The rd1 mouse is a model of retinal degeneration
The rd1 mouse retina has been of interest as a model of inherited retinal degenerative diseases since it was first identified in 1924 . The initial molecular trigger in the rd1 retina, defective PDE6β leading to increased cGMP, is well understood [3, 7, 8]. The final events that underlie cell death have been the target of many investigations (reviewed in ). A virtual black box obscures the molecular processes that link these two events. In order to investigate the earliest events that are downstream of the Pde6b mutation in rd1 photoreceptors, we selected time points starting at P2, which is more than a week after Pde6b gene expression but prior to any identified biochemical or morphological differences, and concluding at P8, prior to the initiation of photoreceptor cell death.
Previous studies in rd1 whole retina during this time window have shown that the cytoplasmic cGMP level is twice the wt level by P6 with an increase of nearly 10-fold by P13 [3, 9]. Electron microscopical studies [17, 23, 24] have identified abnormal pathology in the rd1 retina, including retardation of IS growth as early as P4 , Golgi disruption by P6, and formation of widespread scattered vacuole-like structures by P8 . The mitochondrial matrix begins to disintegrate in photoreceptor IS by P6-8, in some cases prior to the appearance of outer segment disks [17, 23, 24]. At the rod ribbon synapse, a normal dyad configuration is formed with horizontal cells at P7, but the triad configuration incorporating a bipolar process seen in the wt retina at P8 fails to form in the rd1 retina .
Our microarray analysis of differentially expressed genes during this early time frame identified only 2 of 143 genes associated with apoptosis, consistent with our effort to target the earliest molecular changes that may provide the initial trigger for degeneration. Several studies using microarray analysis of rd1 retina at later time points, focusing on initiation of cell death, have been previously reported [25–27]. Using cluster analysis to investigate temporal patterns in groups of genes, Rohrer and colleagues identified differential expression beginning by P10 [25, 26]. Hackam, et al. identified temporally distinct pathways by comparing peak rod degeneration at P14 to early (P35) and later (P50) cone degeneration . Genes involved in transport mechanisms and signaling pathways were differentially expressed at P14 as well as in our study at earlier time points.
The link between the mutant gene and rd1 pathology remains poorly understood
cGMP regulates the cGMP-gated (CNG) ion channel in the mature retina, which regulates Ca2+ and Na+ entry in response to light. Efforts to measure Ca2+ in the degenerating rd1 retina have provided support for the hypothesis that the loss of function of Pde6β induces apoptosis as a result of high levels of Ca2+ influx through the CNG ion channel [3, 28, 29]. This hypothesis is further supported by experiments in which loss of functional CNG channels slows the degeneration in Pde6b mutants [30, 31].
Several observations raise the question as to whether Pde6β may play an unexplored role in photoreceptor development, independent of its role in phototransduction in the mature retina, and whether such a role could contribute to the initiation of cell death. First, Pde6b is expressed at embryonic day 12 (E12) in the mouse retina, much earlier than other genes involved in phototransduction, such as rhodopsin expression that becomes apparent at P5. Pde6b is even expressed prior to Nrl, a transcription factor that activates expression of rod-specific genes and is expressed at P1 . Interestingly, Pde6a and Pde6g are first expressed more than a week after Pde6b at P1 , suggesting that any possible role that Pde6β might play during embryonic development may be through an atypical structural conformation. Other members of the PDE family, including the cone PDE6α’, function as homodimers. The inhibitory gamma subunits are unique to the PDE6 subfamily. Further research is needed to explore the possibility that PDE6β could play a functional role during early photoreceptor development, possibly by forming a homodimer.
Secondly, the doubling of cGMP levels in whole rd1 retina by P6 [3, 9] indicates that Pde6β is functional in wt retina prior to this age, a time at which expression of rhodopsin and other genes associated with phototransduction is first initiated . In addition, pathological changes described above can be observed prior to outer segment differentiation and prior to expression of genes required for assembly of the phototransduction machinery. Finally, although best known in the retina for its role in phototransduction, cGMP is an important signaling molecule throughout the CNS. It functions in signaling pathways involved in neuronal differentiation and gene expression, modulation of neurotransmitter release, learning and memory, brain seizure activity, and neurotoxicity [33–37]. cGMP is known to act on three signaling pathways: cGMP-gated ion channels, inhibitory feedback onto Pde, and activation of phosphokinase G (PKG). Increased phosphorylation of PKG substrates has been observed in the rd1 retina compared to the wt retina at P11, consistent with multiple signaling roles for cGMP in the retina . Although specific substrates for PKG have not been clearly identified in photoreceptors , signaling through PKG is known to regulate gene transcription .
Together, these observations support the hypothesis that Pde6β plays a role in regulation of cGMP signaling during pre- and/or early postnatal photoreceptor differentiation that is independent of its role in phototransduction in the mature outer segment. Our analysis of functional groups of the differentially regulated genes supports this hypothesis. First, only 7 of the differentially expressed genes, excluding the mutant gene, were associated with visual transduction, and most of these were only expressed at the later time points. Secondly, the differentially expressed genes are predominantly in functional groups that are consistent with a role in development and differentiation, including development, transport, cell cycle, signal transduction, transcription, and metabolism. Transport is particularly important during photoreceptor differentiation as the cell must transport membranes and proteins in order to elongate both its axon and inner segment, followed by assembly of the outer segment. Within this category, we identified Rabac1, which was of particular interest as the only gene in the dataset other than the mutant Pde6b gene, identified as downregulated at all four time points examined.
PRA1 is important for vesicular trafficking
PRA1, the protein product of Rabac1, is comprised of 185 amino acids and an estimated molecular mass of 20.6 kDa [11, 12, 40]. Structural studies have identified two integral membrane domains , although fractionation studies have localized PRA1 to both the cytoplasm and the Golgi complex [11, 14, 41, 42]. PRA1 interacts with numerous small GTPases, all of which are prenylated [11–14] and has been proposed to play a role in vesicular trafficking.
Martincic et al.  hypothesized that PRA1 might function in vesicular docking and fusion based on its initial binding partners, Rab3a and VAMP2. Since this initial study five specific hypotheses have been proposed. The observation that PRA1 binds to prenylated GTPases and occurs as a cytoplasmic protein suggested that PRA1 might act as an escort protein, transporting prenylated GTPases through the cell by masking the prenyl moiety . Secondly, PRA1 may function as a Golgi sorting protein by facilitating the insertion of small GTPases into the membranes of transport vesicles and instructing them where to go in the cell . Sivars and colleagues  proposed a third function of PRA1 as a guanine nucleotide dissociation inhibitor (GDI) displacement factor (GDF) that aids in recycling Rabs during vesicular trafficking. An additional role for PRA1 in lipid transport, modulation of lipid homeostasis, and cell migration has been proposed based on proteomic analysis of PRA1 depleted nasopharyngeal carcinoma cells  and further supported by studies implicating PRA1 in the fusion of transport vesicles with the plasma membrane . Finally, evidence supports a role for PRA1 in transport and assembly of viral proteins, although in some cases it may play an inhibitory role [45–49]. These proposed functions for PRA1 are in no way mutually exclusive and in many cases are overlapping.
PRA1 is significantly downregulated during early photoreceptor differentiation in the rd1 retina
We present here the first report describing the expression and localization of PRA1 protein in the developing and mature wt and rd1 mouse retinas. In the developing wt retina, colocalization with GM-130-LIR indicates that PRA1 is localized to the photoreceptor Golgi apparatus as it translocates from the perikarya to the proximal IS during photoreceptor differentiation. PRA1 is also inferred to be in proximity with Rab6 during photoreceptor differentiation. Diffuse PRA1-LIR that does not colocalize with the Golgi marker is seen in the distal IS of wt photoreceptors starting at P8. In the mature retina, PRA1 positive punctae extend up to, but not overlapping with, the proximal end of the OS axoneme as labeled by RP1-LIR. Only very sparse punctae are seen in the OS. Both plexiform layers also contain PRA1 positive punctae independent of GM-130 staining during postnatal retinal development. In the wt OPL, PRA1-LIR appears less intense at P21, suggesting that it may play a role in neurite outgrowth and/or synapse formation that is not required for maintenance in the adult. This observation is of particular interest in light of the failure of the rd1 rod photoreceptors to form a triad synapse with bipolar dendrites . Together, these observations are consistent with a role for PRA1 in vesicular and lipid trafficking from the Golgi to vesicles directed both toward the cilia and the synapse. PRA1-LIR also colocalizes with the Golgi marker in the perinuclear region of most cells in the GCL and the INL.
By both Western blot at P2-P4 and immunohistochemistry at P6-P21, PRA1 expression appears less intense in rd1 compared to age-matched, wt retinas. Mislocalization of PRA1-LIR in the IS layer is seen in the rd1 retina at all ages examined. Compared to wt, rd1 retinas display a less intense, diffuse pattern of staining in the IS with some large punctae distributed throughout. Colocalization of PRA1- and GM-130-LIR is seen in some, but not all, of these IS punctae. This observation is consistent with EM pathology in the rd1 retina showing defects characteristic of vesicular trafficking [17, 23, 24].
In developing mouse retina, the decrease in PRA1-LIR in the rd1 IS layer compared to wt is apparent at all ages examined, and is statistically different by P8. Staining in the residual rd1 outer retina at P21 suggests that PRA1-LIR is also present in cone photoreceptors. The PRA1-LIR intensity in the perinuclear region of the rd1 GCL is also reduced at P21, consistent with a possible role of PRA1 in neurite remodeling and sprouting. Virtually all of the proteins altered in PRA1-depleted nasopharyngeal carcinoma cells that are linked to changes in cell migration  are also known to be important in neurite outgrowth. In the inner retina we found similar average intensities of PRA1-LIR at all time points examined except at P8, most likely an anomalous result due to small sample size. This suggests that the 2–3 fold reduction in Rabac1 mRNA measured by qPCR in whole retina is largely due to loss of expression in photoreceptors, with less overall change in protein expression in the inner retina.
PRA1 may play a role in vesicular trafficking during photoreceptor differentiation
Rod photoreceptors are highly specialized cells that exhibit unambiguous cellular polarity. Polarity of these cells is established during cell differentiation and maintained by the sorting of lipid membranes and proteins to their appropriate targets through a process of vesicular trafficking. Using Xenopus as a model system, four members of the Rab GTPase family, Rab3, Rab6, Rab8, and Rab11, have been identified in vesicular trafficking of rhodopsin from the Golgi to the connecting cilium at the base of the outer segments . Two of these, Rab3 and Rab6, bind directly to PRA1 in yeast two hybrid screens [11–13]. Rab proteins have also been highlighted in proteomics analyses of the bovine rod outer segment  and of the mouse photoreceptor sensory cilium complex . These studies have focused on maintenance of the mature photoreceptor cell. Whether the same Rabs play a role in vesicular sorting during photoreceptor development has not yet been explored.
Defects in vesicular trafficking have been implicated in retinal degenerative diseases. Mutation in Rab escort protein 1, a protein responsible for modifying small GTPases, has been linked to choroideremia, a disease characterized by degeneration of the choroid followed by photoreceptor degeneration . Defects in Rab8 trafficking have been documented in Bardet-Biedl syndrome, a cilliopathy characterized by developmental defects including degeneration of the photoreceptors [53, 54]. The association between vesicular trafficking defects and retinal degenerative disease is consistent with the early pathology observed in the rd1 mouse. PRA1 has been proposed to regulate the recruitment of Rab effector proteins as well as proteins involved in proper tethering and fusion of vesicles to their target membrane, such as VAMP2 [12, 55]. A defect in recruitment by PRA1 of Rab effectors and proteins involved in the downstream events of vesicular trafficking could correlate to the defects in vesicular trafficking reported in rd1 retinas [17, 56].
Alternatively, Figueroa and colleagues  have proposed that PRA1 could act as a chaperone protein to shuttle small GTPases through cells to their target locations. PRA1 was found to bind to other small prenylated GTPases besides Rabs, including RhoA, a small GTPase involved in actin remodeling, and K-Ras and H-Ras, small GTPases involved in cell growth, differentiation, and survival [14, 57]. For K-Ras, the rate of dissociation from the plasma membrane has been shown to be reduced or enhanced by knockdown or overexpression of PRA1, respectively . As with defects in vesicular trafficking, a defect in shuttling small GTPases required for differentiation throughout photoreceptors could correlate to the early pathology observed in the rd1 mouse retina.