INDY—From Flies to Worms, Mice, Rats, Non-Human Primates, and Humans

I’m Not Dead Yet (Indy) is a fly homologue of the mammalian SLC13A5 (mSLC13A5) plasma membrane citrate transporter, a key metabolic regulator and energy sensor involved in health, longevity, and disease. Reduction of Indy gene activity in flies, and its homologs in worms, modulates metabolism and extends longevity. The metabolic changes are similar to what is obtained with caloric restriction (dietary restriction). Similar effects on metabolism have been observed in mice and rats. As a citrate transporter, INDY regulates cytoplasmic citrate levels. Indy flies heterozygous for a P-element insertion have increased spontaneous physical activity, increased fecundity, reduced insulin signaling, increased mitochondrial biogenesis, preserved intestinal stem cell homeostasis, lower lipid levels, and increased stress resistance. Mammalian Indy knockout (mIndy-KO) mice have higher sensitivity to insulin signaling, lower blood pressure and heart rate, preserved memory and are protected from the negative effects of a high-fat diet and some of the negative effects of aging. Reducing mIndy expression in human hepatocarcinoma cells has recently been shown to inhibit cell proliferation. Reduced Indy expression in the fly intestine affects intestinal stem cell proliferation, and has recently been shown to also inhibit germ cell proliferation in males with delayed sperm maturation and decreased spermatocyte numbers. These results highlight a new connection between energy metabolism and cell proliferation. The overrall picture in a variety of species points to a conserved role of INDY for metabolism and health. This is illustrated by an association of high mIndy gene expression with non-alcoholic fatty liver disease in obese humans. mIndy (mSLC13A5) coding region mutations (e.g., loss-of-function) are also associated with adverse effects in humans, such as autosomal recessive early infantile epileptic encephalopathy and Kohlschütter−Tönz syndrome. The recent findings illustrate the importance of mIndy gene for human health and disease. Furthermore, recent work on small-molecule regulators of INDY highlights the promise of INDY-based treatments for ameliorating disease and promoting healthy aging.

Tricarboxylate Citrate Transporter of an Oleaginous Fungus Mucor circinelloides WJ11: From Function to Structure and Role in Lipid Production

The citrate transporter protein (CTP) plays an important role in citrate efflux from the mitochondrial matrix to cytosol that has great importance in oleaginous fungi. The cytoplasmic citrate produced after citrate efflux serves as the primary carbon source for the triacylglycerol and cholesterol biosynthetic pathways. Because of the CTP's importance, our laboratory has extensively studied its structure/function relationships in Mucor circinelloides to comprehend its molecular mechanism. In the present study, the tricarboxylate citrate transporter (Tct) of M. circinelloides WJ11 has been cloned, overexpressed, purified, kinetically, and structurally characterized. The Tct protein of WJ11 was expressed in Escherichia coli, isolated, and functionally reconstituted in a liposomal system for kinetic studies. Our results showed that Tct has a high affinity for citrate with Km 0.018 mM. Furthermore, the tct overexpression and knockout plasmids were created and transformed into M. circinelloides WJ11. The mitochondria of the tct-overexpressing transformant of M. circinelloides WJ11 showed a 49% increase in citrate efflux, whereas the mitochondria of the tct-knockout transformant showed a 39% decrease in citrate efflux compared to the mitochondria of wild-type WJ11. To elucidate the structure-function relationship of this biologically important transporter a 3D model of the mitochondrial Tct protein was constructed using homology modeling. The overall structure of the protein is V-shaped and its 3D structure is dimeric. The transport stability of the structure was also assessed by molecular dynamics simulation studies. The activity domain was identified to form hydrogen bond and stacking interaction with citrate and malate upon docking. Tricarboxylate citrate transporter has shown high binding energy of −4.87 kcal/mol to citric acid, while −3.80 kcal/mol to malic acid. This is the first report of unraveling the structural characteristics of WJ11 mitochondrial Tct protein and understanding the approach of the transporting toward its substrate. In conclusion, the present findings support our efforts to combine functional and structural data to better understand the Tct of M. circinelloides at the molecular level and its role in lipid accumulation.

Phenobarbital Induces SLC13A5 Expression through Activation of PXR but Not CAR in Human Primary Hepatocytes

Phenobarbital (PB), a widely used antiepileptic drug, is known to upregulate the expression of numerous drug-metabolizing enzymes and transporters in the liver primarily via activation of the constitutive androstane receptor (CAR, NR1I3). The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter, plays an important role in intracellular citrate homeostasis that is associated with a number of metabolic syndromes and neurological disorders. Here, we show that PB markedly elevates the expression of SLC13A5 through a pregnane X receptor (PXR)-dependent but CAR-independent signaling pathway. In human primary hepatocytes, the mRNA and protein expression of SLC13A5 was robustly induced by PB treatment, while genetic knockdown or pharmacological inhibition of PXR significantly attenuated this induction. Utilizing genetically modified HepaRG cells, we found that PB induces SLC13A5 expression in both wild type and CAR-knockout HepaRG cells, whereas such induction was fully abolished in the PXR-knockout HepaRG cells. Mechanistically, we identified and functionally characterized three enhancer modules located upstream from the transcription start site or introns of the SLC13A5 gene that are associated with the regulation of PXR-mediated SLC13A5 induction. Moreover, metformin, a deactivator of PXR, dramatically suppressed PB-mediated induction of hepatic SLC13A5 as well as its activation of the SLC13A5 luciferase reporter activity via PXR. Collectively, these data reveal PB as a potent inducer of SLC13A5 through the activation of PXR but not CAR in human primary hepatocytes.

METTL3-Dependent Glycolysis Regulates Dental Pulp Stem Cell Differentiation

N6-methyladenosine (m6A) is a eukaryotic messenger RNA modification catalyzed by methyltransferase-like 3 (METTL3), which is involved in various developmental and disease processes. However, the connection between the epigenetic modification of m6A and glucose metabolism during osteogenesis is still unclear. Here, we show that interference with METTL3 in dental pulp stem cells (DPSCs) inhibits cell proliferation and osteogenic differentiation. Moreover, transcriptome sequencing and metabolic testing were used to explore the mechanism between glucose metabolism and m6A modification in METTL3-knockdown DPSCs. Methylated RNA immunoprecipitation–quantitative polymerase chain reaction and RNA stability assays were used to determine the target genes of METTL3. Mechanistically, METTL3 directly interacts with ATP citrate lyase (ACLY) and a mitochondrial citrate transporter (SLC25A1) and then further affects the glycolytic pathway. M6A-mediated ACLY and SLC25A1 stability depends on the m6A readers IGF2BP2 and IGF2BP2/3, respectively. Our experiments uncovered the potential molecular mechanism of epigenetic modification in osteogenic differentiation, providing new ideas for the clinical application of stem cells and the intervention of metabolic bone diseases.

Growth and Overall Health of Patients with SLC13A5 Citrate Transporter Disorder

We were interested in elucidating the non-neurologic health of patients with autosomal recessive SLC13A5 Citrate Transporter (NaCT) Disorder. Multiple variants have been reported that cause a loss of transporter activity, resulting in significant neurologic impairment, including seizures, as well as motor and cognitive dysfunction. Additionally, most patients lack tooth enamel (amelogenesis imperfecta). However, patients have not had their overall health and growth described in detail. Here we characterized the non-neurologic health of 15 patients with medical records uploaded to Ciitizen, a cloud-based patient medical records portal. Ciitizen used a query method for data extraction. Overall, the patients’ records suggested a moderate number of gastrointestinal issues related to feeding, reflux, vomiting and weight gain and a diverse number of respiratory complaints. Other organ systems had single or no abnormal diagnoses, including liver, renal and cardiac. Growth parameters were mostly in the normal range during early life, with a trend toward slower growth in the few adolescent patients with data available. The gastrointestinal and pulmonary issues may at least partially be explained by the severity of the neurologic disorder. More data are needed to clarify if growth is impacted during adolescence and if adult patients develop or are protected from non-neurologic disorders.

Metabolic flux from the Krebs cycle to glutamate transmission tunes a neural brake on seizure onset

Kohlschütter-Tönz syndrome (KTS) manifests as neurological dysfunctions, including early-onset seizures. Mutations in the citrate transporter SLC13A5 are associated with KTS, yet their underlying mechanisms remain elusive. Here, we report that a Drosophila SLC13A5 homolog, I’m not dead yet (Indy), constitutes a neurometabolic pathway that suppresses seizure. Loss of Indy function in glutamatergic neurons caused “bang-induced” seizure-like behaviors. In fact, glutamate biosynthesis from the citric acid cycle was limiting in Indy mutants for seizure-suppressing glutamate transmission. Oral administration of the rate-limiting α-ketoglutarate in the metabolic pathway rescued low glutamate levels in Indy mutants and ameliorated their seizure-like behaviors. This metabolic control of the seizure susceptibility was mapped to a pair of glutamatergic neurons, reversible by optogenetic controls of their activity, and further relayed onto fan-shaped body neurons via the ionotropic glutamate receptors. Accordingly, our findings reveal a micro-circuit that links neural metabolism to seizure, providing important clues to KTS-associated neurodevelopmental deficits.

Molecular Mechanisms of the SLC13A5 Gene Transcription

Citrate is a crucial energy sensor that plays a central role in cellular metabolic homeostasis. The solute carrier family 13 member 5 (SLC13A5), a sodium-coupled citrate transporter highly expressed in the mammalian liver with relatively low levels in the testis and brain, imports citrate from extracellular spaces into the cells. The perturbation of SLC13A5 expression and/or activity is associated with non-alcoholic fatty liver disease, obesity, insulin resistance, cell proliferation, and early infantile epileptic encephalopathy. SLC13A5 has been proposed as a promising therapeutic target for the treatment of these metabolic disorders. In the liver, the inductive expression of SLC13A5 has been linked to several xenobiotic receptors such as the pregnane X receptor and the aryl hydrocarbon receptor as well as certain hormonal and nutritional stimuli. Nevertheless, in comparison to the heightened interest in understanding the biological function and clinical relevance of SLC13A5, studies focusing on the regulatory mechanisms of SLC13A5 expression are relatively limited. In this review, we discuss the current advances in our understanding of the molecular mechanisms by which the expression of SLC13A5 is regulated. We expect this review will provide greater insights into the regulation of the SLC13A5 gene transcription and the signaling pathways involved therein.

The Role of Citrate Transporter INDY in Metabolism and Stem Cell Homeostasis

I’m Not Dead Yet (Indy) is a fly gene that encodes a homologue of mammalian SLC13A5 plasma membrane citrate transporter. Reducing expression of Indy gene in flies, and its homologues in worms, extends longevity. Indy reduction in flies, worms, mice and rats affects metabolism by regulating the levels of cytoplasmic citrate, inducing a state similar to calorie restriction. Changes include lower lipid levels, increased insulin sensitivity, increased mitochondrial biogenesis, and prevention of weight gain, among others. The INDY protein is predominantly expressed in fly metabolic tissues: the midgut, fat body and oenocytes. Changes in fly midgut metabolism associated with reduced Indy gene activity lead to preserved mitochondrial function and reduced production of reactive oxygen species. All these changes lead to preserved intestinal stem cell homeostasis, which has a key role in maintaining intestinal epithelium function and enhancing fly healthspan and lifespan. Indy gene expression levels change in response to caloric content of the diet, inflammation and aging, suggesting that INDY regulates metabolic adaptation to nutrition or energetic requirements by controlling citrate levels.

Identification of Adeno-Associated Virus Variants for Gene Transfer Into Human Neural Cell Types by Parallel Capsid Screening

Human brain cells generated by in vitrocell programming provide exciting prospects for disease modeling, drug discovery and cell therapy. These applications frequently require efficient and clinically compliant tools for genetic modification of the cells. Recombinant Adeno-associated viruses (AAVs) fulfill these prerequisites for a number of reasons, including the availability of a myriad of AAV capsid variants with distinct cell type specificity (also called tropism). Here, weharnessed a customizable parallel screening approach to assessa panel of natural or synthetic AAV capsid variants for their efficacy in lineage-related human neural cell types.We identified common lead candidates suited for the transduction of directly converted,early-stage induced neural stem cells (iNSCs), induced pluripotent stem cell (iPSC)-derived later-stage, radial glia-like neuralprogenitors,as well as differentiated astrocytic and mixed neuroglial cultures.We then selected a subsetof these candidates for functional validation in iNSCs and iPSC-derived astrocytes, usingshRNA-induced downregulation of the citrate transporter SLC25A1 and overexpression of the transcription factor NGN2 for proofs-of-concept. Our study provides a comparative overview ofthe susceptibility of different human cell programming-derived brain cell types to AAV transduction and a critical discussionof the assets and limitations of the specific AAV capsidscreening approach.