**4. Discussion**

Here, we have presented the generation and initial characterization of a novel mouse model of RP59, where we have achieved global homozygous knock-in of the *K42E Dhdds* mutation specifically associated with RP59 [3–5]. Based upon the clinical presentation of RP59 in human patients [3–5], as well as the demonstrable importance of dolichol-dependent protein glycosylation in maintaining the normal structure and function of the vertebrate retina [11–14], we expected to observe retinal degeneration and retinal thinning in *Dhdds*K42E/K42E mice, particularly in mice homozygous for the K42E mutation. This expectation was also predicated on a preliminary report [23], using a similar K42E mouse knock-in model, that claimed nearly 50% loss of OS length and reduction in ONL thickness by about two-thirds at PN 3 months of age, compared to WT mouse retinas. However, we observed no evidence of retinal degeneration in *Dhdds*K42E/K42E mice up to one year of age. Furthermore, despite the confirmed mutation of *Dhdds*, we found no evidence for defective protein *<sup>N</sup>*-glycosylation in the retinas of these mice. The retinas were labeled robustly with fluor-tagged ConA lectin, irrespective of genotype. These findings are in good agreemen<sup>t</sup> with observations made by Sabry et al. [24] who found normal mannose incorporation into *N*-linked oligosaccharides using either siRNA silencing of *DHDDS* in a HepG2 cell line or in RP59 (severe mutation) patient fibroblasts. The ConA binding observed in our study is further consistent with observations made by Wen et al., who observed that rather than any loss in dolichols, there was an alteration in dolichol chain lengths (increased D17:D18 ratio) in RP59 patients compared to normal human subjects, but without obvious hypoglycosylation of serum transferrin [25]. These findings collectively sugges<sup>t</sup> hypoglycosylation-independent retinal degeneration in RP59, the mechanism of which still remains to be elucidated.

Understanding the pathophysiological and biochemical mechanisms underlying RP59 remains limited due to the lack of a validated vertebrate animal model that faithfully mimics the key hallmarks of the disease. Heretofore, only a zebrafish model of RP59 has been documented, using global knock-down of *DHDDS* expression by injection of morpholino oligonucleotides at the one-cell embryo stage [26]. In that case, the fish exhibited defective photoresponses and their cone outer segments (as assessed indirectly by PNA staining) were dramatically shortened, if not nearly absent. It should be noted that zebrafish have a highly cone-rich retina, unlike humans or mice (which have highly rod-dominant retinas). Also, the reduction and loss of PNA binding in the zebrafish knock-down retinas most likely reflects degeneration and death of cone photoreceptors, with concomitant degeneration and loss of their outer segments, due to their requirement for dolichol. Unlike the zebrafish *Dhdds* knock-down model, the murine RP59 model generated in the present study exhibits robust PNA staining in the outer retina, suggesting persistence of viable cone photoreceptors. In a parallel study (Ramachandra Rao et al., unpublished), we have observed *Dhdds* transcript distribution in all retinal nuclear layers by in situ hybridization, consistent with the fact that all cells require dolichol derivatives to support protein *<sup>N</sup>*-glycosylation. Taken together, these findings sugges<sup>t</sup> that the K42E *Dhdds* mutation does not a ffect cone photoreceptor viability. Recently (Ramachandra Rao et al., manuscript submitted for publication), we also generated a conditional *Dhdds* knockout mouse model, with targeted ablation of *Dhdds* in retinal rod photoreceptors, using a Cre-lox approach; however, unlike the K42E knock-in model, the rod-specific *Dhdds* knockout model exhibits profound, rapid retinal degeneration, with almost complete loss of photoreceptors by PN 6 weeks. Yet, there was no evidence of compromised protein *<sup>N</sup>*-glycosylation prior to the onset of photoreceptor degeneration. In addition, as reported in a companion article in this Special Issue of *Cells* [27], targeted ablation of *Dhdds* in retinal pigment epithelium, (RPE) cells in mice also results in a progressive, but somewhat slower, retinal degeneration.

As pointed out by Zelinger et al. [4], the phenotype of RP59 only involves the retina; there is no observable dysfunction or pathology in other tissues and organs in RP59 patients. Hence, those authors speculated that the K42E mutation, "alters, rather than abolishes, enzymatic function, perhaps either by reducing the level of DHDDS protein or by preventing requisite interactions between DHDDS and a photoreceptor-specific protein" [4]. They also suggested, alternatively, that mutation of DHDDS might result in, "a toxic accumulation of isoprenoid compounds," such as occurs in various forms of neuronal ceroid lipofuscinosis (e.g., Batten disease). While such speculations may turn out to be true, there is no direct empirical evidence extant to support this hypothesis. It is also entirely possible, however, that mutations (whether K42E or others) in DHDDS may a ffect its interactions with its enzymatic partner, Nogo-B receptor (NgBR, encoded by the *Nus1* gene) [8,9], with concomitant alterations in dolichol synthesis and protein *<sup>N</sup>*-glycosylation [28,29]. At present, nothing is known about the expression of Nogo-B receptor or its interactions with DHDDS, specifically in the retina. Our *Dhdds*K42E/K42E mouse line and retinal cell type-specific conditional DHDDS knockout mice o ffer potentially valuable model systems in which to pursue further investigations along these lines. In addition, we are currently pursuing studies employing dual, targeted ablation of DHDDS and NgBR in the retina. (See also the article by DeRamus et al., in this Special Issue of *Cells*, regarding an RPE-specific DHDDS knockout mouse model [27].)

Our findings bring into question the current concept that RP59 is a member of a large and diverse class of diseases known as "congenital disorders of glycosylation" (CDGs) [30,31]. While, in principle, it would be reasonable to consider RP59 as a CDG, due to the associated mutation(s) in DHDDS, there is no direct evidence to demonstrate a glycosylation defect in the human retinal disease or in any animal model of RP59 generated to date. The mechanism underlying the DHDDS-dependent retinal degeneration in human arRP patients remains to be elucidated, but is more complex than simply loss-of-function of DHDDS.

**Author Contributions:** Conceptualization, S.J.P. and S.J.F.; methodology, S.J.P., S.J.F., S.R.R., P.K.; M.N.N.; validation, S.R.R., M.N.N., P.K., S.J.P. and S.J.F.; formal analysis, M.N.N., S.J.P., S.J.F. and S.R.R.; investigation, S.J.P., S.R.R., P.K.; resources, S.J.P. and S.J.F.; data curation, S.J.P. and S.J.F.; writing—original draft preparation, S.J.F.; writing—review and editing, S.J.F., S.J.P., P.K., M.N.N. and S.R.R.; visualization, S.J.F., S.R.R. and S.J.P.; supervision, S.J.P. and S.J.F.; project administration, S.J.P. and S.J.F.; funding acquisition, S.J.P. and S.J.F. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was supported by U.S. Department of Health and Human Services (National Institutes of Health (NIH)/National Eye Institute (NEI)) gran<sup>t</sup> R01 EY029341 to S.J.P. and S.J.F., and NIH/NEI core gran<sup>t</sup> P30 003039 to S.J.P.; a Fight for Sight Summer Student Fellowship to S.R.R.; a Career-Starter Research Grant from the Knights Templar Eye Foundation to S.R.R.; as well as support from the UAB Vision Science Research Center (S.J.P., M.N.N., P.K.) and facilities and resources provided by the VA Western NY Healthcare System (S.J.F., S.R.R.).

**Acknowledgments:** We thank Isaac Cobb for technical assistance with OCT and genotyping. The opinions expressed herein do not reflect those of the Department of Veteran A ffairs or the U.S. Government.

**Conflicts of Interest:** The authors declare no conflict of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results.
