33.3 Results

Mutation rate of IMPDH1 gene had no significance between in adRP patients and in the normal control by exact probabilities in 2 × 2 table (p = 0.232). The mutation frequency of IMPDH1gene in the Han samples was 3.6%. Group distribution χ2 = 1.635, p >0.5. The allelic distribution of the gene was in accordance with HardyWeinberg equilirium.

33.4 Discussion

The identification of IMPDH1 as the causitive gene in the RP10 form of adRP had implicated a nucleotide biosynthesis pathway in a degeneration of the retina (Kennan et al. 2003). The IMPDH1 gene encodes a protein subunit of 514 amino acid residues, with the active IMPDH1 enzyme consisting of a homotetramer of these subunits. Each monomer of the protein possesses two domains; the larger forms a barrel which contains the active site loop and the smaller is comprised of two tandem cystathionine-beta-synthase dimer domains (Nimmesgern et al. 1999). It was difficult to speculate how precisely mutations within the IMPDH1 gene may bring about the pathology. One possibility was that, while not in the active site of the enzyme, the mutation might cause a reduction in the levels of guanine nucleotides available to photoreceptors which it appeared, might be relying almost solely on IMPDH1 for maintaining their guanine nucleotide reserves.

The mutation frequency of IMPDH1 gene was approximately 2–5% of the adRP cases among Americans of European origin and Europeans (Bowne et al. 2006; Bowne et al. 2002; Wada et al. 2005). The mutation frequency of IMPDH1 gene of the Han population in Ganzhou city was similar as this. The evidence implicating the IMPDH1 gene as a cause of dominant RP includes the identification of different missense mutations in different families, the observation that none of these mutations is found among normal controls, and the observation that the mutations perfectly cosegregate with RP. There is no reported comparison of the clinical features of patients with mutations in this gene versus those with mutations in other identified RP genes. The most frequent mutation, Asp226Asn, appeared to cause at least as much loss of rod function as cone function. Patients with this form of RP retain, on average, two to five times more ERG amplitude per unit of remaining visual area than patients with three other forms of dominant RP (Wada et al. 2005). Bowne et al. (2006) reported that IMPDH1 mutations did not alter enzyme activity and demonstrated that these mutants altered the recently identified singlestranded nucleic acid binding property of IMPDH. Subsequent studies are needed to further elucidate the nucleic acid binding property of IMPDH1 and its relevance to photoreceptor biology and retinal disease.

References

Bowne SJ, Sullivan LS, Blanton SH et al (2002) Mutations in the inosine monophosphate dehydrogenase 1 gene (IMPDH1) cause the RP10 form of autosomal dominant retinitis pigmentosa. Hum Mol Genet 11(5):559–568

Bowne SJ, Sullivan LS, Mortimer SE et al (2006) Spectrum and frequency of mutations in IMPDH1 associated with autosomal dominant retinitis pigmentosa and leber congenital amaurosis. Invest Ophthalmol Vis Sci 47(1):34–42

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Gandra M, Anandula V, Authiappan V et al (2008) Retinitis pigmentosa: mutation analysis of RHO, PRPF31, RP1, and IMPDH1 genes in patients from India. Mol Vis 14:1105–1113

Hims MM, Diager SP, Inglehearn CF (2003) Retinitis pigmentosa: genes, proteins and prospects. Dev Ophthalmol 37:109–125

Kennan A, Aherne A, Bowne SJ et al (2003) On the role of IMPDH1 in retinal degeneration. Adv Exp Med Biol 533:13–18

Kennan A, Aherne A, Palfi A et al (2002) Identification of an IMPDH1 mutation in autosomal dominant retinitis pigmentosa (RP10) revealed following comparative microarray analysis of transcripts derived from retinas of wild-type and Rho(–/–) mice. Hum Mol Genet 11(5): 547–557

Nimmesgern E, Black J, Futer O et al (1999) Biochemical analysis of the modular enzyme inosine 5 -monophosphate dehydrogenase. Protein Expr Purif 17(2):282–289

Wada Y, Sandberg MA, McGee TL et al (2005) Screen of the IMPDH1 gene among patients with dominant retinitis pigmentosa and clinical features associated with the most common mutation, Asp226Asn. Invest Ophthalmol Vis Sci. 46(5):1735–1741

Wang Q, Chen Q, Zhao K et al (2001) Update on the molecular genetics of retinitis pigmentosa. Ophthalmic Genet. 22(3):133–154

Yu Y, Yang H, Yu Y et al (2007) Mutations of the IMPDH 1 gene in patients correlated with autosomal dominant retinitis pigmentosa family. Rec Adv Ophthalmol 27(9):649–652

Zhao C, Lu S, Zhou X et al (2006) A novel locus (RP33) for autosomal dominant retinitis pigmentosa mapping to chromosomal region 2cen-q12.1. Hum Genet 119(6):617–623

Part III

Diagnostic, Clinical, Cytopathological and

Physiologic Aspects of Retinal

Degeneration

Chapter 34

Reversible and Size-Selective Opening

of the Inner Blood-Retina Barrier:

A Novel Therapeutic Strategy

Matthew Campbell, Anh Thi Hong Nguyen, Anna-Sophia Kiang, Lawrence Tam, Paul F. Kenna, Sorcha Ni Dhubhghaill, Marian Humphries, G. Jane Farrar, and Peter Humphries

Abstract The inner Blood-Retina-barrier (iBRB) remains a key element in retarding the development of novel therapeutics for the treatment of many ocular disorders. The iBRB contains tight-junctions (TJ’s) which reduce the space between adjacent endothelial cells lining the fine capillaries of the retinal microvasculature to form a selective and regulatable barrier. We have recently shown that in mice, the iBRB can be transiently and size-selectively opened to molecules with molecular weights of up to approximately 1 kDa using an siRNA-mediated approach involving suppression of the tight junction protein, claudin-5. We have systemically delivered siRNA targeting claudin-5 to retinal capillary endothelial cells in mice and through a series of tracer experiments and magnetic-resonance-imaging (MRI), we have shown a transient and size-selective increase in permeability at the iBRB to molecules below 1 kDa. The potential to exploit this specific compromise in iBRB integrity may have far reaching implications for the development of experimental animal models of retinal degenerative disorders, and for enhanced delivery of therapeutic molecules which would normally not traverse the iBRB. Using RNAi-mediated opening of the iBRB, the systemic delivery of low molecular weight therapeutics could in principle, hold real promise as an alternative to repeated intraocular inoculation of compounds. Results demonstrated here in mouse models, should lead to a ‘humanized’ form of systemic delivery as opposed to the hydrodynamic approach used in our work to date.

34.1 Introduction

The human retina has the highest oxygen consumption per weight of any tissue in the body. The high metabolic rate of the neural retina underlines the need for a distinct and regulated blood supply, and this is mediated via the Blood Retinal Barrier

M. Campbell (B)

Ocular Genetics Unit, Department of Genetics, Trinity College Dublin, Dublin 2, Ireland e-mail: matthew.campbell@tcd.ie

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_34,C Springer Science+Business Media, LLC 2010

(BRB). At the inner retina, retinal capillaries arising from the central artery permeate the retina only as far as the inner nuclear layer (INL), with the outer segments of the retina remaining avascular. Very similar in structure and function to the blood brain barrier (BBB), the BRB in the retina allows for the maintenance of neural tissue environments through the regulation of ion concentrations, water permeability, delivery of amino acids and sugars, and by preventing the exposure of the neural tissue to circulatory factors such as antibodies and immune cells (Antonetti et al. 1999). In contrast to the BBB, however, the BRB consists of both an inner blood retinal barrier (iBRB) and an outer blood retinal barrier (oBRB). The iBRB comprises retinal endothelial cells, which line the micro-vessels allowing for the maintenance of blood vessel integrity and preserving the vessel’s homeostasis while the oBRB is made up of retinal pigment epithelial (RPE) cells and Bruch’s membrane, and it acts as a filter to restrict the passage of macromolecules.

Both the iBRB and oBRB contain tight junctions that confer highly selective properties on barrier function. Tight junctions are formed at the apical periphery of endothelial cells of the iBRB (Fig. 34.1). They perform the dual role of creating a primary barrier to the diffusion of solutes through the paracellular pathway, while also maintaining cell polarity as a boundary between the apical and basolateral plasma membrane domains Sakakibara et al. (1997).

Tight junctions are complex structures, which are composed of a series of integral and peripheral membrane proteins. The transmembrane proteins of the tight junction include occludin, junctional adhesion molecule (JAM) and claudins 1–20. These proteins extend into the paracellular space, creating the seal characteristic of the tight junction (Fanning and Anderson 1998; Riesen et al. 2002).

Occludin and the claudins are transmembrane proteins associated with the tight junction and have previously been shown to interact homotypically with proteins on adjacent endothelial cells. In 2003, claudin-5 knockout mice were reported, and were shown to have a compromised BBB. The authors concluded that while removal of claudin-5 compromises the function of the BBB by allowing it to become permeable to molecules of up to approximately 800 Da, the barrier could still form, remaining intact and impervious to larger molecules (Nitta et al. 2003).

Claudin-5 is highly expressed in the plasma membrane of retinal microvascular endothelial cells under normal conditions in vivo while hypoxia significantly reduces the level of claudin-5 in the membrane of these cells. In addition, inhibition of claudin-5 expression using RNAi leads to a reduction of transendothelial electrical resistance in bEND.3 cells even under normoxic conditions (Koto et al. 2007).

It has been proposed that claudin-5 may play a role in the formation of paracellular pores or channels that function in mediating selective ion permeability (Anderson et al. 2001). It is clear however that removal of claudin-5 from the tight junction will cause a size-selective increase in the permeability of the tight junction.

Here, we describe the first report of reversible and controlled RNAi mediated size-selective opening of the paracellular pathway of the iBRB, representing a novel approach for delivery of a wide range of small molecules to the inner retina. This method of reversible iBRB modulation may pave the way for controlled delivery