Gene Expression and Protein Analysis for Understanding of Cellular and Molecular Mechanisms to Prevent Auditory Loss

CHAPTER ONE

Literature review and Introduction

1.1 Hearing loss

The ability to detect vibrations and perceive sound through ears is termed as “Hearing”. Any individual unable to hear within the normal hearing threshold (≥ 25 dB) is said to have the condition of hearing loss (http://www.who.int/mediacentre/factsheets/fs300/en/, 2017).

Table: Classification of Severity of Hearing Impairment (http://www.rehabcouncil.nic.in/writereaddata/hi.pdf, 2012)

Niparko (2012), classified hearing loss as: –

1. conductive hearing loss- caused due to lesions in the tympanic membrane, external auditory canal, or middle ear, prevents the reception of sound to the inner ear;
2. sensorineural hearing loss- caused due to lesions are present either in the inner ear or the auditory nerve; and
3. mixed loss- caused due to chronic infection, extreme head injury, genetic disorders, or during a state of transient hearing loss accompanied with sensorineural hearing loss (Listen Hear! New Zealand, 2017).

Hearing loss is amongst a frequent sensory impairment affecting 1 in 500 new-borns and 1 in 300 children by the age of 4. Approximately, 70% of genetic hearing loss is non-syndromic (i.e., hearing loss is the only phenotype), and 30% syndromic (e.g., additional clinical findings such as changes in pigmentation of the hair, skin, and eyes or individuals with Waardenburg syndrome) (Peters et al., (2002)).

In New Zealand, hearing loss is relatively common as it affects one in six New Zealanders. In 2016, the frequency of hearing loss was estimated to be 880,350 people. This figure represented ~18.9%. Also, it prevalence amongst males was greater (472,961 people) than females (407,388) (Listen Hear! New Zealand, 2017) (https://www.nfd.org.nz/our-work/education-and-prevention/).

Table: The prevalence of hearing loss among the population aged ≥65 years, by Region (DJ Exeter, B Wu, AC Lee, & Searchfield, 2015).

Region	2011	2021	2031	Change (%)  2011–2031	People living in rural areas 2013 (%)
NZ	115,248	161,673	217,245	88.50	14.03
Northland	5,084	7,185	9,422	85.33	50.19
Auckland	30,839	45,326	65,348	111.90	3.98
Waikato	11,130	15,527	20,435	83.60	22.88
Bay of Plenty	8,794	12,033	15,841	80.13	18.33
Gisborne	1,158	1,610	2,140	84.80	25.19
Hawke’s Bay	4,652	6,340	8,088	73.86	12.69
Taranaki	3,455	4,554	5,732	65.90	22.98
Manawatu-Wanganui	7,067	9,364	11,817	67.21	19.29
Wellington	11,974	16,371	21,475	79.35	3.84
Tasman	1,512	2,297	3,082	103.84	41.21
Nelson	1,413	1,983	2,650	87.54	1.90
Marlborough	1,688	2,414	3,082	82.58	23.07
West Coast	1,021	1,453	1,884	84.52	43.42
Canterbury	16,627	23,360	31,231	87.83	16.61
Otago	5,987	8,068	10,325	72.46	20.82
Southland	2,827	3,749	4,711	66.64	30.57

1.2 Anatomy of the human ear

The ear functions as a sensory system to detect sound. It is unguarded to thermal injury not only due to its protruding morphology and site but also of the subcutaneous tissue layers and very thin skin (Bos, Doerga, Breugem, & van Zuijlen, (2016)). The human ear is anatomically composed of three parts: the outer, middle, and the inner ear. The outer and the middle ear works to transmit sound waves while in the inner ear, sound stimuli are transduced to nerve impulses via the auditory nerve (Alters, (2000)). The inner ear consists of the cochlea which contains sensory hair that can be damaged by genetic or environmental factors, and are unable to regenerate naturally. Thus, hearing has to be restored by a cochlea transplant (Introduction to Psychology, (2008)).

Figure: Parts of an ear (“http://keywordsuggest.org/gallery/381978.html,”).

The inner ear represents the sensory organ required for hearing (the cochlea) and balance (the semi-circular canals). The cochlea is snail shaped bony structure (filled with two fluids: –  endolymph and perilymph). The sensory receptor, called as the Organ of Corti is embedded inside the cochlea which clasps the hair cells, which are the nerve receptors for hearing  (“http://www.asha.org/public/hearing/Inner-Ear/,”).

1.3 Underlying causes of hearing loss

The etiology and causes of Hearing loss can be congenital/acquired; sudden/ progressive; and temporary/permanent. Causes of hearing loss according to World Health Organization, 2015 are as follows: –

1) Congenital causes – due to hereditary or non-hereditary factors, prenatal exposure in utero to maternal disease or inappropriate drug use such as aminoglycosides, cytotoxic drugs, antimalarial drugs, and diuretics; severe jaundice in the neonatal period, maternal rubella, syphilis or birth asphyxia, and low birth weight resulting from premature birth.

2) Acquired causes-

1. Noise exposure / Noise-induced hearing loss (NIHL) – single instances of extreme noise, and prolonged exposure to noise (machinery and explosions) can lead, respectively, to sudden or gradual sensorineural hearing loss, because of damage to the sensory cells. NIHL is commonly associated with occupational-related noise in industries such as agriculture, manufacturing and construction and may occur with noisy leisure pursuits.
2. Ageing – presbycusis also called as age-related hearing loss occur progressively with age due to degeneration of the cochlea and/or auditory nerve, due to exposure to high volume of sound for prolonged time;

3. Diseases and disorders – hearing loss resulting from a variety of different conditions, such as chronic ear infections, autoimmune disorders, measles, mumps, meningitis, otitis media, drug-resistant tuberculosis, head or ear injury, and cancer;

4. Physical trauma –caused by injuries either to the ear or the brain.

5. Cerumen accumulation –build-up of cerumen (earwax) or other foreign bodies in the ear canal, can lead to temporary hearing loss (Listen Hear! New Zealand, 2017) (http://www.who.int/mediacentre/factsheets/fs300/en/, 2017).

1.3a Hearing loss at cellular level

Cochlear cells are also undermined if traumatized by auricular overstimulation and compromises its function. Clinical implications of acoustic trauma often include auditory symptoms, like the hearing loss and hearing sensitivity, or problems with clarity. Sometimes patients also experience symptoms like tinnitus (ringing in the ear unrelated to outside sound) and hyperacusis (intolerance to environmental sound). Tinnitus affects 10-12% of general population in New Zealand and about 1%

have a disturbing degree of severity. Patients with a primary complaint of tinnitus showed a prevalence rate of hyperacusis ~ 40%; and those of a primary complaint for hyperacusis showed a prevalence of tinnitus has as 86% (Yang et al., (2016)) (“http://www.tinnitus.org.nz/,”) (Baguley, (2003)).

Cochlear damage by noise can be due to mechanical, biological, and molecular stresses (these include inflammation, oxidative stress, energy exhaustion and excitotoxicity). These stress responses further aggravate cell death by apoptosis and necrosis. Investigations in the past have identified multiple genes associated with transcriptional control and various molecular pathways, such as the JNK pathway, phospho-MEK1/ERK1/2/p90, p38/MAPK signalling pathway, RSK signalling pathway, etc (Yang et al., (2016)).

Congenital deformities such as absence of the middle ear (microtia) are additional conditions to accidents or disease. The most conventional medical care is to substitute the damaged ear with a prosthesis or cartilage (silicone ear implant). However, these techniques are unideal, expensive and involves patient’s customization. Therefore, 3D printing is becoming more relevant in auricular research to create tissue-engineered constructs or prostheses (Shafiee & Atala, (2016)).

Non-syndromatic hearing loss follows a Mendelian inheritance pattern for a single gene mutation. A mutation in transcription factor TFCP2L3 led to non-syndromic AD age-related hearing loss (DFNA28), the function of which is still a mystery (Peters et al., (2002)). 50% cases of hearing impairment arising due to disruption of gap junctions is correlated to yet another gene called Connexin 26 (GJB2) (Shalit & Avraham., (2008)). Autosomal-recessive (AR) inheritance accounts for 80% of nonsyndromic genetic hearing loss, normally prelingual (at birth) while autosomal-dominant (AD) inheritance reckons for the remaining 20% and is mostly post lingual (typically leading to progressive sensorineural hearing loss (SNHL) with unreliable severity, usually beginning at 10 to 40 years (Peters et al., (2002)). X-linked (designated “DFNX#”) and mitochondrial inheritance accounts for 1% to 2% nonsyndromic hearing loss. Mitochondrial inheritance patients tend to incur progressive SNHL and begins at 5-50 years, with variable degree of hearing loss (Chang, (2015)). Examples of genes encoding -components of the hair cells and the nerves (PMCA2 and otoferlin), cytoskeleton proteins (myosin VI, myosin VIIA, and myosin XVA), and proteins significant for potassium recycling in the organ of Corti (connexin, KCNQ4, Pendrin, and Claudin 14) (Shalit & Avraham., (2008)).

1.4 Models used in auditory research and current technology

On the other hand, animal models such as mice (Mus musculus), hamsters (Cricetinae), rats (Rattus rattus), Zebrafish (Danio rerio) are being used to study the genetics of hearing loss. An estimated number of 115 million animals and above are being applied to laboratory experiments each year worldwide. However, the precise number remains unknown (“http://www.hsi.org/,”).

In particular, a Tfcp2l3 mutant zebrafish was developed in 2011 and resulted in impaired optic development and hearing ability (Han et al., (2011)). Knock out or mutant mice have been instrumental in illuminating the functions and process of the inner ear and its membrane formation, signalling pathways, growth factors, and hair cell projections (stereocilia and kinocilia) (Friedman, Dror, & Avraham, (2007)).

With the advances in medical technology, the number of animals used in research every year has increased. Besides ethical concerns, the use of animals in preclinical drug testing is very laborious, time consuming, and expensive. Such disadvantages have propelled researchers to find new substitutes to compensate for the drawbacks and to reduce the number of animals used (Sánchez-Romero, Schophuizen, Giménez, & Masereeuw, (2016)). Another approach for re-establishing hearing could be in “stem cell therapy” which involves, surgical stacking of stem cells within the cochlea which will not only allow fusion with the remaining cochlear cells but develop response to hair cells (https://hearinglosscure.stanford.edu/research/stem-cell-therapy/). Researchers at Stanford University studied expression of ATOH1 gene, that was found to be active in developing hair cells. Their finding emphasized that activation of ATOH1 using various tools of gene therapy in the human ear, could possibly  influence cells to evolve as new hair cells (https://hearinglosscure.stanford.edu/research/gene-therapy/). The researchers have also pioneered a new technology called as, the Volumetric Optical Coherence Tomography Vibrometry, that enables non-invasive imaging of sound-induced vibrations (unusually small (<1 nm) but critical to normal hearing) within the cochlea (https://hearinglosscure.stanford.edu/research/targeted-neural-stimulation/).

Age related degeneration of the sensory cells in a mammalian cochlea broadcast severe symptoms that worsen later in life (Yang et al., (2016)). Hence, it is important to emphasize the understanding of cellular and molecular mechanisms to prevent auditory loss, especially gene expression and protein analysis.

1.5 Vitelline Membrane (layer) VM

 

The vitelline membrane of hen eggs, separating the yolk from the egg white, consists of two distinct layers of different compositions and structures, the inner layer (lamina perivitellina) and the outer layer (lamina extravitellina). The two vitelline layers are synthesised in two different organs: the inner layer is formed in the ovary before ovulation, whereas the outer layer is formed in the upper oviduct after ovulation (Kido, Morimoto, Kim, & Doi, (1992)).

The embryo of Caenorhabditis elegans is surrounded by a concealed inner VM and a distinguished outer chitinous eggshell. When the VM was gently beamed with a laser irradiation of only the eggshell, it had the property of resealing over time. However, this membrane lost the resealing agility when bombarded with larger holes made into the eggshell. This scenario leads to an impaired gastrulation that renders the gut precursor cells inefficient to migrate towards an embryonic halt. This underscores   a critical role for pattern formation of the “micro-environment” throughout the embryo safeguarded  by the unimpaired vitelline membrane (Schierenberg & Junkersdorf, (1992)).

In another study, the VM domain was characterized by the presence of three promptly spaced cysteine residues (CX7CX8C). The VM proteins along with VM domains are integrated into large disulphide linkages during late oogenesis, which are frequently utilized in extracellular matrices to stabilize other non-covalent interactions. The regulation of disulphide bond formation for balancing early elasticity and late stability in the extracellular region may be beneficial for morphogenesis of proper vitelline membrane (Wu, Manogaran, Beauchamp, & Waring, (2010)).

In Drosophila melanogaster, the eggshell is composed of a vitelline membrane that undergoes cross-linking during the oogenesis and the vitelline membrane proteins are bound by disulphide linking. It was observed that when the VM is treated with reducing agents it leads to membrane solubilization. Besides the VMs structural role, it may constitute a repository for products of follicle cells involved in embryonic patterning. Vitelline membrane defects have also been detected in some mutants of the germ-line dependent genes fs (1) polehole [fs (1) ph] and fs (1) Nasrat [fs (1) Nas. Mutations in these genes fall into two major phenotypic classes: having either a fragile eggshell and consequent early embryonic development arrest, or defects only at the termini of the embryo characteristic of genes involved in the Tor signalling pathway (Cernilogar, Fabbri, Andrenacci, Taddei, & Gargiulo, (2001)).

1.6 Vitelline Membrane Outer Protein 1 (VMO1) and history

VMO1 is identified as one of the proteins in the outer layer of egg vitelline membranes, and was first identified by Back et al. (1982). This protein was also accompanied with other proteins such as lysozyme, ovomucin, and a second vitelline membrane outer protein (VMO2), which was indicated by Kido et al. (1992) and was later found to be 100% identical to mature β-defensin-11. All the VM proteins participate and are responsible for maintaining its structural need (Guérin-Dubiard & Nau, (2007)) (Mann, (2008)).

Schäfer et al. (1998) indicated in his research that egg stored under non-refrigerated conditions disintegrate VMO1 and VMO2 proteins, leading to vitelline membrane distortion. This scientific finding can now be an appropriate assumption as to why Guerin-Dubiard et al. (2006) was fortunate to detect VMO1 for the first time. Along with his colleagues, they determined the molecular weight of VMO1 at 17.6 KDa, and was consistent with Schäfer et als’ SDS-PAGE analysis. Also,  VMO1 protein was suggested to have a pI near 10 since it was found to be in the alkaline area of 2-D gel analysis (Guérin-Dubiard & Nau, (2007)). Chromatographic analysis identified VMO-1 as a spot in conjunction with lysozyme. However, there is no experimental evidence for interactions between these two proteins in egg white. (Guérin-Dubiard et al., (2006)).

The crystal structure of VMO1 was originally determined using by the multiple isomorphous replacement anomalous scattering (MIRAS) method at 3 Ǻ resolution. The parent chain of this protein consists of a peculiar three β-sheets folds that forms  Greek key motifs. Sequence analyses revealed the a 53 residue, 3-protein component of Greek key motifs. VMO-I could also synthesize N-acetyl chito-oligosaccharides (n = 14 or 15) from hexa saccharides of N-acetylglucosamine, this activity was found to be similar in comparison to the transferase activity of lysozyme without the hydrolysis activity. The true physiological function yet remains to be poorly understood. (Shimizu, Vassylyev, Kido, Y. U. K. I. O., & Morikawa, (1994)).

1.6a Chicken VMO1

Chick embryo development comprises of three phases. days 0 to 7 denotes the inception of germ layers, the ectoderm, mesoderm, and the endoderm responsible for tissue and organs differentiation during gastrulation. During days 3 and 7, functional organs and extra-embryonic membranes (amnion, allantois, chorion, and yolk sac) are developed. The second phase, from days 8 to 18 marks the development of chorio-allantoic membrane (CAM) and embryo completion. At day 10, the chick embryo is fully formed. The final phase, days 19–21, represents emergence (Cordeiro & Hincke, (2015)).

Proteins present in the eggshell membranes (ESMs) of fertilized eggs during peculiar days of chick embryo development are represented in the figure below. The black circles correspond to the presence of VMO1 gene, emphasizing its role in extracellular structure organization during developmental stage 2 and 3. The ESMs are polymeric scaffolds that accelerate embryo development and offers protection against invading viruses  (Cordeiro & Hincke, (2015)).

Figure: (Cordeiro & Hincke, (2015))

Table: Heat-map illustrating the comparative analysis of the ESM proteins with different characters traced in the fertilized versus unfertilized eggs at various days (0, 3, 7, 11, 15 and 19) of incubation (Cordeiro & Hincke, (2015)).

Intensity of red colour depicts the fold increase in abundance of ESM proteins within fertilized eggs and in comparison, to unfertilized condition. Intensity of green illustrates the fold increase in abundance of ESM proteins for unfertilized eggs, in comparison to embryonated eggs. Intensity of grey shows comparative analysis of the degree of variation in ESM proteins which are present in both fertilized and unfertilized conditions at a similar level.  White cells in the figure indicates the absence of particular proteins at that specific day of incubation. The article also affirms the expression of VMO1 gene at all levels, amongst 17 other genes marked by a circle(Cordeiro & Hincke, (2015)).

Table: Official gene symbols correspond to eggshell membranes proteins from fertilized eggs (Cordeiro & Hincke, (2015))

(Lee et al., (2015)) analysed  the expression pattern and functional activity of VMO-1 in the laying hen oviduct using reverse transcription polymerase chain reaction (RT-PCR), quantitative RT-PCR (qRT-PCR), and RNA interference (RNAi). The group inferred that microRNAs (miRNAs) like 1651-3p, 1552-3p, and gga-miR-1623 had an influential role on the expression of VMO-1 through its 3′-UTR, speculating its posttranscriptional regulation. Some VMO-1 gene knockdown experiments have also revealed repression of ovomucin by VMO1 silencing, while other studies have linked estrogen as an inducible factor for VMO-1 mRNA expression in vitro.

Figure: Expression of VMO-1 in chickens.

In a study conducted by (Lee et al., (2015)), expression of VMO-1 was under examination in numerous organs of both male and female chickens as seen in A. It was evident that VMO1 gene was highly expressed in females’ oviduct. Also, RT-PCR and qRT-PCR analyses of the chicken oviduct comprising of the infundibulum, isthmus, magnum, and shell gland as seen in B, was conclusive in determining the presence of this gene is the “Magnum”.

(Lim & Song, (2015)) proposed that VMO1 holds a crucial contribution in the morphogenesis of oviduct in presence of estrogen and moulting, they also affirmed that the onset of VMO1 expression is associated with carcinogenesis of laying hens. A CA-125 biomarker for diagnosing early stage of ovarian cancer in women was cross-reactive with biomarkers for ovarian cancer in laying hens.

1.6b Mouse/rat Vmo1

Differentially expressed mRNAs products confer cells with specialized functions and structures that could be confined to different cell types or in small copy numbers. The intricate morphology of the mammalian middle ear makes it a complicated model to detect mRNAs. For instance, the sensory cells of the inner ear, the organ of Corti, and outer hair cells, possess less than ∼5% of this tissue. Although in an average cell, about 250,000 mRNAs are exhibited but a typical low-abundance message specific to the hair cells would be expected (at a rate of 1 in 1 million transcripts). Before its discovery, the mouse Vmo1 gene was originally annotated as an in silico-predicted gene, GM741 (GeneID 327956; RefSeq XM_282996) (Peters et al., (2007)).

 

Table: Molecular pathways identified (Yang et al., (2016)).

Molecular pathways	Rat cochlea	Mouse cochlea
Complement and coagulation cascades	+	–
Cytokine receptor interaction	+	+
Chemokine signalling pathway*	+	+
Jak-STAT	+	–
Cell adhesion molecules	+	–
Toll-like receptor signalling pathway	+	+
Glycerophospholipid metabolism	+	–
NOD-like receptor signalling pathway	+	+
p53 signalling pathway	+	–
Adipocytokine signalling pathway	+	–
Fc gamma R-mediated phagocytosis	+	–
Cytosolic DNA-sensing pathway	–	+
RIG-I-like receptor signalling pathway	–	+

Evidence for the presence of Vmo1 in the ear of mouse can from two experiments conducted by (Peters et al., (2007)). First the mRNA was procured from adult liver, kidney, pancreas, retina, brain, testes, inner ear, and genomic DNA from mouse. This was followed by a RT experiment that revealed the presence of Vmo1 in the inner ear. The evidence was further advocated by performing an In situ hybridization, in order to detect the tissue specific localization of mRNA, and was found in the   Reissner’s membrane.

1.6c VMO1 and miscellaneous

Presence of VMO1 have also been reported in various exocrine glands and/or secretions, for example., pancreas, urine, breast,  cerebrospinal fluid, and respiratory secretions, and also in minuscule quantities in human tears while a comparatively  higher in camel tears (Wang et al., (2014)).

Proteins present in the tear film often play an important defensive role to thwart pathogens, while maintaining the integrity of the tear film, and modulating wound healing. In the recent research, proteomics studies have enlightened potential biomarkers in the tear proteins, for treating systemic and ocular diseases. Camels often habitat under harsh environmental conditions and have been demonstrated for the presence of large amount of VMO1 homolog in camel tears (Chen et al., (2011)). Thus, characterizing their tear components can shed some light underlying the mechanisms of tear film stabilization and understanding other variants of this genes’ product.

Figure: Interaction model between VMO1 (red) and lysozyme (blue). This model was beneficial in deciphering how these two molecules associated through various amino acid interactions. This was done using computer-assisted programs. Under this particular  model, Glutamine (Q) 153, Glutamic acid (E) 110, and  Q 70 of VOM1 binds to Arginine (R) 59, R59 or Serine (S) 100, and Aspartic acid (D) 85 of lysozyme through hydrogen bonding (Wang et al., (2014)).

Intraembryonic hematopoietic stem cells cluster in the floor of the dorsal aorta, vitelline membrane, and umbilical arteries. Here, Notch signalling has demonstrated to be functionally important particularly for Yolk Sac-derived haematopoiesis. It acts as a mediator by balancing the mechanisms of proliferation and differentiation of progenitors (lineage restricted intermediate) (Cortegano et al., (2014)).

VMO1 homolog was found to be one of the most highly transcribed genes in two undisputed samples from dog: Bichon fries  and golden retriever at 3548 and 2723 FPKM (fragment per kilo base of exon per million of fragments mapped) respectively (Galibert, Azzouzi, Quignon, & Chaudieu, (2016)).

1.7 Bioinformatics

One study involved the use of two bioinformatics tools specifically to determine the functional relevance of the differentially expressed genes (DEGs): The Database for Annotation, Visualization, and Integrated Discovery (DAVID v 6.7) and the Ingenuity Pathway Analysis (IPA). For DEGs identified in the rat cochleae, over 400 genes were Upregulated amongst which was Vmo1 with 19.08-fold change (Yang et al., (2016)).

Comparative analysis of chicken VMO-1 protein-coding sequence and the human, mouse, rat, and bovine VMO-1 proteins using multiple sequence alignment tool revealed high degree of homology of 55%, 53%, 48%, and 54%, respectively (Lee et al., (2015)).

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