Dimeric ^R25CPTH(1–34) Activates the Parathyroid Hormone-1 Receptor in vitro and Stimulates Bone Formation in Osteoporotic Female Mice

Reviewer #1 (Public Review):

Summary:

In this work, the authors investigate the functional difference between the most commonly expressed form of PTH, and a novel point mutation in PTH identified in a patient with chronic hypocalcemia and hyperphosphatemia. The value of this mutant form of PTH as a potential anabolic agent for bone is investigated alongside PTH(1-84), which is a previously used anabolic therapy. The authors have achieved the aims of the study. Their conclusion, however, that this suggests a "new path of therapeutic PTH analog development" seems unfounded; the benefit of this PTH variant is not clear, but the work is still interesting.

The work does not identify why the patient with this mutation has hypocalcemia and hyperphosphatemia; this was not the goal of the study, but the data are useful for helping to understand that.

Strengths:

The work is novel, as it describes the function of a novel, naturally occurring, variant of PTH in terms of its ability to dimerise, to lead to cAMP activation, to increase serum calcium, and its pharmacological action compared to normal PTH.

Weaknesses:

(1) The use of very young, 8-10 week old, mice as a model of postmenopausal osteoporosis is a major limitation of this study. At 8 weeks, the effect of ovariectomy leads to lack of new trabecular bone formation, rather than trabecular bone loss due to a defect in bone remodelling. Although the findings here provide a comparison between two forms of PTH, it is unlikely to be of direct relevance to the patient population. For example, the authors find an inhibitory effect of PTH on osteoclast surface, which is very unusual. Adding to this concern is that the authors have not described the regions used for histomorphometry, and from their figures (particularly the TRAP stain), it seems that the primary spongiosa (which is a region of growth) has been used for histomorphometry, rather than the secondary spongiosa (which more accurately reflects bone remodelling). Much further detail is needed to justify the use of this very young model, and a section on the limitations of this model is needed. Please provide that section in the revised manuscript.

(2) It is also somewhat concerning that the age range is from 8-10 weeks, increasing the variability within the model. Did the age of mice differ between the groups analysed?

(3) Methods are not sufficiently detailed. For example, the regions used for histomorphometry are not described, there is no information on micro-CT thresholds, no detail on the force used for mechanical testing. Please address this request.

(4) There are three things unclear about the calvarial injection mouse model. Firstly, were the mice injected over the calvariae or with a standard subcutaneous injection (e.g. at the back of the neck)? If they were injected over the calvaria, why were both surfaces measured? Secondly, why was the dose of the R25C-PTH double that of PTH(1-34)? Thirdly, there is no justification for the use of "more intense coloration" as a marker of new bone; this requires calcein labelling to prove it new bone. It would be more reliable to measure and report the thickness of the calvaria. Please address these technical questions.

(5) The presentation of mechanical testing data is not sufficient. Example curves should be shown, and data corrected for bone size needs to be shown. The difference in mechanical behaviour is interesting, but does it stem from a difference in the amount of bone, or two a difference in the quality of the bone? Please explain this matter better in the manuscript.

(6) The micro-CT analysis of the cortical bone in the OVX model is insufficient. Please indicate whether cross-sectional area has increased. Is there an increase in the size of the bones, or is the increase in cortical thickness due to a narrowing of the marrow space? This may help resolve the apparent contradiction between the cortical thickness data (where there is no difference between the two PTH formulations) and the mechanical testing data (where there is a difference). Please explain this matter better in the manuscript.

(7) The evidence that dimeric PTH has a different effect to monomeric PTH is very slim; I am not sure this is a real effect. Such differences take a long time to sort out (e.g. the field is still trying to determine whether teriparatide and abaloparatide are different). I think the authors need to look more carefully at their data - almost all effects are the same. Ultimately, the statement that dimeric PTH may be a more effective anabolic therapy than monomeric PTH are not supported by the data, and this should be removed. There is little to no difference found between normal PTH and the variant in their effects on calcium and phosphate homeostasis or on bone mass. However, the analysis has been somewhat cursory, with insufficient mechanical testing or cortical data presented. Many of the effects seem to be the same (e.g. cortical thickness, P1NP, ALP, vertebral BV/TV and MAR), but the way it is written it sounds like there is a difference. Please remove some of the unfounded claims that you have made in this manuscript.

(8) Statistical analysis used multiple t-tests. ANOVA would be more appropriate.

Reviewer #2 (Public Review):

Summary:

The study conducted by Noh et al. investigated the effects of parathyroid hormone (PTH) and a dimeric PTH peptide on bone formation and serum biochemistry in ovariectomized mice as a model for postmenopausal osteoporosis. The authors claimed that the dimeric PTH peptide has pharmacological benefits over PTH in promoting bone formation, despite both molecules having similar effects on bone formation and serum Ca2+. However, after careful evaluation, I am not convinced that this manuscript adds a significant contribution to the literature on bone and mineral research.

Strengths:

Experiments are well performed, but strengths are limited to the methodology used to evaluate bone formation and serum biochemical analysis.

Weaknesses:

(1) Limited significance of this study:
• this study follows a previous study (not cited) reporting the effect of the dimeric R25CPTH(1-34) on bone regeneration in an osteoporotic dog (Beagle) model (Jeong-Oh Shin et al., eLife 13:RP93830, 2024). It's unclear why the authors tested the dimeric R25C-PTH peptide on a rodent animal model, which has limitations because the healing mechanism of human bone is more similar in dogs than in mice.
• the authors should clarify why they tested the effects of dimeric R25CPTH(1-34) and not dimeric R25CPTH(1-84)?
• The study is descriptive with no mechanism.

(2) Statistics are inadequately described or performed for the experimental design:
• the statistical analysis in Figure 5 needs to be written in a way that makes it clearer how statistics were done; t-test or one-way ANOVA?
• Statistics in Figures 6 and 7 should be performed by one-way ANOVA to compare the mean values of one variable among three or more groups, and not t-test.

(3) Misleading and confused discussion:
• The first paragraph lacks clarity in the PTH nomenclature and the authors should provide a clear statement that the PTH mutant found in patients is likely a monomeric R25CPTH(1-84), considering that there has been no proof of a dimeric form.
• Moreover, the authors should discuss the study by White et al. (PNAS 2019), which shows that there are defective PTH1R signaling responses to monomeric R25CPTH(1-34). This results in faster ligand dissociation, rapid receptor recycling, a short cAMP time course, and a loss of calcium ion allosteric effect.
• The authors should also clarify what they mean by "the dimeric form of R25CPTH can serve as a new peptide ...(lines 328-329)" The dimeric R25CPTH(1-34) induces similar bone anabolic effects and calcemic responses to PTH(1-34), so it is unclear what the new benefit of the dimeric PTH is.

Please address these concerns.