By deletion of Slc13a5 in our mouse models we found that elevated mineral citrate levels reduce bone mass, mineral density and strength. However, we do not know yet how the altered mineral citrate levels alter the hydroxyapatite structure such as crystal leaflet thickness, local calcium levels, collagen crosslinks, water content, organic matter, … This project, in collaboration with Markus Hartmann at the Ludwig Boltzmann Institute – Osteology in Vienna, will elucidate how differences in bone mineral citrate content affect the structure and composition of mineralized bone matrix using techniques such as quantitative Backscattered Electron Imaging (qBEI), Fourier transform infrared imaging (FTIRI) & Raman spectroscopic (RS).
While extensive research has been performed on endocrine regulation of plasma calcium levels by calciotropic hormones such as parathyroid hormone (PTH) and calcitonin with respective hypercalcemic and hypocalcemic functions, the hormonal regulation of citrate homeostasis has largely been ignored in biomedical research. However, since plasma calcium and citrate concentration often occur in concert (to prevent crystal nucleation and growth), a common factor in regulation is assumed. Indeed, parathyroidectomy and hypoparathyroidism mostly results in hypocalcemia and hypocitricemia while PTH administration or hyperparathyroidism evokes hypercalcemia and hypercitricemia in patients. The “citricemic” action of PTH on bone is assumed to arise from promoting osteoclastic bone resorption and thereby releasing calcium and citrate. Despite the association of PTH levels with renal citrate clearance, the molecular mechanisms remain elusive.
Although reduced urinary citrate secretion correlates with kidney stone formation and the prevalence of osteoporotic fractures is higher in patients with kidney stones the molecular mechanisms linking urinary citrate to bone mineral homeostasis have not been elucidated yet. Since more than 80% of the total body citrate is stored in the bone minerals, we believe that the skeleton is major player in controlling systemic citrate levels in response to physiological needs, and thus hormonal cues, not only on the level of osteoclast-mediated bone resorption but also specifically on the level of osteoblast mediated citrate uptake, production and release.
We recently discovered that Slc13a5 expression on osteoblasts is regulated by the calciotropic hormone PTH, which suggests a coupling between skeletal citrate partitioning and calcium homeostasis. Since bone mass deterioration, impairment of mineral homeostasis and disturbed signaling of calciotropic hormones are often associated with chronic kidney disease with kidney stone formation and hypocitraturia, we believe that targeting osteogenic SLC13A5 could be a step forward in understanding mineral diseases and development of treatment.
Reduced expression of the plasma membrane citrate transporter SLC13A5, also known as INDY, has been linked to increased longevity and mitigated age-related cardiovascular and metabolic issues. Citrate, a vital component of the tricarboxylic acid cycle, constitutes 1-5% of bone weight, binding to mineral apatite surfaces. Our previous research highlighted osteoblasts’ specialized metabolic pathway facilitated by SLC13A5, regulating citrate uptake, production, and deposition within bones. Disrupting this pathway impairs mineralization in young mice. Mendelian randomization analysis using UK Biobank data indicated that SNPs linked to reduced SLC13A5 function, lowered osteoporosis risk. Comparative studies of young (10 weeks) and aged (52 weeks) osteocalcin-cre driven osteoblast-specific Slc13a5 knockout mice (Slc13a5cKO) showed that aged females exhibited improved elasticity, while old males demonstrated enhanced bone strength due to reduced SLC13A5 function. These findings suggest that reduced SLC13A5 function could attenuate age-related bone loss, advocating for SLC13A5 inhibition as a potential osteoporosis treatment. This project will further elucidate why inhibiting SLC13A5 function in aged bones has differential effects compared to its inhibition during growth. Further translational studies will assess whether SLC13A5 inhibitors could be used as treatment for low bone mass disorders.
Genetic mutations in SLC13A5, the membrane transporter for extracellular citrate uptake, are associated with early onset epilepsy. Several disease-causing mutations in the citrate transporter SLC13A5 have been discovered and outcomes range from no protein translated to a malfunctioning transporter present in the membrane, all leading to a complete loss of citrate transport. Affected children have elevated citrate levels in blood and cerebrospinal fluid (CSF) and develop seizures within 24 hours after birth and show a developmental delay with mild to severe intellectual disability. This form of epilepsy, is distinct from other epileptic disorders as affected children present with tooth hypoplasia or hypodontia, which is used for initial differential diagnosis. We have intensively studied the role of SLC13A5 in tissue mineralization and discovered a novel metabolic pathway in skeletal cells in which SLC13A5 partitions citrate in bone and regulates osteogenic mitochondrial citrate synthesis and secretion. In collaboration with TESS Research Foundation, CUREepilepsy and Dr. Mary McKenna we will start shedding light on the metabolic causes of SLC13A5 epilepsy to accelerate disease treatment.
Astrocytes and neurons undergo a continuous exchange of metabolites (e.g. glutamine) to 1) provide a carbon skeleton for the generation of neurotransmitters or energy in neurons and 2) prevent overexcitation by neurotransmitter recycling from the synaptic cleft and conversion into glutamine by astrocytes, ensuring a continues availability of glutamine for neurons. We postulate that due to a defective SLC13A5 transporter, astrocytes augment their endogenous mitochondrial citrate production to the expense of glutamine synthesis. This shift in metabolism leads on the one hand to an increased release of citrate in the extracellular space, similar to what we observed in osteoblasts, and on the other hand to reduced trafficking of glutamine to neurons. While the first could be explanatory for the elevated levels of citrate in the CSF in SLC13A5 epilepsy patients that could be toxic (e.g. changing pH; modulating NMDA receptors) and contribute to the seizures, the second would imply an imbalance in excitatory (glutamate) and inhibitory (GABA) neurotransmitter generation by neurons leading to seizures as well. We will evaluate this hypothesis using a combination of innovative techniques such as mass spectrometry and 13C-NMR. The latter technique is the only method that can determine changes in cell specific pathways of [1,6-13C]glucose metabolism, neurotransmitter synthesis and metabolite trafficking between neurons and astrocytes in whole brains. Identifying the molecular pathways and metabolite trafficking in SLC13A5 epilepsy will allow future development of small molecules targeting specific enzymes or transporters to aid in disease treatment and management by reversing the SLC13A5-deficiency induced metabolic changes.
