TSD is caused by a mutation in the lysosomal Hexosaminidase A (HEXA) enzyme resulting in excessive GM2 gangliosides accumulation and progressive neurodegeneration in the central nervous system [30]. Bone structure anomalies including kyphosis, kyphoscoliosis [13, 14], scoliosis, and lordosis[15, 16] were also detected in TSD patients. Similarly, kyphosis was observed in the TSD mouse model (Hexa-/-Neu3-/- mice) [3]. Bone loss-associated disorders like osteoporosis contribute to bone structure anomalies such as kyphosis [31]. In this study, we focused on the detection of bone remodeling process markers in the plasma and femur of Hexa-/-Neu3-/- mice to clarify the basis of kyphosis. Hexa-/-Neu3-/- mice were analyzed by micro-CT imaging techniques to evaluate the secondary effect of GM2 ganglioside accumulation on the trabecular bone microarchitecture. To our knowledge, high-resolution micro-CT imaging was used first time for the evaluation of the 3D bone morphology of the entire mouse skeleton system.
Similar to TSD patients, bone lesions are observed in the other types of lysosomal storage disorders such as Gaucher, Mucopolysaccharidoses, and GM1 gangliosidosis, and in many neurological diseases like multiple sclerosis, neuromuscular dystrophy, Parkinson's, and Alzheimer Disease [1, 4, 32]. Neurophysiological alterations frequently lead to changes in bone physiology, which result in changes in bone microarchitecture, decreased bone strength, and reduced bone mineral density and content. These changes ultimately result in the development of osteopenia/osteoporosis in many neurological diseases[32].
Calcium is primarily involved in the mechanical strength of the skeleton and teeth and metabolic processes [33]. In case of inadequate calcium intake and/or high calcium excretion, calcium levels in the blood stay low, and calcium is automatically released from the bones to maintain blood calcium levels. When it occurs repeatedly, a negative calcium balance triggers porous and fragile bones, and osteoporosis [33]. In this work, all mouse groups have unlimited access to a standard chow diet and water. However, a significant reduction in the Ca level was detected in 5-month-old Hexa-/-Neu3-/- mice compared to age-matched WT and Hexa-/- mice, and 2.5-month-old Hexa-/-Neu3-/- mice (Fig. 1C). The reason for the stimulation of negative calcium balance in Hexa-/-Neu3-/- mice might be related to excessive calcium excretion depending on the accumulation of GM2, GM3, and SM2a in the kidney [3].
OCN is extensively studied as a bone formation marker and its level is directly correlated with the amount of bone formation [29]. Our work showed that the expression level of OCN severely decreased in the femur of 5-month-old Hexa-/-Neu3-/- mice compared to age-matched mice groups (Fig. 2). Similarly, ALP levels in the plasma were significantly decreased in the 2.5- and 5- month-old Hexa-/-Neu3-/- mice compared with age-matched WT mice (Fig. 1). Both are involved in the process of bone mineralization [29]. These results indicated that Hexa-/-Neu3-/- mice have impaired bone formation. Regarding bone resorption, the levels of TRAP protein in the plasma and TRAP mRNA in the femur were significantly enhanced only in 5-month-old Hexa-/-Neu3-/- mice compared with age-matched control groups (Figs. 1 and 2). A reduction in osteoblastic activity and an increase in osteoclastic activity might trigger net bone loss. Our results illuminated the presence of age-dependent increases in the imbalance between resorption and formation in favor of bone resorption. Increases in bone resorption could lead to a progressive negative impact on skeletal structure and cause kyphosis in Hexa-/-Neu3-/- mice.
Lysosomal functions are directly involved in osteoblastic differentiation and bone resorption [34]. Lysosome-related organelles (LROs), specialized lysosomes known as secretory lysosomes, function in bone resorption in osteoclasts. They are also involved in the regulation of exocytosis and extracellular material degradation [35]. The most specific component of LROs is TRAP [34]. Our findings support that the severity of bone lesions found in Hexa-/-Neu3-/- mice correlates with plasma ALP, TRAP, and Ca levels. However, the origin of ALP reduction and TRAP elevation in Hexa-/-Neu3-/- mice is still not clear. The reason for bone lesions might have resulted from a primary defect of bone cells such as osteoblasts and osteoclasts.
Osteoporosis is characterized by an impairment of bone architecture and a decrease in bone mass due to a decrease in the thickness and number of trabecular bones and an increase in the risk of bone fragility and fracture. Micro-CT analysis provides the evaluation of the bone microarchitecture which is important for improving diagnosis and treatment [36, 37]. This study revealed trabecular bone loss in the skeleton of Hexa-/-Neu3-/- mice via micro-CT imaging. An increase in the spacing (Tb. Sp), and a reduction in the trabecular bone thickness (Tb. Th), number (Tb. N), BV/TV, and BMD might indicate osteoporosis and/or osteopenia in Hexa-/-Neu3-/- mice (Fig. 3).
Measurement of 3D bone morphology by micro-CT imaging revealed bone microarchitectural deterioration, decreased trabeculae, and enlarged interspace on the sagittal, dorsal, and ventral views of the Hexa-/-Neu3-/- mice skeleton. Similarly, bone structure anomalies in the TSD patient might indicate osteoporosis. A better understanding of the bone remodeling biology of Hexa-/-Neu3-/- mice led to the discovery of potential therapeutic targets for bone lesions. Living conditions of the TSD patient and mice could be improved by treating osteoporosis via anti-osteoporotic medicines, such as Astaxanthin (AST) and 2,3,5,4’-Tetrahydroxystilbene-2-O-β-D-glucoside (TSG) [36, 37]. Furthermore, teriparatide, a parathyroid hormone analog, might be used to encourage new bone formation by stimulating osteoblastic activity. Bisphosphonates such as alendronate, risedronate, and zoledronic acid might be used to inhibit osteoclast resorption and promote a net gain in bone mass [38].
In this research, we focused on the detection of the marker of bone loss-associated disorders such as osteopenia and osteoporosis in the TSD mouse model. It was demonstrated that elevated plasma TRAP and reduced plasma ALP levels in Hexa-/-Neu3-/- mice were correlated with the severity of bone lesions found in micro-CT analysis. Eventually, a better understanding of the reasons for bone loss in the TSD mouse model would guide therapeutic options for osteoporosis to encourage the life standards of TSD patients.