Fig. 6.2 Southern Blot of telomere lengths of HUVEC that were cultured for 3 months. 1: normal (control);

2:apoptosis group cultured in 0.2 mmol/l

FeSO4/0.0001 mmol/l H2O2;

3:apoptosis group cultured in 0.2 mmol/l

FeSO4/0.005 mmol/l H2O2

Table 6.3 Mean telomere length of human umbilical endothelial cells that were cultured for 1 or 3 months under different oxidation concentrations

6.4 Discussion

Over the past decades, scientists have found that the telomere shortening is present in Hutchinson-Gilford progeria and Down’s syndrome (Vaziri et al. 1993). More recent reports show telomere shortening is associated with age-related diseases (Gilley et al. 2008), even cardiovascular diseases (Kurz et al. 2006; Brouilette et al. 2008), and diabetic nephropathy (Verzola et al. 2008). These studies attempted to elucidate the age-related mechanism between telomere length, age and the environment. We designed this experiment to simplify the detailed, complicated interactions

among the environment and cell, gene-gene interactions, gene-protein interactions, and apoptosis and ageing. We show the environment-telomere-apoptosis interactions through the use of culture time to reflect the telomere age. As expected, telomeres of HUVEC shortened as culture time increased. Importantly, we found that the apoptosis rate was not only affected by oxidative concentrations, but also by the culture time and TML (as an indirect measure of the amount of cell division), indicating that the cells with more cell divisions or with short telomeres are sensitive or susceptible to oxidation. In other words, we hypothesize short-telomere cells are susceptible to apoptotic factors, while long-telomere cells are tolerant to apoptosis. Thus, we speculate the age-related diseases, degenerative diseases might occur at young ages in individuals who have short telomeres, and vice versa. This hypothesis can explain not only the variance of cells in an individual’s organ to apoptosis, but also the differences between individuals. It can provide evidence for research on age-related diseases through the examination of telomere lengths.

Most importantly, this hypothesis also can be applied to non-dividing cells such as neurons or myocardial cells. Horvitz and Herskowitz (1992) and Morrison et al. (1997) elucidated the symmetric and asymmetric divisions for stem cells, especially the division of neuron stem cells. For symmetric division, cell division amounts are the same, while some cells experience more divisions and some have fewer divisions in asymmetric division. Translating this hypothesis to telomere length, asymmetric division in stem cells produces large amounts of cells with a significant variance in telomere lengths resulting from different division times. This is the reason that telomere length shows as a band or range on Southern blots, thus telomere length in non-dividing cells after birth are different, originating from asymmetric division of the stem cells. We have found this to be the case for telomere length variance in separated rabbit retinal ganglion cells (unpublished). Hence the telomere hypothesis may also be applied to age-related neuropathy such as glaucoma, retinal degeneration diseases to suggest an explanation for their sensitivity to apoptosis.

Furthermore, we found that oxidation can enhance the telomere shortening. This is consistent with the report by Toussaint et al. (2000), which showed telomeres are highly susceptible to oxidative stress. De Meyer et al. (2008) also showed that elevated levels of oxidative stress and inflammation further increase the rate of telomere attrition. These data show there is a relationship between telomere length and oxidation, suggesting that we can construct a “bridge” among genes and the environment, and ageing and apoptosis through the use of telomeres.

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Chapter 7

Photoreceptor Guanylate Cyclases and cGMP

Phosphodiesterases in Zebrafish

Ross F. Collery and Breandán N. Kennedy

Abstract Tightly regulated control of cGMP levels is critical for proper functioning of photoreceptors, and mutations in cGMP synthesis or degradation factors can lead to various forms of retinal disorder. Here we review heterogenous human retinal disorders associated with mutant retinal guanylate cyclases (RetGCs) and phosphodiesterase 6 (PDE6), and describe how zebrafish are being used to examine phototransduction components and their roles in these diseases. Though mutations in RetGCs and PDE6 lead to retinal disorders, there is a lack of molecular and biochemical data on routes of subsequent photoreceptor degeneration and visual impairment. Use of animal model systems provides important information to connect in vitro biochemical analyses of mutant genes with clinically observed pathologies of human retinal diseases. Zebrafish are an excellent in vivo system to generate animal models of human retinal disorders and study photoreceptor components, and have already provided valuable data on retinal diseases caused by phototransduction component mutations.

7.1Regulation of cGMP Levels in Photoreceptor Outer Segments

Cyclic GMP (cGMP) is a key second messenger in photoreceptor outer segments (for review, see Kaupp and Seifert 2002), regulating intracellelular calcium ion entry through cyclic nucleotide-gated (CNG) channels (Fig. 7.1). Depletion of cGMP causes these ion channels to close, while photoreceptors continue to efflux ions via other cGMP-independent ion channels. This leads to a drop in intracellular ionic

R.F. Collery (B)

UCD School of Biomolecular and Biomedical Science, UCD Conway Institute of Biomolecular and Biomedical Research, University College Dublin, Dublin, Ireland

e-mail: rcollery@mcw.edu

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_7,

C Springer Science+Business Media, LLC 2010