in the bath solution where neurons were maintained (110 mM NaCl, 4 mM KCl, 1 mM NaH2PO4, 25 mM NaHCO3,

1.5 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose and 20 mM HEPES at pH 7.4 at 37°C). When working with GFP-transfected cells, we found the cells using a fluorescence microscope. Recordings were performed using an Axopatch 200B amplifier (Molecular Devices) and pClamp software (version 8.2). In the current-clamp mode, we recorded resting membrane potential and spontaneous activity.

2.3 Fluorescence microscopy studies Different fluorescence microscopy modalities have been key to the research in this thesis. In our studies, proteins were either fused to various

23 fluorescent proteins or labeled by fluorescent antibodies. Also, cells were loaded with dyes to measure the concentration of intracellular inorganic ions. The sample is illuminated at a specific wavelength and the emission light – which is shifted to higher wavelengths – is collected using a sensitive camera or photomultiplier tube (PMT). To test how mutations and/or ligands affect sodium and calcium homeostasis in neurons we performed live imaging on a widefield microscope. To look for protein distribution we used confocal microscopy. To search for co-localization, we used stochastic optical reconstruction microscopy (STORM) which belongs to a group of super-resolution microscopy techniques. An additional approach to search for protein-protein interactions is fluorescence correlation spectroscopy (FCS), which we used in our study.

2.3.1 Live widefield imaging We used wide-field microscopy to collect light from the entire field of view to perform fast recordings of cellular activity. We used this technique for sodium and calcium imaging with a frequency of 0.4-1 Hz. The sample was either loaded with a dye 30-60 min before the experiment or cells were transfected with a genetically encoded calcium indicator GCaMP 24-48 h beforee th experiment. We used Asante Natrium Green-2 AM for measuring sodium and Fura-2 AM for measuring calcium ions. These dyes contain an AM (acetoxymethyl) ester which makes the dyes electrically neutral, allowing them to easily cross the plasma membrane. When inside the cell, the AM ester is cleaved by esterase, and the dye becomes negatively charged and unable to leave the cytosol. After loading the cells at 37C, they were rinsed 3 times in media to remove excess dye and then mounted onto the microscope. Asante Natrium Green-2 has an excitation maximum at 517 nm and an emission maximum at 542 nm146. Fura-2 AM belongs to a group of ratiometric dyes that show a shift of their excitation peak when either bound to or free of Ca2+. Measuring the ratio between the emitted

24 signal with an emission maximum of 510 nm at two different excitation wavelengths (340 and 380 nm) cancels out differences in experimental parameters such as cell thickness and dye loading. While widefield microscopy usually uses a mercury lamp with a combination of filters, we used a monochromator which gives precise excitation wavelengths. Since Fura-2 AM can be toxic for cells and inhibit NKA147 we also used a genetically encoded calcium indicator GCaMP6f with an excitation maximum at 496 nm and an emission maximum at 513 nm. The low transfection efficiency allowed us to record from individual neurons as we often had just one transfected neuron in the field of view. We mounted a perfusion system to the widefield microscope to allow the rapid addition or removal of chemicals in the perfusate. Usually, we used a pump speed of 2 mL/min which does not stress neurons and gave a fast exchange rate in the perfusion chamber.

2.3.2 Confocal microscopy We used confocal microscopy in situations where we needed to collect signals only from one focal plane. In confocal microscopy, a focused laser beam is scanned across the sample and the signal at each point is collected and used to reconstruct an image of the scanned area. Variation of the size of the pinhole allowed us to collect more or less out-of-focus signal, balancing between the amount of signal collected and the axial resolution. In our projects, this technique was used to determine whether a protein was localized at the plasma membrane. To do so, we collect light only from a plane at the half-height of the cell where we could estimate the localization of the protein. Also, to make a 3D image we scanned plane by plane to form an image stack. We then performed maximum intensity projections which represent the 3D structure of the cell better than a widefield microscopy image.

2.3.3 Stochastic optical reconstruction microscopy (STORM) The resolution of images produced by both widefield and confocal microscopy is limited by diffraction and is dependent on the wavelength used and the numerical aperture of the objective. Even in the best scenario, the resolution cannot be lower than 150-250 nm. Different techniques have been proposed to improve the resolution of light microscopes. Some of them involve specimen preparation such as expansion microscopy. Another approach is to use superresolution microscopy for which the Nobel Prize in Chemistry was awarded in 2014 to Eric Betzig, Stefan W. Hell, and William E. Moerner. We have performed superresolution imaging using STORM in our studies.

STORM is based on the principle that the position of a molecule can be determined with a precision of a few nanometers by fitting the recorded signal from that molecule to a Gaussian distribution model. To do so, we need to make sure that we are fitting the signal from a single molecule since fitting the signal from two or more molecules with a single Gaussian would give the wrong position. To be able to use STORM, photoactivatable fluorescent molecules are used, of which only wa fe are activated during each frame. By repeatedly activating a subset of different molecules and fitting the emission (in our experiments we recorded 20000-25000 frames per image) we could reconstruct an image with a very high resolution down to 10-20 nm148. In our experiments, we used a Zeiss Elyra (Carl Zeiss) microscope equipped with a Plan-Apochromat 100x/1.46 Oil objective and an Andor iXon EM-CCD camera. Two laser lines (488 nm and 642 nm) were used to activate, excite and deactivate Atto-488 and Alexa-647 respectively. A low-wavelength 405 nm laser is used to continuously push molecules into an activated state. We used STORM to judge whether two proteins co-localized on the plasma membrane of neurons or not.

2.3.4 Fluorescence correlation spectroscopy To look for oligomerization of the NKA we used applications of fluorescence correlation spectroscopy (FCS) such as combination of Förster Resonance Energy Transfer and Fluorescence Correlation Spectroscopy (FRET-FCS) and fluorescence cross-correlation spectroscopy (FCCS). FCS detects fluorescence fluctuations from molecules diffusing in a solution or a lipid membrane through a sub femtoliter detection volume and can be used to measure concentrations and diffusion coefficients of molecules as well as molecular interactions. From single color FCS, it is possible to extract not only the average transit time and the average number of molecules in the area but also the brightness of molecules which might indicate an oligomerization state. FRET-FCS detects the acceptor emission from FRET-active oligomers containing both donor and acceptor. In FRET measurements in general, the sensitivity is limited by the amount of background-signal. Background occurs when some of the donor emission is detected in the red channel, i.e. so called cross-talk, and when acceptors to a small extent are excited directly by the 488 nm laser. By using donors and acceptors with an unusually large spectral shift – such as Alexa 488 and Alexa 647 – the background signal from such non-interacting, monomeric donors and acceptors is made very small. Thereby interactions can be detected even in cases where only a small fraction of the available donor and acceptor molecules form FRET-active oligomers. In our ongoing study, we use FRET-FCS and FCCS and two-color labeled α and/or β subunit of NKA to see whether we have a fraction of interacting molecules or not. Since none of the studies of NKA oligomerization have been performed on living cells, our ongoing study will give us the possibility to solve the question of whether two or more NKA interact in real-time and if so, how we can modulate this oligomerization.

2.4 Animal studies To study how a mutation leads to a disease on the organism level, we established a mouse model for one of the studied diseases – rapid-onset dystonia-parkinsonism (RDP). This is a knock-in mouse model, T613M ATP1A3, created by using the CRISPR/Cas9 technology. Since all humans with RDP have this mutation in the heterozygous state, we used heterozygous animals as a model of the disease and their wild-type littermates as control animals. To do this we crossed wildtype male and heterozygous female or vice versa to get a 1:1 ratio between wildtype and heterozygous animals. To see whether the offspring inherit the mutation we performed genotyping of the animals. To characterize abnormalities in animals we used a set of behavior studies that indicate motor, anxiety, or cognitive abnormalities compared to wild-type animals.

2.4.1 Genotyping of animals There are just 2 basepairs (bp) that differ between the sequence of transgenic mutants and wildtype. To test whether an animal carries the mutation, we isolated DNA from tissue (ear clipping) and performed a PCR with the specific primers to amplify a region of the ATP1A3 sequence containing the mutation, giving a 418 bp product size. Then, we cut the product of the PCR using a MlsI restriction enzyme which recognizes the TGGCCA sequence. In the wildtype sequence, there is just one restriction site and, after running the DNA on a gel, we should get bands at 341 and 77 bp. In the mutant sequence, there are two restriction sites and we should get bands at 230, 111, and 77 bp. Since heterozygous animals have one wild- type allele and one mutant allele we should get 4 bands: 341, 230, 111, and 77 bp.

2.4.2 Behavioral tests To fully characterize the phenotype of our mouse model we applied different tests that characterize different aspects of its behavior.

The open-field test was used to analyze the explorative behavior of the animals. Mice were placed in the center of an open square area and their locomotion activity was recorded by a video camera mounted on the top of the apparatus. Then we extracted the total distance traveled by the animal and its velocity. Here we could judge whether the animal was hyperactive or hypoactive.

The elevated plus-maze test was used to measure the anxiety level of the animals. Mice were placed in the center of a four-arm apparatus were two arms were walled and two were without walls. As in the previous test, a video camera recorded the animal movement from the top and we extracted the total time which mice spent in the open and in close arms. Mice spending more time in the open arms indicated lower levels of anxiety.

A forced swim test was used to analyze depression-like behavior. Mice were placed in a plastic cylinder filled with warm water. The etim spent passively floating and actively floating was recorded. Mice spending more time actively floating showed a lower level of depressive-like behavior.

A rotarod test was used to analyze the motor coordination, balance, and grip strength. Mice were placed on a rotating rod at low speed (5 rpm) and after 30 seconds it was accelerated to high speed (40 rpm) for 3 minutes. The test was repeated 3 times per day during 2 consecutive days and we calculated the latency to fall during each round.

A vertical pole test was used to analyze whether the nigrostriatal pathway is affected in mutant animals. Mice were placed on the top of a vertical pole with their heads towards the top. Then the mouse will turn and climb back, returning to the home cage. These times were calculated and presented as an average of five trials after one training trial.

A catwalk test was used to analyze gait, coordination, and motor abnormalities. The mice were placed in the apparatus which is a dark elongated tunnel. The mice were allowed to explore and walk freely in this tunnel, and a video camera recorded them in the center of the tunnel through the bottom glass floor. Three consecutive days of training were required and on the next day, the test was performed.

A passive avoidance test was used to analyze long-time memory. The mice were placed in the light section of the apparatus which contained two sections: a dark and a light section with a closed-door in between. After one minute the door was opened and when the mouse entered the dark section the door was closed, and an electrical current was applied to the floor of the dark section. The next day the mouse was placed again in the light section and the latency to go to the dark section was measured.

3 Summary and Discussion

3.1 Paper I. Ouabain Modulates the Functional Interaction Between Na+,K+-ATPase and NMDA Receptor In this paper, our goal was to study how ouabain regulates structural and functional interaction between NKA and NMDA receptor (NMDAR).

The NMDAR is one of the most popular proteins to study in the neuroscience field. It is characterized by high complexity and it plays an important role in synaptic plasticity and molecular memory. It belongs to the class of ionotropic glutamate receptors. There are two types of glutamate receptors – ionotropic and metabotropic which work as ion channels and GPCRs proteins respectively. The NMDAR is permeable to sodium and calcium ions and the flux of calcium ions plays a role in the regulation of synaptic plasticity. A large influx of calcium via the NMDAR often leads to excitotoxicity which can happen following a stroke.

Previously, my former colleagues and I published a paper describing a functional interaction between the NKA and the NMDAR91. We showed that they co-precipitate using the co-immunoprecipitation method and that the application of ouabain leads to a reduction of the amount of NMDAR subunit protein after one hour. These results together with the results from other groups99,149,150 inspired us to study this interaction in live conditions by using various light microscopy approaches. First, we showed that ouabain administration to a primary culture of hippocampal neurons leads to attenuated NMDAR-dependent calcium influx. We showed that this effect is observed regardless of the method, whether we apply it to the entire coverslip with neurons or locally to an individual neuron. Application of ouabain together with glutamate which activates all types of

31 glutamate receptors and NMDAR-specific antagonist, D-AP5, did not reveal the same effect. We, therefore, concluded that this effect happens via a structural and/or functional interaction between NKA and NMDAR.

In the previous study, it was shown that NKA and the NMDAR co- precipitate151. Co-immunoprecipitation requires that the cells are first lysed and it is possible that the two proteins only interact in the cell lysate and not in their native state. To check whether they co-localize on the plasma membrane we used two approaches. The first one was proximity ligation assay (PLA) where we stained proteins of interest with primary and then with secondary antibodies bound to oligonucleotides. When the proteins are near, the oligonucleotides attached to the secondary antibodies can be ligated by the addition of a ligase enzyme. This will form a circle of DNA which can be used as a primer for DNA polymerase. After rolling circle amplification (RCA), the amplicon can be visualized by microscopy. However, this method only gives us a binary answer about whether the proteins are near or not. To estimate the distance between the NKA and the NMDAR we used STORM microscopy, which allowed us to calculate the distribution of distances between the proteins using the nearest neighbor algorithm. Our data showed that a large fraction of the NKA and the NMDAR are co-localized. Other potential possibilities for how ouabain can regulate the NMDAR could be through membrane potential changes, fast events of internalization, or the involvement of tyrosine kinases. We applied different microscopy and electrophysiological techniques to reject these hypotheses.

Numerous studies show the presence of ouabain and other cardiotonic steroids in mammals26,33,152, indicating that the regulation of the NMDAR by ouabain might be an endogenous pathway. To answer whether this regulation loop might be important for brain tissue, we used a long-time

32 potentiation protocol on acute brain slices in the presence or absence of ouabain. Since the NMDAR is involved in the reduction of LTP, the effect after ouabain treatment suggested that we can see ouabain-induced attenuation of the NMDAR function not only in primary culture but also in brain tissue slices.

Taken together, our data show that the NKA and the NMDAR co-localized on the plasma membrane, and since ouabain affects calcium influx by the NMDAR, we propose that changes of NKA conformation led to changes of NMDAR conformation via a direct protein-protein interaction. Some support for this idea was given in a previous Co-IP study151. We started to investigate the idea by using a GST pull-down assay with cytoplasmic fragments of α-subunits of NKA fused to glutathione S-transferase and found that the second cytoplasmic domain of both the α1 and the α3- subunits of NKA might interact with the NR1 (also known as GluN1) subunit (Figure 3.1), but this should be investigated further. In conclusion, we showed that potential endogenous regulators belonging to the class of cardiotonic steroids attenuate the NMDAR functiona vi protein-protein interactions, and compounds from the CTS family might be considered to be pharmacological tools to treat NMDAR-dependent calcium excitotoxicity.

Figure 3.1. GST pull-down assay for interaction between the α1- and the α3-subunits of NKA and different NMDAR isoforms (NR1, NR2A, and NR2B). NT – N-terminus, CD2 – cytoplasmic domain 2, CD3 – cytoplasmic domain 3, CT – C-terminus.

3.2 Paper II. Ouabain-regulated phosphoproteome reveals molecular mechanisms for Na+,K+-ATPase control of cell adhesion, proliferation, and survival In this paper, our goal was to study how ouabain regulates the phosphoproteome of COS-7 cells.

After the discovery in 1996 in the Askari’s Lab that ouabain triggers signaling cascades via binding to NKA, a lot of papers were published regarding this topic. The most studied cascades are Src/EGFR/ERK,

PI3K/Akt, and IP3R/Ca2+ oscillations which were discussed in detail in Chapter 1. Usually, researchers focused on one or two signaling cascades at a time, but it would be interesting to study how all these signaling cascades interplayed with each other. In most of the studies performed to date, researchers used inhibitory analyses which might not only affect the signaling cascade of interest but also affect the entire system as well. Recently, many high-throughput technologies such as mass spectrometry have been developed which allows us to study many proteins in one sample

34 at the same time153. During signaling cascades, many proteins undergo changes in their phosphorylation status which activate, enhance, or deactivate them, and these changes propagate the signal to the next player in the cascade. Phosphoproteomics is a very powerful tool to study this aspect of cell signaling as it allows us to resolve a snapshot of the phosphorylation status of proteins in a sample154.

In this study, we used COS-7 fibroblast cells which are derived from the kidney of an African green monkey. This cell line has been widely used, for example, to show that ouabain triggers calcium oscillations via protein- protein interactions between NKA and the IP3R71. We have shown that the treatment of COS-7 cells with ouabain leads to calcium oscillations without affecting the basal intracellular sodium level, indicating that the application of a selected concentration of ouabain (100 nM) is not enough to change the ionic gradient in COS-7 cells. In our phosphoproteomic study, we choose two-time points – 10 and 20 minutes – since in this time range the signaling effect in phosphorylation is detected in most previous studies. W e quantified 15348 phospho-sites in proteins corresponding to 3937 protein-coding genes while using proteomic analysis we identified proteins from 7108 protein-coding genes. On a protein level, it was just a little change while we found 1941 and 1484 phospho-sites significantly regulated after 10 and 20 min of ouabain-treatment respectively. Interestingly, 99% of the sites were on serine or threonine while tyrosine contains less than 1% of all the significantly regulated phospho-sites.

We classified 6 different clusters based on the different temporal regulation patterns observed. Gene Ontology enrichment analysis showed that four of the clusters were enriched by proteins involved in cell adhesion and cell-cell junction. The role of ouabain in regulating tight junctions has been previously shown155 which supports our findings. Also, we found

35 proteins involved in cytoskeletal re-organization (clusters 1-3) and cell division (cluster 4). Interestingly, we found a decreased phosphorylation level of ERK1 which is activated by ouabain in many studies156–158. However, we analyzed the time-course of ERK activation and found that ERK phosphorylation follows a more complex model – it is activated 5 minutes after ouabain treatment and deactivates thereafter. We did not observe Src activation or deactivation after 10 and 20 minutes of ouabain treatment which brings another question to the debate whether an NKA/Src complex exists. We also found that there was an enrichment of significantly regulated phospho-sites on calcium-regulated protein kinases which might shed light on the regulation of calcium oscillations found in COS-7 cells treated with ouabain.

Taken together, we created the first database of ouabain-triggered phosphoproteomics on the COS-7 cell line which could help understand the whole picture of ouabain-triggered signaling cascades. Several studies show the effect of ouabain on gene expression based on RNA sequencing159–161, however, these downstream effects happen 4-16 hours after ouabain application. Here we tried to catch earlier processes occurring 10-20 minutes after ouabain application on the phosphorylation levels of proteins. We found that ouabain leads to the phosphorylation or dephosphorylation of more than 1000 proteins which could be studied in the future.

3.3 Paper III. Severe epileptic encephalopathy caused by a de novo germline Trp931Arg mutation in ATP1A1 that converts Na,K- ATPase into an ion channel In this paper, our goal was to find the cause of death of a 10 months old patient with a mutation in the ATP1A1 gene.

Studying the NKA function is important not only for fundamental science but also for its medical importance. During the last 20 years, it has been shown that mutations in the α-subunit of NKA lead to several neurological diseases. Monoallelic diseases caused by mutations in ATP1A1 were first described in 2018132. An infant at the Karolinska hospital who died at 10 months due to therapy-resistant epilepsy was found to have a Trp931Arg mutation in ATP1A1. After the whole exome sequencing of the patient and both parents we, found that the mutation was de novo. Using multiple alignments of different sequences from several vertebrates we showed that this position is highly conserved. On the protein level, this mutation results in a one amino acid change from an aromatic hydrophobic tryptophan to a positively charged arginine. Since this amino acid is located in the 8th transmembrane domain, changing a hydrophobic amino acid to a hydrophilic amino acid might affect the protein structure. Using the ouabain viability assay we showed that the viability of cells transfected with the mutant α1 subunit is lower than wild-type cells, indicating that there is a reduced or abolished activity of the pump. Transfection of neurons with the mutated ATP1A1 leads to membrane depolarization and may explain epilepsy observed in the patient.

We wanted to investigate how a reduction in protein activity results in the depolarization of the neurons. Since the mutation is located in the transmembrane domain, we predicted that it may affect the folding of the protein. To test whether the mutant protein is present in the plasma membrane to the same extent as the wild-type protein we tagged it with pH-sensitive GFP (pHluorin) and transfected neurons with it. We found that while the wild-type protein is mostly present in the plasma membrane there is a large fraction of the mutant protein which is not trafficked to the plasma membrane. To demonstrate that a fraction of the mutant proteins

37 is present in the plasma membrane we performed additional staining of transfected neurons with anti-GFP antibodies which only bind to the extracellular pHluorin. We could conclude that although there was mislocalization of a fraction of the proteins, another fraction was present in the plasma membrane. Additional energy is required by the cells to degrade the mislocated proteins and may compromise the health of the cells. Staining of patient samples showed expression of α1 in the brain, but, since the patient was heterozygous for the mutation, the staining may have only shown the wildtype allele.

We wanted to analyze in greater detail how this mutation affects the protein using an electrophysiological approach on Xenopus oocytes and a molecular dynamics study. We found that there is a leak current in cells transfected with the mutant protein, and introducing different monovalent cations such as Na+, K+ or Cs+ contributes to this leakage, showing that the leakage is not sodium specific. At the same time, we observed that there is water accumulation around the mutated amino acid in the E1 state of the mutant compared to the wild-type protein. This might be due to the tryptophan-to-arginine mutation since the positively charged arginine could attract water, and this water pathway might be complemented with an ion leakage as we recorded by electrophysiology.

Taken together, we have shown that a mutation in the ATP1A1 gene found in a 10 months old patient leads to therapy-resistant epilepsy, and have found a potential mechanism explaining the observed pathophysiology. Although the α1 subunit of NKA is expressed in the entire body, the patient only showed symptoms related to brain function, indicating that the dysfunction of one allele only affects nervous tissue. This is not surprising since heterozygous knockout mice of the α1 subunit were viable and did not show severe pathologies117. We have shown that this mutation converts

NKA to a non-specific ion channel that is permeable to different monovalent ions and this may contribute to the epileptic attacks observed in the patient. A separate study published in 2018 showed that other mutations in the α1 subunit lead to a cation leak136, indicating that the leak may be a common result of mutations in the transmembrane helixes of the α1 subunit of NKA.

3.4 Paper IV. Mechanisms by which the T613M mutation causes mobility and gait disturbances in Rapid-Onset Dystonia- Parkinsonism In this paper, our goal was to study how a T613M mutation in the α3 subunit of NKA affects protein, neuronal cell, and central nervous system function.

Rapid-onset dystonia-parkinsonism (RDP) was first described in 1993 and described as a monoallelic disease that can be attributed to different mutations in the ATP1A3 gene in 2004. It is characterized by an abrupt onset of dystonic symptoms, often with bradykinesia and gait instability, and usually associated with speech and swallowing problems. The mutation p.T613M is the most common mutation found in ATP1A3 and is located in the third and largest cytoplasmic domain. To describe how this mutation leads to such a severe disease we decided to do a translational study.

First, as in our previous study, we characterized whether this mutation affects NKA localization in the plasma membrane. We found that the T613M mutation in the α3-subunit does not affect NKA localization in the plasma membrane as the mutant protein was present in the plasma membrane to the same extent as the wild-type protein. Changes in

39 extracellular pH almost completely abolished the signal meaning the signal was mostly coming from the plasma membrane. We checked the membrane localization of three other mutations known to cause RDP and found that two of them (I274T and F780L) have mostly membrane signal while a large fraction of the D801Y mutated ATP1A3 was not present in the plasma membrane (Figure 3.2).

A I274T NKAα3-pHluorin B F780L NKAα3-pHluorin C D801Y NKAα3-pHluorin

1 2 1 2

1 2

3 3 2 1 3 2 3 4 1

5 4 4 5 3 5 4 5 4 5

0.05% 0.1 % 0.1% 20 μm 20 μm 10 μm 10 μm 10 μm Figure 3.2. Membrane expression of different mutations in NKAa3 characterized for RDP. A, B, C. Representative pictures of hippocampal neurons in culture expressing I274T, F780L, and D801Y NKAα3-SEP respectively. Calibrated fluorescent beads are indicated with white triangles. Fluorescence intensity profiles through the soma and dendrites along the lines indicated.

We used molecular dynamic simulations to investigate the effect of disease-related mutations and revealed that this mutation affects the nucleotide-binding site and leads to its reorganization which probably affects the organization of the entire protein structure. This may also explain the reduced activity of mutated NKA which has been shown in two studies123,162. Using live sodium imaging, we showed that this mutation leads to increased basal intracellular sodium and a reduction of the maximum recovery rate of sodium. Interestingly, other mutations (I274T, F780L, and D801Y) which are specific for RDP showed the same tendency. However, T613M showed the most prominent difference compared to wildtype which is an indication as to why T613M is the most common

40 mutation causing RDP. The electrophysiological analysis showed that T613M-expressing neurons have a higher resting membrane potential and that they are prone to respond to depolarization with higher frequency action potential firing. Taken together, we show that the reduced activity of the T613M mutant leads to altered sodium homeostasis and depolarization of neurons.

To understand how this mutation manifests itself at the level of the whole organism we’ve been working with heterozygous animals of a knock-in mouse model. We found that heterozygous mutant animals have lower weight and a higher rate of spontaneous deaths compare to wild-type animals. Since most of the RDP patients developed their symptoms after triggers, we tried to mimic these triggers in our model using immobilization stress, cold-water, and alcohol injection. We did not succeed in triggering RDP but two mice spontaneously showed symptoms of dystonia without the application of a trigger. We have shown that animals are hyperactive and have motor problems which lead to differences in gait control. We investigated this further and found that there are fewer motor neurons in the spinal cord which show ultraslow afterhyperpolarization, a process which is NKA-dependent112,113.

Taken together, we performed a translational study and found that a mutation in the ATP1A3 gene leads to reorganization of the protein structure, alters sodium homeostasis, and depolarized cells. In a mouse model, this mutation results in hyperactive animals with motor problems and lacking ultraslow afterhyperpolarization in spinal cord motor neurons. This study may be complemented with the analysis of neuronal circuits in the spinal cord and the expression of genes involved in the regulation of afterhyperpolarization. RDP was first described almost 30 years ago but there is no treatment so far. Research has mostly been focused on the brain

41 while we have shown that the spinal cord is also affected by the mutation. This research opens up a new field of study concerning RDP’s effect on spinal cord function and the potential for treatment which might be beneficial for those patients with problems regarding gait and motor coordination. At the same time, this study sheds light on the specific function of the α3-subunit of NKA in the nervous system which is still mostly unknown.

4 Ongoing study

Although more and more becomes known about the interaction of NKA with other proteins, the interactions within the NKA molecule are not fully understood. It is well known that NKA consists of two subunits: α and β. The third subunit, also known as the FXYD protein, is a regulatory subunit, however, NKA forms a minimal functional unit as a dimer of the α- and β subunits without the FXYD protein163. During the last decades of the 20th century, it has been debated whether NKA exists as a protomer (α-β-FXYD heterotrimer) or as an oligomer of protomers (diprotomer or tetraprotomer). Several studies have indicated that NKA exists as an oligoprotomer, using two-dimensional membrane crystals8, high- performance gel chromatography9, cross-linking approach10, etc. Moreover, even before the crystal structure of NKA was solved, researchers predicted two functional ATP-binding sites on the NKA molecule which with the new data showed just one ATP-binding site could indicate that two α subunits interact with each other164. One of the pioneers in the NKA research field, Professor Amir Askari (23.12.1930-15.01.2020), who first described the role of NKA in cell signaling, in a recent review, which pointed out controversial issues in this field, highlighted the potential NKA oligomerization as a forgotten but important question which needs to be solved165. All of the studies mentioned above have been done by applying biochemistry to lysed cells, but not at a cellular level. In this study, we are eager to unravel this more than 30 years old mystery by using a combination of modern techniques such as non-canonical amino acids (ncAA)-labeling and Fluorescence Correlation Spectroscopy (FCS) to track this potential interaction in live cells. Our goal was to look at the potential oligomerization of labeled α- and β- subunits of NKA by live-imaging of the cell membrane. Using a fluorescent

43 protein-tagged approach has a limited capacity for this project, because the large size of GFP (238 a.a., 27 kDa) might disrupt potential dynamic interactions between subunits. Also, since NKA is a very abundant protein and transfection of the constructs leads to overexpression it is hard to track single molecules. Using antibody labelling would not work since α- subunits have very small extracellular domains and although β has a big extracellular domain, binding of antibodies might also change the nature of potential oligomerization.

In collaboration with Dr. Simon Elsässer’s group, we applied a technique to use genetically encoded non-canonical amino acids which were specifically labeled by click-chemistry with a small organic dye molecules cross-linked with tetrazine derivatives (0.7-1.2 kDa). This method is based on the concept of genetic code expansion where an exogenous pair of tRNA and aminoacyl-tRNA synthetase (aaRS) recognizes one of the stop codons (usually the amber stop codon, UAG) and specifically incorporates non- canonical amino acids instead of terminating the translation (Fig. 4.1). Nowadays there are several different tRNA/aaRS pairs166 for different amino acids and stop codons and there is a possibility to incorporate two different amino eacids in th same protein molecule by combining two different stop codons in the RNA sequence167.

Figure 4.1. Scheme of non-canonical amino acid incorporation into the protein. Usually, when translation reaches any of three stop codons on an mRNA sequence it terminates translation (scenario A). However, by using an orthogonal pair tRNA/aaRS and with supplementation of ncAA (star), aaRS synthesizes ncAA-tRNA which recognizes the amber stop codon (UAG) and incorporates ncAA to the protein (scenario B).

To this end, we have cloned coding sequences of the α1 and the β1 subunits to the vector containing pyrrolysine-tRNA (PylT) which recognizes an amber codon and transfects cells together with another vector containing pyrrolysine-tRNA synthetase (RS), specific for this tRNA and ncAA. The PylT/RS pair was isolated from archaeon Methanosarcina mazei and is widely used for this technique. To decide the positions for the codon change we looked at multiple alignments of protein sequences of the α1 and the β1 subunits from different species among vertebrates and picked several amino acids which we deemed not important for the protein function (Fig 4.2A). We looked only at the extracellular part of the proteins because labelling only them, with non-permeable dyes, would allow us to track only proteins located in the plasma membrane. Those positions were supposed to be on the surface of protein molecules and not be involved in the secondary structure of the polypeptides, and be as close as possible to the start codon to have the smallest truncated side product (Fig. 4.2B). We

45 chose three amino acids for α1 and two for the β1 subunits to be replaced by ncAA based on their variability between different species and their localization. For an additional construct for α1 the amber stop codon was inserted in the position wherein GFP was inserted in an earlier study, without disrupting protein function168. An additional construct for β1 is in the C-terminus for which a side product is a properly functional β1. We added a HA-tag to the N-terminus of the α-subunit and to the C-terminus of the β-subunit to discriminate between exogenous and endogenous proteins.

Figure 4.2. Position selection on the α1 and the β1 subunits of NKA. A – multiple alignments of the first and the second extracellular loops of the α1 subunit and the beginning and the end of the extracellular domain of the β1 subunit. Positions of variable amino acids selected for mutations are highlighted by a red rectangular, positions for insertions are highlighted by a red line. B – 3D structure of NKA based on the 2ZXE crystal structure, yellow – α1 subunit, cyan – β1 subunit, and red – positions of the mutations/insertions. 1 - T121TAG, 2 - E123TAG, 3 - E314TAG, 4 - 317insTAG (all – α1). 5 – L64TAG, 6 – K65TAG, 7 - 304insTAG (all – β1).

Cells were transfected with two plasmids: the first one contains the α or the β subunit and four tandem repeats of PylT with the h7SK promoter for PolII transcription and the second contains the aaRS coding sequence. After that the non-canonical amino acid trans-cyclooct-2-ene-L-lysine (TCOK) was supplemented to the culture media. Two days after transfection cells were incubated with pyrimidyl-tetrazine conjugated with AF488 or AF647 which lead to the strain-promoted inverse electron- demand Diels-Alder cycloaddition (SPIEDAC) reaction between pyrimidyl-tetrazine and ncAA. To test whether we successfully incorporated ncAA and obtained a labeled α or β subunit we ran electrophoresis and looked for a fluorescent signal from α and β (Fig. 4.3A and 4.4A). A signal was detected for amber mutants in a TCOK (ncAA) dependent manner but no signal for other lanes. To test whether we had truncations resulting from translation termination at the internal amber codon (side product) we did a western blot with antibodies against the HA- tag (Fig. 4.3B and 4.4B) and showed that while we observed a correct product for all of the constructs except the 304insTAG construct (which might be explained by close location of the HA-tag and cross-linked dye) we had strong bands of side products for the two α-subunits mutants – for E314TAG and 317insTAG. For those two mutants, side products were recognized by anti-α1 antibodies (Fig. 4.3C).

Figure 4.3. Analysis of α1 constructs with an amber stop codon. A – electrophoresis to check labeled ncAA, B – Western blot for anti-HAtag antibodies, C – Western blot for anti-α1 antibodies. Lanes: 1 - α1 WT with HAtag, 2-3 – positive control with InsR plasmid without and with TCOK aa, 4-5 – α1 T121TAG without and with TCOK aa, 6-7 – Α1 E123TAG two different clones with TCOK aa, 8-9 – α1 E314TAG without and with TCOK aa, 10-11 – α1 317+TAG without and with TCOK aa.

Figure 4.4. Analysis of β1 constructs with an amber stop codon. A – electrophoresis to check labeled ncAA, B – Western blot for anti-HAtag antibodies, C – Western blot for anti-β1 antibodies. Lanes: 1-2 – positive control with the CRFR1 plasmid without and with TCOK aa, 3 - β1 WT with HAtag, 4-5 – β1 L64TAG without and with TCOK aa, 6-7 – β1 K65TAG without and with TCOK aa, 8-9 – β1 304insTAG without and with TCOK aa.

To test the membrane staining we looked at cells by confocal microscopy. We found that three constructs for the α1 subunits (T121TAG, E123TAG, E314TAG) and two for the β (L64TAG, K65TAG) had good membrane staining (Fig. 4.5A-C, 4.5E-F). Two constructs – α1 317insTAG and β1 304insTAG did not reveal clear membrane staining (Fig. 4.5D and 4.5G). As a negative control, we used cells transfected with the wild-type α1 and β1 subunits without amber stop codon and PylT/RS pair. We found a weak

48 non-specific background signal which was not localized to the membrane (Fig. 4.5H-I). Together these observations suggested to use T121TAG, E123TAG, and E314TAG for α1 and L64TAG and K65TAG for β1.

Figure 5. Analysis of membrane localization of the Pyrimidyl-tetrazine- AF488 labeled HEK cells. A-I. Representative pictures of HEK cells expressing T121TAG (A), E123TAG (B), E314TAG (C), 317insTAG (D), and WT (H) α1 or L64TAG (E), K65TAG (F), 304insTAG (G) and WT (I) β1.

We then performed FCCS and FRET-FCS measurements with dual labeled HEK cells with the selected 5 constructs. We labeled cells with Pyrimidyl- tetrazine-AF488 (5 µM) and Pyrimidyl-tetrazine-AF647 (5 µM) in the ratio 1:1 expecting equal labeling of these dyes. We compared wild-type conditions and 50 nM ouabain application. We choose a ouabain

49 concentration of 50 nM in order not to inhibit exogenous NKA but to bind to a few molecules while inhibiting more than 50% of endogenous NKA, since human α1 has three orders of magnitude higher affinity to ouabain compared to that of rat α1.

A typical recorded FCCS curve is presented in Fig 4.6A. No cross- correlation was observed for any of the five studied constructs, neither with nor without ouabain, indicating either that no NKA-oligomeris were present, or that they were present but at too low concentration compared to the green- and red-fluorescently labelled monomeric NKA. However, we did observe FRET-FCS curves indicating that a small fraction of the NKA molecules oligomerized. The fact that FRET-FCS clearly indicated that oligomers were present, while FCCS did not, indicates that only a small fraction of the available donors and acceptors formed oligomers, since FRET-FCS is less sensitive than FCCS to the presence of large fractions of non-interacting monomers169. Moreover, we observed that for the T121TAG and the E314TAG α1, addition of ouabain enhanced the FRET- FCS signal indicating that ouabain triggers oligomerization of exogenous NKA (Fig. 4.6B and Table .4.1) We did not see the same effect for E123TAG, possibly due to the disruption of the ouabain binding site since amino acids of the first intracellular loop of the α1 subunit participate in the ouabain- binding site59, and dye-binding to the amino acid could mask them. We did not observe the FRET-FCS signal increase for the β1-subunits which could have two possible explanations. First, β1 could be binding to endogenous α1 which is more than 70% inhibited at this ouabain concentration, and too high ouabain concentration may affect the interaction differently compared to low concentration. Second, if α-α oligomerization is possible, the two β1 subunits would be on opposite sites of this complex and therefore the distance could be too large for FRET.

A B

Figure 4.6. FCCS and FRET-FCS analysis. A. Green- and red autocorrelation curves and cross-correlation curve for the T121TAG α1 construct with 50nM ouabain added. B. FRET- FCS autocorrelation curves for the T121TAG α1 construct with and without 50nM ouabain.

Table 4.1. FRET-FCS signal presented in CPM (counts per molecule) for the acceptor dye. Control (cpm) 50 nM ouabain (cpm) α1 T121TAG 0.041 0.20 α1 E123TAG 0.072 0.051 α1 E314TAG 0.045 0.30 β1 L64TAG 0.05 0.11 β1 K65TAG 0.042 0.047

To continue this project, we need to apply different ouabain concentrations to get a broader view of the potential oligomerization. It is important because an increase in activity has been reported at low ouabain concentration170, which may be because if ouabain binds to only one protomer in an oligomer it results in enzyme activation, while if it binds to both protomers the enzyme is inhibited. Also, since NKA-related monoallelic diseases have been observed on heterozygous states, and keeping in mind that a heterozygous knock-out mice model did not reveal similar symptoms, this difference may be explained by the interaction between mutated NKA and wild-type NKA resulting in a pathological state

51 of NKA encoded by the wild-type allele. This potential explanation of the different symptoms between knock-out and knock-in models should be studied further, and the model system allows us to track this interaction by dual-labeling of the wild-type and mutant proteins using non-canonical amino acids.

5 Conclusions and future perspective

Na+,K+-ATPase was discovered more than 60 years ago but interest in it has significantly increased in recent years. Firstly, this is because genes encoding α subunits have been attributed to be the cause of several neurological diseases such as alternating hemiplegia of childhood, rapid- onset dystonia-parkinsonism, familial hemiplegic migraine type 2, Charcot-Marie-Tooth Type 2, and others. Secondly, NKA was found to be a signal transducer in mammalian cells and the ligand, cardiotonic steroids, were found to be synthesized endogenously and proposed to be hormones in mammals. Thirdly, in the past decade, it has been shown that NKA interacts with many membrane and cytoplasmic proteins and can regulate or be regulated by these proteins. This regulation affects not only ion homeostasis and secondary active transport but also synaptic transmission, tight junctions, endocytosis, and other important processes for mammalian cells. In this thesis, I have covered these three aspects from a biophysical perspective.

5.1 Neurological diseases and Na+,K+-ATPase In 2018, 2003, and 2004 it was found that mutations in ATP1A1, ATP1A2, and ATP1A3 respectively are responsible for different neurological disorders which are monoallelic diseases and found in the heterozygous state. Currently, there is no available therapy and genome editing might be beneficial to completely cure these diseases; however, it is not available yet. To find other therapies, it is very important to know whether the mutation results in loss-of-function or gain-of-function. In the case of loss-of- function, it might be enough to increase the expression of the wild-type allele or find a way to increase the protein activity. For mutations resulting in a gain-of-function, the protein encoded by the mutant allele should be

53 removed or, if this is not possible, to compensate for the side effects of the mutant allele. Lingrel and colleagues made knockout mice models of the ATP1A1, ATP1A2, or ATP1A3 genes and showed that all three heterozygous animals survived. Although there were several differences with wild-type animals, heterozygous animals did not have any neurological symptoms. This evidence indicates that those mutations in the aforementioned diseases are not just loss-of-function but more likely gain-of-function. It is therefore of great importance to study these mutations in vitro and in vivo to find out which part of the nervous system is affected and find potential treatments.

One of the potential issues with the mutated protein may be that it doesn’t properly fold. Since NKA is one of the most abundant proteins in eukaryotic cells, proteolysis of a lot of misfolded proteins may have a high energetic cost. To test this possibility, we tagged the α1 and α3-subunits of NKA with an extracellular pHluorin and quantified the amount of protein in the plasma membrane compared to the cytoplasm. We concluded that the T613M mutation in the α3-subunit is present in the plasma membrane toe th same extent as wildtype. For the W931R mutation in the α1-subunit, we found that there is a large fraction of the protein that is not in the plasma membrane which might partially explain the pathology. This method is very robust and can be used to test whether mutant NKA subunits are plasma membrane-localized in the future. Molecular dynamic simulation is a very powerful tool that can predict how a mutation affects protein structure. For the T613M mutant in the α3-subunit, it was shown that there is an instability in the protein close to the ADP-binding site while for the W931R mutant it has been shown that this mutation might convert the pump to a channel. There is a correlation between the severity of symptoms and the location of mutations in the transmembrane helixes. However, the effect of a mutation may be different even if the mutation is

54 on the same site but leads to the insertion of different amino acids. It is therefore important to perform molecular dynamic studies for each mutation since each mutation is characterized by different phenotypes. After finding out how the mutation affects protein folding, structure, and function it is also important to study the consequences of the mutation in neuronal cells. To do this, we have applied different techniques – electrophysiology to check whether there is membrane depolarization or hyperpolarization and whether there is an ion leak. The mutation affects sodium ion homeostasis over the membrane we used, a fluorescent dye that can be used to read out the sodium concentration within the cell. We challenged the neuronal cells by removing extracellular potassium or adding NMDA and measured the rate of recovery. The combination of these approaches allows us to study how the mutant protein affects the electrophysiological properties of the neuronal cell and could be very useful to predict whether the cell is in a pathological state.

The spectrum of diseases caused by mutations in α-subunits of NKA is very broad and it is unlikely that a common therapy will be suitable for the different mutations. Studying these mutations individually on the molecular and cellular level will allow us to identify pathological processes that are specific for each mutation and design specific treatments in the future.

5.2 Na+,K+-ATPase as a signal transducer It was shown over twenty years ago that ouabain and other compounds from the cardiotonic steroid family can start a signaling cascade by binding to the NKA. This discovery revolutionized the NKA field as it was previously believed that NKA functioned only as a pump. Around the same time, it was shown that compounds from the cardiotonic steroids family

55 may be present in mammals. These two pieces of evidence shifted the paradigm that ouabain acts only as an inhibitor of the NKA and instead identified ouabain as a hormone with NKA being its receptor.

Since that time, groups began studying the signaling cascades activated by ouabain-NKA but usually just one pathway at a time. Currently, several high-throughput technologies allow us to look at transcriptional, and protein expression states simultaneously. We applied phosphoproteomics for the first time to ouabain treated cells and made a database of proteins that are phosphorylated or dephosphorylated 10-20 minutes after the addition of ouabain. Using this approach, we made a snapshot of the phosphorylation status of proteins at two-time points allowing us to predict all the potential pathways that ouabain can influence. We chose COS-7 cells (which can be classified as non-differentiated cells) to catch processes that might be common for all cell types. It would be interesting to perform additional studies to determine the specific signaling cascades activated in differentiated cell types such as neurons, glia, and cardiomyocytes or the involvement of different α-subunits of NKA.

5.3 Na+,K+-ATPase and its interactome More recently, it was shown that NKA can interact with many proteins and reciprocally regulate their functions. This is especially interesting as NKA interacts with many receptors and transporters involved in synaptic transmission and plasticity. In our study, we characterized the functional interaction between NKA and one of the most important proteins for the molecular biology of memory – the NMDAR. Previous studies showed their functional coupling using biochemical methods. Although earlier published data was convincing, we wondered how fast ouabain could regulate the NMDAR’s function via NKA. Using numerous microscopy

56 techniques, we observed the interaction between NKA and NMDAR in real- time. We demonstrated that the effect of ouabain on the NMDAR activity is fast and showed the two proteins co-localize on the plasma membrane. Further studies might reveal a more complete picture of the NKA interactome using a combination of high-throughput proteomic analysis and confirmed using superresolution microscopy.

While it has been shown that NKA interacts with many proteins, it is still an open question whether it exists in the plasma membrane as a protomer (α-β-FXYD in the complex) or a dimer, or even a tetramer of protomers. There are a lot of studies that claim this, but it has never been shown in a live cell. By combining non-canonical amino acids labeling with FRET-FCS measurements we have shown that ouabain triggers oligomerization of NKA.

Na+,K+-ATPase was discovered in 1957 by Nobel Laureate Jens Skou but it conceals many unexplored mysteries and secrets. By applying modern biophysical methods we have taken one step forward to uncover the secrets of this important mammalian protein – the Na+,K+-ATPase.

6 Acknowledgments

During the last years, SciLifeLab has started to become my second home (except the fact that I never slept there). My time there was similar to calcium oscillations – sometimes it was full of happiness, joy and inspiration and sometimes I was on the other side of the oscillation pattern. Now I am on the upper part of the curve and I hope to reach a plateau or even go to a higher level, like an excited fluorescent molecule, albeit without the following emission step. But before continuing to the next challenge I would like to thank all the people who were with me during this journey.

A big thank you to my main supervisor, Hjalmar Brismar, who supported me during this journey, supervised me at different stages in my career and, taught me physics and microscopy, which, as a biologist, was not always easy. I am so proud that thanks to you I am a doctoral student in physics even with my biological background.

A big thank you to my main co-supervisor, Anita Aperia, who always inspired me with enthusiasm, desire to open new horizons in science, and by new ideas. Thanks forl al of your supervision and for all the scientific discussions which I am so proud of. You always made me think about science from a biological perspective and prevented me from getting too deep into the microscopy

Thank you to my co-supervisor, Stefan Wennmalm, for introducing me to another angle of microscopy, FCS, very nice scientific discussions and for helping me wrap up my thesis in a very short time.

Great thanks to my collaborator, Mia Lindskog, and her former and current Lab members (Salvatore Magara, Erika Vazquez-Juarez, Ipsit Srivastava and Benjamin Portal) for all of our work together and help me discover the field of electrophysiology! Your team helped me with almost all projects presented in the thesis!

Thanks to my collaborator, Per Svenningsson, and his Lab members (Vasco Sousa and Xiaoqun Zhang) for guiding me how to perform animal behavioral tests and moreover how to analyze the data!

Thanks to my international collaborator, Gareth Miles, and his former and current members, Laurence Picton and Frankie Sorrell, who inspired me to look at the spinal cord more carefully and shifted the focus of our project!

Big thanks to my collaborator, Erdinç Sezgin, who always inspired me how to do science and for our fantastic talks about it. It was always easy to follow your explanations about difficult topics. I hope to collaborate with you in the near future!

Thanks to Simon Elsässer, Johannes Heimgärtner and Birthe Meineke for the great work together and trying to solve a very fundamental question using your awesome technique!

I would like to thank my collaborator, Minako Hoshi, for the excellent quality of scientific discussion, fantastic research questions and very warm relationship during this time!

I would like to thank all my colleagues who worked with me, especially Magnus Andersson, Rebecca Howard, Sofia Ygberg and Martin Paukar for a very pleasant time working together!

Thanks to Jacopo Fontana, we ate a bushel of salt together, participated in several projects together and are still friends after this journey! I will always be a big fan of Italian football and especially FC Napoli. I would very much like to thank my Russian friends and former Lab members, Nina Illarionova and Georgiy Khodus, who showed me Sweden, Stockholm and helped find the “KGB bar” at midnight (it was permanently closed). Big thanks to all former members from the Lab! Lena Scott, Linda Westin, Thomas Liebmann, Nicolas Fritz and Daniel Jans, it was so nice to work with you and spend free time together! My special thanks to Lill-Britt Svensson for your kindness and helping a lot during these years! Special thanks to my big Brazilian friend, Paula Kinoshita. I never thought that we would have a chance to work together again and… we ended up working together twice in Stockholm!

Thanks to Steven Edwards, who was my PhD-mate even you were accidentally registered as a doctoral student three months later than me. We’ve had a lot of joy talking about different things and it is always nice to be friends with a native English speaker and, maybe more importantly, the possessor of a British sense of humor. Thanks to Liang Zhang, who told me how I should love Argentina, Messi and Hot-Pot. Thanks for your Chinese lessons which will help me in the future! Thanks to Maximilian Senftleben and Linnea Nordahl for making a great new team in the Lab and the time spent together! Thanks to Katja Schach who joined the lab for an internship and, after just a few months has a preliminary manuscript. I am waiting for the invitation to your Nobel Prize Ceremony.

Thank you, David Unnersjö-Jess, for making a very warm and happy environment and your songs at defense parties which I unfortunately will not receive thanks to COVID. When you are in Germany I feel that your spirit is still here (especially when I open my drawer at the lab bench). Thank you, Hans Blom, for being always super helpful with microscopy and for being critical which always improve my research. Thanks to Ilaria Testa, Giovanna Coceano and Francesca Pennacchietti for a lot of scientific and non-scientific talks, for discussions about Kebab pizza ingredients and for allowing me to discover chocolate salami! Thanks to the beer team including Daniel Morgan, Deniz Seçilmis and Miguel Castresana. It was always fun and joyful to spend time with you!

I would like to thank all my current and former colleagues from Gamma- 3: Alexander, Ana A, Ana O, Andrea, Andreas, Annemarie, Björn, Chiara, Daniel S, Daria, Dirk, Elham, Elina, Evgeniya, Federico, Florian, Francisca, Hanna, Hao, Ingeborg, Jes, Jonatan, Kalai, Karl, Karolin, Kristoffer, Kyra, Laura, Linnea, Luciano, Lucie, Ludwig, Marina K, Marina Z, Marta, Martina, Martyna, Markus, Minttu, Miroslav, Niklas, Otto, Patrick, Per, Quentin, Sara, Sarah, Taras, Tong, Valentina, Xavi and Ying. It was really nice to work with you, guys!

Thanks to all my friends who supported me during this time. A special thanks to my Mom, Elena, who has supported me from a distance all this time. Thank you for all your help and love during my life! I am so proud that you are my Mom! And last, but not least, thank you to my wife, Natasha, who has been by my side this entire time and supported me daily, always cheering me up and definitely increasing the level of my life oscillations! I love you!

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