UBAP2 contributes to radioresistance by enhancing homologous recombination through SLC27A5 ubiquitination in hepatocellular carcinoma.

Radiotherapy stands as an effective method in the clinical treatment of hepatocellular carcinoma (HCC) patients. However, both primary and acquired radioresistance limit its clinical application in HCC. Therefore, investigating the mechanism of radioresistance may provide other options for treating HCC. Based on single-cell RNA sequencing (scRNA-seq) and HCC transcriptome datasets, 227 feature genes with prognostic value were selected to establish the tSNE score. The tSNE score emerged as an independent prognostic factor for HCC and correlated with cell proliferation and radioresistance-related biological functions. UBAP2 was identified as the most relevant gene with the tSNE score, consistently elevated in human HCC samples, and positively associated with patient prognosis. Functionally, UBAP2 knockdown impeded HCC development and reduced radiation resistance in vitro and in vivo. The ectopic expression of SLC27A5 reversed the effects of UBAP2. Mechanically, we uncovered that UBAP2, through the ubiquitin-proteasome system, decreased the homologous recombination-related gene RAD51, not the non-homologous end-joining (NHEJ)-related gene CTIP, by degrading the antioncogene SLC27A5, thereby generating radioresistance in HCC. The findings recapitulated that UBAP2 promoted HCC progression and radioresistance via SLC27A5 stability mediated by the ubiquitin-proteasome pathway. It was also suggested that targeting the UBAP2/SLC27A5 axis could be a valuable radiosensitization strategy in HCC.

Molecular basis of inhibition of the amino acid transporter B0AT1 (SLC6A19).

The epithelial neutral amino acid transporter B0AT1 (SLC6A19) is the major transporter for the absorption of neutral amino acids in the intestine and their reabsorption in the kidney. Mouse models have demonstrated that lack of B0AT1 can normalize elevated plasma amino acids in rare disorders of amino acid metabolism such as phenylketonuria and urea-cycle disorders, implying a pharmacological approach for their treatment. Here we employ a medicinal chemistry approach to generate B0AT1 inhibitors with IC50-values of 31-90 nM. High-resolution cryo-EM structures of B0AT1 in the presence of two compounds from this series identified an allosteric binding site in the vestibule of the transporter. Mechanistically, binding of these inhibitors prevents a movement of TM1 and TM6 that is required for the transporter to make a conformational change from an outward open state to the occluded state.

Long-term effects of luteolin in a mouse model of nephropathic cystinosis.

In infantile nephropathic cystinosis, variants of the CTNS gene cause accumulation of cystine in lysosomes, causing progressive damage to most organs. Patients usually present before 1 year of age with signs of renal Fanconi syndrome. Cysteamine therapy allows cystine clearance from lysosomes and delays kidney damage but does not prevent progression to end-stage kidney disease, suggesting that pathways unrelated to cystine accumulation are also involved. Among these, impaired autophagy, altered endolysosomal trafficking, and increased apoptosis have emerged in recent years as potential targets for new therapies. We previously showed that luteolin, a flavonoid compound, improves these abnormal pathways in cystinotic cells and in zebrafish models of the disease. Herein, we have investigated if prolonged luteolin treatment ameliorates kidney damage in a murine model of cystinosis. To this end, we have treated Ctns-/- mice from 2 to 8 months with 150 mg/kg/day of luteolin. No significant side effects were observed. Compared to untreated animals, analyses of kidney cortex samples obtained after sacrifice showed that luteolin decreased p62/SQSTM1 levels (p <0.001), improved the number, size, and distribution of LAMP1-positive structures (p <0.02), and decreased tissue expression of cleaved caspase 3 (p <0.001). However, we did not observe improvements in renal Fanconi syndrome and kidney inflammation. Kidney function remained normal during the time of the study. These results indicate that luteolin has positive effects on the apoptosis and endo-lysosomal defects of cystinotic proximal tubular cells. However, these beneficial effects did not translate into improvement of renal Fanconi syndrome.

Hyperekplexia: A Single-Center Experience.

BACKGROUND: Hyperekplexia is a rare neurogenetic disorder that is classically characterized by an exaggerated startle response to sudden unexpected stimuli. This study aimed to determine clinical and genetic characteristics of our patients with hyperekplexia. METHODS: The age of onset and diagnosis, familial and perinatal history, clinical course, complications, metabolic screening tests, magnetic resonance imaging (MRI), medications, neuropsychometric evaluations, and gene mutations of patients diagnosed with hyperekplexia were reviewed retrospectively. RESULTS: All hyperekplexia patients had displayed neonatal excessive startle response and muscle stiffness, which we accepted as the major form of the disorder. Sixteen patients had mutations in genes associated with hyperekplexia. The ages at clinical diagnosis and genetic confirmation ranged from newborn to 16 years old and from 2.5 to 19 years, respectively. Nine patients (56.25%) were initially misdiagnosed with epilepsy. Seven patients (43.75%) carried a diagnosis of intellectual disability, defined here as a total IQ <80. Delayed gross motor development was detected in 4 patients (25%), and speech delay was reported in 3 (18.75%). Mutations in GLRA1 (NM_000171.4) and SLC6A5 (NM_004211.5) were identified in 13 (81.25%) and 3 patients (18.75%), respectively. Fifteen of the 16 patients (93.75%) showed autosomal recessive inheritance. Only 1 patient (6.25%) showed autosomal dominant inheritance. CONCLUSION: Although hyperekplexia is a potentially treatable disease, it can be complicated by delayed speech and/or motor acquisition and also by intellectual disability. This study shows that hyperekplexia is not always a benign condition and that all patients diagnosed with hyperekplexia should be evaluated for neuropsychiatric status and provided with genetic testing.

Structure and function of the SIT1 proline transporter in complex with the COVID-19 receptor ACE2.

Proline is widely known as the only proteogenic amino acid with a secondary amine. In addition to its crucial role in protein structure, the secondary amino acid modulates neurotransmission and regulates the kinetics of signaling proteins. To understand the structural basis of proline import, we solved the structure of the proline transporter SIT1 in complex with the COVID-19 viral receptor ACE2 by cryo-electron microscopy. The structure of pipecolate-bound SIT1 reveals the specific sequence requirements for proline transport in the SLC6 family and how this protein excludes amino acids with extended side chains. By comparing apo and substrate-bound SIT1 states, we also identify the structural changes that link substrate release and opening of the cytoplasmic gate and provide an explanation for how a missense mutation in the transporter causes iminoglycinuria.

MiR-21-5p modulates LPS-induced acute injury in alveolar epithelial cells by targeting SLC16A10.

Sepsis is a systemic inflammatory response syndrome resulting from the invasion of the human body by bacteria and other pathogenic microorganisms. One of its most prevalent complications is acute lung injury, which places a significant medical burden on numerous countries and regions due to its high morbidity and mortality rates. MicroRNA (miRNA) plays a critical role in the body's inflammatory response and immune regulation. Recent studies have focused on miR-21-5p in the context of acute lung injury, but its role appears to vary in different models of this condition. In the LPS-induced acute injury model of A549 cells, there is differential expression, but the specific mechanism remains unclear. Therefore, our aim is to investigate the changes in the expression of miR-21-5p and SLC16A10 in a type II alveolar epithelial cell injury model induced by LPS and explore the therapeutic effects of their targeted regulation. A549 cells were directly stimulated with 10 µg/ml of LPS to construct a model of LPS-induced cell injury. Cells were collected at different time points and the expression of interleukin 1 beta (IL-1ß), tumor necrosis factor-α (TNF-α) and miR-21-5p were measured by RT-qPCR and western blot. Then miR-21-5p mimic transfection was used to up-regulate the expression of miR-21-5p in A549 cells and the expression of IL-1ß and TNF-α in each group of cells was measured by RT-qPCR and western blot. The miRDB, TargetScan, miRWalk, Starbase, Tarbase and miR Tarbase databases were used to predict the miR-21-5p target genes and simultaneously, the DisGeNet database was used to search the sepsis-related gene groups. The intersection of the two groups was taken as the core gene. Luciferase reporter assay further verified SLC16A10 as the core gene with miR-21-5p. The expression of miR-21-5p and SLC16A10 were regulated by transfection or inhibitors in A549 cells with or without LPS stimulation. And then the expression of IL-1ß and TNF-α in A549 cells was tested by RT-qPCR and western blot in different groups, clarifying the role of miR-21-5p-SLC16A10 axis in LPS-induced inflammatory injury in A549 cells. (1) IL-1ß and TNF-α mRNA and protein expression significantly increased at 6, 12, and 24 h after LPS stimulation as well as the miR-21-5p expression compared with the control group (P < 0.05). (2) After overexpression of miR-21-5p in A549 cells, the expression of IL-1ß and TNF-α was significantly reduced after LPS stimulation, suggesting that miR-21-5p has a protection against LPS-induced injury. (3) The core gene set, comprising 51 target genes of miR-21-5p intersecting with the 1448 sepsis-related genes, was identified. This set includes SLC16A10, TNPO1, STAT3, PIK3R1, and FASLG. Following a literature review, SLC16A10 was selected as the ultimate target gene. Dual luciferase assay results confirmed that SLC16A10 is indeed a target gene of miR-21-5p. (4) Knocking down SLC16A10 expression by siRNA significantly reduced the expression of IL-1ß and TNF-α in A549 cells after LPS treatment (P < 0.05). (5) miR-21-5p inhibitor increased the expression levels of IL-1ß and TNF-α in A549 cells after LPS stimulation (P < 0.05). In comparison to cells solely transfected with miR-21-5p inhibitor, co-transfection of miR-21-5p inhibitor and si-SLC6A10 significantly reduced the expression of IL-1ß and TNF-α (P < 0.05). MiR-21-5p plays a protective role in LPS-induced acute inflammatory injury of A549 cells. By targeting SLC16A10, it effectively mitigates the inflammatory response in A549 cells induced by LPS. Furthermore, SLC16A10 holds promise as a potential target for the treatment of acute lung injury.

Residual Cystine Transport Activity for Specific Infantile and Juvenile CTNS Mutations in a PTEC-Based Addback Model.

Cystinosis is a rare, autosomal recessive, lysosomal storage disease caused by mutations in the gene CTNS, leading to cystine accumulation in the lysosomes. While cysteamine lowers the cystine levels, it does not cure the disease, suggesting that CTNS exerts additional functions besides cystine transport. This study investigated the impact of infantile and juvenile CTNS mutations with discrepant genotype/phenotype correlations on CTNS expression, and subcellular localisation and function in clinically relevant cystinosis cell models to better understand the link between genotype and CTNS function. Using CTNS-depleted proximal tubule epithelial cells and patient-derived fibroblasts, we expressed a selection of CTNSmutants under various promoters. EF1a-driven expression led to substantial overexpression, resulting in CTNS protein levels that localised to the lysosomal compartment. All CTNSmutants tested also reversed cystine accumulation, indicating that CTNSmutants still exert transport activity, possibly due to the overexpression conditions. Surprisingly, even CTNSmutants expression driven by the less potent CTNS and EFS promoters reversed the cystine accumulation, contrary to the CTNSG339R missense mutant. Taken together, our findings shed new light on CTNS mutations, highlighting the need for robust assessment methodologies in clinically relevant cellular models and thus paving the way for better stratification of cystinosis patients, and advocating for the development of more personalized therapy.

MFSD12 depletion reduces cystine accumulation without improvement in proximal tubular function in experimental models for cystinosis.

Cystinosis is an autosomal recessive lysosomal storage disorder, caused by mutations in the CTNS gene, resulting in an absent or altered cystinosin (CTNS) protein. Cystinosin exports cystine out of the lysosome, with a malfunction resulting in cystine accumulation and a defect in other cystinosin-mediated pathways. Cystinosis is a systemic disease, but the kidneys are the first and most severely affected organs. In the kidney, the disease initially manifests as a generalized dysfunction in the proximal tubules (also called renal Fanconi syndrome). MFSD12 is a lysosomal cysteine importer that directly affects the cystine levels in melanoma cells, HEK293T cells, and cystinosis patient-derived fibroblasts. In this study, we aimed to evaluate MFSD12 mRNA levels in cystinosis patient-derived proximal tubular epithelial cells (ciPTECs) and to study the effect of MFSD12 knockout on cystine levels. We showed similar MFSD12 mRNA expression in patient-derived ciPTECs in comparison with the control cells. CRISPR MFSD12 knockout in a patient-derived ciPTEC (CTNSΔ57kb) resulted in significantly reduced cystine levels. Furthermore, we evaluated proximal tubular reabsorption after injection of mfsd12a translation-blocking morpholino (TB MO) in a ctns-/- zebrafish model. This resulted in decreased cystine levels but caused a concentration-dependent increase in embryo dysmorphism. Furthermore, the mfsd12a TB MO injection did not improve proximal tubular reabsorption or megalin expression. In conclusion, MFSD12 mRNA depletion reduced cystine levels in both tested models without improvement of the proximal tubular function in the ctns-/- zebrafish embryo. In addition, the apparent toxicity of higher mfsd12a TB MO concentrations on the zebrafish development warrants further evaluation.NEW & NOTEWORTHY In this study, we show that MFSD12 depletion with either CRISPR/Cas9-mediated gene editing or a translation-blocking morpholino significantly reduced cystine levels in cystinosis ciPTECs and ctns-/- zebrafish embryos, respectively. However, we observed no improvement in the proximal tubular reabsorption of dextran in the ctns-/- zebrafish embryos injected with mfsd12a translation-blocking morpholino. Furthermore, a negative effect of the mfsd12a morpholino on the zebrafish development warrants further investigation.

SFXN1-mediated immune cell infiltration and tumorigenesis in lung adenocarcinoma: A potential therapeutic target.

BACKGROUND: Sideroflexin 1 (SFXN1), a mitochondrial serine transporter implicated in one-carbon metabolism, is a prognostic biomarker in lung adenocarcinoma (LUAD). However, its role in LUAD progression remains elusive. This study aimed to investigate the functional significance of SFXN1 in LUAD and evaluate its potential as a therapeutic target. METHODS: We analyzed SFXN1 expression and its diagnostic and prognostic value in LUAD using the Pan-cancer TCGA dataset. In vitro assays (CCK-8, cell cycle, EDU, wound-healing, and transwell) were employed to assess the role of SFXN1, complemented by in vivo experiments. RNA sequencing elucidated SFXN1-mediated cellular functions and potential mechanisms. Bulk RNA-seq and scRNA-seq data from TCGA and GEO were used to investigate the correlation between SFXN1 and the tumor immune microenvironment. RT-qPCR, Western blot, and IHC assays validated SFXN1 expression and its impact on the immune microenvironment in LUAD. RESULTS: SFXN1 was upregulated in LUAD tissues and associated with poor prognosis. RNA-seq and scRNA-seq analyses revealed increased SFXN1 expression in tumor cells, accompanied by decreased infiltration of NK and cytotoxic T cells. SFXN1 knockdown significantly reduced cell proliferation and migration, and the inhibition of ERK phosphorylation and CCL20 expression may be the molecular mechanism involved. In vivo, targeting SFXN1 decreased Tregs infiltration and inhibited tumor growth. CONCLUSIONS: Our findings suggest that SFXN1 may be a potential therapeutic target for LUAD treatment.

The Differential Effect of a Shortage of Thyroid Hormone Compared with Knockout of Thyroid Hormone Transporters Mct8 and Mct10 on Murine Macrophage Polarization.

Innate immune cells, including macrophages, are functionally affected by thyroid hormone (TH). Macrophages can undergo phenotypical alterations, shifting between proinflammatory (M1) and immunomodulatory (M2) profiles. Cellular TH concentrations are, among others, determined by TH transporters. To study the effect of TH and TH transporters on macrophage polarization, specific proinflammatory and immunomodulatory markers were analyzed in bone marrow-derived macrophages (BMDMs) depleted of triiodothyronine (T3) and BMDMs with a knockout (KO) of Mct8 and Mct10 and a double KO (dKO) of Mct10/Mct8. Our findings show that T3 is important for M1 polarization, while a lack of T3 stimulates M2 polarization. Mct8 KO BMDMs are unaffected in their T3 responsiveness, but exhibit slight alterations in M2 polarization, while Mct10 KO BMDMs show reduced T3 responsiveness, but unaltered polarization markers. KO of both the Mct8 and Mct10 transporters decreased T3 availability and, contrary to the T3-depleted BMDMs, showed partially increased M1 markers and unaltered M2 markers. These data suggest a role for TH transporters besides transport of TH in BMDMs. This study highlights the complex role of TH transporters in macrophages and provides a new angle on the interaction between the endocrine and immune systems.

Omic Studies on In Vitro Cystinosis Model: siRNA-Mediated CTNS Gene Silencing in HK-2 Cells.

Cystinosis is an autosomal recessive disease caused by mutations in the CTNS gene encoding a protein called cystinosine, which is a lysosomal cystine transporter. Disease-causing mutations lead to accumulation of cystine crystals in the lysosomes, thereby causing dysfunction of vital organs. Determination of the increased leukocyte cystine level is one of the most used methods for diagnosis. However, this method is expensive, difficult to perform, and may yield different results in different laboratories. In this study, a disease model was created with CTNS gene-silenced HK2 cells, which can mimic cystinosis in cell culture, and multiomics methods (ie, proteomics, metabolomics, and fluxomics) were implemented at this cell culture to investigate new biomarkers for the diagnosis. CTNS-silenced cell line exhibited distinct metabolic profiles compared with the control cell line. Pathway analysis highlighted significant alterations in various metabolic pathways, including alanine, aspartate, and glutamate metabolism; glutathione metabolism; aminoacyl-tRNA biosynthesis; arginine and proline metabolism; beta-alanine metabolism; ascorbate and aldarate metabolism; and histidine metabolism upon CTNS silencing. Fluxomics analysis revealed increased cycle rates of Krebs cycle intermediates such as fumarate, malate, and citrate, accompanied by enhanced activation of inorganic phosphate and ATP production. Furthermore, proteomic analysis unveiled differential expression levels of key proteins involved in crucial cellular processes. Notably, peptidyl-prolyl cis-trans isomerase A, translation elongation factor 1-beta (EF-1beta), and 60S acidic ribosomal protein decreased in CTNS-silenced cells. Additionally, levels of P0 and tubulin α-1A chain were reduced, whereas levels of 40S ribosomal protein S8 and Midasin increased. Overall, our study, through the utilization of an in vitro cystinosis model and comprehensive multiomics approach, led to the way toward the identification of potential new biomarkers while offering valuable insights into the pathogenesis of cystinosis.

Functional analysis of the CTNS gene exonic variants predicted to affect splicing.

Cystinosis is a severe, monogenic systemic disease caused by variants in CTNS gene. Currently, there is growing evidence that exonic variants in many diseases can affect pre-mRNA splicing. The impact of CTNS gene exonic variants on splicing regulation may be underestimated due to the lack of routine studies at the RNA level. Here, we analyzed 59 exonic variants in the CTNS gene using bioinformatics tools and identified candidate variants that may induce splicing alterations by minigene assays. We identified six exonic variants that induce splicing alterations by disrupting the ratio of exonic splicing enhancers/exonic splicing silencers (ESEs/ESSs) or by interfering with the recognition of classical splice sites, or both. Our results help in the correct molecular characterization of variants in cystinosis and inform emerging therapies. Furthermore, our work suggests that the combination of in silico and in vitro assays facilitates to assess the effects of DNA variants driving rare genetic diseases on splicing regulation and will enhance the clinical utility of variant functional annotation.

Extrarenal complications of cystinosis.

Cystinosis is a rare autosomal recessive disease with an incidence 1 per 100,000-200,000 live births. It is caused by pathogenic variants of the cystinosin (CTNS) gene that lead to impaired cystine transport from lysosomes to cystosol, resulting in cystine accumulation in lysosomes and subsequent cellular dysfunction. The initial manifestation, cystine accumulation in proximal tubular cells (PTCs), causes renal Fanconi syndrome, which presents with proximal renal tubular acidosis and generalized dysfunction of the proximal tubule, including the presence of polyuria, glycosuria, phosphaturia, aminoaciduria, tubular proteinuria, growth retardation, and rickets. Eventually, glomerular involvement, glomerular proteinuria, focal segmental glomerulosclerosis (FSGS), and progression to kidney failure occur. Although the kidneys are the first organs affected, and play a key role in morbidity and mortality, extrarenal multiorgan involvement can occur in patients with cystinosis, which is seen not only in adults but in early ages in untreated patients, patients with insufficient treatment, and in those that don't comply with treatment. The treatment of cystinosis consists of supportive treatment for Fanconi syndrome, and specific lifelong cystine-depleting therapy using oral cysteamine. There is strong evidence that as early as possible, initiation and ongoing appropriate therapy with cysteamine are essential for delaying the progression to kidney failure, end-organ damage, and extrarenal involvement. The present review aimed to evaluate the extra renal complications of cystinosis.

Neurologic involvement in cystinosis: Focus on brain lesions and new evidence of four-repeat (4R-) Tau immunoreactivity.

Nephropathic cystinosis is a rare autosomal recessive storage disorder caused by CTNS gene mutations, leading to autophagy-lysosomal pathway impairment and cystine crystals accumulation. Neurologic involvement is highly variable and includes both neurodevelopmental and neurodegenerative disturbances, as well as focal neurologic deficits. By presenting longitudinal data of a 28-year-old patient with a large infratentorial lesion, we summarized the pathology, clinical and imaging features of neurological involvement in cystinosis patients. Brain damage in form of cystinosis-related cerebral lesions occurs in advanced disease phases and is characterized by the accumulation of cystine crystals, subsequent inflammation with vasculitis-like features, necrosis, and calcification. Epilepsy is a frequent comorbidity in affected individuals. Steroids might play a role in the symptomatic treatment of "stroke-like" episodes due to edematous-inflammatory lesions, but probably do not change the overall prognosis. Lifelong compliance to depleting therapy with cysteamine still represents the main therapeutic option. However, consequences of CTNS gene defects are not restricted to cystine accumulation. New evidence of four-repeat (4R-) Tau immunoreactivity suggests concurrent progressive neurodegeneration in cystinosis patients, highlighting the need of innovative therapeutic strategies, and shedding light on the crosstalk between proteinopathies and autophagy-lysosomal system defects. Eventually, emerging easily accessible biomarkers such as serum neurofilament light chains (NfL) might detect subclinical neurologic involvement in cystinosis patients.

Event-related potential (ERP) evidence for visual processing differences in children and adults with cystinosis (CTNS gene mutations).

BACKGROUND: Cystinosis, a rare lysosomal storage disease caused by mutations in the CTNS gene, is characterized by cystine crystallization and accumulation within multiple tissues, including kidney and brain. Its impact on neural function appears mild relative to its effects on other organs during early disease, but since therapeutic advances have led to substantially increased life expectancy, neurological implications are of increasing interest, necessitating deeper understanding of the impact of cystinosis on neurocognitive function. Behavioral difficulties have been reported in cystinosis in the visual domain. Very little is known, however, about how the brains of people living with cystinosis process visual information. This is especially interesting given that cystine accumulation in the cornea and posterior ocular structures is a hallmark of cystinosis. METHODS: Here, high-density scalp electrophysiology was recorded to visual stimuli (during a Go/No-Go task) to investigate visual processing in individuals with cystinosis, compared to age-matched controls. Analyses focused on early stages of cortical visual processing. RESULTS: The groups differed in their initial cortical response, with individuals with cystinosis exhibiting a significantly larger visual evoked potential (VEP) in the 130-150 ms time window. The groups also differed in the associations between neural responses and verbal abilities: While controls with higher IQ scores presented larger neural responses, that relationship was not observed in cystinosis. CONCLUSIONS: The enlarged VEP in cystinosis could be the result of cortical hyperexcitability and/or differences in attentional engagement and explain, at least partially, the visual and visual-spatial difficulties described in this population.

Evaluation of the efficacy of cystinosin supplementation through CTNS mRNA delivery in experimental models for cystinosis.

Messenger RNA (mRNA) therapies are emerging in different disease areas, but have not yet reached the kidney field. Our aim was to study the feasibility to treat the genetic defect in cystinosis using synthetic mRNA in cell models and ctns-/- zebrafish embryos. Cystinosis is a prototype lysosomal storage disorder caused by mutations in the CTNS gene, encoding the lysosomal cystine-H+ symporter cystinosin, and leading to cystine accumulation in all cells of the body. The kidneys are the first and the most severely affected organs, presenting glomerular and proximal tubular dysfunction, progressing to end-stage kidney failure. The current therapeutic standard cysteamine, reduces cystine levels, but has many side effects and does not restore kidney function. Here, we show that synthetic mRNA can restore lysosomal cystinosin expression following lipofection into CTNS-/- kidney cells and injection into ctns-/- zebrafish. A single CTNS mRNA administration decreases cellular cystine accumulation for up to 14 days in vitro. In the ctns-/- zebrafish, CTNS mRNA therapy improves proximal tubular reabsorption, reduces proteinuria, and restores brush border expression of the multi-ligand receptor megalin. Therefore, this proof-of-principle study takes the first steps in establishing an mRNA-based therapy to restore cystinosin expression, resulting in cystine reduction in vitro and in the ctns-/- larvae, and restoration of the zebrafish pronephros function.

The SLC6A15-SLC6A20 Neutral Amino Acid Transporter Subfamily: Functions, Diseases, and Their Therapeutic Relevance.

The neutral amino acid transporter subfamily that consists of six members, consecutively SLC6A15-SLC620, also called orphan transporters, represents membrane, sodium-dependent symporter proteins that belong to the family of solute carrier 6 (SLC6). Primarily, they mediate the transport of neutral amino acids from the extracellular milieu toward cell or storage vesicles utilizing an electric membrane potential as the driving force. Orphan transporters are widely distributed throughout the body, covering many systems; for instance, the central nervous, renal, or intestinal system, supplying cells into molecules used in biochemical, signaling, and building pathways afterward. They are responsible for intestinal absorption and renal reabsorption of amino acids. In the central nervous system, orphan transporters constitute a significant medium for the provision of neurotransmitter precursors. Diseases related with aforementioned transporters highlight their significance; SLC6A19 mutations are associated with metabolic Hartnup disorder, whereas altered expression of SLC6A15 has been associated with a depression/stress-related disorders. Mutations of SLC6A18-SLCA20 cause iminoglycinuria and/or hyperglycinuria. SLC6A18-SLC6A20 to reach the cellular membrane require an ancillary unit ACE2 that is a molecular target for the spike protein of the SARS-CoV-2 virus. SLC6A19 has been proposed as a molecular target for the treatment of metabolic disorders resembling gastric surgery bypass. Inhibition of SLC6A15 appears to have a promising outcome in the treatment of psychiatric disorders. SLC6A19 and SLC6A20 have been suggested as potential targets in the treatment of COVID-19. In this review, we gathered recent advances on orphan transporters, their structure, functions, related disorders, and diseases, and in particular their relevance as therapeutic targets. SIGNIFICANCE STATEMENT: The following review systematizes current knowledge about the SLC6A15-SLCA20 neutral amino acid transporter subfamily and their therapeutic relevance in the treatment of different diseases.

A Slc38a8 Mouse Model of FHONDA Syndrome Faithfully Recapitulates the Visual Deficits of Albinism Without Pigmentation Defects.

Purpose: We aimed to generate and phenotype a mouse model of foveal hypoplasia, optic nerve decussation defects, and anterior segment dysgenesis (FHONDA), a rare disease associated with mutations in Slc38a8 that causes severe visual alterations similar to albinism without affecting pigmentation. Methods: The FHONDA mouse model was generated with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology using an RNA guide targeting the Scl38a8 murine locus. The resulting mice were backcrossed to C57BL/6J. Melanin content was measured using spectrophotometry. Retinal cell architecture was analyzed through light and electron microscopy. Retinal projections to the brain were evaluated with anterograde labelling in embryos and adults. Visual function was assessed by electroretinography (ERG) and the optomotor test (OT). Results: From numerous Slc38a8 mouse mutant alleles generated, we selected one that encodes a truncated protein (p.196Pro*, equivalent to p.199Pro* in the human protein) closely resembling a mutant allele described in patients (p.200Gln*). Slc38a8 mutant mice exhibit wild-type eye and coat pigmentation with comparable melanin content. Subcellular abnormalities were observed in retinal pigment epithelium cells of Slc38a8 mutant mice. Anterograde labeling experiments of retinal projections in embryos and adults showed a reduction of ipsilateral fibers. Functional visual analyses revealed a decreased ERG response in scotopic conditions and a reduction of visual acuity in mutant mice measured by OT. Conclusions: Slc38a8 mutant mice recapitulate the phenotype of patients with FHONDA concerning their normal pigmentation and their abnormal visual system, in the latter being a hallmark of all types of albinism. These mice will be helpful in better understanding the pathophysiology of this genetic condition.

Lysosomal cystine export regulates mTORC1 signaling to guide kidney epithelial cell fate specialization.

Differentiation is critical for cell fate decisions, but the signals involved remain unclear. The kidney proximal tubule (PT) cells reabsorb disulphide-rich proteins through endocytosis, generating cystine via lysosomal proteolysis. Here we report that defective cystine mobilization from lysosomes through cystinosin (CTNS), which is mutated in cystinosis, diverts PT cells towards growth and proliferation, disrupting their functions. Mechanistically, cystine storage stimulates Ragulator-Rag GTPase-dependent recruitment of mechanistic target of rapamycin complex 1 (mTORC1) and its constitutive activation. Re-introduction of CTNS restores nutrient-dependent regulation of mTORC1 in knockout cells, whereas cell-permeant analogues of L-cystine, accumulating within lysosomes, render wild-type cells resistant to nutrient withdrawal. Therapeutic mTORC1 inhibition corrects lysosome and differentiation downstream of cystine storage, and phenotypes in preclinical models of cystinosis. Thus, cystine serves as a lysosomal signal that tailors mTORC1 and metabolism to direct epithelial cell fate decisions. These results identify mechanisms and therapeutic targets for dysregulated homeostasis in cystinosis.

The SLC38A5/SNAT5 amino acid transporter: from pathophysiology to pro-cancer roles in the tumor microenvironment.

SLC38A5/SNAT5 is a system N transporter that can mediate net inward or outward transmembrane fluxes of neutral amino acids coupled with Na+ (symport) and H+ (antiport). Its preferential substrates are not only amino acids with side chains containing amide (glutamine and asparagine) or imidazole (histidine) groups, but also serine, glycine, and alanine are transported by the carrier. Expressed in the pancreas, intestinal tract, brain, liver, bone marrow, and placenta, it is regulated at mRNA and protein levels by mTORC1 and WNT/ß-catenin pathways, and it is sensitive to pH, nutritional stress, inflammation, and hypoxia. SNAT5 expression has been found to be altered in pathological conditions such as chronic inflammatory diseases, gestational complications, chronic metabolic acidosis, and malnutrition. Growing experimental evidence shows that SNAT5 is overexpressed in several types of cancer cells. Moreover, recently published results indicate that SNAT5 expression in stromal cells can support the metabolic exchanges occurring in the tumor microenvironment of asparagine-auxotroph tumors. We review the functional role of the SNAT5 transporter in pathophysiology and propose that, due to its peculiar operational and regulatory features, SNAT5 may play important pro-cancer roles when expressed either in neoplastic or in stromal cells of glutamine-auxotroph tumors.NEW & NOTEWORTHY The transporter SLC38A5/SNAT5 provides net influx or efflux of glutamine, asparagine, and serine. These amino acids are of particular metabolic relevance in several conditions. Changes in transporter expression or activity have been described in selected types of human cancers, where SNAT5 can mediate amino acid exchanges between tumor and stromal cells, thus providing a potential therapeutic target. This is the first review that recapitulates the characteristics and roles of the transporter in physiology and pathology.