PuF mRNA Expression in Mouse Retinal Degeneration

Molecular Cell Biology Research Communications 4, 20 –25 (2000) doi:10.1006/mcbr.2000.0250, available online at http://www.idealibrary.com on

Identification by Array Screening of Altered nm23-M2/PuF mRNA Expression in Mouse Retinal Degeneration Stephen E. Jones, Catherine Jomary, John Grist, Hannah J. Stewart, and Michael J. Neal Retinitis Pigmentosa Research Unit, Department of Pharmacology and Therapeutics, The Rayne Institute, GKT, St. Thomas’ Hospital, London SE1 7EH, United Kingdom

Received August 23, 2000

has been underscored by findings that the homologous gene in humans is also affected in certain cases of recessive RP (2, 3). In rd/rd mice, failure to form the functional PDE holoenzyme results in early postnatal elevation of retinal cGMP levels, which may exert a toxic effect on the developing photoreceptors. More recent studies indicate that the photoreceptors most probably die apoptotically (4 – 6), leaving the inner retinal layers morphologically unaffected for at least the first 3 postnatal months. To gain insight into the mechanisms underlying differential survival and death of retinal neurons in this model, we have employed techniques to identify altered patterns of gene expression occurring at different stages in the degenerative process. The present study reports our findings using differential screening of gridded gene arrays to profile gene expression in rd and control mouse retinas at early postnatal timepoints.

In the rd/rd mouse model of inherited retinal degeneration, the majority of photoreceptors die apoptotically between postnatal age (P)10 and 20 days, during which period the inner retina appears morphologically unaffected. To examine mRNA changes associated with the degeneration, we performed differential screening of 588 arrayed murine cDNAs using probes reverse-transcribed from P8 predegenerative and control mouse retinal RNAs. We detected altered expression of the gene encoding nm23-M2, a member of the family of nucleoside diphosphate kinases implicated in diverse processes including metastasis suppression and transcriptional regulation. Retinal nm23 mRNA levels increased during degeneration while control levels decreased over age-matched time-points. In situ hybridization showed the high level of expression at P20 in rd/rd was concentrated in the retinal ganglion cells. Previous studies have indicated upregulation of the stress-response related gene ␣B-crystallin in the rd/rd inner retina, and increased nm23 levels may be a component of this response to photoreceptor loss and altered retinal architecture. © 2000 Academic Press Key Words: apoptosis; array screening; nm23; PuF; retinal degeneration; retinal ganglion cell.

TISSUES, MATERIALS, AND METHODS Tissues. Homozygous rd/rd mice and nondegenerative C57BL/6 (“C6”) control mice were the source of tissues for RNA extraction and sectioning for in situ hybridization. The animals were maintained in a 12 h light, 12 h dark cycle, and were killed by cervical dislocation and enucleated during the light phase. The rd/rd mice, in which the rd locus is closely linked to the light ear (le) character (7), were maintained in an inbred colony at St. Thomas’ Hospital. The C57BL/6 animals were obtained from Harlane UK (Bicester, UK).

The retinal degeneration (rd) mouse is the one of the best characterized animal models of inherited blindness of the type classed as retinitis pigmentosa (RP; reviewed in (1)). In mice homozygous for the recessive rd allele, degeneration of the photoreceptor cells occurs rapidly during the first 3 postnatal weeks, while heterozygotes retain morphologically unaffected retinas. Early studies indicated a defect in retinal cyclic nucleotide metabolism, and subsequently the gene for the ␤-subunit of the visual transduction enzyme cGMP phosphodiesterase (PDE-␤) was identified as carrying the mutation(s) responsible for the degeneration (1 and references therein). The importance of the rd model 1522-4724/00 $35.00 Copyright © 2000 by Academic Press All rights of reproduction in any form reserved.

RNA extraction and Northern blot analysis. Total RNA was extracted from frozen tissues using the RNeasy system (Qiagen GmbH, Hilden, Germany) or according to a protocol associated with the differential array screening procedure (Clontech Laboratories Inc., Palo Alto, CA). For Northern analysis, 1.5 or 3 ␮g RNA samples were processed as previously described (8). Isolated cDNA inserts or PCR products (⬃20 – 60 ng) in low melting point agarose (GibcoBRL Life Technolo20

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gies Ltd., Paisley, UK) were labelled with ␣-[ 32P]dCTP using the Rediprime kit (Amersham Life Science Ltd.) according to the manufacturer’s instructions, and hybridizations performed in the presence of 50% formamide at 42°C overnight, followed by stringent washing and autoradiography or phosphoimaging using a Cyclone Storage Phosphor System (Packard Instrument Co., Meriden, CT). The probes were stripped off between hybridizations. Differential screening of gridded gene arrays. Gridded arrays of 588 murine partial cDNAs were obtained from Clontech Laboratories Inc. and processed in accordance with the manufacturer’s protocols. Total RNA samples (5 ␮g) from P8 C57BL/6 and rd/rd retinas were reverse transcribed in the presence of ␣-[ 32P]dATP to produce complex probes. Following hybridization to the membranes at 68°C overnight, stringent washings and autoradiography, the patterns of spots were compared both by direct visual inspection of the films and after computer-based image enhancement. Genes were then identified by reference to a key and to the online database available through the Clontech website, http://atlasinfo.clontech.com/ver2/enter.htm.

FIG. 1. Autoradiographs of differential screening of 588 paired, arrayed murine cDNAs by hybridization with complex probes reverse transcribed from control C57BL/6 and degenerative rd/rd mouse P8 retinal RNA. Circled are signals from nm23-M2 (upper panel) and adenomatous polyposis coli (lower panel) cDNAs.

clone of nm23-H2, and subsequent hybridization of the probes to cryostat sections of murine retinas, was performed using the colour in situ kit (Amersham Pharmacia Biotech) in accordance with the manufacturer’s recommendations. Following hybridization overnight at 55°C in the presence of 50% formamide, stringent washing, and RNaseA treatment, the hybridized probes were detected by incubation with alkaline phosphatase-conjugated anti-fluorescein antibody and development in the presence of nitroblue tetrazolium/ 5-bromo-4-chloro-3-indolyl phosphate (NBT/BCIP) substrates. A deep purple-brown reaction product indicates hybridization; this is visualized in the black and white figure as areas of dark coloration present in antisense but not sense probings.

Generation of cDNA probes. Approximately 1 ␮g of total RNA from human retina was reverse transcribed in 20 ␮l reactions with 1.25 units AMV reverse transcriptase (GIBCO-BRL Life Technologies) and random hexamer primers (Pharmacia, St. Albans, UK) for 60 min at 42°C, followed by heat inactivation of the enzyme. PCR amplification of an nm23-H2 partial cDNA, corresponding to bases 41–530 of the published sequence (9) (GenBank Accession No. L16785), was then performed using primers HPUF.1: 5⬘-CGCAGGACCATGGCCAACCT-3⬘ and HPUF.2: 5⬘-AGACTGCTGTTGTGTCCACC-3⬘ in 50 ␮l reactions containing 10 pmol each primer, 200 mM dNTPs and 1.0 unit Taq DNA polymerase (GIBCO-BRL Life Technologies), with the following conditions: 94°C 3 min, then 40 cycles of (94°C 45 s, 57°C 45 s, 72°C 60 s) and a final step of 72°C 5 min. A PCR product of the expected size (490 bp) was cloned into the pCR2.1-TOPO vector (Invitrogen, Leek, The Netherlands) according to the manufacturer’s protocol to give clone pHPUF.TA5. For the generation of riboprobes, the nm23 insert was subcloned into the vector pBluescript SK (Stratagene, La Jolla, CA) to give clone pBS.PuF3. Cloning and orientation of the cDNAs was confirmed by partial sequence analysis (MWG-Biotech GmbH, Ebersberg, Germany); the cloned sequence has ⬃89% identity with the homologous region of the mouse nm23-M2 cDNA. The control probe was a cloned cDNA of rat glyceraldehyde phosphate-3-dehydrogenase (GAPD), a generous gift of Dr. B. O’Hara (Stanford University, CA).

RESULTS AND DISCUSSION By gene expression profiling using a gridded array of murine partial cDNAs, we sought to identify altered expression of genes associated with the onset of photoreceptor death in the rd/rd mouse model of inherited retinal degeneration. We selected retinas from control and dystrophic animals at postnatal day 8 (P8), since at this time ultrastructural analyses have indicated the first signs of photoreceptor cell death, with swelling of mitochondria and vacuolar inclusions in the rod inner segments in the rd/rd homozygote (1 and references therein). By screening of the Atlas array of 588 murine partial cDNAs with complex probes derived from P8 rd/rd and control mouse retinas, we were able to visualize approximately 140 paired signals (24% of the total) in both cases (Fig. 1). Only a few signals were distinctly differential, and of these the most notable

In situ hybridization. Labelling of sense and antisense riboprobes generated from linearized restriction endonuclease enzyme digestions of the pBluescript 21

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was confirmed. Interestingly, this differential pattern progressively reversed over the period spanning the degeneration, such that by P17-20, nm23 mRNA levels were higher in rd/rd versus control (Fig. 2, bottom panel). Similar patterns of expression of nm23 mRNA in rd/rd retinas with age were confirmed in repeat Northern analyses of RNA extracts of tissues from different mouse litters (not shown), such that at P20, rd/rd values were ⬃1.4- to 1.9-fold higher than control values. To determine the cellular localization of expression of the nm23 gene in the mouse, we performed in situ hybridization using a colour reaction protocol. Representative results, from P8 and P20 rd/rd and C6 retinal sections probed with antisense and sense control riboprobes, are shown in Fig. 3. The changes in retinal architecture with development and degeneration were in accord with those previously reported by ourselves and others (1, 8): briefly, at P8 the differentiating neural layers in C6 and rd/rd are morphologically similar, but by P20 there has been almost complete loss of the photoreceptor outer segments (PR) and outer nuclear layer (ONL) from the rd/rd retina compared with the normal maturation of the C6 retina. The in situ analysis showed that in C6 retinas at P8 (Fig. 3A), there was moderate hybridization signal at the inner nuclear and ganglion cell layers (INL and GCL, indicated by a white asterisk and black arrows respectively), while at P20, the signal was weakly localized primarily to the GCL (Fig. 3E, arrowed). In contrast, rd/rd retinas gave very little signal at P8 with the antisense riboprobe (Fig. 3C); some sections displayed a weaker pattern of distribution similar to that in C6. However, at P20 in rd/rd, strikingly intense signals were obtained specifically localized to the ganglion cells at P20 (Fig. 3G, arrowed). The sense riboprobe gave minimal nonspecific signal (Figs. 3B, 3D, and 3H) with the exception of some slight binding at the photoreceptor outer segments in C6 P20 sections (Fig. 3F). The presence of dark pigment in the retinal pigment epithelium tended to obscure the colour reaction product and further studies will be required to determine expression in this cell layer. The progressive loss of photoreceptor cells that occurs during inherited retinal degenerative diseases such as retinitis pigmentosa (RP) generally has little effect in its early stages on the histomorphology of the inner retinal layers. In one of the best-studied models of RP, the retinal degeneration (rd) mouse, rod photoreceptors undergo rapid postnatal degeneration, declining from approximately 10 rows of cell nuclei in the outer nuclear layer to only one row by P20. The residual photoreceptors are largely cone cells, which die more slowly over the subsequent 3 months. The inner retinal layers remain morphologically normal during this time, although early studies of rd/rd retinas indicated that between 3 and 6 months of age, there is an

FIG. 2. Northern blot analysis of nm23 and GAPD mRNA expression in control C57BL/6 (C6) and rd/rd mouse retinal total RNA (3 ␮g/lane). Postnatal ages are shown in days. The histogram shows the ratio of nm23 levels in rd/rd to C6 normalized to GAPD levels at each timepoint.

corresponded to cDNAs for adenomatous polyposis coli protein (APC; Fig. 1, lower panel, circled) which was higher in rd/rd, and nm23-M2 (also called nucleoside diphosphate kinase B; Fig. 1, upper panel, circled), which gave a higher signal with C6 control. Other candidate differentially expressed genes were zonulaoccludens-1 (ZO-1) and c-erbA oncogene, apparently higher in rd/rd; and cAMP response element binding protein (CRE-BP1), higher in C6. The limited number of clearly differential signals between rd/rd and C6 at P8 may indicate that very few changes in gene expression are induced at this predegenerative stage; alternatively, this could reflect (i) the small sample size of detectable genes; (ii) the limited sensitivity of the technique in detecting low abundance transcripts; or (iii) the possibility that the main molecular changes associated with triggering of photoreceptor apoptosis occur posttranscriptionally, for example in protein cleavage or subcellular relocalization. Probing of a Northern blot of rd/rd and C6 total retinal RNAs with a murine APC partial cDNA failed to confirm the differential signal obtained with the array (data not shown). However, Northern analysis of retinal nm23 expression using a human nm23-H2 cDNA as probe showed a distinctive pattern of expression (Fig. 2). Following normalization of nm23 mRNA levels (top panel) to GAPD mRNA expression (middle panel), higher expression of nm23 in C6 retinas at P8 22

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FIG. 3. In situ hybridization analysis of nm23 mRNA expression in C57BL/6 and rd/rd mouse retinal cryosections using antisense (A, C, E, G) and sense (B, D, F, H) riboprobes. Specific signal is identified by dark-coloured reaction product present only in the antisense probings. The presence of pigment in the retinal pigment epithelium at the upper edge of the sections masks the hybridization signal. Asterisk (A) and arrows (A, E, G) indicate signal at the inner nuclear and ganglion cell layers respectively. GCL, ganglion cell layer; INL, inner nuclear layer; ONL, outer nuclear layer; PR, photoreceptors. Bar ⫽ 100 ␮m.

though the mechanisms are unclear, and the relationship between increased expression and metastasis suppression is controversial. Biochemically, the nm23 gene products function as nucleoside diphosphate kinases (NDPKs), 17–20 kDa polypeptides which are capable of forming homo- or heterohexamers in all combinations. The holoenzymes maintain the cellular nucleotide pool through their “housekeeping” NDPK activity. However, alternative roles of nm23 appear to be independent of such catalytic activities. These include transcriptional activation of the c-myc gene in vitro by binding to the PuF element of the c-myc promoter (9), and the inhibition of cell motility through serine phosphorylation and phosphotransferase activity (18). Modulated expression during development and differentiation has also been described, especially in the developing neural system (19). nm23 proteins also appear to interact with GTP-binding (G) proteins, and therefore may influence signal transduction pathways (20). Recently, the Drosophila homologue DRnm23 has been shown to induce apoptosis when overexpressed in cultured myeloid cells independently of its NDPK activity (21). The multiple functions ascribed to the members of the nm23 family, and the present uncertainty as to the molecular mechanisms of these activities, inevitably complicate interpretation of the significance of the altered patterns of nm23 expression we observed in rd/rd mouse retina. Regarding the low levels of nm23 mRNA at P8 in rd/rd, it may be that some elements of developmentally-regulated gene expression are delayed, corresponding to the retarded differentiation,

alteration in the number and a 10 –20% size reduction in the retinal ganglion cells (RGCs) (10). The changes that result from photoreceptor degeneration include loss of synaptic connectivity, as well as activation of glial cell proliferation. Thus, the progressive apoptotic death of the photoreceptors leads in turn to restructuring of retinal architecture and further cell demise, perhaps in part due to loss of trophic factors produced (or induced) by the photoreceptors themselves. These changes are associated with alterations in retinal gene expression, including upregulation of the genes for the apoptosis marker protein clusterin (8, 11), the transcription factor c-Fos (12), the neurotrophic factor neurturin (13), and basic fibroblast growth factor (bFGF/FGF2) (14). Further, the retinal gliosis results in increased expression of Mu¨ller cell genes, in particular that encoding glial fibrillary acidic protein (15, 16). The present study shows that normal spatiotemporal patterns of nm23-M2 mRNA expression are disrupted during the critical period spanning the degeneration in rd/rd mice. nm23-M2, also known as nucleoside diphosphate kinase-B and with a coding sequence identical to that of the transcription factor PuF, is the second murine member of a family of nm23 genes, of which 6 have now been identified in humans (see (17) for review). nm23-M1 was first isolated as a gene showing expression levels which inversely correlated with the metastatic potential of murine melanoma cell lines, and was therefore postulated to be a metastasis suppressor gene. Accumulating evidence suggests that both nm23-M1 and its human homologue nm23-H1 play important roles in controlling oncogenesis, al23

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phy (31), further analysis of the role of thyroid hormone and THRs in retinal degenerative disease may be warranted. Beyond this, a wider and more sensitive survey of gene expression patterns at both the transcriptome and proteome levels will likely be required to identify the molecular triggers causing photoreceptor apoptosis.

synaptogenesis and cone maturation in this strain (22, 23). The later increased nm23 expression particularly in the retinal ganglion cells (RGCs) of the mutant retina could reflect increased metabolic activity, perhaps due to stress/survival-related responses. Of potential relevance is the report of a 16 kDa member of the nm23 protein family which has been shown to function as a regulator of the molecular chaperone, heat-shock cognate (or constitutive) protein-70 (Hsc70) (24). The NDPK-like cytosolic protein p16 may act to dissociate unfolded proteins from Hsc70, and its expression is induced on cell stress in vitro in parallel with Hsc70 and the related Hsp70. Our own studies have shown the heat-shock like protein ␣B-crystallin, which is similar to Hsp27, is increased in expression at the protein and mRNA levels in rd/rd retinas, and the protein is particularly localized to the retinal ganglion cells (25), raising the possibility that nm23 proteins interact directly with a range of stress-induced proteins, perhaps to facilitate protein catabolism. RGCs may be particularly sensitive to the stresses arising from changes in the outer retinal architecture: recent studies have demonstrated in a rat model of RP that the regenerative capacity and dendritic transport of the RGCs is compromised following photoreceptor loss (26). Whether the long-term survival of RGCs or other retinal cells is affected by overexpression of nm23 remains to be established. We are presently examining whether nm23 proteins are induced by apoptotic stress in vitro, and whether they colocalize with or bind to ␣B-crystallin in vitro or in vivo. Evidence linking nm23 with activation of c-myc expression in vitro (9) and the widely established role of c-myc in apoptosis (see e.g., Ref. 27 for review) suggested a second scenario in which coordinated expression of these genes might be occurring, with effects on the survival or death of inner retinal cells. However, our unpublished investigations showed no alterations in the level of total c-myc protein in dystrophic vs control retinas by Western blot analysis, and retinal c-myc mRNA levels were not detectable by Northern analysis or standard RT-PCR. It is possible that the interaction of nm23 with the c-myc promoter is not a physiologically significant phenomenon (28). A further possibility for the abnormal pattern of nm23 expression in the rd retina has emerged from recent reports which suggest an association between overexpression of thyroid hormone receptors (THRs) and negative regulation of nm23-H1 (e.g., 29). Interestingly, our original differential screening revealed higher expression of c-erbA (thyroid receptor alpha) mRNA at P8 in rd/rd retina compared with the control, at a time when nm23 expression is low. In view of the established role of the thyroid hormone system in the control of apoptosis (e.g., 30), and circumstantial evidence such as that associating increased prevalence of thyroid disease in patients with RP or gyrate atro-

ACKNOWLEDGMENTS This work was supported by the Iris Fund for Prevention of Blindness, the British Retinitis Pigmentosa Society, the Guide Dogs for the Blind Association, and the National Eye Research Centre. We also thank Shafath Rehman for some of the retinal RNA preparations, and the Rayne Management Committee for the provision of research facilities.

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