Citrate is essential for the metabolism and development of neurons. A membrane transport protein called SLC13A5 plays a central role in this process and has previously been linked to a particularly severe form of epileptic encephalopathy. Building on data from the recently completed RESOLUTE and REsolution flagship projects, scientists at CeMM have comprehensively studied the function and structure of the membrane transporter SLC13A5, experimentally investigating 38 mutant variants. Their findings, published in Science Advances (DOI 10.1126/sciadv.adx3011 ) shed new light on the mechanisms of this disease and lay the foundation for further research into epilepsy and other disorders.
Citrate, the negatively charged ion of citric acid, is a key component in the metabolism of every cell. In the citric acid cycle—often referred to as the "hub" of cellular metabolism—organic substances are broken down to generate chemical energy, while also producing various precursors for the biosynthesis of fatty acids and critical signaling molecules involved in inflammation and cell development.
In neurons, citrate plays an especially important role. As a so-called "neuromodulator," it influences neuronal activity and is therefore present in relatively high concentrations in the cerebrospinal fluid. Accordingly, neurons express high levels of the SLC13A5 transporter to facilitate citrate uptake. When this transporter is not fully functional, it can lead to SLC13A5 Citrate Transporter Disorder—a severe form of epilepsy associated with impaired brain development (scientifically referred to as developmental epileptic encephalopathy, DEE). This condition is caused by mutations in the SLC13A5 gene. However, until now, little was known about which mutations are involved, how they affect the molecular function of the transporter, and how they influence disease progression.
Ten Thousand Mutations Analyzed
To address this knowledge gap, scientists at CeMM Research Center for Molecular Medicine of the Austrian Academy of Sciences performed a technique called "deep mutational scanning" (DMS), analyzing the effect of nearly ten thousand different genetic mutations on the function of the SLC13A5 transport protein. The dataset was further enriched by computational analyses of protein stability, and 38 mutated SLC13A5 variants were selected for experimental investigation. This approach revealed several molecular mechanisms linked to the manifestation of the disease. These included differences in transporter production levels in neurons, their precise localization in the cell membrane, and the actual rate of citrate transport.
"With these results, we were able to identify and characterize disease-causing variants of the SLC13A5 transporter," explains co-first author Wen-An Wang, summarizing the main findings of the study. "In addition, by computationally analyzing the mutant variants, we assessed protein stability across different conformations and established an evolutionary conservation score for all variants," adds co-first author Evandro Ferrada, now at the University of Valparaíso in Chile.
"Our work highlights the importance of systematically investigating the effects of genetic variants. Especially in rare diseases such as SLC13A5 citrate transporter deficiency, a specific form of epilepsy, this approach helps us uncover molecular disease mechanisms," emphasizes senior author Giulio Superti-Furga. "At the same time, we gain valuable insights into the impact of variants that also occur in the general population – an important step toward a more comprehensive understanding of genetic diversity and its impact on human health."
The scientists involved in the study were supported by the REsolution consortium, the successor to RESOLUTE—a large-scale project led by Giulio Superti-Furga at CeMM that functionally mapped the entire SLC transporter family and helped decode the "logistics of the cell." Patient data were provided by the TESS Research Foundation, which is dedicated to advancing research on SLC13A5 citrate transporter deficiency.
