Ectopic expression of tyrosine hydroxylase in the pigmented epithelium rescues the retinal abnormalities and visual function common in albinos in the absence of melanin

Authors


Address correspondence and reprint requests to Lluís Montoliu, Centro Nacional de Biotecnología (CNB-CSIC), Department of Molecular and Cellular Biology, Campus de Cantoblanco, C/Darwin 3, 28049 Madrid, Spain. E-mail: montoliu@cnb.uam.es

Abstract

Albino mammals have profound retinal abnormalities, including photoreceptor deficits and misrouted hemispheric pathways into the brain, demonstrating that melanin or its precursors are required for normal retinal development. Tyrosinase, the primary enzyme in melanin synthesis commonly mutated in albinism, oxidizes l-tyrosine to l-dopaquinone using l-3,4-dihydroxyphenylalanine (L-DOPA) as an intermediate product. L-DOPA is known to signal cell cycle exit during retinal development and plays an important role in the regulation of retinal development. Here, we have mimicked L-DOPA production by ectopically expressing tyrosine hydroxylase in mouse albino retinal pigment epithelium cells. Tyrosine hydroxylase can only oxidize l-tyrosine to L-DOPA without further progression towards melanin. The resulting transgenic animals remain phenotypically albino, but their visual abnormalities are corrected, with normal photoreceptor numbers and hemispheric pathways and improved visual function, assessed by an increase of spatial acuity. Our results demonstrate definitively that only early melanin precursors, L-DOPA or its metabolic derivatives, are vital in the appropriate development of mammalian retinae. They further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities.

Abbreviations used
BH4

tetrahydrobiopterin

dpc

days post coitum

DiI

1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate

GTP-Ch1

mouse GTP-Cyclohydrolase 1 protein

GTP-Ch1

mouse GTP-Cyclohydrolase 1 gene

L-DOPA

l-3,4-dihydroxyphenylalanine

LCR

locus control region

OCA1

oculocutaneous albinism type 1

RPE

retinal pigment epithelium

Tyr

mouse tyrosinase protein

Tyr

mouse tyrosinase gene

Th

mouse tyrosine hydroxylase protein

Th

mouse tyrosine hydroxylase gene

The mammalian albino retina develops abnormally. Patterns of cell production are distorted and delayed. At maturity, the central region is underdeveloped, rod photoreceptor numbers are reduced and hemispheric pathways at the optic chiasm are disrupted, such that the uncrossed projection is reduced in favour of the crossed (Jeffery 1997). These retinal abnormalities result in significant visual impairment (Kinnear et al. 1985). Oculocutaneous albinism type 1 (OCA1) (Oetting et al. 2003), a common form of albinism, results from mutations in the tyrosinase gene (Tyr), which encodes the enzyme catalysing the initial steps in melanin synthesis in melanocytes and retinal pigment epithelium (RPE) cells (Beermann et al. 1992; Gimenez et al. 2003). The introduction of functional Tyr transgenes in albino animals rescues all retinal abnormalities (Schedl et al. 1993; Jeffery et al. 1994, 1997; reviewed in Giraldo and Montoliu 2002), demonstrating that it plays a critical role in retinal development via melanin synthesis.

An early melanin precursor, l-3,4-dihydroxyphenylalanine (L-DOPA), appears to signal cell cycle exit. L-DOPA was first suggested to act as an anti-tumour agent (Wick 1977). Thereafter, L-DOPA has been implicated as a significant metabolite regulating normal cell production in RPE cells (Akeo et al. 1989, 1994) and neural retina (Ilia and Jeffery 1999). Tyrosinase (Tyr; monophenol dihydroxyphenylalanine: oxygen oxidoreductase; EC 1.14.18.1) oxidizes l-tyrosine to l-dopaquinone, leading to pigment production (Solano and Garcia-Borron 1998). L-DOPA acts as an intermediate product and is formed in a subsequent biochemical step within the melanin biosynthetic pathway (Riley 1999). Another independent but biochemically related enzyme, tyrosine hydroxylase (Th; EC 1.14.16.2), involved in the first step of catecholamine synthesis (Nagatsu et al. 1964), can also catalyse the conversion of l-tyrosine to L-DOPA (that is subsequently converted to dopamine by L-DOPA decarboxylase, EC 4.1.1.28), but cannot further oxidize L-DOPA to l-dopaquinone (Flatmark 2000), and hence cannot be involved in melanin synthesis (Zucca et al. 2004). Th is present in dopaminergic cells throughout the body (Banerjee et al. 1992; Min et al. 1996), but it is neither present in the RPE nor in melanocytes (Kagedal et al. 2004). It has also been shown that Tyr can provide L-DOPA to catecholaminergic cells for peripheral dopamine synthesis in tyrosine hydroxylase (Th)-deficient mice in young (Rios et al. 1999), but not in adult animals (Eisenhofer et al. 2003).

Here, we have used albino mice to generate transgenic animals expressing Th ectopically in the RPE to ask whether the production of an early precursor in the melanin pathway, L-DOPA or a later metabolite, is sufficient to rescue the retinal abnormalities and visual function found in albinism in the absence of pigment.

Experimental procedures

Construction of the TyrTH transgene

A 3.7 kb fragment containing the locus control region (LCR) of the mouse Tyr from pTyr14:E6 and a 2.4 kb 5′ fragment of the mouse Tyr promoter from pHSTyrTET were cloned into pHD2WOP (Gimenez et al. 2004), which provided the polyadenylation signal from SV40. A Th chimeric minigene was prepared by combining a rat Th cDNA fragment, exons 2–11, from p151g (Leonard et al. 1987) (kindly provided by A. Muñoz, IIB, Madrid, Spain), and two genomic DNA fragments from the mouse Th locus, obtained by PCR, corresponding to exon 1–2 and exons 11–13, including introns. The resulting Th minigene and corresponding deduced amino acid sequences are described in GenBank AY855842. A complete GTP-cyclohydrolase 1 (GTP-Ch1, EC 3.5.4.16) coding sequence was built from two partial mouse cDNA clones (IMAGE 1451309 and 475302) and hooked to the Th minigene sequence using an internal ribosomal-entry site (IRES) element from the encephalomyocarditis virus (ECMV) (Mountford et al. 1994) thereby generating the final plasmid construct TyrTH. Cloning details are available upon request.

Cell culture conditions and stable transfection of melan-c cells

Albino mouse melanocyte melan-c cells (Bennett et al. 1989) (kindly provided by D. Bennett, St. George's Hospital Medical School, London, UK) were cultured at 37°C in 10% CO2 in RPMI-1640 medium (Invitrogen S.A., Barcelona, Spain) complemented with 10% fetal calf serum (Invitrogen), 100 U/mL penicillin, 100 µg/mL streptomycin (Invitrogen) and 200 nm 12-O-Tetradecanoylphorbol 13-acetate (Sigma, Sigma-Aldrich-Química, Madrid, Spain). Melan-c cells were stably co-transfected with the TyrTH construct and a selection plasmid carrying the neomycin resistance cassette (pTKneo) using the Transfast Kit (Promega, Promega Biotech Ibérica, S.L., Madrid, Spain), according to the supplier. G418 was added 48 h after transfection at a final concentration of 1000 µg/mL (Invitrogen), and the selection was applied for 60 days. G418-resistant clones were combined and analysed in pool, resulting in the mixed melan-c/TyrTH cell line.

Rat phaeochromocytoma PC12 cells (ATCC number: CRL-1721) were used as a suitable positive control for the immunocytochemical analyses, due to their high endogenous Th expression (not shown). PC12 cells were cultured at 37°C, 5% CO2 in RPMI-1640 medium complemented with 10% fetal calf serum, 5% horse serum, glutamine 2 mm (Invitrogen), 100 U/mL penicillin and 100 µg/mL streptomycin.

Reverse transcription polymerase chain reaction analysis of TyrTH-transfected melan-c cells

Total RNA from cells was prepared as described (Schedl et al. 1993; Montoliu et al. 1996) and retrotranscribed and amplified using the Titan One Tube RT–PCR System (Roche Applied Science, Roche Diagnostics S.L., Barcelona, Spain) with specific oligonucleotides. Tyr expression was detected using primers 363 (Oligo A: 5′-TTCAAAGGGGTGGATGACCG-3′, nucleotides 313–332, GenBank X12782) and 364 (Oligo B: 5′-TGACACATAGTAATGCATCC-3′, nucleotides 654–635, GenBank X12782), as described before (Schedl et al. 1993; Montoliu et al. 1996). Th expression was detected using primers TH4 (5′-GGGCTGCAGAGACAGAACTCGG-3′, nucleotides 3590–3611, GenBank X53503: underlined bases correspond to a new PstI restriction site, shown in bold, not present in the original sequence, that was introduced for cloning purposes) and cDNAa (5′-CGGGTCTCTAAGTGGTGAATTTTGGC-3′, nucleotides 717–692, GenBank L22651). PCR conditions for Tyr and Th products were identical and as described (Schedl et al. 1993). The corresponding specific PCR products for Tyr (342 bp) and Th (390 bp) were analysed in standard 1.5% agarose electrophoresis gels stained with ethidium bromide (0.5 µg/mL).

Immunocytochemical analysis of TyrTH-transfected melan-c cells

Immunocytochemistry was performed as follows: cells were grown and fixed on round φ13 mm coverslips in 4% paraformaldehyde in phosphate-buffered saline for 15 min at 25°C and then permeabilized with 0.05% Triton X-100 (Sigma) in phosphate-buffered saline for 10 min at 4°C. After blocking with 10% fetal calf serum in phosphate-buffered saline for 1 h at 25°C, cells were incubated with 1 : 250 goat polyclonal antibody against Th (αTH sc-20, Santa Cruz Biotechnology, Santa Cruz, CA, USA) in 2% fetal calf serum, 0.05% Triton X-100 in phosphate-buffered saline for 45 min at 25°C. Cells were washed three times in phosphate-buffered saline and then incubated with 1 : 500 rabbit polyclonal antibody against Tyr, αPEP7 (Jimenez et al. 1989) (a generous gift from V. Hearing, NIH, Bethesda, MD, USA) in 2% fetal calf serum in phosphate-buffered saline, for 45 min at 25°C, as described (Lavado et al. 2005a). The appropriate secondary antibodies were used to reveal specific signals: Cy3-labelled donkey anti-goat (dilution, 1 : 500; Amersham Biosciences Europe GmbH, GE Healthcare, Barcelona, Spain) and Alexa 488-labelled goat anti-rabbit (dilution, 1 : 500, Molecular Probes, Invitrogen, Barcelona, Spain), for 1 h at 25°C. Cell nuclei were stained with 1 : 200 TO-PRO3 (Molecular Probes) in phosphate-buffered saline. Coverslips with cells were mounted in polyvinyl alcohol (PVA) mounting medium with DABCO (anti-fading agent, Fluka, Sigma). All preparations were examined in a confocal microscope (Axiovert 200, Carl Zeiss AG, Oberkochen, Germany) and images were processed with Laser Sharp 2000 program (Bio-Rad Laboratories, Hercules, CA, USA).

Generation of TyrTH transgenic mice

TyrTH construct was excised from vector sequences with NotI and microinjected into the pronuclei of fertilized oocytes of albino outbred NMRI mice (Harlan Interfauna Iberica, S.L., Barcelona, Spain), as reported before (Montoliu et al. 1996). Up to six transgenic founder mice were identified by Southern blot analysis. Three transgenic lines (#6764, #6775, #6930), containing six, four and two copies of the TyrTH construct, were established. The transgenic mouse line with highest TyrTH eye-specific expression levels and significant Th enzymatic activity in eye extracts corresponds to #6775. This line was used in all the analyses described in this work. No correlation was detected between transgene expression levels and copy numbers.

YRT2 transgenic mice, carrying the entire mouse Tyr locus within a 250 kb yeast artificial chromosome and shown to be undistinguishable from wild-type mice (Schedl et al. 1993; Jeffery et al. 1994, 1997), and non-transgenic NMRI mice have been used throughout as control pigmented and albino animals, respectively. All experiments complied with local and European legislation concerning vivisection and the experimentation and use of genetically modified organisms. Statistical analyses were performed using SPSS.

Analysis of TyrTH transgene expression

Total RNA was extracted from various organs of two individuals per group (Montoliu et al. 1996). The reverse transcription reaction and real-time PCR quantification were as described (Gimenez et al. 2003), using SYBR Green kit (Applied Biosystems, Warrington, UK) and Th specific primers: THA, 5′-GCGTCGGAAGCTGATTGC-3′, bases 606–623, within exon 6, and THB, 5′-TCCCGGCAGGCATGG-3′, bases 752–738, within exon 7 (nucleotide positions as in GenBank AY855842). Real-time PCR data were expressed by the number of Th molecules per number of β-actin molecules.

Immunohistochemistry

The immunodetection of Th was undertaken at +18.5 days post coitum (dpc) and in adults. Adult mice were killed with CO2, perfused with 4% paraformaldehyde in phosphate-buffered saline and their eyes removed. Fetuses were collected and their heads fixed in 4% paraformaldehyde. Fetal heads and adult eyes were cut in a cryostat (Leica Microsistemas S.A., Barcelona, Spain) and 15-μm transverse sections were collected. Subsequent steps occurred at 25°C. All sections were treated with 2.5 g/L KMnO4 for 30 min followed by 10 g/L oxalic acid for 5 min for depigmentation (Foss et al. 1995). They were re-fixed in 4% paraformaldehyde for 10 min, washed in phosphate-buffered saline, treated with glycin 100 mm (Merck KGaA, Darmstadt, Germany) for 30 min, permeabilized with 0.01% Triton-X100 for 10 min then blocked with 10% fetal calf serum for 1 h. Sections were then incubated with rabbit polyclonal specific antibodies against Tyr (αPEP7, 1 : 250, Jimenez et al. 1989) and Th (dilution 1 : 250; Chemicon International Inc., Temecula, CA, USA) in 2% fetal calf serum, 0.01% Triton-X100, in phosphate-buffered saline for 1 h, washed in phosphate-buffered saline with 0.01% polyoxyethylene-sorbitan monolaurate (Tween 20, Sigma) and then incubated for 1 h with secondary antibodies: Alexa 488-labelled (Tyr) and Alexa 594-labelled (TH) goat anti-rabbit (Molecular Probes, 1 : 500). Cell nuclei were counterstained with TO-PRO3 (1 : 200). Sections were mounted and viewed as described for immunocytochemistry.

Th enzymatic activity

Th enzymatic activity was recorded following the described assays for Tyr (Lavado et al. 2005a), with a modified reaction solution. Reaction volume included: 10 µL of a cofactors solution [5 mm tetrahydrobiopterin (BH4) (Sigma), 50 mmβ-mercaptoethanol (Sigma) in 1 m Tris-HCl pH = 7], 10 µL of l-[3,5-3H]Tyrosine mix [450 µL of l-tyrosine 262 µm (Serva Electrophoresis GmbH, Heidelberg, Germany) in 10 mm sodium phosphate buffer pH =6.8 and 50 µL l-[3,5-3H]Tyrosine (1 mCi/mL, 46 Ci/mmol, Amersham)], 5 µL of 10 mm sodium phosphate buffer pH = 6.8 and 25 µL of eye protein extracts (Lavado et al. 2005a). The mixture was incubated for 14–16 h at 37°C and then stopped by adding 450 µL 1% trichloroacetic acid (Merck). A small amount (∼50 mg) of adsorbing substrate [1 : 1 charcoal activated (Merck) and Celite® 545 (Fluka)] was added, mixed for 30 min at room temperature and centrifuged to remove any organic material. Tritiated water from clear supernatants (100 µL) was measured in a β-scintillation counter (Beckman Coulter Inc., Fullerton, CA, USA) using liquid scintillation cocktail (CytoScint, ICN, Costa Mesa, CA, USA), as an indirect assessment of L-DOPA production. This analysis proved to be a highly reliable and reproducible method for estimating L-DOPA production. Th catalyses the following reaction in the presence BH4 (Nagatsu et al. 1964; Flatmark 2000):

image

Note: The tritiated molecule of water produced in the reaction contains one tritium and one protium.

Th enzymatic assays in cells were performed as described for whole eye extracts. Cell extracts were prepared (Lavado et al. 2005b) from 1 × 106 rat phaeochromocytoma PC12, melan-c, melan-c/TyrTH or mouse melanoma B16 cells. Tyr enzymatic activity (melanogenic assay) and melanin contents were recorded as described (Lavado et al. 2005a,b).

Labelling of retinal pathways

Anterograde labelling of optic fibres with 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate (DiI) (Molecular Probes) was undertaken in +18.5 dpc mouse fetuses of each animal group, as described (Gimenez et al. 2004). The optic chiasm and tract were exposed and photographed using a fluorescence microscope (MZFL, Leica). Retrograde labelling of optic fibres was undertaken at the same stage essentially as described (Godement et al. 1987), using 40 TyrTH transgenic and 61 albino mouse fetuses labelled with DiI.

Mitotic profile and photoreceptor outer segment counts

Retinal mitotic figures were counted in 5-µm horizontal sections from +18.5 dpc mouse fetuses fixed with Carnoy's fluid, embedded in Historesin (Leica) and stained with 1% cresyl violet as described (Gimenez et al. 2001). The section containing the optic nerve head was identified and the sections retained directly above and below were used. Five individuals and both eyes were used per group resulting in n = 20 measurements, as described (Gimenez et al. 2005). Outer segments counts were undertaken on 1–2 mm Historesin sections in each of the groups as described (Jeffery et al. 1997; Gimenez et al. 2005). Ten eyes were used per group.

Optomotor experiments

Spatial acuity was measured in a series of optomotor experiments following the experimental procedures described by Abdeljalil et al. (2005). Experimental device for optomotor texts was custom made, following the descriptions made by Schmucker et al. (2005). During the experiments, mice were placed in a clear transparent acrylic glass cylinder (diameter: 15 cm, height: 15 cm) that was placed in the middle of a rotating drum. The custom-made drum was built of white paper (12 g/m2) supported by aluminium wires; it had a diameter of 33 cm and an height of 30 cm. Vertical black and white stripes were placed inside the drum. Black stripes were printed with a 600-dpi laser print. The drum provided the animals with a drifting vertical square-wave patterns when rotated in vertical axis. Light stimulation was achieved by ultra-light white LED (Ref. 310–6707, RS-Amidata, Barcelona, Spain) focused on the external face of the drum. Inside the drum, homogeneous luminance of the black and white stripes was measured with a luminance meter (Mavo-Monitor USB, Gossen, Nürnberg, Germany); luminance of white stripes was measured as 30 cd/m2 and black stripes as 0,1 cd/m2. The drum was rotated with an electric motor (LKB, 2115 multiperpex, Izasa, Spain). Spatial frequency could be varied by adjusting the angular speed of the electric motor, ranging from 0.05 to 0.5 cyc/deg. The direction of the rotation could be changed by reversing direction of the motor. The best optomotor responses were obtained for an angular speed of the stripe pattern at 50 deg/s.

The animals were placed into the glass cylinder and experimental tests were delayed until the animals had adapted to their new environment and finished their exploratory or self-cleaning behavior. Behavioral responses were measured as head tracking movements induced by drum rotation (Abdeljalil et al. 2005). Animals were videotaped with a Sony CCD AVC D5CE, digital video camera (Sony, Tokyo, Japan) and scored by two different observers. In all the experiments, the directional preference of the head movement was correlated with the drift direction of the stripes. To minimize the habituation to the optomotor response, the direction of rotation of the drum was reversed randomly. Angular running speeds were also changed in a random order. All mice were tested at four different spatial frequencies (0.15, 0.25, 0.33 and 0.50 cyc/deg). A test of 5 min in total was performed for each spatial frequency. Three groups of animals were used: albino, TyrTH transgenic and pigmented mice. Although the tests were performed on six animal of each group, just three albino animals, five TyrTH transgenic and five pigmented mice were analysed, as the other animals did not reach the desirable adaptation to their new environment after 5 min of test performance. All three groups showed clear differences in terms of visual perception (spatial acuity). Under experimental conditions, the rotation of the drum covered with black and white stripes should elicit head movements in mice at the same angular speed as the drum rotations. For all animals tested, as the spatial frequency of the stripes was increased, a threshold was reached beyond which no tracking movements of the head were detected. Mean responses for a series of three to six animals of each mice group were plotted against spatial frequency. Responses at different spatial frequencies were compared by analysis of variance (Kruskal–Wallis one-way anova test). The significant level was set at 5%.

Results

Functional analysis of TyrTH in vitro

The TyrTH construct was prepared, containing a minigene version of Th under the control of promoter and regulatory regions of the mouse Tyr(Fig. 1). A full-length cDNA encoding GTP-cyclohydrolase 1 (GTP-Ch1, EC 3.5.4.16) was also integrated in the TyrTH construct to trigger the synthesis of tetrahydrobiopterin (BH4), a cofactor that is required for Th activity (Nagatsu et al. 1964; Bencsics et al. 1996; Flatmark 2000).

Figure 1.

TyrTH construct. Schematic view of TyrTH construct, linearized with NotI, used in melan-c cells (Fig. 2) and for the generation of transgenic mice (Figs 3–6). From left to right the elements shown in the construct are: 5′ upstream regulatory sequences (LCR) and promoter from the mouse Tyr (light grey boxes), Th minigene (exons in black, introns as white boxes, the corresponding exons represented in each box are indicated below), IRES (striped box), the coding region of the mouse GTP-Ch1 gene (dark grey box) and the polyadenylation signal from SV40 (light grey). The start of transcription is indicated with an arrow. Not drawn to scale. The GTP-Ch1 gene codes for the key enzyme in the synthesis of tetrahydrobiopterin (BH4), which is the natural cofactor of Th (Nagatsu et al. 1964; Flatmark 2000). The GTP-Ch1 gene is included in the TyrTH construct to guarantee the potential Th enzymatic activity in retinal pigment epithelium (RPE) cells (Bencsics et al. 1996). IRES, internal ribosomal-entry site.

First, to ascertain the function of the TyrTH construct in vitro, prior to the in vivo analysis in mice, we stably transfected it into albino mouse melanocytes (melan-c cells, Bennett et al. 1989) that carry the Tyr albino allele (Tyrc), a reported point-mutation within the coding region that results in a non-functional protein and hence they can not produce melanin (Jackson and Bennett 1990). TyrTH/melan-c cells did not show any traces of pigment. However, Th expression was specifically recorded in them (Fig. 2a) and Th protein detected using specific antibodies (Fig. 2b). Immunocytochemical analysis confirmed the presence of the Th protein in TyrTH/melan-c cells (Fig. 2c). Finally, specific Th enzymatic activity was recorded in TyrTH/melan-c cells (Fig. 2d), above the background. Moreover, Tyr enzymatic activity (melanogenic assay, Fig. 2e) and the quantification of melanin contents (Fig. 2f) confirmed the lack of any pigment traces in TyrTH/melan-c cells, suggesting that the active Th protein from the TyrTH construct was not able to sustain the melanin pathway downstream of L-DOPA in melanocytes.

Figure 2.

Functional analysis of the TyrTH construct in stably transfected albino mouse melanocytes. (a) Reverse transcription (RT)–PCR analysis of Th mRNAs with the oligonucleotide pair TH4/cDNA in rat phaeochromocytoma PC12 cells (lane 1, positive control), TyrTH-transfected melan-c cells (lane 2), non-transfected melan-c cells (lane 3) and non-template control (lane 4, negative control). M stands for a molecular-weight marker (1kb-ladder, Invitrogen). As a control, the RT–PCR analysis of Tyr mRNAs with the oligonucleotide pair 363/364 is also shown in mouse melanoma B16 cells (lane 5, positive control), TyrTH-transfected melan-c cells (lane 6), non-transfected melan-c cells (lane 7) and non-template control (lane 8, negative control). (b) Immunodetection of Th in protein cell extracts by standard western blot analysis using αTH sc-20 antibody (Santa Cruz Biotechnology). Lane 1, non-transfected melan-c cells (negative control); lane 2, TyrTH-transfected melan-c cells; lane 3, rat phaeochromocytoma PC12 cells (positive control). The Th-specific band is seen at the approximately apparent molecular mass of 59 kDa in lanes 2 and 3. (c) Immunocytochemical analysis. TyrTH stably transfected albino mouse melan-c melanocytes stained with antibodies against Tyr, Th and the corresponding merged picture showing co-expression of Tyr and Th in the same cells. In albino mouse melanocyte melan-c cells (Bennett et al. 1989), carrying the Tyrc mutant allele (Jackson and Bennett 1990), the transcriptional status of the locus is not affected and the non-functional Tyrc protein produced can still be detected (Toyofuku et al. 2001) with standard antibodies against Tyr (i. e. αPEP7et al. 1989). Cell nuclei are shown in blue (TO-PRO3). Scale bar = 5 µm. Enzymatic activities of albino mouse melanocyte cells stably transfected with the TyrTH construct: (d) Th enzymatic activity in albino mouse melanocyte melan-c, TyrTH-transfected melan-c and rat phaeochromocytoma PC12 cells; (e) Tyr enzymatic activity (melanogenic assay) in melan-c, TyrTH-transfected melan-c and mouse B16 melanoma cells; (f) melanin contents in melan-c, TyrTH-transfected melan-c and mouse B16 melanoma cells recorded by spectrophotometry. White bars indicate phenotypically albino cells, black bars indicate wild-type pigmented cells. Bars indicate mean ± SD, n = 5. (t-Student's paired test, **p < 0.01, ***p < 0.001, ****p < 0.0001). Th and Th, mouse tyrosine hydroxylase protein and gene, respectively; Tyr and Tyr, mouse tyrosinase protein and gene, respectively.

Functional analysis of TyrTH in vivo

Next, three transgenic mouse lines were generated with the TyrTH construct. All transgenic animals remained phenotypically albino in coat and eye colour. Only the line having the highest expression of the TyrTH construct in the eye was fully analysed (Fig. 3a). The TyrTH transgenic mice expressed Th specifically in RPE cells, without any significant expression at other locations, as seen in developmental retinae from +18.5 dpc mouse fetuses (Fig. 3b) and adult retinae (Fig. 3c). A comparable pattern of expression was accounted for the GTP-Ch1 cDNA compound of the TyrTH transgene (not shown). The Th protein product encoded by the TyrTH transgene was enzymatically active, showing significant Th activity (and hence, L-DOPA production) in fresh protein eye extracts, as compared to that found in albino non-transgenic animals (Fig. 3c).

Figure 3.

Analysis of the TyrTH construct in vivo: specific expression of Th in the RPE of transgenic mice and enzymatic activity. (a) Real-time PCR expression analysis of TyrTH transgenic and albino mice. Relative values are given as number of Th mRNA molecules per β-actin molecules in the following organs: B, brain; L, liver; S, dorsal skin; E, eye. Recordable values for brain and liver were low, but similar between transgenic and non-transgenic animals, and below the scale used in this figure. Values represent the mean of two animals per group. (b) Immunohistochemical analysis in +18.5 dpc mouse fetuses. Central retinal sections from albino (panels 1, 4), TyrTH transgenic (panels 2, 5) and pigmented (panels 3, 6) +18.5 dpc mouse fetuses are shown. In each case the ventricular margin (RPE) is to the left. Nuclei are shown in blue. Panels 1–3 are stained with an antibody against Tyr (shown in green), panels 4–6 are stained with an antibody against Th (shown in red). The RPE labelled with an antibody against Th is indicated by an arrow (panel 5). Scale bar = 6 µm. (c) Immunohistochemical analysis in adult mice. Central retinal sections from albino (panels 1, 4), TyrTH transgenic (panels 2, 5) and pigmented (panels 3, 6) adult mice. In each case, the RPE is displayed above. The RPE labelled with an antibody against Th is indicated by an arrow (panel 5). Nuclei are shown in blue (most apparent stripe of nuclei corresponds to the outer nuclear layer, ONL). Panels 1–3 are stained with an antibody against Tyr (shown in green), panels 4–6 are stained with an antibody against Th (shown in red). Scale bar = 6 µm. (d) Th enzymatic activity in fresh protein eye extracts from albino and TyrTH transgenic mice. Th enzymatic activity background levels observed in albino non-transgenic mice are likely to derive from other Th-containing cells found in the eye, including amacrine cells. Bars represent the mean ± SD of five animals per group. Mann–Whitney test (*p < 0.05, p = 0.0143). RPE, retinal pigment epithelium; Th and Th, mouse tyrosine hydroxylase protein and gene, respectively; Tyr and Tyr, mouse tyrosinase protein and gene, respectively.

Analysis of photoreceptor cell counts in TyrTH transgenic mice

Photoreceptor outer segments were counted in albino, TyrTH transgenic and pigmented animals. No differences were observed between TyrTH transgenic and pigmented mice; however, both groups were significantly different from albino animals. Albinos had approximately 25% fewer photoreceptors than either of the other groups (Fig. 4). The vast majority of photoreceptors (∼97%) are rods generated in a period spanning from late prenatal through to early neonatal stages (Carter-Dawson and LaVail 1979; Young 1983). In albino rodents there is an abnormal elevation in the number of mitotic figures over this time followed by an elevated period of cell death (Ilia and Jeffery 1999; Gimenez et al. 2005). Counts of mitotic figures in the retinae of the three groups at +18.5 dpc revealed a significant (anovapost hoc with Bonferroni correction, n = 20/group, p < 0.0001) elevation of ∼25% in their number in albinos (mean ± SD, 52.6 ± 7.7) compared with pigmented animals (39.7 ± 4.7), similar to that reported before (Ilia and Jeffery 1999; Gimenez et al. 2005). The numbers found in the TyrTH transgenic mice (35.4 ± 9.0) were significantly lower (as above, p < 0.0001) than that found in albinos, but similar to that found in pigmented mice (as above, NS, p = 0.261).

Figure 4.

Photoreceptor outer segments numbers in adult albino, TyrTH transgenic and pigmented mice. This bar graph depicts the number of photoreceptor outer segments counted in 100 µm strips in comparable central retinal regions. Albinos have significant lower numbers than in either of the other two groups. Bars represent the mean ± SD of 10 eyes per group. White and black bars indicate phenotypically albino and pigmented animals, respectively. anova multiple comparison test with post hoc Bonferroni correction (****p < 0.0001). OS, outer segment.

Analysis of chiasmatic pathways in TyrTH transgenic mice

Anterograde tracing of chiasmatic pathways with DiI in mouse fetuses (+18.5 dpc) revealed similar patterns to those found in the analysis of photoreceptor numbers. In all pigmented mice, a clear uncrossed chiasmatic pathway could be identified in the optic tract, whereas in albino animals this was reduced in size and could be seen in only ∼15% of the labelled chiasms (Fig. 5), consistent with the smaller uncrossed pathway in hypopigmented animals (Lund 1965). Clear uncrossed pathways could be identified in ∼87% of the TyrTH transgenic mice. When this pathway was present it appeared similar to that seen in pigmented animals (Fig. 5). The marked differences found between the albino and TyrTH mice were reinforced by counts of ganglion cells retrogradely labelled with DiI from the ipsilateral optic tract. Significantly more cells (∼25%) with uncrossed pathways were found in the TyrTH animals (418.4 ± 44.3) when compared with albinos (315.7 ± 31.5) (Mann–Whitney, TyrTH n = 5, albinos n = 7, p = 0.0045), with ganglion cell counts within the range of similar type analyses (Herrera et al. 2004). In both groups of animals the majority of cells were located appropriately in the temporal retina.

Figure 5.

Optic pathway labelling at +18.5 dpc albino, TyrTH transgenic and pigmented fetuses. Unilateral anterograde labelling with DiI. Ventral-lateral view of the optic tract ipsilateral to the eye in which the fluorescent tracer was placed. Uncrossed fibres in the optic tract (OT) are clearly present in TyrTH transgenic (13 out of 15 mice) and pigmented (5 out of 5) animals (arrows). The uncrossed projection was either absent or very small (2 out of 13) in the albino animals. Scale bar = 0.5 mm. DiI, 1,1′-dioctadecyl-3,3,3′,3′-tetramethylindocarbocyanine perchlorate.

Analysis of the optomotor response in TyrTH transgenic mice

To assess the visual functional improvement in TyrTH transgenic mice, an experiment for testing the optomotor response was undertaken, following described methods (Abdeljalil et al. 2005). Curves representing the optomotor response for all three groups of animals (albino, TyrTH transgenic and pigmented mice) are shown in Fig. 6. Albino animals showed a limited number of head movements, even for low spatial frequencies (2.0 ± 1.0 head movements/min at 0.15 cyc/deg). Head movements were observed for a spatial frequency of 0.25 cyc/deg, and almost none were observed for 0.33 cyc/deg. No response was measured for 0.5 cyc/deg. When pigmented mice were tested, a significant increase in the number of head movements was measured for 0.15, 0.25 and 0.33 cyc/deg, as compared with albino mice, reaching values of 12.2 ± 2.6, 9.4 ± 2.1 and 6.6 ± 2.7 head movements/min. As expected, no significant increase in the number of head movements were observed for a spatial frequency of 0.50 cyc/deg, as this value is close to the spatial resolution limit for mice (Gianfranceschi et al. 1999; Abdeljalil et al. 2005). When the optomotor test was performed on TyrTH transgenic mice, the degree of visual function was significantly higher than in albino mice, although lower than in pigmented mice (Fig. 6). The number of head movements at 0.15 and 0.25 cyc/deg was 6.0 ± 1.5 and 5.2 ± 1.2, respectively. For spatial frequencies of 0.33 and 0.50 cyc/deg, no significant differences with responses from albino animals were observed.

Figure 6.

Spatial acuity in albino, TyrTH transgenic and pigmented animal groups. Quantification of the optomotor response, expressed as the number of head movements/min and as a function of four different spatial frequencies (0.15, 0.25, 0.33 and 0.5 cyc/deg) in albino (open circles), TyrTH transgenic (grey circles) and pigmented mice (black circles). The number of individuals analysed per group is indicated. Results are expressed as mean ± SD. At 0.5 cyc/deg, differences among all three groups were statistically not significant. At 0.33 cyc/deg, the differences between TyrTH transgenic and albino were not significant but pigmented animals were significantly different from TyrTH transgenic (p = 0.01) and albino mice (p < 0.05). At 0.25 cyc/deg, differences between TyrTH transgenic and albino were significant (p < 0.05) and pigmented animals were again significantly different from TyrTH transgenic (p = 0.01) and albino mice (p < 0.05). Finally, at 0.15 cyc/deg, differences between TyrTH transgenic and albino were also significant (p < 0.01) and pigmented animals were significantly different from TyrTH transgenic (p < 0.01) and albino mice (p < 0.05). Data were analysed by the non-parametric Kruskal–Wallis one-way anova test.

Discussion

In this study, we have examined whether the ectopic expression of Th inserted in the absence of functional Tyr in albino mouse RPE cells could correct retinal abnormalities commonly associated with albinism. We directed the expression of the TyrTH transgene to RPE cells by using a combination of promoter and regulatory elements from the mouse Tyr locus that have previously been employed (Gimenez et al. 2004; Montoliu et al. 2004).

During development, hypopigmented animals have abnormal spatio-temporal patterns of cell proliferation in the retina, and at maturity they have reductions in photoreceptor numbers and abnormal chiasmatic pathways (Jeffery 1997). A key element regulating normal retinal development may be L-DOPA, an early upstream melanin intermediate product. One of the key problems of albino retinal development is that cells do not leave the cell cycle at appropriate times, resulting in elevated levels of mitosis and extended waves of cell proliferation during the period when rod production takes place (Young 1983; Ilia and Jeffery 1999). L-DOPA produced during melanin synthesis or a later metabolite may be a significant factor in signalling cell cycle exit. Its addition to retinal cell cultures (Akeo et al. 1989, 1994) and in in vitro whole ocular preparations (Ilia and Jeffery 1999) results in reductions in the levels of mitosis. However, it has not been possible to expose the developing neural retina in vivo to a source of L-DOPA from an appropriate origin throughout development, as it is the aim of this work.

Here we have ectopically expressed Th in the RPE of transgenic albino mice that lack functional Tyr. In the TyrTH transgenic mice, L-DOPA is produced (Fig. 2d), but at this level, the potential synthetic pathway of melanin is truncated, as it can not be oxidized further towards l-dopaquinone (Flatmark 2000; Zucca et al. 2004), and hence no pigment is produced (as it was first observed in cells, Figs 2e and f), leaving the animal phenotypically albino. However, these TyrTH transgenic mice display corrected photoreceptor numbers and also normal chiasmatic pathways, similar to the patterns seen in pigmented animals, but in the absence of pigment.

The RPE is required for the development and maintenance of the neural retina (Raymond and Jackson 1995). During development, gap junctions are transiently formed between the RPE and the neural retina, as it has been shown in frogs (Hayes 1976), birds (Hayes 1977) and primates (Townes-Anderson and Raviola 1981). Further, these cellular interactions appear to play a crucial role, locally, during development (Gimenez et al. 2005). Therefore, it is likely that L-DOPA (or its related metabolites), produced within RPE cells by Tyr activity as an intermediate product, could influence the cellular fate of the developing neural retina. In our study, we have produced L-DOPA within the same RPE cells by Th, and showed the correction of albino retinal abnormalities in the absence of pigment.

We initially tried to assess the levels of L-DOPA in mouse eye extracts using a High Performance Liquid Chromatography (HPLC) approach (A. Lavado, L. Campa, F. Artigas and L. Montoliu, unpublished). Although we could demonstrate significant differences in L-DOPA levels between albino and pigmented mouse eye extracts, as reported previously (Ilia and Jeffery 1999), we did not find significant differences in L-DOPA contents from eye extracts of TyrTH transgenic mice and albino non-transgenic littermates (not shown). This can be explained as a result of: (i) limited amounts of L-DOPA being produced in RPE cells that are titrated out in whole-eye extracts; (ii) subsequent conversion (i.e. oxydation) of the L-DOPA produced into other related metabolites or (iii) a combination of the two. In contrast, using an alternative methodology (measuring tyrosine hydroxylase enzymatic activity as a function of the amount of tritiated water produced from radioactively labelled l-tyrosine, as described in methods, Figs 2 and 3) we could infer L-DOPA production in TyrTH transgenic mice, as compared with albino non-transgenic counterparts.

From the optomotor test data, we can conclude that visual function in TyrTH transgenic mice is significantly better than in the albino mice. Spatial resolution of pigmented mice is close to standard for other pigmented strains (see, for comparison, Abdeljalil et al. 2005). Ectopical expression of Th in the RPE of TyrTH transgenic mice also induces a significant recover of spatial vision, although their resolution does not reach that of control values.

Development of the uncrossed chiasmatic pathway occurs earlier, between approximately +13.5 dpc to +18.5 dpc (Colello and Guillery 1990), and it is unclear what role L-DOPA or a later metabolite may play in its formation. However, as Tyr is present in the RPE from +10.5 dpc (Beermann et al. 1992; Gimenez et al. 2003) it is likely that it has a role. Further, the physiological relevance of early pigment intermediates, as L-DOPA or its related metabolites, in functions independent of melanin synthesis and in eye development has been reported in several studies (Libby et al. 2003; Page-McCaw et al. 2004). Another possibility is that Tyr L-DOPA production in the RPE provides the neural retina, before Th is available, with a substrate for the synthesis of other catecholamines (i.e. dopamine) (Kubrusly et al. 2003). The effects of dopamine in the adult and developing retina have been well documented (Lankford et al. 1988; Varella et al. 1999; Guimaraes et al. 2001; Witkovsky 2004). Furthermore, exogenously applied dopamine has been shown to extend mitosis in developing albino rat retinae, but not in pigmented animals (Kralj-Hans et al. 2006).

We demonstrated in these transgenic TyrTH albino mice that photoreceptors numbers, chiasmatic pathways and visual spatial acuity are significantly different from that found in non-transgenic albinos, but similar to that present in pigmented mice. These results strongly support the notion that L-DOPA or one of its metabolic derivatives plays an important role in normal retinal development, and whose absence is responsible for the deficits found in albinism. They could also suggest further experiments for the development of innovative therapeutical approaches to address visual abnormalities commonly associated with hypopigmented genetic conditions, such as OCA1. Finally, our results further highlight the value of substituting independent but biochemically related enzymes to overcome developmental abnormalities.

Acknowledgements

This work was supported by funds from the Spanish Ministry of Science and Technology (SMCT) Bio97-0628, Bio 2000-1653, Bio 2003-08196 and from Comunidad Autónoma de Madrid (CAM) 08.5/0046/2003 and CAM GR/SAL/0654/2004 to LM, from The Wellcome Trust to GJ and from SMCT (SAF04-05870-C02-01) to PV. The authors are most grateful to Vincent Hearing, Francisco Solano and José Carlos García-Borrón for critical revision of the manuscript; to Francesc Artigas, Dorothy Bennett and Fernando Rodríguez de Fonseca for help and useful comments throughout the project; and to Soledad Montalbán, Patricia Cozar, Marta Cantero, Juan José Lazcano, Leticia Campa, Rima Barhoum, Núria Forns and Sylvia Gutiérrez for technical assistance.

Ancillary