Ординатура / Офтальмология / Английские материалы / Retinal Degeneration Disease_Hollyfield, Anderson, LaVail_1999

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senescence and apoptosis (Jacobs et al. 1999). It was demonstrated that Bmi1 loss leads to an impairment in neural stem cell (NSC) proliferation and renewal (Molofsky et al. 2003). Another recent study showed that Bmi1 is crucial in the expansion of cerebellar granular cells both in vivo and in vitro (Leung et al. 2004). However, the effect of Bmi1 loss in other brain regions remains to be addressed, as well as the question whether the action of Bmi1 is intrinsic to the NSCs or due to the stem cell niche.

Our recent study (Zencak et al., submitted) provided evidence that Bmi1 is expressed in neural progenitor cells (NPCs), and that the distribution of the Bmi1-positive cells in vivo is similar to what would be expected for NPCs and NSCs. More precisely, Bmi1+ cells were observed mostly in the sub-ventricular zone (SVZ), and more dispersed in the corpus callosum and in the cortex at birth. Because of the action of Bmi1 on the ink4a/arf locus, Bmi1 loss was expected to result either in a decrease in proliferation or in an increase in apoptosis. BrdU incorporation analysis was performed on newborn brains after a short pulse injection of BrdU 30 minutes prior to sacrifice, and on adult brains (at P30) after a two-day treatment with BrdU. Interestingly, Bmi1 loss led to a decrease in proliferation in the newborn cortex, as well as in the newborn and P30 dorso-lateral corner of the SVZ, while no significant change was observed in the number of apoptotic cells (evaluated by TUNEL analysis). This observation was strongly accentuated in vitro with a 10-fold reduction of NSC colony formation from primary cortical cultures in conditions allowing NSC proliferation and renewal. When we tested self-renewal by replating and dissociating individual primary colonies, we observed an almost complete failure of Bmi1-/- NSCs to self-renew. In addition, by using an RNAi approach, we showed that the effect of Bmi1 loss was intrinsic to NSCs and not due to surrounding cells, such as the stem cell niche. In summary, Bmi1 is intrinsically required for NSC proliferation and self-renewal.

3. INCREASED PRESENCE OF GLIAL CELLS IN THE BMI1-/- BRAIN

The reduced proliferation in vitro and in vivo could result in an altered cell pattern in vivo. Surprisingly, no significant difference was observed in the distribution of neurons and oligodendrocytes. However, an increased number of astrocytes was observed at birth in the marginal zone of the cortex during the early stages of astrocyte appearance, while a massive gliosis was detected in the young adult Bmi1-/- brain. Interestingly, the increased astrocytic population appeared to proliferate normally in vivo, as assessed by BrdU incorporation. Taken together, our recent study showed that Bmi1 is intrinsically required for neural stem cell renewal, leading to a reduced proliferation in vivo and to an increased astroglial population that retains the ability to proliferate.

4. BMI1 LOSS AND NEUROPROTECTION IN RD1 MICE

4.1. Introduction

The increased glial population in the brain led us to investigate the effect of Bmi1 loss on Müller glia in the retina. Several studies suggest a role of the Müller cells in retinal neuroprotection (Ooto et al. 2004; Garcia and Vecino 2003). In this view, we hypothesized an increased presence or activity of Müller cells in the Bmi1-/- retina and analyzed the effect

of Bmi1 deletion in rd1 mice, on an inbred FVB genetic background. Rd1 mice are currently used as a model of human RP, a group of inherited retinal dystrophies untreatable at present. Rd1 mice are characterized by an early severe degeneration of the outer nuclear layer (ONL) due to a mutation of the Phosphodiesterase-6-beta (Pde6b) gene. In these mice, a complete loss of rod photoreceptors is observed at 3 weeks of age, followed by a death of cone photoreceptors. To date, ciliary neurotrophic factor (CNTF) treatment slowed moderately the photoreceptor cell death in Rd1 mice as revealed by histological analyses (LaVail et al. 1998), while glial cell line-derived neurotrophic factor (GDNF) injections could in some cases lead to recordable ERGs with a slight delay of the degeneration (Frasson et al. 1999). In the present study we tested whether an increased glial activity was present in the Bmi1-/- mice and if such increase could protect a degenerating retina.

4.2. Materials and Methods

4.2.1. Animals

Bmi1-knockout mice on a FVB (Rd1) inbred genetic background were generated and handled as previously described (van der Lugt et al. 1994; Jacobs et al. 1999). All mice carried the Pde-6b mutation characteristic of Rd1 mice (FVB background), and were wildtype (Bmi1+/+), heterozygous (Bmi1+/-) or homozygous (Bmi1-/-) for the Bmi1-knockout allele. In the text, Bmi1+/+ mice are mentioned as Rd1 mice.

4.2.2. Immunohistochemistry and Antibodies

Immunostainings were performed on eye cryostat sections from newborn (P0) or perfusion-fixed adult (P30) mice. Primary antibodies included rabbit antiserum, rabbit polyclonal anti-CRALBP (1/1000, gift of J.Saari), mouse monoclonal anti-Rho4D2 (1/40, gift of D.Hicks), rabbit polyclonal anti-Recoverin (1/500, Chemicon). They were revealed by fluorescence using the appropriate FITCor Cy3-conjugated secondary antibody. Primary antibodies were incubated overnight at 4°C.

4.2.3. Electroretinogram Recording (ERGs)

Rd1-Bmi1-/- and control (Rd1-Bmi1+/+ or +/-) mice were tested in scotopic and photopic conditions at different ages ranging from P16 to P35. They were dark-adapted overnight before recording scotopic responses and then light-adapted for 5 min. before recording the photopic responses.

4.3. Results

To analyze the presence of Müller cells, we analyzed CRALBP expression by immunohistochemistry in the Rd1 and Rd1-Bmi1-/- retina in adult mice (P30). Confirming our hypothesis, Rd1- Bmi1-/- retinas displayed a more intense CRALBP immunoreactivity compared to Rd1 (Figure 31.2A-B) this should be translated in a quantitative way. The DAPI dye used as counterstaining evidenced another important difference: the Rd1-Bmi1-/- retinas presented typically 7 to 8 rows of photoreceptors with the characteristic condensed chromatin (Figure 31.2C-D), while Rd1 mice had a completely dystrophic outer nuclear layer

Figure 31.2. Histology of the Rd1 and Rd1-Bmi1-/- adult retina. A,B, CRALBP immunohistochemistry on P30 Rd1 and Rd1-Bmi1-/- respectively. Note the stronger CRALBP immunoreactivity in Rd1-Bmi1-/- retina compared to Rd1, with the characteristic pattern of Müller glia (arrows). C,D, DAPI counterstaining corresponding respectively to A and B. Note the presence of photoreceptors with the typically condensed chromatin in the Rd1-Bmi1-/- ONL, while in (D). ONL = outer nuclear layer, dONL = dystrophic outer nuclear layer, INL = inner nuclear layer, GCL = ganglion cell layer. Magnification: 200¥.

(ONL). Immunohistochemical characterization of the cells present in the Rd1-Bmi1-/- ONL revealed rod photoreceptor features (data not shown). Moreover, the length and the shape of the outer segments in the Rd1-Bmi1-/- retina were similar to functional photoreceptors.

To test the visual function of Rd1 and Rd1-Bmi1-/- mice, we recorded ERG responses in scotopic and photopic conditions at several stages from P16 to P35. As expected, Rd1 mice displayed a slight response at P16, which disappeared with the progression of the retinal degeneration. On the contrary, ERG recordings in Rd1-Bmi1-/- mice showed a clear response at all tested ages to both single flash and flicker stimuli, improving from P16 to P35 (data not shown). Nevertheless, the shape of the ERG response was unusual, probably due to Pde6b loss of function. No significant response was observed in photopic vision in neither group.

Taken together, these results show that Bmi1 loss provided a consistent delay in the rod photoreceptor degeneration in the Rd1 mice to an extent unattained to date, with the presence of functional rod photoreceptors at 35 days of age. The reduced viability of the mice prevented us from investigating the effect of Bmi1 in Rd1 mice in the long term.

4.4. Discussion and Perspectives

Our work provides evidence that Bmi1 loss strongly delays rod photoreceptor degeneration in Rd1 mice, a model of human RP. The reported observation of an ONL with functional photoreceptors at 30 and 35 days of age has never been described in Rd1 mice so far, and may open new perspectives for the treatment of retinal degeneration once the mechanism underlying the delayed degeneration is identified.

The stronger intensity of the CRALBP staining in the absence of Bmi1 may reflect an increased activity of Müller glia, which remains to be quantified. Considering the role of Müller cells in neuroprotection, this could explain the rescue of photoreceptors. On the other hand, we can hypothesize an involvement of the Retinoblastoma (Rb) protein, a critical indirect downstream target of Bmi1 and recently described to play a critical role in rod photoreceptor development (Zhang et al. 2004). Both hypotheses require further investigation currently in progress.

5. ACKNOWLEDGEMENTS

We would like to thank Dana Hornfeld and Muriel Jaquet for editing help. This work was supported by the Swiss National Science Foundation, the ProVisu Foundation, the Velux Foundation, and the French Association Against Myopathies.

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abnormally high levels of cGMP. Therefore, such events will likely result in retinal functional and structural abnormalities. Recently, genetic defects in transcriptional mechanisms that control the expression of several retina-specific genes have been linked to different types of retinal disorders (Bessant et al., 1999; Freund et al., 1997; Freund et al., 1998; Haider et al., 2000; Swain et al., 1997). Given the detrimental effect mutations in the protein-coding region of the b-PDE gene have on photoreceptor integrity, it is crucial to understand the molecular events that mediate the precise regulation of expression of this gene in rod photoreceptors in the human retina.

Recent advances in the field of regulation of gene expression indicate that genes are differentially expressed according to their interplay with particular sets of transcription factors. Therefore, rod photoreceptor-specific expression of a gene is likely to be regulated by a unique set of transcription factors specific for rods, rather than a single rod-specific transcription factor. Our long-term interest in understanding the mechanisms of rod-specific regulation of the b-PDE gene expression has led us to continue the investigation of transcriptional and, more recently, post-transcriptional control of this gene utilizing a combination of in vitro, ex vivo and in vivo approaches. The results of these studies reviewed in this chapter will contribute to our pursuit of knowledge of how to maintain the expression of the b-PDE gene in rod photoreceptors at physiological levels, and to decelerate or prevent the development of certain forms of retinal degenerations.

2. TRANSCRIPTIONAL STUDIES

Formation of the preinitiation transcription complex involves coordinated interactions of RNA polymerase II with an array of basal transcription factors at the basal promoter sequences in the upstream regions of genes. The basal level of transcription is supported by general transcription factors and is enhanced by the action of activator proteins that interact with specific DNA elements. In addition, activated transcription may be repressed by the action of repressor proteins that also interact with specific DNA regulatory sequences. In previous studies, we reported our initial results on the transcriptional control mechanisms that take place in the human b-PDE 5¢-flanking region. Mutational analysis of the b-PDE promoter tested both in vitro and ex vivo, and confirmed by the generation of transgenic Xenopus expressing mutant b-PDE promoter/GFP fusion constructs in vivo, revealed a minimal promoter region, from -93 to +53, that supports high levels of rod-specific transcription (Lerner et al., 2001). Two enhancer elements were localized within this minimal promoter, bAp1/NRE and b/GC that interact with nuclear factors and activate transcription from the b-PDE promoter.

The functionally important b/GC element is homologous to the consensus GC box that binds members of the Sp family of transcription factors including Sp1, Sp3 and Sp4. These nuclear factors share similar structural features and have highly conserved DNA binding domains that allow them to bind with identical affinity to the consensus GC box (Hagen et al., 1992). However, while Sp1 and Sp3 are ubiquitously expressed, Sp4 is expressed predominantly in the CNS. This prompted us to further test its abundance in the adult retina, and to evaluate its activation properties on the rod-specific b-PDE promoter under defined conditions in direct comparison to Sp1 and Sp3.