Hutchinson-Gilford Progeria Syndrome (HGPS) is a rare senescence syndrome affecting the cardiovascular, musculoskeletal (MSK), and integumentary systems. Although patients appear normal at birth, manifestations of HGPS appear within the first year of life, including growth deficiency, alopecia, loss of subdermal fat, and sclerodermatous-like changes to the skin (Ullrich et al., 2012). However, it is the rapidly progressive changes in the cardiovascular and cerebrovascular systems that lead to premature death during the second decade of life (Capell et al., 2008). Some of the most significant consequences of HGPS involving the MSK system include skeletal dysplasia with joint contractures, in addition to craniofacial abnormalities that become more evident around 2 years of age and progress with time (Merideth et al., 2008). These features include thinning of the calvarium, mottling of the skull in the frontal, parietal, and sphenoid regions, and widening cranial sutures. Other prevalent cranial abnormalities in HGPS include orbital hypotelorism, resulting in prominent eyes, a narrow nasal bridge with a broad-tipped nose, and a large skull relative to facial size (Ullrich et al., 2012). HGPS children also exhibit other craniofacial abnormalities, such as thin zygomatic arches and J-shaped sellas.

Most cases of HGPS are caused by a sporadic autosomal dominant mutation in the LMNA gene (c.1824C > T, p. G608G) (De Sandre-Giovannoli et al., 2003; Eriksson et al., 2003). Prelamin A, the precursor protein of Lamin A, undergoes normal post-translational processing of its C-terminal region. Sequential post-translational modifications include transient farnesylation of the cysteine at the C-terminal CaaX motif, proteolytic cleavage of the last three amino acids (aaX) by the metalloproteinase ZMPSTE24, and carboxymethylation. Finally, ZMPSTE24 cleaves the last 15 amino acids in the C-terminal region, removing the farnesyl group (Eriksson et al., 2003). These post-translational modifications lead to a mature, unfarnesylated Lamin A protein (Gargiuli et al., 2018; Harhouri et al., 2018), which localizes to the inner nuclear membrane and broadly influences nuclear structure and function (Broers et al., 2006). In HGPS, the G608G mutation activates a cryptic splice site, thereby deleting 50 amino acid residues containing the second endoproteolytic cleavage site and resulting in a truncated, permanently farnesylated protein termed “progerin” or LMNAΔ50 (Hernandez et al., 2010). Persistent farnesylation causes progerin accumulation at the inner nuclear membrane and difficulties reassembling the daughter nuclei properly after mitosis (Goldman et al., 2004).

Various murine models have been developed to mimic the bone abnormalities observed in Hutchinson-Gilford Progeria Syndrome (HGPS). The ZMPSTE24^−/− mouse was one of the first, displaying musculoskeletal (MSK) abnormalities such as kyphosis, spontaneous fractures by 6–7 weeks, and reduced zig-zag cranial sutures. This model mirrors some bone issues seen in progeria patients (Bergo et al., 2002). The Lmna^G609G/G609G mouse model was created recently, showing skeletal deformities such as lordo-kyphosis, joint immobility, and skull abnormalities (Osorio et al., 2012; Gargiuli et al., 2018). This model also highlights smaller lower incisors and delayed bone mineralization, including maxillary, mandibular, and calvarial structures. In vitro and in vivo studies of these mice reveal altered differentiation of bone cell populations and post-natal bone loss linked to defective Wnt signaling and reduced osteoclast activity (Cabral et al., 2023). The progression and characteristics of bone abnormalities in these murine models offer valuable insights into the mechanisms underlying HGPS and may guide therapeutic strategies to manage bone dysplasia in affected patients.

We have previously reported the generation of our transgenic mouse model carrying a human BAC harboring the common HGPS dominant-negative mutation (LMNA ^G608G), which makes it possible to test novel therapeutic approaches in the context of the human gene, RNA, and protein sequences. Whereas single-copy transgenic mice (LMNA ^G/+) do not develop pathologic changes outside of the vascular system before 20 months of age, double-copy mice faithfully recapitulate the vascular, dermal, and musculoskeletal phenotypic features of HGPS in just a few months (Varga et al., 2006; Cabral et al., 2021). A general phenotypic description by Cabral et al. notes reduced femoral trabecular bone volume (Tb BV/TV) by 5 months of age; a more detailed characterization of the long bone phenotype in LMNA ^G608G/G608G mice has been published by Cubria et al. Specifically, the authors noted reduced cortical thickness (Ct Th) with normal BMD and no significant difference in trabecular volume (Tb BV/TV) compared to age-matched wild-type mice. The different findings by Cabral et al. and Cubria et al. in the same mouse model may be due to a difference in the number of mice analyzed and the age of the mice (5 months versus 8 months/endpoint). However, the higher SMI in the trabecular bone of LMNA ^G/G mice suggested a more rod-like structure that is characteristic of normally aged bone. Furthermore, femoral head articular cartilage volume and GAG content were significantly decreased, as occurs in the early stages of osteoarthritis. These previous studies did not, however, focus on the cranial bones.

Inasmuch, no mouse model has accurately reproduced the craniofacial changes found in humans with HGPS to test new therapeutic strategies and their potential impact on the natural course of the disease. Therefore, in the present study, we aimed to characterize the craniofacial abnormalities in the homozygous G608G mouse model.

Genotyping was performed using genomic DNA isolated from mouse tail cuts using the Red Extract-n-Amp tissue PCR kit (Sigma-Aldrich, St. Louis, MO, United States), then amplified by heminested PCR using forward (GenoCh4-F1, 5′-CAAACAAGTACATATCATAGGC-3′) and reverse (GenoCh4-R1, 5′-ATGATAGTGACAGGTATACGG-3′) primers external to the BAC insertion site on chromosome 4 and an additional reverse primer (GenoBAC-R2 5′-ATTCTAGTGGAGGGAGACAG-3′) located within the BAC sequence. Thermal cycling conditions included an initial denaturation at 95°C, then 35 cycles at 94°C, 58°C and 72°C for 45 s each. Genotype was detected by electrophoretic separation of 493bp (wild-type) and 233bp (transgene) amplification products.

All animal care was conducted at the National Institutes of Health (NIH) following Institutional Animal Care and Use Committee (IACUC) approval. Mice grew at a reduced rate for approximately 4 months and normally ate until 7 months of age, when attentiveness and activity began to decline. This caused progressive deterioration until eventual death before 8 months of age.

HGPS mice and age-matched WT littermates were imaged at the same time points of 2, 4, 6, and 8 months. The mean age at death for HGPS mice was 39.4 weeks (SD = 6.2 weeks). Each group consisted of 5 mice.

Using whole-body CT scans, we characterized craniofacial abnormalities in a homozygous G608G mouse model. A series of ten measurements at the skull’s superior, inferior, and lateral aspects were taken at the four time points (2, 4, 6, and 8 months) to determine several differences observed between HGPS and age-matched WT littermates at the end of the study. These include internasal width I, interzygomatic distance, sagittal cranial, palatine length, and intermolar distance. HGPS mice exhibited a significant increase in bone volume by 4 months compared to their earlier measurements (p = 0.021). By 8 months, their bone volume was 21.2% higher than those of WT mice, although not statistically significant (p = 0.170). The increased bone volume may be linked to greater cortical thickness, which was 16.6% thicker in HGPS mice at 8 months, although this also did not reach statistical significance (p = NS 0.224). Sagittal cranial length increased similarly in both groups (HGPS 3.02% vs. age-matched WT littermates 3.15%, p = NS 0.44). HGPS mice had a significantly smaller internasal width at two (p = 0.008) and 8 months (p = 0.03). Interzygomatic distance increased more in age-matched WT littermates (5.39% vs. HGPS 3.07%, p = 0.03). HGPS mice showed a significant decrease in sagittal cranial length by 6 months (p = 0.003). HGPS mice had significantly lower intermolar distances at 6 and 8 months (p = 0.04 and p = 0.01, respectively). These findings reflect the progressive nature of HGPS and are consistent with the observation that craniofacial abnormalities become more prominent with time. It should also be noted that INWI differed between HGPS mice and age-matched WT littermates at the earlier 2-month time point.

Our G608G homozygous mouse model showed significant decreases in various parameters (INWI, IZD, SC, PL, and ID) compared to age-matched WT mice, as mentioned above. However, we did not detect a consistent and significant reduction in all craniofacial measurements in HGPS mice. Thus, despite our more rigorous quantification of craniofacial features in the homozygous G608G mouse model, it remains difficult to effectively conclude an overall skull size reduction as reported in the homozygous G609G model by Osorio et al. In addition, most craniofacial abnormalities in our animal model without treatment were noted at later stages of disease progression (6–8 months follow-up). These findings are consistent with clinical descriptions of HGPS in humans, wherein abnormalities present around 2 years of age and worsen with time (Merideth et al., 2008). In addition, some of the most distinct craniofacial features of HGPS in humans include prominent eyes (due to orbital hypotelorism) and a narrow nasal bridge with a broad-tipped nose (Ullrich et al., 2012). These descriptions are accurately reflected in our HGPS G608G mice, which exhibited significantly decreased internasal width (INWI) compared to age-matched WT littermates; however, no observations were recorded about prominent eyes in this model. Moreover, our model is consistent with descriptions of maxillary hypoplasia in humans with HGPS Field (Hennekam, 2006) since our HGPS mice exhibited lower 8-month averages in palatine length (shorter snout) and intermolar distance (narrower face) compared to age-matched WT littermates.

Despite extensive clinical characterization of the musculoskeletal and craniofacial phenotypes of HGPS, relatively little is known about the molecular mechanisms underlying these abnormalities (Schmidt et al., 2012). Recent in vitro studies have linked the accumulation of progerin with altered cellular mechanisms that can explain the bone and cartilage tissue changes seen in HGPS disease (Schmidt et al., 2012; Gargiuli et al., 2018). More specifically, the osteoblast-specific expression of the HGPS mutation increases DNA damage and adversely affects the Wnt signaling pathway, impacting bone and cartilage configuration (Day et al., 2005). Defects in the Hippo pathway and RUNX2 gene also contribute to musculoskeletal abnormalities; however, Wnt/beta-catenin derangements appear to be more central to developing the HGPS phenotype (Chen et al., 2008). Further, fibroblasts from the LMNA Δ9 progeria mouse model and hair follicle stem cells in ZMPSTE24-deficient mice have shown reduced Wnt/β-catenin signaling (Espada et al., 2008; Hernandez et al., 2010). This alteration leads to mesenchymal stem cell dysfunction (Harhouri et al., 2018) and abnormal expression of extracellular matrix genes with specific roles in skeletal and cartilage development (Day et al., 2005; Chen et al., 2008; Hernandez et al., 2010). In the teeth, these changes perturb dentin formation and cause severe dental abnormalities involving the incisors (Gargiuli et al., 2018). In addition, histopathologic analysis of a mouse model generated by Eriksson et al. demonstrated dramatic effects on bone microstructure, including hypocellular red bone marrow, widespread loss of osteocytes, and defects in mineralization (Eriksson et al., 2003). This mouse model also revealed fractured incisors, disturbed dentin formation, and the presence of secondary inflammatory reactions around the fracture sites. Bone biomarkers such as alkaline phosphatase, osteocalcin, and Collagen type I were also downregulated (Schmidt et al., 2012). More importantly, such disturbances in osteoblast differentiation were only observed after long-term expression of the HGPS mutation. Even though we did not assess for dental abnormalities of the incisors, blood levels of bone biomarkers, or perform a histopathological analysis of the craniofacial structures, our findings in the HGPS mice were also observed at later time points, where disease progression phenotypes are more evident.

Other laminopathy animal models have been generated to recapitulate bone microstructural abnormalities observed in HGPS patients. The first of these was the ZMPSTE24^−/− mouse model, which exhibits MSK abnormalities such as kyphosis, spontaneous bone fractures by 6–7 weeks of age, significant micrognathia, and a reduction of the zig-zag appearance of the cranial sutures (Bergo et al., 2002). More recently, the Lmna ^G609G/G609G homozygous mouse was generated, exhibiting joint immobility, skeletal deformities in the vertebral column (lordo-kyphosis), skulls with a marked size reduction, and smaller lower incisors (Osorio et al., 2012; Gargiuli et al., 2018). Our recent publication describing the bone phenotype in this mouse model may provide some insight into HGPS bone dysplasia (Cabral et al., 2023); although intramembranous and endochondral bone formation proceeds by different mechanisms, we demonstrated altered differentiation and functioning of long bone cell populations in vitro and in vivo. Specifically, we noted delayed maxillary, mandibular, and calvarial mineralization with increased craniofacial and mandibular cartilage content in newborn mice. Post-natal bone loss in these mice was associated with increased growth plate chondrocyte TUNEL staining, an adipogenic gene expression pattern with reduced Wnt signaling in osteoblasts and reduced osteoclastogenesis resulting from osteoblast-secreted factors (presumably OPG).

Detailed molecular studies characterizing gene expression and intracellular signaling pathways in LMNA ^G608G/G608G bone cell populations have not been published but are the subject of a current investigation. Alternative HGPS mouse models have consistently demonstrated a combination of decreased bone formation and resorption that is attributed to reduced numbers of osteoblasts, osteocytes, and osteoclasts in vivo (Lmna ^−/−, Lmna ^HG/HG, Zmpste24 ^−/−, and G608G tissue-specific tetop-LA^wt+;Sp7-tTA⁺). Our prior analysis revealed significantly reduced bone turnover in Lmna ^G609G/G609G femora, which is attributed to defective osteoblast differentiation associated with an adipogenic gene expression signature and inhibition of osteoclastogenesis by osteoblast-secreted factors, presumably OPG (PMC1513052, 3081846, 130584, 19587107, 3460452). Thus, defects in bone remodeling are a common mechanism in the bone phenotype of HGPS mice.

Importantly, the development and growth of the cranium occur by two distinct processes. The first, endochondral ossification, requires deposition and replacement of a pre-existing cartilage model by bone and occurs mainly in the skull base. Endochondral bone formation, therefore, requires vascular invasion of the tissue, followed by the introduction of osteoclasts to resorb the pre-existing cartilage matrix. The second growth process, intramembranous ossification, requires neural crest-derived mesenchymal cells to directly differentiate into osteoblasts that deposit and mineralize the extracellular matrix, mainly in the cranium and facial skeleton. Intramembranous bones of the skull are joined by a fibrous matrix housing pluripotent progenitor cells that are continuously remodeled to allow for cranial enlargement. Furthermore, ossification of the sutures is concurrent with cessation of remodeling and cranial growth. We, therefore, highlight alterations to the normal processes of bone remodeling that are critical for the proper development of craniofacial skeletal architecture (Holmbeck and Szabova, 2006; Thilander, 1995) and that ours is the first study to link previously reported cellular defects in HGPS to craniofacial alterations demonstrated here.

One limitation of this study is the relatively small sample size (n = 5) of each transgenic mouse group. This low sample size may have increased measurement variability, which could account for the absence of significant differences in some craniofacial parameters. While our animal study has a sufficient sample size based on the degrees of freedom (Charan and Kantharia, 2013), a larger sample could more reliably predict differences in outcome parameters. Another limitation was the potential for unreliable results if different investigators carried out measurements. This was mitigated by assigning a single investigator to carry out these measurements consistently. Three-dimensional measurements were also pre-defined using the craniofacial landmarks to ensure precision. Previous studies have also found that 3D-CT volume rendering has small interobserver and intra-observer variation among measurements (Rocha Sdos et al., 2003; Cavalcanti et al., 2004).

In summary, the long bone phenotype in Lmna ^G609G/G609G mice is associated with a growth plate defect and low bone turnover condition that can be described as osteochondrodystrophy. Although these findings have not yet been confirmed in the G608G transgenic mouse model, our unpublished data has shown significantly reduced deposition of ECM and hydroxyapatite by G608G homozygous calvarial osteoblasts in vitro (manuscript in production). Thus, our work provides a unique opportunity for future studies in other animal models of HGPS to combine molecular, biomarker, and histopathological analyses with our proposed 3D cephalometric measurements. This combined strategy can contribute to a new understanding of potential disease mechanisms affecting the craniofacial structures in HGPS and improve and optimize therapeutic strategies. Most cephalometric measurements were decreased in the HGPS mice compared to age-matched WT littermates. These changes became more apparent as the animals aged, consistent with descriptions of disease progression in pediatric patients with progeria. Ultimately, this insight motivates future studies to test emerging therapeutic strategies on larger animal cohorts and ameliorate craniofacial abnormalities in humans with HGPS.