Ординатура / Офтальмология / Английские материалы / Recent Advances in Retinal Degeneration_LaVail, Hollyfield, Anderson _2008

activation of the JNK-p53-Bax pro-death pathway and by increasing levels of the pro-apoptotic molecule ceramide from sphingomyelin hydrolysis (Fig. 1). In some neurons, Trk receptors compete for binding with adaptor proteins to p75NTR and block the apoptotic signal.

p75NTR is expressed primarily in Muller glia and at low levels in RGC. During photoreceptor degeneration in rd1 mice, p75NTR protein is upregulated in Muller glia and in the ONL (Nakamura et al., 2005). Reducing p75NTR levels by crossing p75NTR-/- and p75NTR+/- mice with retinal degeneration rd1 mice did not alter the rate of photoreceptor death (Nakamura et al., 2005). Similarly, Rohrer et al showed that lack of p75NTR-/- did not reduce apoptosis from light toxicity (Rohrer et al., 2003). These studies demonstrate that neurotrophin-independent death pathways primarily mediate photoreceptor death in p75NTR-/- mice. However, these experiments are complicated by data showing that the p75NTR-/- mice express a fragment of p75NTR that could potentially retain death-inducing activity (von Schack et al., 2001). Further, p75NTR+/- retinas had reduced light damage (Rohrer et al., 2003) and a p75NTR blocking antibody had a small neuroprotective effect for light damage (Harada et al., 2000), indicating that p75NTR – dependent apoptosis may play a role in the retina.

The pro-death outcome of p75NTR binding depends on the specific interacting adapter proteins and their downstream pathways. p75NTR also promotes neuronal survival in response to neurotrophins by regulating NF-kB, which induces survival or death signals (Kalb, 2005). p75NTR also activates the Akt pro-survival pathway through PI3K signaling (Roux et al., 2001). Unexpectedly, Trk receptors can induce death signals in some situations. For example, cortical neuronal cultures undergo necrosis with BDNF and TrkB activation (Kim et al., 2003), BDNF increases susceptibility to excitotoxicity in spinal cord neurons (Fryer et al., 2000) and medulloblastoma cell lines undergo apoptosis from TrkA receptor activation by NGF (Muragaki et al., 1997). The mechanism of death-promoting Trk signaling may be through activation of MAPK and PI3K pathways. That these pathways also promote survival in some situations illustrates the complexity of the death and survival choices induced by neurotrophins. It will be intriguing to determine whether these non-traditional activities for neurotrophin receptors play a role in the retina.

4 Regulation of Neurotrophin Expression

Neurotrophins are expressed locally in the retina, by Muller glia, microglia and other cells, or are transported retrogradely by RGCs. Retrograde transport of neurotrophins, which is regulated by p75NTR, has not been explored during photoreceptor degeneration. At the transcriptional level, the regulation of BDNF is the best described of the neurotrophins and has proven to be very complex. The BDNF gene has four 5’ non-coding exons and each exon has its own promoter elements and cell-specific expression patterns. Promoter III has been investigated in detail and contains numerous transcription factor binding elements, including sites for USF1/2, CREB and Brn3c (Timmusk et al., 1993). Since CREB is activated by Trk

binding, the regulation of BDNF transcription by CREB suggests positive feedback mechanisms that may sustain the activity of BDNF.

Several signaling networks leading to transcription factor binding to the BDNF promoter have been characterized. For example, neuronal calcium signaling induces BDNF through binding of upstream stimulatory factors (USF) 1 and 2 to Ca2+- responsive E-box elements (Chen et al., 2003), and dopamine-induced transcription involves cAMP/CREB binding. Understanding the regulation of the BDNF promoter in the retina would provide insight into its activity during retinal degeneration.

Neurotrophin release from astrocytes is regulated differentially by signals from adjacent neurons. For example, in basal forebrain astrocyte cultures BDNF transcripts but not NGF are increased by KCl, Ca2+ and the muscarinic agonist carbachol, both BDNF and NGF are increased by glutamate, and NT3 is not regulated by any of these stimuli (Wu et al., 2004). In rat Muller glia cultures, glutamate upregulated expression and secretion of all four neurotrophins (Taylor et al., 2003).

In the retina, neurotrophins increase expression of other growth factors, which may influence the efficacy of therapeutic delivery strategies. In rat Muller glia cultures, microglia-derived GDNF and CNTF increased BDNF and bFGF, and BDNF increased CNTF and bFGF levels (Harada et al., 2002). Injection of NGF in retinal degeneration RCS rats induced BDNF, bFGF, TGF , VEGF and neuropeptide Y (Lenzi et al., 2005). In contrast, exogenous NGF decreased bFGF expression by activating p75NTR (Harada et al., 2000). Withdrawal of bFGF-mediated survival signals from Muller glia was proposed to contribute to photoreceptor death (Harada et al., 2002). Therefore, protection by neurotrophins may be indirect by inducing the release of additional secreted factors or by stimulating other protective trophic pathways. This effect could amplify the protective response, possibly by recruiting other glia that are not proximal to the injury site.

In our laboratory, we investigate cellular pathways that regulate neurotrophin expression during degeneration. One example is the Wnt pathway, an essential signaling cascade that mediates retinal development and stem cell proliferation and is a critical regulator of cell survival in degenerative conditions of the brain such as Alzheimers disease. Wnt signaling regulates growth factor expression in several tissues, including NT3 in the limb bud (Patapoutian et al., 1999), GDNF during kidney development and TGF during adult chondrogenesis (Tuli et al., 2003). The Wnt pathway is active during photoreceptor death in the rd1 mouse (Yi et al., submitted), suggesting that Wnt signaling may regulate neurotrophin expression and activity during retinal degeneration.

5 Regulation of Receptor Activity

The signaling response to neurotrophin injections in the retina is transient (Wahlin et al., 2001). Cellular mechanisms down-regulate the signal in order to precisely control neurotrophin signaling for the proper time period and in the correct location. One regulatory mechanism is the degradation of Trk receptors through liganddependent ubiquitination (Arevalo et al., 2006). p75NTR may regulate the ubiq-

uitination of Trk receptors. Additionally, stimulation of the target cell by cAMP, Ca2+ or electrical activity induces exocytosis of intracellular vesicles containing Trk receptors into the plasma membrane (Meyer-Franke et al., 1998). Other sources of regulation include tyrosine phosphatases to terminate the signaling, differential expression of the adaptor proteins and enzymes, membrane sorting of Trk receptors and association with Trk splice variants. How these regulatory mechanisms contribute to the regulation of Trk receptors during retinal degeneration is an important topic that requires further exploration.

6 Conclusions

Over-expression of neurotrophins and other growth factors could elevate the threshold for apoptosis and halt or delay degeneration. Delaying blindness by even a few years would have a meaningful impact on quality of life. Furthermore, delivering a combination of growth factors, or using a factor that upregulates multiple endogenous growth factors, may be a more effective photoreceptor protector than growth factor delivery alone. This improved rescue would be similar to combining BDNF with its TrkB receptor which protected more ganglion cells than BDNF alone (Cheng et al., 2002). Growth factor mediated rescue will be applicable to all photoreceptor degenerations, regardless of the primary cause or genetic mutation. Much effort has focused on the downstream signaling cascades induced by neurotrophins. However, a key question is the identity of the upstream signaling pathways that regulate growth factor expression in Muller glia and other cells. Elucidating the molecular mechanisms and signaling pathways that regulate neurotrophin expression and activity will be important for taking advantage of the therapeutic potential of these important molecules and may lead to the identification of novel treatments.

Acknowledgments Funding was provided by a Research for the Prevention of Blindness Career Development Award, the Karl Kirchgessner Foundation, a Fight for Sight Grant-in-Aid and the International Retina Research Foundation. Institutional support was provided by an unrestricted grant to Bascom Palmer Eye Institute from the Research to Prevent Blindness and an NEI core grant (P30 EY014801).

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Involvement of Guanylate Cyclases in Transport of Photoreceptor Peripheral Membrane Proteins

Sukanya Karan, Jeanne M. Frederick and Wolfgang Baehr

Guanylate cyclase 1 (GC1) is present in mouse rod and cone outer segments while guanylate cyclase 2 (GC2) is present only in rods. Accordingly, deletion of GC1 (gene symbol Gucy2e) affects predominantly cones while knockout of GC2 (gene symbol Gucy2f ) has no major effect on rod and cone physiology since GC1 can substitute for the loss of GC2. Simultaneous inactivation of GC1 and GC2 abolishes rod and cone phototransduction, generating a phenotype affecting viability of both rods and cones, and resembling human Leber Congenital Amaurosis. While studying the GC single and double knockout mice, we observed that GCAP1, GRK1, cone PDE and cone transducin subunits were either severely reduced or absent in GC1-/- cone outer segments, while GCAP1, GCAP2 and rod PDE6 subunits were downregulated and/or absent in GCdko rods. Based on the absence of several peripheral membrane-associated proteins in GC1-/- and GCdko outer segments, we have developed a model in which GCs serve a role in transport of peripheral membrane proteins from the inner segment, where biosynthesis occurs, to the outer segment.

1 Introduction

In photoreceptors, guanylate cyclases are essential components responsible for the production of cGMP, the internal transmitter of phototransduction. In darkness, the cytoplasm of the rod contains high levels of cGMP (1–10 M) maintaining a number of cGMP-gated (CNG) cation channels in an open state. Within milliseconds after a light flash, CNG channels close and photoreceptor cells hyperpolarize. CNG channel closure terminates the influx of cations (mostly Na+ and Ca2+), while the Na2+, Ca2+/K+ exchanger (NCKX) continues to extrude Ca2+. The consequence of channel closure and continued NCKX activity is a rapid drop in internal free Ca2+, and formation of Ca2+-free guanylate cyclase-activating proteins (GCAPs)

S. Karan

John A. Moran Eye Center, University of Utah, Salt Lake City, UT 84132, Tel: 801-585-7621, Fax: 801-585-7686

e-mail: sukanyat15@yahoo.com

in chicken (rd chicken), caused also by a null mutation in the GC1 gene (SempleRowland et al., 1998), mimics the human LCA phenotype. GC1-/- mice were generated by the laboratory of David Garbers several years ago by replacing a portion of exon 1 with a neo cassette (Yang et al., 1999). Surprisingly, the null mutation in the mouse GC1 gene produced a slowly progressing cone dystrophy, but not a rod/cone dystrophy as observed in LCA. In contrast to cones, the GC1-/- rods remain viable and responsive to light, most likely due to the presence in GC2. The number of cones in 4- and 5-week-old GC1-/- mice gradually decreased by 6 months, forming a from the inferior retinal region with relatively few survivors to the superior region where more cones survived (Coleman et al., 2004).

4 Knockout of GC2

In human, no retina disease has been linked to a mutation in the GC2 gene on the X-chromosome. Since a GC1 null allele in human causes a severe rod/cone dystrophy and GC2 cannot substitute for the loss of GC1, a recessive disease based on a GC2 null allele seems rather unlikely. To elucidate the role of GC2 in the mouse retina, we deleted the GC2 gene by targeted recombination (Baehr et al., 2007). We replaced a portion of exon 2, which contains the translation start codon ATG, by the neo cassette, thereby eliminating the peptide leader sequence which is important for translocation of GC2 into the ER, and also a part of the mature N-terminus of GC2. This construct is unable to initiate translation of a GC2 polypeptide. GC2-/- photoreceptors function nearly as well as WT in electroretinography and single rod cell recordings, and retina histology is normal (Baehr et al., 2007).

5 GC Double Knockout Retinas

GC double knockouts (GCdko) mice were generated by crossbreeding GC1-/- and GC2-/- mice (Baehr et al., 2007). The GCdko mice were fertile and developed normally, thereby excluding a vital role for GC1 or GC2 in embryonic development. An immunoblot of GC1-/-, GC2-/- and GCdko retinal lysates using polyclonal antibodies raised against the hinge domain present in both GC1 and GC2 (Tucker et al., 1999) revealed that GC2 is less abundant than GC1 and that both GCs are absent in the GCdko retina (not shown). Further, immunoblots with antiGC1 and anti-GC2 antibodies showed that the GC1 expression level is maintained in GC2-/- retina, and that the GC2 expression level is also maintained in GC1-/- retina (Baehr et al., 2007). At 2 months of age, outer segment lengths are approximately 50–70% of normal suggesting that degeneration progresses relatively slowly in GCdko mice (Fig. 1). At six months of age, the GCdko ONL contains only four-to-six rows of nuclei, and rod outer segment lengths are severely reduced in superior/inferior and nasal/temporal quadrants.

unaffected. Interestingly, rod arrestin protein levels were elevated relative to WT. The downregulations of GCAPs and PDE in double knockout retinas are likely posttranslational since Guca1a (encoding GCAP1), Guca1b (encoding GCAP2), and Pde6a (encoding PDE6subunit) RNA concentrations in WT and GCdko are identical, as evidenced by semiquantitative real-time RT/PCR (Baehr et al., 2007). Independent microarray analysis confirmed that RNA levels of these and other phototransduction genes are unchanged (results not shown). In GC1-/- and GCdko cones, myristoylated cone T , farnesylated cone T , geranylgeranylated cone PDE ’, and farnesylated GRK1 were undetectable by immunocytochemical analysis (not shown).

The posttranslational downregulation of these proteins could result from several mechanisms, including proteolytic degradation, protein instability, cGMP dependence, and/or lack of vesicular transport. Concerning proteolytic degradation, GCAPs have been shown to be susceptible to proteolysis in low free Ca2+ in vitro (Rudnicka-Nawrot et al., 1998), conditions presumed to be predominant in the GCdko and GC1-/- photoreceptor cells where cation channels are closed. In the Ca2+-bound form, GCAPs assume a compact conformation which is resistant to proteolysis, whereas in the Ca2+-free form, GCAPs open up and become accessible to proteases. Thus, proteolytic degradation of GCAPs in low [Ca2+] could provide a mechanism for GCAP downregulation. However, transducin and PDE subunits are Ca2+ insensitive and do not change their conformation depending on Ca2+. PDE subunits can be expressed stably (although inactive) in tissue culture in the absence of GC, Ca2+, or cGMP (Qin and Baehr, 1994). It seems unlikely that low occupation of non-catalytic cGMP binding sites (GAF domains) (Mou and Cote, 2001) present on catalytic subunits influences PDE stability. However, we cannot exclude a functional deficit of an unidentified cGMP-dependent pathway essential for protein stabilization.

7 GCS and Rhodopsin as Key Molecules in Vesicular Transport

Common to all “destabilized” proteins (GCAPs, rod PDE) or proteins absent from cone outer segments (cone Tα, cone Tγ , GRK1, cone PDE ’) is their membrane association, either directly via prenyl or acyl anchors (PDE subunits, GRK1) or indirectly by interaction with integral membrane proteins (GCAPs and GCs). Since membrane-associated proteins are firmly bound to the membrane surface and do not diffuse, they must be transported to the ROS post-synthesis. To explain downregulation, we therefore favor a model in which vesicular transport of peripherally membrane-associated PDE subunits and GC-associated GCAPs is impaired in GC1-/- and GCdko photoreceptors. Failure of transport of essential outer segment proteins disrupts phototransduction, renders outer segments non-functional and labile. Presumably, ER-associated proteins that are not competent for vesicular transport are eventually degraded by the ER quality control machinery, a mechanism similar to that degrading mutant or misfolded integral membrane proteins that cannot exit the ER (Frederick et al., 2001).