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Centre of New Technologies, University of Warsaw Medical University of Warsaw
Ranju Kumari
Investigation of the physiological role of TMCC2 in inner ear sensory hair cells and the erythropoietic system using a novel knockout mouse line.
Badanie fizjologiczej roli TMCC2 w komórkach rzęsatych ucha wewnętrznego i układzie erytropoetycznym przy użyciu nowej linii myszy
knockout.
PhD thesis completed in Laboratory of Molecular Basis of Synaptic Plasticity Centre of New Technologies, University of Warsaw
Supervisor:
Prof. dr hab. Dominika Nowis
Auxiliary Supervisor:
Dr. Piotr Kaźmierczak Warsaw, 2022
2 Oświadczenie kierującego pracą
Oświadczam, że niniejsza praca została przygotowana pod moim kierunkiem
i stwierdzam, że spełnia ona warunki do przedstawienia jej w postępowaniu o nadanie stopnia doktora nauk biologicznych w zakresie dyscypliny (wpisać zgodnie ze
wskazaniem przy wszczęciu przewodu: biologia, ekologia lub biotechnologia).
Data Podpis kierującego pracą
Oświadczenie autora pracy
Świadom odpowiedzialności prawnej oświadczam, że niniejsza rozprawa doktorska została napisana przeze mnie samodzielnie i nie zawiera treści uzyskanych w sposób niezgodny z obowiązującymi przepisami.
Oświadczam również, że przedstawiona praca nie była wcześniej przedmiotem procedur związanych z uzyskaniem stopnia doktora w innej jednostce.
Oświadczam ponadto, że niniejsza wersja pracy jest identyczna z załączoną wersją elektroniczną.
Data Podpis autora pracy
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This work and scholarship of Ranju Kumari was funded by the National Science Centre (OPUS 2016/21/B/NZ3/03643) Laureate: Dr. Piotr Kaźmierczak
National Science Centre (2019/35/B/NZ6/00540) Laureate: Prof. dr hab. Dominika Nowis
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LIST OF PUBLICATIONS
1. Kumari R. and Kaźmierczak P. (2022) Modeling congenital dyserythropoietic anemia in genetically modified mice. Acta Haematologica Polonia. DOI:
10.5603/AHP.a2022.0003
2. *Ranju Kumari, *Tomasz M. Grzywa, Milena Małecka-Giełdowska, Karolina Tyszkowska, Robert Wrzesień, Olga Ciepiela, Dominika Nowis, and Piotr Kaźmierczak. Ablation of Tmcc2 gene impairs erythropoiesis in mice. International Journal of Molecular Sciences 2022 (Accepted)
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ACKNOWLEDGEMENTS
I would like to thank:
My supervisor, Dr. Piotr Kaźmierczak for providing me the opportunity to work in his group.
I am grateful for the invaluable scientific supervision, guidance, insightful discussions and advice throughout my research.
Professor Dominika Nowis from Medical University of Warsaw for acting as the doctoral thesis supervisor and for collaboration on the role of TMCC2 in erythropoiesis.
Dr. Magdalena Dziembowska from Centre of New Technologies for allowing me to work in her laboratory and providing me constant support for a successful completion of thesis.
Tomasz M. Grzywa from Medical University of Warsaw for collaboration on the role of TMCC2, assistance in data analysis and performing the flow cytometry experiments.
Karolina Tyszkowska and Dr. Robert Wrzesień of Central Laboratory of Experimental Animal, Medical University of Warsaw for maintaining the mouse colony and sharing important observations.
Dr. Julita Nowakowska for assistance with transmission electron microscopy.
Dr. Jakub Gruchota and Dr. Andrzej Dziembowski for assistance with generating the Tmcc2 knockout mice.
Jacek Miłek, Bożena Kuźniewska, Marta Magnowska, Patrycja Wardaszka, Aleksandra Stawikowska, Marta Wiatrowska, Piotr Ostapczuk, Joanna Chmielewska, Małgorzata Kreżel, Dr. Łukasz Samluk, members of Laboratory of Molecular Basis of Synaptic Plasticity for a nice and pleasant atmosphere at work.
My friends, Nirlep, Kinney, Sorabhi, Shiladitya, Praveen, Sam, Marta, Yasamin, Madhur, Mugdh, Swati, Bala, Shri, Akanksha, Ved, Santhosh, Michael, Nelka, Shreshth, Iga, Rottzy, Karthika, Swaraj, Mana, Habib, Rohit, Sagar, Neha and Gurnishan for motivating me and lifting up my mood.
My parents, Ram Bhagat and Kamla for constant support, encouragement and belief in me which is beyond expression.
6 Table of Contents
LIST OF PUBLICATIONS………...4
ACKNOWLEDGEMENTS………...……5
ABBREVIATIONS………...9
ABSTRACT………...12
STRESZCZENIE………...14
1 INTRODUCTION………...16
1.1 Transmembrane and coiled-coil 2 (TMCC2)………...16
1.2 Introduction to the hair cell function in hearing………...17
1.2.1 Structure of ear………...17
1.2.2 Structure and function of apical hair bundle………...19
1.2.3 Congenital sensorineural deafness…..……….21
1.2.3.1 Myosin7a……….……….21
1.2.3.2 Myosin15a……….………...22
1.2.3.3 Myosin6……….………...23
1.2.4 Genes involved in hair bundle maintenance.………23
1.2.4.1 EPS8……….………24
1.2.4.2 EPS8L2……….24
1.2.5 Tip link proteins………...25
1.3 Introduction to the process of erythropoiesis………...26
1.3.1 Erythropoiesis………...26
1.3.2 Different type of anemias……….28
1.3.3 Congenital dyserythropoietic anemia (CDA)………...28
1.3.3.1 CDA type I………...29
1.3.3.2 CDA type II...30
1.3.3.3 CDA type III...31
1.3.3.4 Transcription factor related CDA……….32
1.3.3.4.1 CDA type IV………32
1.3.3.4.2 X-linked thrombocytopenia with or without dyserythropoietic anemia………...34
1.3.3.5 CDA variants………34
1.3.4 Enucleation………...35
2 BACKGROUND & AIM OF THE STUDY...……….39
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3 MATERIALS AND METHODS……….40
3.1 Generation of Tmcc2 null mice………40
3.2 Genotyping………...40
3.2.1 DNA electrophoresis………41
3.3 Construction of DNA vector………41
3.3.1 DNA electrophoresis and gel extraction………...42
3.3.2 PCR product and DNA vector purification and digestion………42
3.3.3 Ligation of DNA fragments……….42
3.3.4 Preparation of competent bacteria for plasmid amplification………..43
3.3.5 Competent bacteria transformation……….…….43
3.3.6 Isolation of plasmid DNA from bacterial culture and DNA concentration….44 3.4 Western blotting of mouse brain extracts to detect TMCC2 protein………...44
3.5 Immunofluorescent staining in Tmcc2 knockout mice………45
3.6 Sample collection……….………46
3.7 Bilirubin assay……….……….46
3.8 Flow Cytometry………...………...46
3.9 Blood smear staining………47
3.10 Statistical analysis………47
4 RESULTS……….48
4.1 The role of TMCC2 in hearing……….48
4.1.1 Generation of Tmcc2 knockout mice………48
4.1.2 Detection of TMCC2 using different antibodies in transfected cells………...48
4.1.3 Confirmation of successful disruption of Tmcc2 gene……….50
4.1.4 Expression of TMCC2 in hair cells of mice……….51
4.1.5 Morphology of the hair bundles and the presence of CDH23 in stereocilia…53 4.1.6 Localization of apical markers – EPS8 and EPS8L2………...54
4.1.7 Localization of TMCC2 in hair cells……….………...54
4.1.8 Analysis of supranumerary OHCs in Tmcc2-/- mice……….55
4.1.9 Expression of TMCC3, a close paralog of TMCC2……….58
4.1.10 Summary of the findings regarding TMCC2 in hair cells………59
4.2 The role of TMCC2 in erythropoiesis and enucleation………60
4.2.1 Characterization of Tmcc2-/- knockout pups……….60
4.2.2 Analysis of mice weight………..……….61
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4.2.3 Evaluation of RBCs morphology in mice………62
4.2.4 Evaluation of blood cells using CBC of mice pups..………...64
4.2.5 Flow cytometry analysis of erythrocyte precursors of mice pups……...…….67
4.2.6 Evaluation of blood cells using CBC of adult mice……….69
4.2.7 Flow cytometry analysis of erythrocyte precursors of adult mice……..…….70
4.2.8 Analysis of blood samples using TEM of mice…….………...72
4.2.9 Summary of the findings regarding the role of TMCC2 in erythropoiesis and enucleation………...74
5 DISCUSSION………...76
6 CONCLUSIONS………...90
REFERENCES……….91
APPENDIX 1 – DECISION OF THE FIRST LOCAL ETHICS COMMITTEE………….109
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ABBREVIATIONS
1. APP – amyloid-β protein precursor 2. ApoE – apolipoprotein E
3. ARF6 – adenosine 5’-diphosphate-ribosylation factor 6 4. ASF1A – histone H3/H4 chaperone anti-silencing 1 5. BAC – bacterial artificial chromosome
6. BM – bone marrow
7. BSA – bovine serum albumin
8. BFU-E – burst forming unit erythroid 9. CAR- contractile actin ring
10. Cas9 – CRISPR associated protein 9 11. CBC – complete blood count
12. CC – coiled coil
13. CD169 – sialoadhesin (SIGLEC1) 14. CD71 – transferrin receptor
15. CDA – congenital dyserythropoietic anemia 16. CDA type I – CDA I
17. CDA type II – CDA II 18. CDA type III – CDA III 19. CDAN1 – codanin-1 20. CDH23 – cadherin 23 21. CDIN1 – C15orf41
22. CECs – CD71+TER119+ erythropoietic precursor cells 23. CFU-E – colony forming unit erythroid
24. CIT – citron rho-interacting serine/threonine kinase 25. CPC – chromosomal passenger complex
26. CRISPR – clustered regulatory interspaced short palindromic repeats 27. DIAPH3 – synonymous with mDia2 and DIAP3
28. ENU – N-ethyl-N-nitrosourea 29. ER – endoplasmic reticulum
30. F4/80 - adhesion G protein-coupled receptor E1 precursor (ADGRE1) 31. HA – hemagglutinin
32. HATs – histone acetyltransferases
10 33. HCT – hematocrit
34. HDACs – histone deacetylases 35. HGB – hemoglobin concentration 36. HP1α – heterochromatic protein 1 α 37. HSCs – hematopoietic stem cells 38. IHCs – inner hair cells
39. IMPC – International Mouse Phenotyping Consortium 40. IMSR – International Mouse Strain Resource
41. IPSCs – induced pluripotent stem cells 42. KOMP – Knockout Mouse Program 43. macroR – macrocytic red blood cells
44. MARCH8 – membrane associated ring finger (C3HC4) 8 45. MCH – mean corpuscular hemoglobin
46. MCHC – mean corpuscular hemoglobin concentration 47. MCSs – membrane contact sites
48. MCV – mean corpuscular volume 49. MFI – mean fluorescence intensity 50. microR – microcytic red cells
51. MKLP1 – mitotic kinesin-like protein 1 52. MLC2 – myosin regulatory light chain 2 53. MYB – myleblastosis oncogene
54. MYO7A – myosin 7a
55. Myo7ash1/sh1 – shaker-1 homozygotes 56. MYO15A – myosin 15a
57. nRBCs – nucleated RBCs 58. OHCs – outer hair cells 59. PCDH15 – protocadherin 15 60. PCP – planar cell polarity 61. PFA – paraformaldehyde
62. PolyE/ OrthoE – polychromatic to orthochromatic erythroid maturation stage 63. RBCs – red blood cell
64. RDW – red cell distribution width
65. RDW-CV – red cell distribution width-coefficient of variation 66. RDW-SD – red cell distribution width-standard deviation
11 67. ROCK – rho kinase
68. SDS-PAGE – sodium dodecyl sulphate-polyacrylamide gel electrophoresis 69. shRNA – short hairpin interfering RNA
70. SNPs – single nucleotide polymorphisms 71. SPF – specific-pathogen free
72. TBST – tris-buffered saline with 1% tween 73. TEM – transmission electron microscopy 74. TMCC1 – transmembrane and coiled-coil 1 75. TMCC2 – transmembrane and coiled-coil 2 76. TMCC3 – transmembrane and coiled-coil 3 77. TSB – total serum bilirubin
78. USH1C – harmonin
79. VCAM1 - vascular cell adhesion molecule 1 80. Vitamin B12 – folate and cobalamin
81. VPA – valproic acid
82. WES – whole exome sequencing 83. WGS – whole genome sequencing
84. XLTDA – X-linked thrombocytopenia with or without dyserythropoietic anemia
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ABSTRACT
Genetically modified animal models have proven to be indispensable for progress towards the comprehensive understanding of the genetic and the molecular basis of life and of disease.
Mice are a particularly useful model organism due to the relative feasibility of gene manipulation and the degree of their genetic proximity to humans is sufficient to draw conclusions meaningful in the context of human physiology. Despite continuous concerted effort of laboratories and international consortia and tremendous research progress, many genes have not been inactivated yet and the phenotypic characterization of the existing knockout strains is often limited. This creates an opportunity for discovery of new functions of genes, the proteins they code and their potential link to human disease. I took a candidate gene approach and focused on the transmembrane and coiled-coil domain 2 (Tmcc2) gene that encodes a product belonging to a small, poorly characterized family of proteins with a predicted role in intracellular vesicular transport and an expression pattern suggesting a function in the inner ear, the brain and the hematopoietic system. I analyzed the phenotype of a newly generated knockout mouse line that carries an inactivating deletion in the Tmcc2 gene. Contrary to the initial expectation, I found that TMCC2 is not required for the development and maintenance of the inner ear sensory hair cells. Inner and outer hair cells are polarized and positioned normally within the organ of Corti. Their hair bundles are properly organized. Subcellular localization of such important hair cell proteins as cadherin 23 (CDH23), myosin 6 (Myo6), and myosin 7a (Myo7a) appears to be normal. The asymmetric distribution of EPS8 and EPS8L2 between the longer and the shorter stereocilia is also preserved and the knockouts react to sound. Instead, TMCC2 turns out to be indispensable for normal erythropoiesis. The knockout pups develop severe anemia that closely resembles a group of congenital dyserythropoietic anemias (CDAs). Visibly paler and smaller than their heterozygous littermates, knockout neonates have drastically reduced red blood cell (RBC) count, macrocytosis and a significantly elevated fraction of nucleated RBCs (nRBCs). Strikingly, multinucleated RBCs with up to 12 nuclei are present in their blood.
Intrusions of cytoplasm into the nucleus and characteristic double membranes are evident in the Tmcc2-/- nRBCs analyzed under the transmission electron microscope. The relative content of CD71+TER119+ erythropoietic precursors (CECs) in the blood, the bone marrow and the spleen of the knockout mice is comparable to that of the heterozygotes but the fractions of enucleated precursors and erythrocytes are markedly reduced. Interestingly, adult
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Tmcc2 knockouts have a slightly elevated RBC count and no macrocytosis. Such change in phenotype is likely a consequence of extramedullary compensation as even in adult knockouts erythropoiesis remains abnormal, with a severe shift in the relative content of different stage erythroid precursors in the bone marrow that indicates a defect of maturation at the transition from the polychromatic to the orthochromatic stage. Based on the phenotype observed in the Tmcc2 knockout mice, I propose that the human ortholog (TMCC2) is a candidate CDA causative gene. While it is not possible to unequivocally predict which type of CDA would be caused by the so far unidentified TMCC2 mutations, the phenotype of the knockout mice has the cellular and ultrastructural characteristics that resemble CDA type 2, 3 and 4 rather than type 1. Additionally, the fact that the impairment of erythropoiesis is the only readily noticeable phenotype of the Tmcc2-/- mice challenges the often expressed notion that the main role of the TMCC2 protein in vertebrates is in the brain.
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STRESZCZENIE
Badania wykorzystujące genetycznie zmodyfikowane modele zwierzęce okazały się niezbędne w dążeniu do wszechstronnego zrozumienia genetycznych i molekularnych podstaw życia i chorób. Myszy są szczególnie użytecznym organizmem modelowym ponieważ potrafimy stosunkowo łatwo modyfikować ich geny a stopień ich genetycznego podobieństwa do ludzi jest wystarczający, aby móc wyciągać wnioski istotne dla zrozumienia fizjologii człowieka. Pomimo ciągłych wspólnych dążeń laboratoriów i międzynarodowych konsorcjów oraz znacznego postępu w badaniach naukowych, wiele genów nie zostało jeszcze dezaktywowanych a zakres przebadania fenotypu istniejących szczepów nokautowych jest często ograniczony. Stwarza to szansę na odkrycie nowych funkcji genów i kodowanych przez nie białek oraz ich potencjalnego związku z chorobami człowieka.
Przyjmując podejście oparte na weryfikacji roli genu o potencjalnym związku z rozwojem konkretnych zaburzeń genetycznych skupiłam sie na genie Tmcc2 (ang. transmembrane and coiled-coil domain 2), który koduje produkt należący do małej, słabo zbadanej rodziny białek o przewidywanej roli w wewnątrzkomórkowym transporcie pecherzykowym i ekspresji wskazującej na możliwą funkcję w uchu wewnętrznym, mózgu i układzie krwiotwórczym.
Przenanalizowałam fenotyp wytworzonej w tym celu linii nokautów mysich, w której genomie znajduje się delecja inaktywująca gen Tmcc2. Wbrew przewidywaniom odkryłam, że białko TMCC2 nie jest niezbędne do rozwoju i utrzymania zmysłowych komórek włoskowatych ucha wewnętrznego. Zarówno wewnętrzne jak i zewnętrzne komórki rzęsate są spolaryzowane i normalnie rozmieszczone w organie Cortiego. Ich wiązki rzęsek są prawidłowo zorganizowane. Wewnątrzkomórkowa lokalizacja takich ważnych dla funkcjonowania komórek rzęsatych białek jak kadheryna 23 (CDH23), miozyna 6 (MYO6) i miozyna 7a (MYO7A) jest prawidłowa. Zachowana jest też asymetria rozmieszczenia białek EPS8 i EPS8L2 między rzęskami dłuższych i krótszych rzędów a myszy nokautowe reagują na dźwięk.
Białko TMCC2 okazało się za to niezbędne dla normalnego przebiegu erytropoezy.
Wkrótce po urodzeniu u osesków nokautów rozwija się głęboka niedokrwistość przypominająca grupę wrodzonych niedokrwistości dyserytropoetycznych (ang. CDA).
Oseski nokautów są wyraźnie bledsze i mniejsze niż ich heterozygotyczne rodzeństwo, mają znacznie zmniejszoną liczbę czerwonych krwinek, makrocytozę i istotnie zwiększoną liczbę jądrzastych czerwonych krwinek. Zaskakująca jest obecność w ich krwi wielojądrzastych
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czerwonych krwinek zawierających nawet 12 jąder komórkowych. Wtręty cytoplazmatyczne w jądrze i charakterystyczne podwójne błony komórkowe są widoczne w jądrzastych erytrocytach z myszy Tmcc2-/- oglądanych pod transmisyjnym mikroskopem elektronowym.
Względna zawartość CD71+TER119+ prekursorów czerwonych krwinek (CECs) we krwi, szpiku i śledzionie nokautów jest porównywalna do tej w heterozygotach, ale udział prekursorów i erytrocytów, które skutecznie utraciły jądro komórkowe jest znacznie mniejszy u nokautów. Co ciekawe, dorosłe nokauty Tmcc2 mają nieznacznie zwiększoną liczbę erytrocytów i nie przejawiają makrocytozy. Taka zmiana fenotypu może być spowodowana erytropoezą pozaszpikową, ponieważ nawet u dorosłych nokautów sam przebieg erytropoezy jest wciąż zaburzony, na co wskazuje znaczna różnica względnej zawartości prekursorów erytrocytów na różnych stadiach dojrzewania zaobserwowana w szpiku, która wskazuje na wstępowanie zaburzenia dojrzewania tych komórek na etapie przejścia od erytroblastów wielobarwliwych do kwasochłonnych. Na podstawie zaobserwowanego fenotypu myszy nokautowych pod względem genu Tmcc2 proponuję rozważenie możliwego związku ortologicznego genu ludzkiego (TMCC2) z wrodzoną niedokrwistością dyserytropoetyczną.
Chociaż nie można jednoznacznie przewidzieć, jaki dokładnie typ CDA może być wywołany przez wciąż nieodkryte mutacje TMCC2, to fenotyp myszy nokautowych ma komórkowe i ultrastrukturalne cechy przypominające CDA typu 2, 3 i 4 bardziej niż typ 1. Ponieważ zaburzenia erytropoezy są jedynym wyraźnym fenotypem zaobserwowanym w myszach linii Tmcc2-/-, należy poddać w wątpliwość często wyrażane w literaturze oczekiwanie, że u kręgowców białko TMCC2 swoje najważniejsze funkcje spełnia w mózgu.
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1 INTRODUCTION
1.1 Transmembrane and coiled-coil 2 (TMCC2)
Transmembrane and coiled-coil 2 (TMCC2) belongs to a family of proteins that share common structural motifs (two transmembrane domains and two coiled-coil domains) with two other members TMCC1 and TMCC3. The longest isoform of TMCC2 is a 709 amino acid protein with the sequence highly conserved among humans and rodents. Despite little is known about the protein, it was found that TMCC2 is a neuronal protein primarily localized to the intracellular membrane compartment, mainly in the endoplasmic reticulum (ER).
TMCC2 is reported to interact with apolipoprotein E (ApoE) and amyloid-β protein precursor (APP) and modify the Aβ production rate (Hopkins, Sáinz-Fuertes, and Lovestone 2011). The TMCC2 protein sequence does not have any recognizable functional motifs or domains apart from two transmembrane segments and coiled-coil domains. Dementin, the sole member of the TMCC family in Drosophila melanogaster, can interact with both human APP and an orthologue of APP, APPL expressed in Drosophila (Hopkins 2013). Hence, the interaction between the APP protein families and Dementin is conserved evolutionarily. Hopkins et al.
reported that mutations in dementin results in the neurodegeneration and accumulation of APPL, resembling the pathological features of Alzheimer’s disease (Hopkins 2013).
TMCC1 helps in the regulation of ER membrane organization and may act as a mediator for the attachment of ribosomes to the ER, and localized in the rough ER (Zhang et al. 2014). RPL4 and RPS6, ribosomal proteins interact with the small tandem coiled-coil domains of TMCC1 in the cytoplasmic region which in turn help the ribosomes to attach to the ER membrane (Zhang et al. 2014). TMCC1 was shown to interact with other family members in homo- or hetero- dimer binding with TMCC1, TMCC2 and TMCC3 (Zhang et al. 2014). According to RT-PCR analysis, TMCC3 is highly expressed in the brain, spinal cord and testis. Using proteomics approach, TMCC3 is expressed in tissues of hind brain, developing lung and mesenchyme of developing tongue with high expression in pancreatic juice, lung, testis, placenta and stomach (Sohn et al. 2016). The wide- and high-expression suggests that TMCC3 may be a requisite for the normal functioning of these tissues. Sohn et al. used different constructs of TMCC3 to characterize the protein interactions deleting first coiled-coil region or both of the coiled-coil regions. It was reported that the second coiled- coil domain is essential for dimer formation and in general coiled-coil domains are required for the normal oligomerization (Sohn et al. 2016).
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A close paralog of TMCC2, TMCC1 has an important function in endosome fission.
Hoyer et al. used a targeted random biotin ligase strategy which helped in the identification of TMCC1 as an ER membrane protein that plays a role in the regulation of ER-associated endosome fission at ER membrane contact sites (MCSs). The localization of TMCC1 is unusual where it is separated to peripheral ER tubules and is kept out from the nuclear envelope (Hoyer et al. 2018). The depletion of TMCC1 resulted in a defect in cargo delivery from late endosomes to the Golgi. However, no morphological malformations of endosomes or disturbances of the biogenesis of sorting domains were observed after depleting TMCC1.
TMCC1 was shown to play a role in the stabilization of ER MCS on endosome buds with cargo-sorting buds. Hoyer et al. showed that TMCC1 has a specific function in the process of ER-associated endosome fission (Hoyer et al. 2018).
Ludwig et al. identified a blood-specific isoform of TMCC2, the expression of which is induced during the process of terminal erythropoiesis and may act as an important regulator of erythropoiesis as knockdown of TMCC2 in erythrocyte precursors cultured in vitro lead to defects in their proliferation and differentiation (Ludwig et al. 2019).
1.2 Introduction to the hair cell function in hearing
The auditory signal transduction transpires in cochlea that converts sound-induced vibrations into electrical signals through a process known as mechanotransduction. These signals traverse through the brain stem to the brain and are interpreted by the auditory cortex.
Cochlear signal transduction necessitates the decomposition of acoustic stimuli into their component frequencies and meticulously transmitting their temporal distribution and relative intensities. The characteristic feature of cochlear transduction is susceptivity, rapid detection and versatility. The inner ear detects vibrational signals of less than a nanometer and amplifies them for better detection. The amplification of the signal offers the human ear a dynamic range of detection and perception of sounds from 20 Hz to 20 kHz. The cochlea is responsible for precisely discriminating relatively similar tones, and different frequencies are analyzed effectively. The minimal latency is crucial in the detection of cochlear transduction signal, happening within tens of microseconds. The mechanotransduction apparatus is responsible for sound detection and amplification (Dallos 1996).
1.2.1 Structure of ear
The mammalian ear has three major components: the outer, middle and inner ear. The outer ear is responsible for gathering the auditory signal and channels it through the ear canal
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to the tympanic membrane, also known as eardrum. Eardrum ensures the faithful transmission of the vibrations to the middle ear ossicles. Middle ear ossicles are three tiny bones, the malleus, incus and stapes, which are directly linked to the oval window of the inner ear. The signal is transmitted faithfully matching the impedances of the incompressible fluid in the inner ear and compressible air in the ear canal after the gain in pressure of the tympanic membrane to oval window. The inner ear is composed of three different compartments filled with fluid: the scala vestibuli, the scala tympani and the scala media (Slepecky 1996). A continuous cochlear duct is formed by the scala vestibuli and the scala tympani with the oval and round windows providing contact with the middle ear. Cochlear fluids receive the vibrations and travel down from base to apex in the scala vestibuli, next to the scala tympani and finally reaching the round window, which vibrates freely inside of the middle ear in the space filled with air. The center of the cochlea constitutes the scala media, segregated by Reissner’s membrane lining the scala vestibuli and by the basilar membrane lining the scala tympani. The fluid compartment is not continuous and is separated through membranes, but they are coupled acoustically, and the pressure wave flowing through Reissners’s membrane also affects the basilar membrane. The basilar membrane constitutes majorly of extracellular matrix materials and mechanical properties of the membrane change depending on the position; at the base, the membrane is narrow and stiff and then towards the apex, it widens and its compliance increases. The mechanical characteristics of the basilar membrane play a fundamental role in determining the position along the organ of Corti where the sound wave evokes vibrations of the highest amplitude (Von Békésy 1960). Similarly, the high frequency peak of the auditory stimuli responds at the base, while peak responses of the lower frequencies are near the apex. The organ of Corti resides on the basilar membrane, which is home for the sensory cells of the auditory system, named the hair cells (Slepecky 1996). The hair cells are microvilli-like protrusions known as stereocilia rising from apical surfaces (Fig.1). The human organ of Corti contains approximately 16,000 hair cells arranged into a single row of inner hair cells (IHCs) and three rows of outer hair cells (OHCs) (Fig.1).
The primary sensory cells responsible for hearing are the IHCs. Approximately 90-95% of the cochlear afferent innervation contacts the IHCs. The outer hair cells have a specialized role of signal amplification through the active change in the length of their cell bodies and are controlled by efferent synapses.
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Figure 1. Hair cells of inner ear. Anatomic representation of cochlear hair cells with single row of inner hair cells (IHCs) and three row of outer hair cells (OHCs) lining the tonotopic axis of the cochlea. The diagram showing morphological difference between IHCs and OHCs. The structure of IHCs are pear-shaped with a central nucleus and apical stereocilia.
OHCs are cylindrical with a basal located nucleus and apical stereocilia.
1.2.2 Structure and function of apical hair bundle
The sensory cells have a definitive structure called the hair bundles that provide the function of mechanotransduction to the auditory system (Hudspeth and Jacobs 1979).
Mechanotransduction is a sensitive process, and hair bundles respond to mechanical stimuli with deflections of less than one nm. The “hairs” are microvilli-like structures named stereocilia, which are derived from tubulin and are, in fact, not true cilia. The composition of stereocilia is parallel actin filaments and actin bundling proteins fimbrin and espin are cross- linked into stiff paracrystalline arrays. One hair cell contains 20-300 stereocilia, with one true cilium per cell after the tallest row of stereocilia, known as the kinocilium. Kinocilium is not critical for mechanotransduction and plays a role in the development of the stereocilia.
Mature mammalian hair cells lack kinocilia (Hudspeth and Jacobs 1979). The hair bundles have a characteristic arrangement of the stereocilia, which are assembled in multiple rows based on increasing height, resulting in the formation of symmetric bilateral structure. The base of the stereocilia is tapered, containing a few dozen actin filaments at the base compared to the hundreds of actin filaments present above the tapered region. The actin filaments at the base of each stereocilium form a rootlet that extends into the cuticular plate, a dense network of actin filaments present in the apical part of the cell. Several kinds of protein crosslinks connect the stereocilia together to form a cohesive unit (Goodyear et al. 2005). Tip links
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connect the tip of a shorter stereocilium to the side wall of its taller neighbor, lateral links extend the shafts of the stereocilia, and ankle links couple the stereociliary bases. The mechanical stimuli result in the movement of the hair bundle, the movement of the stereocilia is without flexing, pivoting at the base (Crawford and Fettiplace 1985; Howard and Ashmore 1986). The shape of the hair bundle is variable depending upon the type of hair cell, and the mechanical properties of the bundle are adapted to the physiological role of the cell. The bundle shape in the mammalian cochlea has distinctive characteristics: bundles of inner hair cells are flat or U-shaped, while those of outer hair cells are V- or W-shaped. The tonotopic position is one factor that defines the variable structure of the hair bundle. The length of the hair bundles increases from base to apex; this difference in height of outer hair cells is accompanied by decrease in the number of stereocilia (Lim 1986). Lin et al. elucidated the mechanism of the complex architecture of the hair bundle (Lin, Schneider, and Kachar 2005).
The actin filaments are renewed within 48 hours, and the turnover of the actin in the bundles is highly coordinated (Schneider et al. 2002). The flux rate of actin subunits is proportional to the height of the stereocilia; the taller stereocilia have speedy replacement compared to the shorter ones ensuring the coordinated replacement. The actin filaments are cross-linked by espin, and it is expressed in both auditory and vestibular hair cells for the formation of bundle (H. Li et al. 2004). The turnover of the stereocilia core is synchronous as espin is replaced at the same rate as actin (Rzadzinska et al. 2004).
The process of mechanotransduction starts with the vibrations in the basilar membrane which stimulates the hair cells residing on them. A small fluid-filled compartment called subtectorial space lies in between the apical surface of the hair cells and the tectorial membrane. The tectorial membrane is a fibrous and gelatinous extracellular matrix, which are linked directly to the OHCs through the hair bundles. The hair bundles are activated by shear caused by the movements induced by the vibrations of the basilar membrane and the overlying tectorial membrane. While the activation of the inner hair cells is induced by the motion of the fluid present at their apical surfaces. Similar to the functioning of the basilar membrane, hair cells are stimulated depending on the specific frequencies. High frequencies excite the hair cells at the base, while low-frequency stimuli activate the hair cells at the apex.
The mechanical properties of the basilar membrane dictate the specific frequency to which the hair cells respond and augmented by the hair cell mechanical and electrical properties.
21 1.2.3 Congenital sensorineural deafness
The study of the genes involved in hearing loss has improved our understanding of the molecular mechanisms involved in the development and regulation of the auditory system.
The major component in the stereociliary core is actin filaments, but in fact, myosin isoforms are critical for hair-bundle formation and proper functioning of the cochlea (Libby and Steel 2000). Myosins are an important class of actin associated proteins. Myosins are motor proteins that move along actin filaments by hydrolyzing ATP. Myosins are separated into 18 different classes; type II or conventional myosins that plays a role in formation of actin filaments, and remaining unconventional (non-filament forming) myosins (Sellers 2000;
Berg, Powell, and Cheney 2001). In hair cells, six distinct myosins are expressed (Gillespie, Wagner, and Hudspeth 1993; Hasson et al. 1997; Liang et al. 1999; Lalwani et al. 2000;
Walsh et al. 2002; Donaudy et al. 2003). Three of these myosins, Myo6, Myo7a and Myo15a are suspected to be involved in hair bundle maintenance and maturation (I. A. Belyantseva, Boger, and Friedman 2003; T. Self et al. 1998; Tim Self et al. 1999). Mutant mice with mutations in Myo6, Myo7a and Myo15a have abnormalities in stereocilia, vestibular balance disorder, congenital deafness displaying the vital need of these myosins for normal functioning of mammalian ear (Avraham et al. 1995; Gibson et al. 1995; Probst 1998). The mutations in these three myosins not only disrupt the function of inner ear in mice but also leads to human congenital deafness causing Usher’s syndrome type IB by MYO7A and non- syndromic deafness named DFNA22 and DFNB37 caused by MYO6, DFNB3 by MYO15 and DFNA11 by MYO7A (Well et al. 1995; Melchionda et al. 2001; Ahmed et al. 2003; A. Wang 1998; X.-Z. Liu et al. 1997). Hence, studying the role of unconventional myosins for better understanding in hair cells could be used for finding treatments for human hereditary deafness.
1.2.3.1 Myosin 7a
Myo7a is a hair cell marker also expressed in mammalian hair cell, where it is localized in the cytoplasm and all over the hair bundle (Hasson et al. 1997). Myo7a mutation in mouse leads to splayed with short stereocilia, which suggests it plays a role in maintenance of bundle structure and linking of stereocilia (T. Self et al. 1998; Gibson et al. 1995; Ernest 2000). MYO7A is a motor protein responsible for the regulation of the growth of stereocilia.
Myo7a mutant mice result in hearing loss, extreme lengthening of stereocilia and disruption of hair bundle morphology (Gibson et al. 1995; X.-Z. Liu et al. 1997; Weil et al. 1997).
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MYO7A has a role in the regulation of F-actin rearward flow as during the absence of Myo7a, the process of actin treadmilling increases (Prosser et al. 2008). MYO7A is likely involved in the transport of proteins and hindering the process would restrict the assembly of actin. One potential cargo protein is twinfilin-2, which helps in the capping and severing of the actin filaments and interacts with MYO7A (Palmgren et al. 2001; Paavilainen et al. 2007;
Rzadzinska et al. 2009). Myo7a-deficiency in mice leads to the disruption of the function of twinfilin, which results in the mislocalization of twinfilin instead of stereociliary tips (Peng et al. 2009; Rzadzinska et al. 2009). The length of stereocilia is decreased by overexpression of twinfilin-2, suggesting a functional connection to Myo7a (Peng et al. 2009; Rzadzinska et al.
2009). The expression of whirlin and espin-1 is more abundant in the longest stereocilia of the hair bundle, while in the shortest stereocilia, twinfilin-2 protein expression is more pronounced (Delprat et al. 2005; Peng et al. 2009; Rzadzinska et al. 2009; Salles et al. 2009).
The length of stereocilia is dependent on the corresponding expression of proteins, including whirlin, espin-1, twinfilin-2 and some other actin-binding proteins.
1.2.3.2 Myosin 15a
Myosin 15a (MYO15A) is an essential protein required for the maintenance and development of the hair bundle and is localised at the tips of the stereocilia (I. A.
Belyantseva, Boger, and Friedman 2003). The expression of MYO15A in stereocilia starts as early as the formation of a staircase array of the hair bundle (I. A. Belyantseva, Boger, and Friedman 2003). MYO15A expression is directly proportional to the length of the stereocilia (Rzadzinska et al. 2004). In bundle development, MYO15A provides an important function by delivering protein cargo to the tips of stereocilia. MYO15A interacts with the bundle protein whirlin (Inna A. Belyantseva et al. 2005; Delprat et al. 2005). MYO15A and whirlin are colocalized at the stereociliary tips (Kikkawa et al. 2005; Delprat et al. 2005), and the loss of the Myo15a gene leads to severe hearing impairment and unusually short stereocilia (Mburu et al. 2003). The localization of whirlin is perturbed in the Myo15a null mice (Kikkawa et al. 2005; I. A. Belyantseva, Boger, and Friedman 2003); restoration of MYO15A expression in these hair cells resulted in the transfer of endogenous whirlin to the tips of stereocilia (I. A. Belyantseva, Boger, and Friedman 2003). Hence, MYO15A and whirlin are two important proteins needed for the development and maintenance of the hair bundle.
23 1.2.3.3 Myosin 6
MYO6 is one of the earliest expressed hair cell specific proteins in the mouse cochlea and an essential protein for the inner ear. MYO6 is expressed on the embryonic day 13 (E13) in inner ear (Montcouquiol et al. 2003). Myo6 was proposed to act as an anchor and help in the maintenance of the structure of stereocilia in the inner and outer hair cells (Tim Self et al.
1999). The study of mouse mutants has helped gather the information concerning different protein functions in inner ear. Myo6 mutant was found that arose due to spontaneous mutation in a colony at the Jackson Laboratory were called Snell’s waltzer mutant (Avraham et al. 1995). With time, the emphasis was on the mouse model for multiple alleles as different mutation might result in new phenotype, help recognizing new functions of the same protein.
Another mouse mutant Tailchaser, generated using N-ethyl-N-nitrosourea (ENU) contains a dominant mutation (de Angelis et al. 2000). Tailchaser was found using a large-scale mutagenesis screen, upon recognition mutant mouse has deafness and shown circling and with time a gradual decline of hearing and balance function was observed (de Angelis et al.
2000; Kiernan et al. 1999). The phenotype of Tailchaser mutant mouse was similar to the dominant forms of nonsyndromic deafness in humans. The stereocilia bundles were analysed using a scanning electron microscopy in Tailchaser, V-shaped pattern of stereocilia was absent at birth, and later the hair bundles were disordered and degenerated by adulthood. A missense mutation was found in Myo6 in Tailchaser and helped in the elucidation of MYO6 function with new Myo6 allele (Hertzano et al. 2008).
1.2.4 Genes involved in hair bundle maintenance
A precise mechanism that controls the length of individual stereocilia is necessary to maintain the staircase-like morphology of the hair bundle. Such molecular feedback mechanism acts mainly through the control of actin nucleation, polymerization and stability of actin filaments (McGrath, Roy, and Perrin 2017).
The actin filaments within each stereocilium are crosslinked by such proteins as espin-1, espin-3A, fascin-2, plastin-1 and XIRP2 (Sekerková et al. 2006; Shin et al. 2010;
Taylor et al. 2015; Francis et al. 2015; Scheffer et al. 2015). Plastin-1, fascin-2 and XIRP2 are functionally vital for the maintenance of stereocilia (McGrath, Roy, and Perrin 2017).
24 1.2.4.1 EPS8
Other proteins known to control the length of the stereocilia are EPS8 and EPS8L2, which function in the bundling and capping of actin filaments (Hertzog et al. 2010; Zampini et al. 2011). EPS8 has capping and bundling activity and can be switched by activating capping activity by Abi-1 and bundling activity by IRSp53 (Disanza et al. 2004; 2006). In hair cells, EPS8 is primarily localized at the tips of the longer row of stereocilia while EPS8L2 at the tips of the shorter row (McGrath, Roy, and Perrin 2017; Zampini et al. 2011; Uri Manor et al. 2011; Furness et al. 2013). The asymmetric distribution of the two EPS8 family members is reflected in their distinct functions in the maintenance and regulation of stereociliary growth and hair bundle structure. EPS8 is transported by MYO15A to the tips of stereocilia (Inna A. Belyantseva et al. 2005; Uri Manor et al. 2011). The lack of EPS8 protein in the mice leads to the abnormal growth of the mechanosensitive hair bundle and profound deafness, even though the knockout hair cells retain the ability to transduce mechanical stimuli. Reduced size of stereocilia in EPS8 knockout mice is mostly seen in the tallest row and IHCs are affected more substantially than OHCs. Eps8L2 knockout mice showed gradual degradation of hair bundle structure which started after the progression of hearing loss (Zampini et al. 2011).
1.2.4.2 EPS8L2
There are numerous cytoskeletal proteins in the hair bundles and mutations in the genes of these proteins cause different defects ranging from morphological to functional (Drummond et al. 2012; Petit and Richardson 2009; Martin Schwander, Kachar, and Müller 2010). The motor proteins are MYO6, MYO7A and MYO15 also participate in the regulation of stereociliary length or hair bundle structure (Zampini et al. 2011; Uri Manor et al. 2011; Kros et al. 2002; Stepanyan and Frolenkov 2009). MYO15A is responsible for the localization of EPS8 at the tips of stereocilia (Uri Manor et al. 2011). The localization of EPS8L2 is not dependent of EPS8 and MYO15A. EPS8 has stronger actin binding potential relative to EPS8L2 in longer stereocilia provided by MYO15A or MYO3A or MYO7A might be responsible for the differential translocation of EPS8L2 (U Manor and Kachar 2008). On the other hand, EPS8L2 can translocate by passive diffusion across the length of stereocilia and gather at the tips and bind to actin filament barbed ends or some other binding partners.
The deletion of Eps8L2 resulted in normal growth of hair cell bundles during development.
The staircase architecture of the hair cell bundle with mechanosensory tip links are intact in
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Eps8L2 KO mice. The MET currents were normal during early stages of development with no deficits in the basolateral membrane development in cochlear hair cells. The cochlear hair bundles degrade gradually in Eps8L2 KO mice. Eps8L2 KO mouse is associated with progressive hearing loss and mostly affects the higher frequencies. In adult Eps8L2 KO mice there is a substantial disorganisation of the hair bundles and a 30% reduction in length of the longer stereocilia, whereas the short and intermediate stereocilia have a broad distribution of length with some of them abnormally long. The loss of OHC shorter and intermediate stereocilia is more visible with age. It has been proposed that the modest phenotype of the Eps8L2 KO mouse might be caused by the functional redundancy with Eps8L1 and Eps8L3, Even though there is no evidence for the expression of the EPS8L1 and EPS8L3 in hair cells (Scita et al. 1999). EPS8 and EPS8L2 have a different function even though they are structurally similar. EPS8 helps in the growth of stereocilia whereas EPS8L2 is involved in the stabilization of hair bundles after formation. Simultaneously, EPS8 and EPS8L2 are responsible for the normal development and maintenance of the hair bundle structure.
Remarkably, a recent study revealed that several mouse lines with mutations in the genes that are required for the process of mechanotransduction display abnormal stereociliary width and length phenotypes and mislocalization of asymmetric stereociliary markers, such as the EPS8 and EPS8L2 proteins (Krey et al. 2020). Similar effect can be obtained when mechanotransduction is chemically blocked with tubocurarine. Therefore, mislocalization of the two hair cell expressed EPS8 family members could be a visible sign of general problems with the maintenance of hair bundle structure and of dysfunctional mechanotransduction.
1.2.5 Tip link proteins
The tip links are essential for the process of mechanotransduction. Tip links can be disrupted by decreasing the Ca2+ concentration to a sub-micromolar level outside the cell, which results in the loss of mechanical sensitivity of a hair cell (Assad, Shepherd, and Corey 1991). This disruption of mechanotransduction led to the hypothesis that the mechanotransducer channel is present at the stereociliary tips instead of the base. The hypothesis was later confirmed by high-resolution, high-speed calcium imaging (Beurg et al.
2009). The formation of tip link involves several proteins, including cadherin 23 (CDH23) and protocadherin 15 (PCDH15) (Siemens et al. 2004; Kazmierczak et al. 2007; Müller 2008;
Alagramam et al. 2011). CDH23 parallel dimer forms the upper portion of the tip link, while the parallel dimer of PCDH15 forms the lower portion (Narui and Sotomayor 2018) interacting with a complex of TMIE and LHFPL5 (also known as TMHS). TMIE and
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LHFPL5 are multipass transmembrane proteins closely linked to the mechanotransducer channel complex (Xiong et al. 2012; Bo Zhao et al. 2014). PCDH15 has multiple isoforms, generated by alternative splicing with variable spatial and developmental distributions in hair bundles. PCDH15-CD2 isoform plays a critical role for mature tip links (Pepermans and Petit 2015). CDH23 lies at the upper end of the tip-link further connected to harmonin (USH1C) and myosin 7a (MYO7A) (Fettiplace and Kim 2014). The mechanotransduction is hampered if there are mutations in CDH23, harmonin or MYO7A (Palma et al. 2001; Grillet et al. 2009;
Yu et al. 2017).
Apart from the cochlea, hair cells are also present in the vestibular organs, where they detect movement of the head, thus mediating the sense of balance. The vestibular system constitutes three semicircular canals, providing angular accelerations detection, and the saccule and utricle, functions in the detection of linear accelerations. The mechanism of mechanotransduction in the cochlear and vestibular hair cells is similar, despite the differences in hair bundle morphology and the frequencies of the stimuli they respond to.
1.3 Introduction to the process of erythropoiesis 1.3.1 Erythropoiesis
Erythropoiesis is a process that allows generation of nearly 2 million red blood cells (RBCs) every second in the human body. Human erythropoiesis is a finely regulated multi- step process, initiating with multipotent hematopoietic stem cells (HSCs) in the bone marrow (BM), which gives rise to the mature red blood cells (RBCs) (Orkin 2000; Caulier and Sankaran 2022). During erythropoiesis, HSCs differentiate into the megakaryocytic-erythroid progenitor, which further differentiates into burst-forming unit erythroid (BFU-E). BFU-Es are the first erythroid-committed precursors entirely dedicated to the erythroid line (Gregory and Eaves 1977). BFU-Es are replicating and differentiating further into the colony-forming unit erythroid (CFU-E). Committed erythroid lineage differentiation process is called terminal erythropoiesis.
Erythroid maturation involves slow and constant accumulation of hemoglobin, condensation of the nucleus and progressive reduction in cell size (Granick and Levere 1964).
CFU-Es are nucleated precursors which differentiate into proerythroblasts, and proerythroblasts further differentiate into basophilic, polychromatophilic and orthochromatic erythroblasts. This phase later leads to enucleation, resulting in the formation of a reticulocyte and a pyrenocyte (expelled nucleus).
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At the final stage of erythroid development, reticulocytes expel the unnecessary organelles, such as the Golgi apparatus, the endoplasmic reticulum (ER), the ribosomes and the mitochondria thus transforming into erythrocytes. During this stage, erythrocytes attain the biconcave shape enabled through substantial remodeling of the cell membrane (Gifford et al. 2006).
Erythroblastic islands provide niches to erythroblasts for terminal differentiation of erythroid cells. Erythroblastic islands are composed of a macrophage located in the center and surrounded by nearly 30 erythroid cells at different maturation stages (S. H. Lee et al.
1988). Erythroblastic islands are conserved throughout mammalian evolution. Hemoglobin synthesis takes places in the erythroid cells from CFU-E to reticulocyte stages of terminal differentiation (M. Bessis 1958; M. C. Bessis and Breton-Gorius 1962). The nucleus extruded during the process of enucleation is engulfed by the central macrophage (Seki and Shirasawa 1965; Ehud Skutelsky and David Danon 1972; M. Bessis, Mize, and Prenant 1978).
Furthermore, the central macrophage provides the direct transfer of iron for heme synthesis to erythroid progenitor cells (M. C. Bessis and Breton-Gorius 1962; Leimberg et al. 2008). The exact roles of the central macrophages are a subject of current intense research and even the previously proposed molecular signature of combination of vascular cell adhesion molecule 1 (VCAM1), CD169 (sialoadhesin, SIGLEC1), and mouse macrophage surface marker F4/80 (adhesion G protein-coupled receptor E1 precursor, ADGRE1) (Waddell et al. 2018) that were used to define these cells turned out to be too broad, while erythropoietin receptor (EPOR) expression proved a novel common feature in mouse and human erythroblastic island macrophages (W. Li et al. 2019).
The process of erythropoiesis has a steady pace, and nearly 1% of the cells in blood circulation are cleared and replaced by newly synthesized cells on a daily basis (Dzierzak and Philipsen 2013). The typical lifespan of RBC is around 120 days, and they are constantly scrutinized in the blood circulation by resident macrophages in the liver and spleen (Crosby 1959). At the time of RBC surveying, the spleen can recognise any abnormal changes in RBCs and remove abnormal and aged RBCs (Crosby 1957).
In some conditions, our body is not able to produce enough healthy red blood cells.
The condition when a person has low number of red blood cells is called anemia. The typical symptoms include pale skin, feeling cold and fatigued. These symptoms may demonstrate that not enough oxygen is supplied to the organs to carry out their function.
28 1.3.2 Different type of anemias
Different kinds of anemias are caused by varying factors and require different treatment (Cazzola 2022). The most common type is iron deficiency anemia which results from the shortage of iron in the body. In peripheral blood of individuals with iron deficiency predominate small microcytic red blood cells, with a thin rim of pink cytoplasm. Some red blood cells have pencil shaped structure. The size of normal RBCs in iron deficiency condition are similar to the size of a nucleus of a small lymphocyte. Another kind of anemia is caused by deficiency of such vitamins as folate and cobalamin (vitamin B12). Under microscope, peripheral blood of patients with these vitamin deficiencies is enriched in large macrocytic RBCs and neutrophils, a type of white blood cells, having multilobed and hypersegmented nuclei. Patients with vitamin deficiency can also have reduced number of white blood cells and platelets if went undiagnosed for a long time. Leukemia and myelofibrosis, are conditions frequently associated with anemia development caused by hematopoiesis disturbances in the BM. In leukemic patients, the morphology of red blood cells varies across different types of this disease. In hypoplastic anemia, red blood cells loose the biconcave shape and are thinner, with translucent appearance (Majumder et al. 2006). In myelodysplastic syndrome patients, red blood cells with thorn and horn like shapes (acanthocytes and echinocytes) are observed (Majumder et al. 2006). A rare type of genetic disorders caused by ineffective erythropoiesis are congenital dyserythropoietic anemias (CDAs). As the genetic complexity of CDAs started to be unravelled it became apparent that these conditions are particularly interesting to study in the context of erythropoietic cell intrinsic molecular mechanisms that underlie the process of erythropoiesis. Mutations that lead to different forms of CDAs provide insight into the processes that a cell must undergo on its way to becoming an erythrocyte.
1.3.3 Congenital dyserythropoietic anemia (CDA)
Congenital dyserythropoietic anemias (CDAs) belong to a group of rare genetic disorders identified by ineffective erythropoiesis that mostly leads to monolinear cytopenia (erythropenia). Initially, the key diagnostic features of CDAs were erythroid hyperplasia with binucleated or multinucleated late erythroblasts observed in the BM. However, these features are not entirely CDAs-specific, and they can be observed in conditions like erythropoietic stress induced by iron deficiency and preterm birth, resulting in certain difficulties while
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diagnosing CDAs. Over time, CDA causing mutations were identified in several genes, which greatly facilitated diagnosis. Despite this progress, mechanistic studies of the pathophysiology of different types of CDAs have been severely hampered by the lack of reliable animal models. CDAs comprise a group of hypoproliferative anemias, which can be categorized into five types: CDA types I, II, III, the transcription-factor-related CDAs and the CDA variants.
1.3.3.1 CDA type I
CDA type I (CDA I) is associated with mutations in two genes: Codanin-1 (CDAN1) and CDIN1 (C15orf41) (Dgany et al. 2002; Babbs et al. 2013). The mode of inheritance of CDA I is autosomal recessive, with 90% of the cases caused by bi-allelic mutations. There are 51 known mutations in CDAN1 and six in CDIN1 causing CDA I (Roy and Babbs 2019;
Babbs et al. 2013; Russo et al. 2019; Palmblad et al. 2018). Some of the basic findings in patients with CDA I include short stature, chest deformities, skeletal abnormalities as well as other congenital abnormalities (e.g. 10% of the patients showing distal limb anomalies) not unique to CDA I (Roy and Babbs 2019; Amir et al. 2017). The hallmarks of CDA I are severe or moderate macrocytic anemia and reticulocytopenia (Roy and Babbs 2019). During erythroid differentiation, the late binucleated erythroblasts with nuclei at different stages of maturation account for 2.4% to 10% of erythroid cells (Gambale et al. 2016; Heimpel et al.
2010). In the intermediate erythroblasts, there is an incidence of thin internuclear chromatin bridges in between the nuclei (Heimpel et al. 2010). Such internuclear chromatin and cytoplasmic bridges are seen in 79% of CDA I patients (Resnitzky et al. 2017). The heterochromatin is abnormally dense when observed under an electron microscope. Small translucent vacuoles cause the characteristic “spongy” or “Swiss-cheese” appearance of the nucleus (Resnitzky et al. 2017; Heimpel et al. 2010).
CDAN1 is a ubiquitously expressed protein, the function of which is still not sufficiently understood. E2F1 transcription factor regulates the expression of CDAN1. The expression of CDAN1 increases during the S-phase and decreases during mitosis (Noy-Lotan et al. 2009). CDAN1 plays a role in nucleosome assembly and disassembly by binding the histone H3/H4 chaperone anti-silencing 1 (ASF1A). ASF1A is important for changes in chromatin structure during replication (Tamary et al. 2010). ASF1A forms a complex by binding histone H3/H4 and importin-4 in the cytoplasm; then this complex transfers the histone dimers to the nucleus where chromatin assembly factors are attached (Alvarez et al.
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2011; Campos et al. 2010; De Koning et al. 2007; Jasencakova et al. 2010). CDAN1 binds directly to ASF1A and ASF1B through its N-terminal domain. This binding may hinder the progression to the S-phase of the cell cycle through a dominant negative effect on the function of the ASF1 complex (Ask et al. 2012). The dissociation of CDAN1 and ASF1A is important in order to deliver the histones in the nucleus for chromatin assembly. As a matter of fact, in CDA patients with CDAN1 mutation, there is a dysregulation of ASF1A. CDAN1 is known to interact with CDIN1, which further interacts with ASF1B, a homolog of ASF1A (Ewing et al. 2007). Heterochromatin protein HP1α (CBX5) is an essential crosslinker of heterochromatin responsible for chromosomal stiffness that facilitates proper alignment of chromosomes during cell division (Strom et al. 2021). In CDA I erythroblasts, there is an accumulation of HP1α in the Golgi apparatus during the intermediate erythroblast maturation stage (Raffaele Renella et al. 2011). CDAN1 is also known to partially co-localize with SEC23B, the mutations of which result in CDA II, indicating a possible molecular connection between two major types of CDAs. The loss of CDAN1 and CDIN1 is lethal, but changes in their sequences affect erythroid cells.
1.3.3.2 CDA type II
One of the most frequent forms of CDA is CDA II, caused by the mutations in the SEC23B gene. It is an autosomal recessive disorder resulting from mutations in both alleles of the SEC23B gene (Schwarz et al. 2009; Bianchi et al. 2009). The clinical findings in CDA II include a variable magnitude of anemia, which is normocytic, and a normal or mildly elevated reticulocyte count. Some of the basic features of CDA II are splenomegaly and jaundice caused by hemolysis. The clinical manifestation of the symptoms is variable, ranging from no symptoms in 10% of the patients to transfusion dependency in 20% of the patients (Russo et al. 2014; Bianchi et al. 2016). In bone marrow, there is an incidence of binucleated cells with both nuclei at the same stage of erythroid maturation and an increase in the number of erythroid precursor cells. One typical morphological feature of CDA II is nearly 10% of binucleated erythroblasts accumulated together, with more than 2% of the cells showing nuclear disintegration (Heimpel et al. 2010). In CDA II patients, the endoplasmic reticulum forms vesicles loaded with proteins and positioned just below the plasma membrane, which results in a discontinuous double-membrane observed in electron microscopy (Gambale et al. 2016). One biochemical feature occurring in 95% of the CDA II patients is the occurrence of hypoglycosylated band 3 in erythrocytes (Russo et al. 2014).
Occasionally, SEC23B mutation is not found, but the patients show biochemical features and
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BM morphology typical for CDA II, indicating that there is another unidentified CDA II locus (Achille Iolascon et al. 1998).
SEC23B is a component of COPII complex and plays a role in intracellular vesicle trafficking in eukaryotic cells. The COPII complex is involved in the transport of correctly folded cargo to the Golgi apparatus from the ER, deformation of the membrane, and accumulation of the secretory cargo (De Matteis and Luini 2011). The expression of SEC23B is ubiquitous; still, the known mutations in SEC23B cause defects primarily in the erythroid lineage (Schwarz et al. 2009; Satchwell et al. 2013). One hypothesis explaining such specificity of the phenotype might be either the tissue-specific expression of SEC23B during erythroid differentiation in humans (Schwarz et al. 2009; Satchwell et al. 2013) in conjunction with the inability of a homologous protein SEC23A to compensate for the loss of SEC23B specifically in the human erythropoietic cells, or might be the need of full function and high levels of a specific COPII component to correctly transport the erythroid-specific cargoes, such as the Band 3 protein (Russo et al. 2013). Another hypothesis could be loss-of- function resulting in impaired cytokinesis in CDA II erythroblasts caused by the disruption of the midbody assembly (Gambale et al. 2016). It has been found that SEC23B is involved in autophagy (Ishihara et al. 2001). This associates the well established importance of autophagy for the last stage of reticulocyte maturation to the COPII proteins involvement in the process of autophagy (Griffiths et al. 2012; Jeong et al. 2018). The clinical presentations of the compound heterozygotes for missense and nonsense mutations are severe (A. Iolascon et al. 2010). There are no null mutations for homozygous or compound heterozygotes; the mutation might be lethal (A. Iolascon et al. 2010). The clinical presentation of CDA II is mild when two hypomorphic alleles are present. This might be linked to the compensatory effect of SEC23A (Russo et al. 2013). Sec23gt/gt deficient mice (Sec23bgt/gt) die prenatally due to degeneration of tissues such as pancreases and salivary glands but have no signs of anemia (Tao et al. 2012). The disparate presentation in SEC23B null allele in mouse and humans might be linked with hypomorphic mutations found in humans or a shift in the function of SEC23 paralogs (Tao et al. 2012).
1.3.3.3 CDA type III
CDA type III (CDA III) is associated with KIF23 gene mutations and is the rarest classical form of CDA (Achille Iolascon et al. 2013). The mode of inheritance is autosomal dominant and known patients are primarily found in two unrelated families, one Swedish, and
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another one is American (Griffiths et al. 2012). A novel mutation is found in KIF23 gene, c.2747C>G (p.P916R), which is present in both Swedish and American families. The clinical manifestations of CDA III range from normal erythrocyte count to moderate anemia, mild relative reticulocytopenia, normal or slightly elevated mean corpuscular volume, signs of hemolysis and jaundice. Splenomegaly does not occur. Occasional giant multinucleated erythroblasts and increased count of erythroid precursor cells are detected in the bone marrow preparations examined under a light microscope. In CDA III patients, the morphological features of the erythroblasts are iron-laden mitochondria, autophagic vacuoles, clefts with heterochromatin, and myelin figures in the cytoplasm observed under electron microscope.
Some of the patients in the Swedish family suffered from visual disturbances, degeneration of the macula, had angioid streaks, monoclonal gammopathy, and multiple myeloma (Liljeholm et al. 2013).
CDA III is caused by mutations in KIF23 encoding mitotic kinesin-like protein 1 (MKLP1), playing an essential role as mitotic protein in ensuring precise separation of the cells during cell division (Liljeholm et al. 2013). MKLP1 is known to interact with ARF6 (adenosine 5’-diphosphate-ribosylation factor 6), which forms an extended b sheet, which further is described to interact with the surface membrane at the cleavage furrow. The complex of ARF6-MLKP1 plays a key role in cytokinesis by linking the membranes at cleavage furrow and microtubule bundle (Makyio et al. 2012). KIF23 mutations may hinder the function of the protein in cytokinesis, leading to the generation of giant multinucleated erythroblasts (Liljeholm et al. 2013). Interestingly, the loss of ARF6 can also lead to the formation of binucleated and multinucleated cells (Lind et al. 1995).
1.3.3.4 Transcription factor related CDA
Transcription-factor-related CDA is a subgroup comprising CDA IV and X-linked thrombocytopenia with or without dyserythropoietic anemia (XLTDA).
1.3.3.4.1 CDA type IV
CDA IV is an autosomal dominant inherited disorder caused by mutations in the erythroid- specific transcription factor 1 (KLF1). As of August 2020, only nine CDA IV patients had been described (Arnaud et al. 2010; Jaffray et al. 2013; Ortolano et al. 2018; Ravindranath et al. 2018; Kohara et al. 2019; Belgemen-Ozer and Gorukmez 2020). The clinical manifestations of CDA IV are severe anemia with signs of hemolysis, slightly elevated or
