73.3.2 The Cngb3 Mutant Dog and Gene Therapy

One autosomal recessive canine disease that occurs naturally in the Alaskan Malamute has been found to be related to CNGB3 mutations. Its phenotype is similar to human achromatopsia, and is characterized by day-blindness and absence of retinal cone function (Sidjanin et al. 2002). Since CNGB3 mutations account for 50% of human achromatopsia, this dog provides a valuable large animal model for exploring disease mechanisms and evaluating potential genetic therapeutic intervention (Sidjanin et al. 2002).

AAV-mediated gene therapy in this canine model of achromatopsia has achieved major therapeutic effect (Komaromy et al. 2008). ERG restoration has been observed and maintained for over 14 months in the CNGB3 Malamute following a subretinal injection of an AAV5 vector containing human CNGB3 cDNA controlled by a truncated human red cone opsin promoter (Komaromy et al. 2008).

73.4 Prospects for Achromatopsia Gene Therapy

AAV mediated gene transfer of the corresponding wild type gene corrects cone functional deficiencies in two mouse models and one dog model representing all three (Gnat3,Cnga3 and CNGB3) genetic forms of human achromatopsia. Such intervention effectively restores cone system function as demonstrated by ERGs and/ or by visually elicited behavior. These data suggests achromatopsia may be a viable candidate for gene-based therapy.

Acknowledgment This research is supported by NIH grants, EY018331, EY13729, EY11123, NS36302, EY08571, EY007758 and FFB, MVRF, RPB, Lions of Central NY.

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Chapter 74

Function and Mechanism of CNTF/LIF

Signaling in Retinogenesis

Kun Do Rhee and Xian-Jie Yang

Abstract Ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF) exhibit multiple biological effects in the developing vertebrate retina. CNTF/LIF inhibits rod photoreceptor, and promotes bipolar cells and Muller glia differentiation. In addition, CNTF/LIF has been shown to have proliferative and apoptotic effects. Moreover, LIF also inhibits retinal vascular development. CNTF/LIF signaling components CNTFRα, LIFRβ, gp130, and a number of STAT proteins are expressed in the retina. CNTF/LIF activates Jak-STAT, ERK, and Notch pathways during retinal development. Perturbation of CNTF induced signal transduction reveals that different combinations of CNTF/LIF signaling pathways regulate differentiation of retinal neurons and glia. Gene expression studies show that CNTF/LIF affects retinogenesis by regulating various genes involved in transcription, signal transduction, protein modification, apoptosis, protein localization, and cell ion homeostasis. Most past studies have deployed ectopic expression or addition of exogenous CNTF/LIF, thus further analysis of mice with conditional mutations in CNTF/LIF signaling components will allow better understanding of in-vivo functions of CNTF/LIF associated signaling events in retinogenesis.

74.1 Introduction

The mature vertebrate retina consists of seven major neuronal cell types generated from a common progenitor pool. Lineage tracing analyses using retrovirus infection and fluorescent dye single cell injection have shown that retinal progenitor cells are multipotent and cell fates are specified during or after their terminal mitosis (Wetts and Fraser 1988; Turner et al. 1990). Molecular genetic studies have further

K.D. Rhee (B)

Jules Stein Eye Institute, Molecular Biology Institute, University of California, Los Angeles, CA, USA

e-mail: kdrhee@ucla.edu

Medicine and Biology 664, DOI 10.1007/978-1-4419-1399-9_74,C Springer Science+Business Media, LLC 2010

demonstrated that retinal cell fates are regulated by both cell intrinsic and extrinsic mechanisms. A proposed model suggests that progenitors pass through a series of intrinsically determined competence states, and under each state, progenitors are capable of giving rise to a limited subset of cell types under the influence of extrinsic signals (Livesey and Cepko 2001). Various signal molecules have been shown to influence retinal cell differentiation either positively or negatively (Levine et al. 2000; Yang 2004). Among them, ciliary neurotrophic factor (CNTF) and leukemia inhibitory factor (LIF), members of the interleukin (IL)-6 family of cytokines, exhibit multiple biological effects in the developing vertebrate retina.

CNTF/LIF family of cytokines bind to a tripartite receptor complex consisting of two single membrane spanning beta-receptor subunits and an additional alpha receptor specific for certain members of the cytokine family (Ip, 1998). CNTF/LIF triggers multiple intracellular signaling events, including activation of the Janus kinases (Jak)-Signal Transducer and Activator of Transcription (STAT), the extracellular signal-regulated kinases (ERK) (Boulton et al. 1994) and the phosphatidylinositol 3 kinase (PI3K)-Akt pathways (Oh et al. 1998).

74.2Effects of CNTF/LIF on Photoreceptor and Bipolar Neuron Differentiation

CNTF/LIF has been shown to strongly inhibit differentiation of rod photoreceptors in both mouse and rat retinas (Ezzeddine et al. 1997; Neophytou et al. 1997; Kirsch et al. 1998; Schulz-Key et al. 2002; Sherry et al. 2005; Elliott et al. 2006). Both CNTFRα and LIFRβ knockout retinas display increased proportions of rod photoreceptors when cultured as explants for 10 days in-vitro starting from postnatal day 0 (Ezzeddine et al. 1997). In contrast, in a chicken retinal culture, CNTF appears to promote the differentiation of a subclass of cone photoreceptors (Fuhrmann et al. 1995; Xie and Adler 2000). Consistent with the observed influence of CNTF/LIF on photoreceptor development in-vitro, retinas of transgenic mice that misexpress LIF from the alpha crystallin promoter during embryonic development show dose-dependent disruption of photoreceptor differentiation (Graham et al. 2005).

In addition to their effects on photoreceptor development, CNTF/LIF also regulates other late born retinal cell types. Concomitant to suppression of rod differentiation, CNTF/LIF promotes the expression of several bipolar cell markers in the postnatal retina (Ezzeddine et al. 1997; Schulz-Key et al. 2002; Bhattacharya et al. 2004; Zahir et al. 2005). By tracking cell birthdates and analyzing proportions of different cell markers, Ezzeddine et al. (1997) concluded that the bipolar marker-positive cells are derived from cells that would normally give rise to rod photoreceptors. This result indicates that CNTF/LIF affects the differentiation process of immature postmitotic neuronal precursors. They also observed that once rod cells begin to express rhodopsin protein, they become increasingly resistant to the inhibitory effects of CNTF. Consistent with this observation, SchulzKey et al. (2002) showed that retinal explants treated with CNTF contain bipolar