SLC13A5 Clinical Overview
Dr. Porter discussed the common symptoms of SLC13A5 Deficiency in children, including seizures in the first days of life, a distinct movement disorder, speech difficulties, and tooth abnormalities. There is no or minimal brain structural problem in the brain MRI. This disease does not appear to be a progressive, degenerative condition. The EEG background can be normal. Seizure burden varies widely in both type and frequency. The children are very sociable. Epilepsy is one small component of the disorder. Movement and motor control as well as cognitive delays are prominent.
SLC13A5 Research Overview
The TESS Foundation has pushed SLC13A5 research a long way. We went from having nothing a few short years ago, to having antibody, iPSCs, and animal models. These tools are now enabling researchers to uncover the role of SLC13A5 in disease.
Structural Role for Osteoblast Derived Citrate in Bone
Bone stiffness and ability to resist fractures is offered by its unique structure consisting of crystals of carbonated apatite embedded in a collagenous matrix. Approximately 80% of total body citrate is stored in the skeleton where it is strongly bound to calcium crystals and defines the mechanical properties of the bone. Despite the enormous potential of citrate in controlling bone quality, the source of citrate and the mechanism of citrate incorporation in the bone minerals remain elusive.
The citrate transporter Slc13a5 is highly expressed in bone by late mineralizing osteoblasts. Indeed, radioactive C-Citrate uptake shows a 4-fold increase in the later stages of osteogenic differentiation. Culturing osteoblasts in the presence of the SLC13A5 inhibitor PF-06761281 or genetic deletion of Slc13a5 abolishes C-Citrate uptake but causes a compensational increase in citrate production from glucose by the TCA cycle in the mitochondria and release in the mineral matrix.
Both inhibitor treated and Slc13a5-/- osteoblasts fail to perform proper “nucleation” of the minerals causing a diffuse appearance of the mineral matrix, attributed to increased mineral citrate content. A mouse model with global deletion of Slc13a5 shows defective tooth enamel and bone development, similar to what has been described in patients with SLC13A5 mutations. Moreover, conditional deletion of Slc13a5 in mature osteoblasts specifically causes significantly weaker bones.
We here expose an entirely new metabolic pathway that controls the deposition of citrate into bone, and identify SLC13A5-mediated citrate uptake as a valid mechanism to further investigate for improving bone strength in low bone mass disorders.
SLC13A5 as a novel pharmacologic target for metformin and its relevance to the antidiabetic efficacy of the drug
Metformin is the first-line treatment for type 2 diabetes. Inhibition of hepatic gluconeogenesis is the primary contributor to its anti-diabetic effect. Metformin inhibits complex I and a-glycerophosphate shuttle, and the resultant increase in cytoplasmic NADH/NAD+ ratio diverts glucose precursors away from gluconeogenesis. These actions depend on metformin-mediated activation of AMP kinase (AMPK). Here we report on a hitherto unknown mechanism. Metformin inhibits the expression of the plasma membrane citrate transporter NaCT in HepG2 cells and decreases cellular levels of citrate. 5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR), an AMPK activator, elicits a similar effect. The process involves a decrease in maximal velocity with no change in substrate affinity. The decrease in NaCT expression is associated with decreased mRNA levels. AMPK inhibits mTOR, and the mTOR inhibitor rapamycin also decreases NaCT expression. The transcription factor downstream of AMPK that is relevant to cAMP signaling is CREB; decreased levels of phospho-CREB seem to mediate the observed effects of metformin on NaCT. Citrate is known to suppress glycolysis by inhibiting phosphofructokinase-1 and activate gluconeogenesis by stimulating fructose-1,6-bisphophatase; therefore, the decrease in cellular levels of citrate would stimulate glycolysis and inhibit gluconeogenesis. These studies uncover a novel mechanism for the anti-diabetic actions of metformin.
The clinical significance of these findings is many-fold. Metformin is known to cross the blood-brain barrier. This raises the question as to whether chronic use of metformin in diabetic patients lead to suppression of SLC13A5 expression in the brain? What would be the impact of such an effect in heterozygous carriers of loss-of-function mutations in SLC13A5? SLC13A5 is also related to Alzheimer’s disease because it contributes to the cytoplasmic citrate that is needed for the synthesis of the neurotransmitter acetylcholine. What would be the effect of metformin-induced suppression of SLC13A5 expression and function in the brain in heterozygous carriers of SLC13A5 mutations in terms of Alzheimer’s disease? These are the questions that need to be addressed in the light of our findings that SLC13A5 is a pharmacologic target for metformin.
Classification of SLC13A5 Deficiency Mutations and Assays for Small Molecule Drug Screen
David Sauer, Jennifer Marden, Jinmei Song, Alexandra Huberfeld and Da-Neng Wang
Mutations that cause SLC13A5 Deficiency can abolish the citrate transport activity of the NaCT protein in several ways. Type I mutations affect the protein’s (Ia) integrity or (Ib) folding, whereas Type II mutations (IIa) abolish citrate binding or (IIb) block conformational changes needed for the transport activity. For Types Ib and IIb mutations, small molecule intervention is a possibility. Searching for such a small molecule drug requires an efficient method of screening. E. coli can potentially provide an effective platform for such screening. Under aerobic conditions E. coli cells cannot grow with citrate as the sole carbon source. This suggests the possibility that, if NaCT can be expressed in the bacterial cells, E. coli growth on citrate can be used to monitor NaCT protein function. Such a platform can be used to screen small molecules which rescue the activity of misfolded NaCT mutants. We validated this approach with an NaCT homolog, the bacterial citrate transporter CitS. However, to express a human membrane protein in E. coli is challenging, and we are still in the process of expressing NaCT. In the meantime, we made significant progress in understanding how various NaCT mutations affect its transport activity.
Tools to study SLC13A5 basic science
Our group is working on different tools to study SLC13A5 basic science and to develop compounds specifically targeting liver SLC13A5 to cure metabolic diseases.
Current available tools out of Andreas Birkenfeld’s lab and collaborator labs are whole body knock out, tissue specific knock outs, knock down/siRNAs and cell lines. Recent data from a metabolic disease model are presented further supporting the role of SLC13A5 in liver energy metabolism and inflammation.
A new approach was started recently to exploit available data for potential SLC13A5 activating compounds. First data and plans are presented.
Dr. Beltran discusses the development of patient-derived induced Pluripotent Stem Cells (iPSCs). She also provides an update on using CRISPR/Cas9 to create isogenic controls.
Patient-derived iPSCs and NPCs
Autosomal recessive mutations in SLC13A5, a gene encoding sodium-dependent citrate cotransporter, cause severe neonatal epileptic encephalopathy. Due to a lack of understanding of the mechanistic connection between the citrate transporter dysfunction and seizures with other neurological problems, we collaborate in generating induced pluripotent stem cells (iPSC), neuronal progenitor cells (NPC), and ultimately cortical neuron cultures from the patients’ somatic cells to examine functional human neurons with genetic alterations in SLC13A5.
Here I summarize our progress in establishing NPC lines from iPSC lines that Dr. Adriana Beltran, University of North Carolina derived from patients and their families. Seven iPSC lines from the patients and two repaired iPSC clones at c.655G>A of TESS#2 patient line (Tess_001_01_FA) were successfully converted into NPC lines. To initiate the NPC conversion, the iPSC were dispersed into single cells with Gentle Cell Dissociation Reagent and plated on Matrigel coated d35 dish at 200,000 cells/cm2 density in Neural Induction Medium (NIM) supplemented with SMAD inhibitors (NIM + SMADi, (StemCell Technology). The procedure in a monolayer culture protocol took around two to three weeks for conversion.