Novel Approaches to Studying SLC13A5 Disease

Abstract

The role of the sodium citrate transporter (NaCT) SLC13A5 is multifaceted and context-dependent. While aberrant dysfunction leads to neonatal epilepsy, its therapeutic inhibition protects against metabolic disease. Notably, insights regarding the cellular and molecular mechanisms underlying these phenomena are limited due to the intricacy and complexity of the latent human physiology, which is poorly captured by existing animal models. This review explores innovative technologies aimed at bridging such a knowledge gap. First, I provide an overview of SLC13A5 variants in the context of human disease and the specific cell types where the expression of the transporter has been observed. Next, I discuss current technologies for generating patient-specific induced pluripotent stem cells (iPSCs) and their inherent advantages and limitations, followed by a summary of the methods for differentiating iPSCs into neurons, hepatocytes, and organoids. Finally, I explore the relevance of these cellular models as platforms for delving into the intricate molecular and cellular mechanisms underlying SLC13A5-related disorders.

Keywords: NaCT; SLC13A5; hepatocytes; iPSCs; neurons; organoids.

Figure 1

Diagram illustrating potential disease-causing mutations in SLC13A5. (A) SLC13A5 gene locus with exons sequentially numbered from 1 to 12. Here, the variants are shown at their specific locations within the exons, designated as c.148T > C; “c.” denotes the coding DNA sequence, “148” specifies the nucleotide position, and “T > C” describes the nucleotide substitution (a thymine replaced by cytosine). (B) SLC13A5 protein model displaying the resultant amino acid substitutions in the protein caused by the variants described in A. The amino acid changes in the protein are denoted by an asterisk (*), with the “P68Q” signifying a change from the amino acid proline (P) to glutamine (Q). The ‘N’ refers to the free amine end, known as the ‘N-terminus’, while ‘C’ denotes the carboxyl terminus, referred to as the ‘C-terminus’, of the protein. The image in (B) was adapted from [30,33,34].

Figure 2

Research workflow using induced pluripotent stem cell (iPSCs) derived cellular models. Illustration of the research procedures for iPSC disease modeling and therapeutic investigations, incorporating both patient-derived and healthy control samples. The process begins with the collection of somatic cells, such as skin fibroblast, blood, or urine cells, from either a patient or a healthy individual, followed by the reprogramming of these samples into iPSCs. Genetic editing techniques such as clustered regularly interspaced short palindromic repeats (CRISPR) may be utilized to create isogenic controls. This involves either correcting pathogenic variants in patient-derived iPSCs or introducing such variants into iPSC-derived from healthy individuals. Next, these iPSCs can be guided to differentiate into disease-relevant cell types, such as neurons and hepatocytes, using either 2D or 3D differentiation protocols. Multiple in vitro assays are then applied to these cell types, aiming to replicate disease phenotypes across functional, morphological, and biochemical aspects. Once a robust and reproducible in vitro disease phenotype is established, the drug discovery phase can commence.

Figure 3

Maintenance and induction of pluripotency. This figure illustrates the regulation of stem cell-like properties, often referred to as the “stemness” phenotype. In embryonic stem cells (ESCs), the network of transcription factors that maintain stemness is active, while the transcription factors that drive differentiation are inactive. By artificially activating the stemness transcription factor network in mature, specialized cells, we can induce a state of pluripotency, creating induced pluripotent stem cells (iPSCs). These iPSCs exhibit characteristics similar to those of ESCs. The transition to this pluripotent state is a dynamic process that can be triggered by various combinations of transcription factors. Notable examples include the Yamanaka factors (OCT4, SOX2, KLF4, and MYC) [20] and the Thomson factors (OCT4, SOX2, NANOG, and LIN28) [21].

Figure 4

Diagram detailing the methods for differentiating induced pluripotent stem cells (iPSCs) into brain and liver cells. (A) Method to differentiate iPSCs into brain cells, including neurons and astrocytes, using a combination of small molecules and both 2D and 3D cultures. The process involves various stages where specific markers are used to confirm cell identity: OCT4 and NANOG for the loss of pluripotency; SRY-Box Transcription Factor 1 (SOX1) and paired box 6 (PAX6) for early neuronal progenitors; T-Brain-1 (TBR1) and Special AT-rich sequence-binding protein 2 (SATB2) for cortical layer neuronal progenitors. Pan-neuronal markers like microtubule-associated protein 2 (MAP2), βIII-tubulin, and synaptophysin, along with postsynaptic density 95 (PSD95), are used to evaluate the health and structure of neuronal networks. Glial fibrillary acidic protein (GFAP) is used to identify astrocytes. (B) Differentiation of iPSCs into hepatocytes. Sex-determining region Y-box 17 (SOX17) and forkhead box A2 (FOXA2) serve as markers for the definitive endoderm stage, while hepatocyte nuclear factor 4 alpha (HNF4a), alpha-fetoprotein (AFP), albumin (ALB), and cytokeratin 18 (CK18) indicate hepatic specification. Mature hepatocytes are identified by their expression of enzymes like tryptophan-oxygenase, tyrosine amino-transferase, and various cytochrome enzymes. The diagram shows the estimated timeline for these processes, from induction (Day 0) to mature neurons (Day 50) and hepatocytes (Day 21), and assays used to evaluate the iPSC-derived progeny functionality.

Figure 5

Proposed human model systems to study SLC13A5 deficiency. (A) Patient samples with SLC13A5 deficiency are reprogrammed to create induced pluripotent stem cells (iPSCs) that retain the genetic characteristics of the disease, which are crucial for understanding the genetics affecting disease manifestation. Isogenic control lines, genetically identical except for the disease specific mutation, can be created using CRISPR gene editing prior to their differentiation into brain and liver cells. (B) of SLC13A5 iPSC-derived brain and liver cells can be used to examine the neurological aspects and metabolic consequences, as well as their potential interactions.