Aus der Klinik für Hals-Nasen-Ohrenheilkunde der Medizinischen Hochschule Hannover
Microarray-based screening system to investigate the activity of Connexin 26 under different conditions and mutations
Dissertation zur Erlangung des Doktorgrades der Medizin in der Medizinischen Hochschule Hannover
vorgelegt von Hongling Wang aus Tianjin, China
Hannover 2019
Angenommen vom Senat der Medizinischen Hochschule Hannover am
Gedruckt mit Genehmigung der Medizinischen Hochschule Hannover
Präsident: Prof. Dr. med. Michael P. Manns Betreuerin der Arbeit: Prof. Dr. med. Athanasia Warnecke Referent /Referentin: PD Dr. Carsten Zeilinger
Tag der mündlichen Prüfung: 06. 07. 2021
Prüfungsausschussmitglieder:
Vorsitz Prof. Dr. med. Dr. h. c. Martin Ptok 1. Prüfer/in Prof. Dr. med. Burkard Schwab 2. Prüfer/in PD Dr. med. dent. Ingmar Staufenbiel
Table of content 1. Introduction
1.1 Hearing loss and connexin 26 1 1.2 Microarray technology 8 2. Goal of the study 10 3. Published paper 11 4. Results and Discussion 23
5. References 30
6. Curriculum vitae 35
7. Acknowledgements 36
8. Declaration 38
1. Introduction
1.1 Hearing loss and connexin 26
Hearing, one of the five human senses, is essential for language development and even cognition12. Consequently, impairment or loss of hearing has been shown to be associated with cognitive decline3 and this is independent of age4,5,6. Thus, hearing loss severely reduces the quality of life of the affected patients at any age. For the society, unaddressed hearing loss produces an annual cost of over $750 billion globally according to the World Health Organization.
Hearing loss is one of the most common sensory deficit diseases: One to three out of every 1000 newborns, about 4% of people younger than 45 years old, and 29% of the ones aged 65 and above are diagnosed with hearing loss7. For natural hearing sensation, a healthy peripheral and central auditory system are mandatory. Auditory stimuli are mechanically transmitted through the external auditory canal to the tympanic membrane and ossicular chain. This results in a piston-like movement of the stapes into the scala vestibuli and subsequently induces movement of the fluid in the perilymphatic space, thereby, leading to a displacement of the basilar membrane. The sensory epithelium containing the hair cells (the organ of Corti) is located on the basilar membrane8,7. Displacement of the basilar membrane results in a deflection of the stereocilia bundle, thereby leading to hair cell transduction. The hair bundles are located at
the apical end of the hair cells and represent the mechanosensitive organelles: The hair bundles consist of a kinocilium and several stereocilia that are linked to each other at their tip with so called tip links9. The tip links are one of the key players in the conversion of mechanical stimuli into electrochemical signals thereby enabling hearing. The molecular composition of tip links - consisting of two members of the cadherin family, cadherin 23 (CDH23) and protocadherin 15 (PCDH15)- enables their function10. Deflection of the hair cells towards the longest stereocilium leads to opening of the mechano-sensitive potassium channels allowing an influx of potassium along the concentration gradient from the endolymph into the cells. This influx leads to a change of the potential and thereby to an opening of the voltage-sensitive calcium channels11,12. Calcium ions are able to enter and thereby further depolarize the hair cells. Once depolarized, the hair cells release glutamate, which in turn activates the postsynaptic AMPA receptors of the spiral ganglion neurons12,13. In summary, through a series of signal transduction, inner hair cells (IHC) transfer outside acoustic information to the primary auditory neurons, the spiral ganglion neurons.
The endolymph in the scala media contains a high concentration of potassium (K+) contributing to the positive endocochlear potential (EP, +110 to +120 mV). The stria vascularis generates the endocochlear potential by actively releasing K+ ions into the endolymph. To avoid
K+-toxicity and maintain hair cell function, the expelled K+ around the hair cells needs to be removed14. Deiters cells, the supporting cells surrounding the outer hair cells, are responsible for taking up these K+ ions that escape from the hair cells.An active transport of K+ depends on the epithelial gap junction network15–18 that connects the type II and type IV fibrocytes of the spiral ligament and the stria vascularis. This epithelial gap junction network is based on the cytoarchitecture of the organ of Corti, the supporting cells, the epithelial cells and the outer sulcus cells. From the stria vascularis, K+ is actively pumped into the endolymph of the scala media. In the stria vascularis, the ion transport is warranted by a Na-K-Cl cotransporter, a Na+ /K+ -ATPase and conexins16–20. This process is termed K+-recycling (see Figure 114) and among the many different mutations associated with hearing loss are mutations of potassium channels as well as of connexins (the molecules that form the gap junctions)16,20–23.
between low (<500 Hz), middle (501–2000 Hz) and high frequency (>2000 Hz) hearing loss24.
The causes of hearing loss can also be divided into genetic (hereditary) and acquired. The later includes effusions and infections of the middle ear, trauma, tumor, immune-mediated, systemic, idiopathic, degenerative and age-related hearing loss. Several ototoxic drugs such as aminoglycoside antibiotics, salicylates, loop diuretics and antineoplastic drugs can cause deafness7.
Among all the causes, genetic and hereditary factors are the most common cause of hearing loss and affect probably more than half of the patients suffering from hearing loss7. The occurrence of both conductive and sensorineural hearing loss is possible. Hereditary hearing loss can be classified into syndromic and non-syndromic hearing loss24. Syndromic hearing loss is less common than non-syndromic and includes more than 400 syndromes. It is defined as hearing loss or deafness that co-segregates with other features to form a recognizable constellation of findings known as a syndrome25.
Non-syndromic hearing loss (NSHL) in the absence of other phenotypic manifestations accounts for more than 70 % of the inherited hearing impairment. It can be inherited in an autosomal recessive (75–80 %), autosomal dominant (20–25 %) or in rare instances as an X-linked or
mitochondrial pattern of inheritance (1–2 %)26. With increasing age, the prevalence of autosomal dominant and mitochondrial inheritance increases while that of autosomal recessive inheritance decreases27.
Thus, hearing loss is a complex disease involving a large number of genetic and environmental factors28. It is well-accepted that mutations in a total of 127 genes can cause NSHL: 46 genes correspond to autosomal dominant NSHL, 76 genes to autosomal recessive NSHL and 5 genes to X-linked NSHL29. Among them, three of these genes (GJB2, GJB3, and GJB6) encode for connexin proteins (Connexin 26, Connexin 30, and Connexin 31, respectively), and have been found to be involved not only in dominant and recessive NSHL but also in syndromic forms of deafness28,30.
Gap junctions are composed of homologous proteins termed connexins that admit the passage of small molecules (up to 1 kDA in size) independent of their charge31. Thus, a rapid transport of ions and small molecules between cells is possible via gap junctions28,32. There are two types of connexins, alpha and beta, named GJA or GJB followed by a number. Connexins are expressed in many different tissues33. About 50–
80 % of the cases of autosomal recessive congenital deafness are caused by mutations of Connexin 26 (Cx26). Of these mutations, 70 % are due to loss of a single nucleotide guanine (G), leading to a premature termination of the protein translation through a frameshift mechanism34.
It is known that 9 mutations of GJB2 are involved in dominant non-syndromic deafness, 92 mutations of GJB2 are involved in recessive non-syndromic deafness, 10 mutations of GJB2 are involved in unknown non-syndromic deafness with unknown mode of inheritance and 8 mutations of GJB2 areinvolved in syndromic deafness33.
To identify the mutations in patients, we can test patients’ blood samples by amplification refractory mutation system PCR (ARMS PCR) and restriction fragment length polymorphism (RFLP) method for already reported common mutations, and single-strand conformation polymorphism (SSCP) analysis, heteroduplex analysis (HA) and next-generation sequencing (NGS) for novel mutations. However, there is no stable test available to give information about the functional state of the connexins. Thus, it is still difficult to detect whether and how mutated Cx26 are influencing the function of the connexin hemichannels or the gap junctions, especially in cases of novel hitherto unknown mutations.
Heterozygous compound mutations in patients can also cause late onset hearing loss35, and we do not really understand in depth the functions of connexins and the influence of diseases and mutations.
In order to understand the pathophysiology of hearing loss, especially in cases of late onset hearing loss, information on the state of the function of the connexons is required. Connexins consist of four transmembrane helices and three loops. Connexons are hemi-channels formed by the
biomolecules, such as nucleic acids, proteins, glycans and peptides, as well as cells38. According to the surface chemistry of microarrays, on which proteins are immobilized through covalent or non-covalent bonds, or physical and chemical hybrid approaches, microarrays can be named by the reactive surfaces coated such as aldehyde, N-hydroxysuccinimide (NHS)-ester, epoxide, histidine, glutathione-S-transferase (GST) or nitrocellulose39,40,41. The piezoelectric non-contact nanoprinters used in microarray technology provide us with the possibility of transferring very small amounts of living cells (1200 cells or fewer) to microscope slides for monitoring of the applied cells42,43. Living cell microarrays allow up to 16 independent screenings on one array, and printing of cells ensures close cell-cell interactions providing an in vivo-like microenvironment42. Automotive microarrays enable the simultaneous analysis of proteins, vesicles or cells, and parallel testing of inhibitors, and are therefor time- and cost-effective.
Microarray technology can also be used to detect the function of connexons expressed on the surface of cells or microsomes and, therefore, can be applied to deafness-related basic research to accurately determine the function of Cx26 and its deficiency degree. Furthermore, microarray technology can detect the influence of environmental factors and compounds on Cx26 function, thereby, providing an accurate and reliable method for the screening of drugs that might improve Cx26 function.
2. Goal of the study
The purpose of this thesis is to investigate the function of Cx26, which is closely related to deafness, and to explore the responses of wild type Cx26 and mutant Cx26 to temperature, Ca2+ concentration, pH and other influencing factors. Furthermore, the ultimate goal is to explore the biological mechanisms related to deafness. Based on our results, we conclude that thermo-sensitivity is an important function of Cx26 and further research may be required to investigate whether thermo-sensitivity, under specific conditions, may be associated with the late-onset of hearing loss and other connexin-related diseases.
Furthermore, microarray-based technologies may be applied to screen for new treatment targets leading to novel therapies for deafness.
4. Results and Discussion
Connexons are a large family of more than 20 members, which are distributed on all cells of the human body44. They play an important role in regulating metabolism, internal environment stability, proliferation and differentiation of cells32. Therefore, the study of connexons is of great clinical significance. The exact mechanism, by which mutant Cx26 leads to the loss of hair cells, is not clear, but it is likely to be related to the signaling molecules exchange between cells and the interaction of cochlear hair cells with the surrounding environment45. Different environmental conditions such as temperature, ion concentration, pH or voltage will affect the opening and closing functions of connexons.
Previous studies aimed to analyze such influencing factors on wild-type connexons, but we are not yet aware of the sensitivity of the mutant type of connexon to these influencing factors46,47. In the present study, we used a microarray-based technique to detect the function of wild-type connexons and mutant connexons at the cellular and microsome levels.
The temperature-dependent opening of the hemichannel human-Cx26 (hCx26) was first studied by two electrode voltage-clamp (TEVC) measurements under controlled temperature after heterologous expression of the hCx26 gene in Xenopus laevis oocytes as shown before47. The
temperature effect on the deactivating currents gives an opposite temperature dependency.
In this study, we used a novel microarray-based technique, which allows the simultaneous monitoring of different mutations on dye transport mediated by Cx26. Lucifer Yellow (LY) is a fluorescent dye, which can freely pass gap junctions and connexons48, so that we can measure the permeability of Hela cells and microsomes expressing wild-type or mutant connexons by detecting connexon opening and closing with LY.
The microsomes are spherical particles formed by broken membrane organelles from cells after mechanical homogenization. Isolation of microsomes from the nucleus, mitochondria, peroxisome and other organelles by differential centrifugation can not only retain the structural characteristics of the membrane but also keep the biological activity and function similar to living cells49. Hela cells and microsomes expressing wild-type Cx26 and the mutated Cx26 were spotted on a chip, and the uptake of LY was observed by fluorescence.
In our experiments, first we observed the uptake of LY by Hela cells expressing wild-type Cx26 under different temperature conditions by detecting the LY fluorescence signal intensity on the chip. The chips signal at cold temperature (4 degree) was much weaker than at warm
temperature (37 degree). This result is similar to a previous study demonstrating the temperature sensitivity of wild-type Cx2647.
We performed the same experiment again on Hela cells expressing mutant Cx26 (Cx26 R184P, Cx26 L90P, Cx26 F161S). L90P with the mutant of T to C transition at nucleotide 269 of the coding sequence changes a leucine at codon 90 of the second transmembrane domain to a proline is associated with non-syndromic deafness50. R184P with G to C transition at nucleotide 551 of the coding sequence changes a arginine at codon 184 of the second extracellular loop to a proline is also associated with non-syndromic deafness51.F161S lies in the second extracellular loop with a change phenylamine to serine52. All three mutations of Cx26 mentioned above lead to hereditary hearing loss. Under warm temperature conditions, the Hela cells with the mutant Cx26 showed weaker fluorescence signals than the Hela cells with the wild-type Cx26.
This result demonstrated that Hela cells expressing the mutant Cx26 uptake less LY than Hela cells expressing wild-type Cx26. Similar results were observed on microsomes expressing wild-type Cx26 and mutant Cx26. Moreover, similar results were observed on the level of the purified protein. The same experiment has been repeated on liposomes reconstituted by purified hemichannel (Cx26K188N) that were expressed in E. coli with amino acid exchange in K188 to N188.
Our experiments confirmed that the LY signal of Hela cells and microsomes expressing Cx26 with mutations was weakened. Several mutations of Cx26 can affect the channel opening and thereby limiting the ability to transmit information between neighboring cells or between the cells and its environment. With the herein presented and investigated microarray, the state of the channel opening and closure under various conditions was visible. The experimental results showed that the opening and closing function of connexons differed depending on the type of mutations. In general, different types of Cx26 mutations resulted in different structures or different hemichannel pore size: Some mutant hemichannels are completely closed and others preserving part of the connexon function.
Under 4°C cold condition, there was almost no LY uptake by Hela cells and microsomes expressing wild-type Cx26. Therefore, the LY fluorescence signals were both extremely weak. This is corroborated by the results obtained from previous research47. The same experiment was performed on Hela cells expressing mutant Cx26 and the result showed that the opening function was almost lost where connexons were in a closed state. The microsome experiment also reached the same conclusion as the cell experiment.
In low temperature environments, all types of Cx26 are basically unable to take up LY and the hemichannel is closed. As the temperature increases, the wild-type Cx26 half-channel is open. The mutant Cx26 is partially open, but the open function is decreased when compared to wild-type Cx26.
These results are similar with previous results that Cx26 hemichannels are inactive at a non-physiological temperature below 23°C47. However, this is not observed for Cx4653. How other connexin hemichannels react under temperature changes has not been investigated yet. Mutations in Cx26 influence the hemichannel activity drastically.
Carbenoxolone (CBX) is a known gap junction blocker that inhibits the function of gap junction and the opening of connexons. CBX intercalates into the plasma membrane and binds to connexons and therefore induces a conformational change which results in closure of the channel54. The effects of different concentrations of CBX on wild-type Cx26 and mutant Cx26 were examined by microarray experiments. The experimental results showed that wild-type Cx26 exhibited a dose-dependent sensitivity to CBX. We tested wild-type Cx26 and three different mutant types (Cx26 R184P, Cx26 L90P, Cx26 F161S). Mutant Cx26 R184P and L90P were less sensitive to CBX than wild-type Cx26. For F161S, we
could not find a dose-dependent effect of CBX. In this regard, microsome and cell experiments showed the same trend.
High concentrations of external free Ca2+ can inhibit the hemichannel function47 and were also used in our experimental setting to investigate the activity of the connexons. With the increase of Ca2+ concentration, hCx26 wild-type Cx26 showed a concentration-sensitive curve for the uptake ability of LY, but this was not observed in any of the mutant Cx26.
Under normal physiological conditions, the human body can regulate the function of gap junction by extracellular free Ca2+ concentration. Thereby, the transmission of intercellular ions, metabolites, signaling molecules, etc. is regulated. Depending on the type of mutation, Cx26 can lose its sensitivity to extracellular free Ca2+ concentration. When the concentration of extracellular free Ca2+ changes, connexons cannot transmit information in time leading to various diseases31.
Previous experiments regarding connexons were based on cells47,44. In our experiments, we, for the first-time, used microsomes to test the connexon function. By yielding the same results as with the use of cells, our experiments confirmed the feasibility of microsomes for the testing of connexon function. This is an important finding, since microsomes show several advantages when compared to cells. Microsomes are more stable than cells. In addition, microsomes are easy to store and can be stored for
long time periods allowing higher flexibility in the planning and performance of screenings. Furthermore, by using microsomes as a test object, the concentration is easier to adjust.
Our experiments provide an accurate and reliable method for the investigation of the channel function of Cx26. We found differences in the channel function between the tested mutant Cx26 and wild-type Cx26.
With this experimental setting, we can screen for chemically-engineered and natural compounds that can affect connexon function in future investigations. Hopefully, with the aid of this screening system, we can identify compounds that can even restore connexin functions. This is clinically relevant since connexins, a big homologous family with similar functions, control many physiological functions and their mutations or dysfunctions can be associated with various diseases such as cancer, kidney, cardiac and skin diseases. Our screening technology can be therefore used for leveraging research on all diseases linked to connexons.
Future research in our laboratory will therefore concentrate on using the herein presented microarray screening system to identify novel compounds that can influence the function of connexins. In addition, the same technology will be also applied to develop a screening system for other relevant channels or receptors.
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7. Acknowledgement
I would like to express my sincere gratitude to all people who helped me in this research work and life during my study. Thank all of you so much.
My deepest gratitude goes first and foremost to my both supervisors Prof.
Dr. med. Athanasia Warnecke from the ENT department of Hannover Medical School and PD. Dr. Carsten Zeilinger at BMWZ in Leibniz University Hannover. Thanks so much to Athanasia for providing me the so precious opportunity to study besides you and to learn in clinic, you offered me many valuable suggestions on my research, your constant encouragement and guidance supported me facing all difficulties during my study time. Thank you very much to dear Carsten, you taught me a lot of knowledge of biochemistry and experimental technique. You always gave me important advice to lead me to the right way during my research work. I got a lot from your immense professional knowledge. You have walked me through all the stages of my research and writing thesis.
Without your consistent and illuminating instruction, my project could not have reached these results. I also must thank you for your great help in my daily living.
I would like also to thank Dr. Melanie Steffens, Dr. Jennifer Schulze and Dr. Kirsten Wissel for their contribution to this research. My sincere
thanks also goes to Dr. Frank Stahl and Dr. Johanna Walter for leading me and solving many difficulties in the practical lab work. I also thank so much PD Dr. med. Martin Durisin who helped me during my internship in the ENT clinic. I am also grateful to Dozens of people who also have helped me. Dr. Marvin Peter, Dr. Qing Yue, PhD students Sona Mohammadi-Ostad-Klayeh, Lu Fan and Master student Daniel Landsberg.
I express my sincere thanks to all of you.
Last but not the least, I wish to thank my family, my parents, my loving husband Zexu Zhang and my cute daughter Yoyo, who have been assisting, supporting, and caring for me all my life. You are the source of my life.
8. Declaration
I declare that the thesis submitted to Medical School Hannover for doctoral with the title
Microarray-based screening system to investigate the activity of Connexin 26 under different conditions and mutations
was performed in the Clinic of Otorhinolaryngology and the BMWZ, under the supervision of
Prof. Dr. med. Athanasia Warnecke and PD. Dr. Carsten Zeilinger and was carried out without any other help than listed above.
The opportunity for the present doctoral procedure has not been communicated to me commercially. In particular, I did not turn on any organization for the support or the preparation of the thesis, neither completely or partially.
So far, this work has not been submitted at any German or foreign university as a thesis. Furthermore, I assure that I have not yet acquired the requested title yet.
Part of this work has been published as follows:
Wang H, Stahl F, Scheper T, Steffens M, Warnecke A, Zeilinger C.
Microarray-based screening system identifies temperature-controlled activity of Connexin 26 that is distorted by mutations. Sci Rep. 2019 Sep 19;9(1):13543. doi: 10.1038/s41598-019-49423-3.
Hannover, _______________
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(Signature)
