Unraveling novel pathogenic mechanisms mediating PLS3-linked osteoporosis

This doctoral thesis aimed to unravel the pathogenesis mechanisms of PLS3-related osteoporosis. Since the discovery in 2013 that genetic defects in PLS3 lead to bone fragility, the mechanistic details of this process have remained obscure. To provide an overview of the functional roles of PLS3 in key bone cell types and proposed hypotheses, in Chapter 2, we systematically reviewed the protein structure of PLS3, in relation to its functional domains. This review suggested that PLS3 may influence bone mechanotransduction, intercellular communication among osteocytes, bone matrix mineralization, and the regulation of bone resorption. In order to investigate this complexity, we examined PLS3 in different bone cell models. Knockdown of pls3 in zebrafish showed a role in bone development. By rescuing the skeletal deformities in pls3-knockdown zebrafish using ACTN proteins containing similar structural domains with PLS3, we demonstrated that they have functional homology to PLS3 in Chapter 3. Furthermore, through osteogenic transdifferentiation of fibroblasts from PLS3 patients, we observed that these patient-derived cells showed increased transcriptional expression of WNT2 compared to those healthy donors. Additionally, RNA sequencing analysis revealed that PLS3 deficiency broadly impacts the transcriptome of osteocytes, affecting multiple bone-related pathways. We observed distinct differences in the mechanoadaptive responses between PLS3 knockdown osteocytes and control cells in Chapter 4. Specifically, Pls3 knockout osteocytes exhibited delayed elevation of nitric oxide levels and reduced changes in cellular alignment in response to fluid shear stress stimulation. Furthermore, transcriptome sequencing revealed that more than half of the signaling pathways enriched within the top 15 were no longer present following PLS3 knockout. We also successfully generated a novel Pls3 knockdown model. At 12 weeks of age, these mice exhibited significantly reduced cortical bone thickness and biomechanical properties, aligning well with our expectations. After five sessions of axial loading, cortical bone modeling was less pronounced in Pls3 knockout mice compared to wild-type littermates, and the beneficial effects of axial loading on trabecular bone remodeling were weaker in male Pls3 knockout mice than in WT mice. Transcriptome sequencing revealed that a single axial loading session induced a more substantial response in WT mice, as evidenced by twice the number of significantly regulated genes compared to Pls3 knockout mice. These findings collectively indicate that PLS3 deficiency negatively impacts osteocyte and bone responsiveness to mechanical stimuli, potentially during the mechanotransduction phase, resulting in widespread downstream disruptions in gene expression patterns. To investigate the lifelong effect of PLS3 mutation in bone and contribute an in vivo therapeutic model, we characterized the bones of Pls3 knockout mice from 8 to 21 months of age in Chapter 5. Micro-CT analysis revealed significantly reduced cortical bone thickness in Pls3 KO mice compared to WT controls. Furthermore, Pls3 KO mice exhibited compromised bone mechanical properties. These results demonstrate that the absence of PLS3 continuously impairs bone remodeling throughout middle to advanced age in mice. Extra-skeletal clinical manifestations in PLS3 patients exhibit considerable diversity, we aim to identify patterns through retrospective studies, and we investigated the medical care use of PLS3 patients by matching data from 17 PLS3 patients registered in the Amsterdam UMC Genome Database with the cohort from Statistics Netherlands (CBS) in Chapter 6. Our findings indirectly confirm that clinical manifestations of PLS3 deficiency primarily occur in childhood and have a strong genetic basis. Additionally, our results suggest that patients with PLS3 deficiency may have an increased risk of developing rheumatic disorders and hearing impairments. Overall, PLS3 mutations disrupt bone mechanoresponsiveness and alter broader bone metabolism pathways. The integrity of the PLS3 protein structure safeguards the bone homeostasis process. PLS3 mutations affect these patients for a lifelong time and may have multisystem effects.