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Abstract Background Mutations in the SLC5A7 gene cause congenital myasthenia, a rare genetic disorder. Mutation points in the SLC5A7 gene differ among individuals and encompass various genetic variations; however, exon deletion variants have yet to be reported in related cases. This study aims to explore the clinical phenotype and genetic traits of a patient with congenital myasthenic syndrome due to SLC5A7 gene variation and those of their family members. Case presentation We describe a case of a Chinese male with congenital myasthenic syndrome presenting fluctuating limb weakness. Genetic testing revealed a heterozygous deletion mutation spanning exons 1–9 in the SLC5A7 gene. QPCR confirmed a deletion in exon 9 of the SLC5A7 gene in the patient’s mother and brother. Clinical symptoms of myasthenia improved following treatment with pyridostigmine. Conclusion Exons 1, 5, and 9 of the SLC5A7 gene encode the choline transporter’s transmembrane region. Mutations in these exons can impact the stability and plasma membrane levels of the choline transporter. Thus, a heterozygous deletion in exons 1–9 of the SLC5A7 gene could be the pathogenic cause for this patient. In patients exhibiting fluctuating weakness, positive RNS, and seronegativity for myasthenia gravis antibodies, a detailed family history should be considered, and enhanced genetic testing is recommended to determine the cause.

Background: Mutations in the SLC5A7 gene cause congenital myasthenia, a rare genetic disorder. Mutation points in the SLC5A7 gene differ among individuals and encompass various genetic variations; however, exon deletion variants have yet to be reported in related cases. This study aims to explore the clinical phenotype and genetic traits of a patient with congenital myasthenic syndrome due to SLC5A7 gene variation and those of their family members. Case presentation: We describe a case of a Chinese male with congenital myasthenic syndrome presenting fluctuating limb weakness. Genetic testing revealed a heterozygous deletion mutation spanning exons 1–9 in the SLC5A7 gene. QPCR confirmed a deletion in exon 9 of the SLC5A7 gene in the patient's mother and brother. Clinical symptoms of myasthenia improved following treatment with pyridostigmine. Conclusion: Exons 1, 5, and 9 of the SLC5A7 gene encode the choline transporter's transmembrane region. Mutations in these exons can impact the stability and plasma membrane levels of the choline transporter. Thus, a heterozygous deletion in exons 1–9 of the SLC5A7 gene could be the pathogenic cause for this patient. In patients exhibiting fluctuating weakness, positive RNS, and seronegativity for myasthenia gravis antibodies, a detailed family history should be considered, and enhanced genetic testing is recommended to determine the cause. [ABSTRACT FROM AUTHOR]

MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA) and intracellular choline levels, but it is independent of gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells. [ABSTRACT FROM AUTHOR]

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium- dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis–Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 μM and a Vmax of 61.0 pmol/oocyte/ h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is thought to be a series of neuroinflammatory diseases caused by abnormal deposits of amyloid-β (Aβ) and tau protein in the brain as part of its etiology. We focused on Aβ aggregation and M1 and M2 microglial polarity in microglia to search for novel therapeutic agents. It has been reported that the inhibition of choline uptake via choline transporter-like protein 1 (CTL1) in microglia preferentially induces M2 microglial polarity. However, the role of the choline transport system on the regulation of microglial M1/M2 polarity in AD is not fully understood. Licochalcones (Licos) A–E, flavonoids extracted from licorice, have been reported to have immunological anti-inflammatory effects, and Lico A inhibits Aβ aggregation. In this study, we compared the efficacy of five Licos, from Lico A to E, at inhibiting Aβ1-42 aggregation. Among the five Licos, Lico E was selected to investigate the relationship between the inhibition of choline uptake and microglial M1/M2 polarization using the immortalized mouse microglial cell line SIM-A9. We newly found that Lico E inhibited choline uptake and Aβ1-42 aggregation in SIM-A9 cells in a concentration-dependent manner, suggesting that the inhibitory effect of Lico E on choline uptake is mediated by CTL1. The mRNA expression of tumor necrosis factor (TNF-α), a marker of M1 microglia, was increased by Aβ1-42, and its effect was inhibited by choline deprivation and Lico E in a concentration-dependent manner. In contrast, the mRNA expression of arginase-1 (Arg-1), a marker of M2 microglia, was increased by IL-4, and its effect was enhanced by choline deprivation and Lico E. We found that Lico E has an inhibitory effect on Aβ aggregation and promotes polarity from M1 to M2 microglia via inhibition of the CTL1 function in microglia. Thus, Lico E may become a leading compound for a novel treatment of AD. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high‐affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis.

Background: Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in microglia, and its association with microglial M1/M2 polarization. Methods: The immortalized mouse microglial cell line SIM-A9 was used for [3H]choline uptake and expression analysis of choline transporters. The association between the choline uptake system and the M1/M2 polarization of microglia was also analyzed. Results: Choline transporter-like protein (CTL) 1 and CTL2 were highly expressed in SIM-A9 cells, and CTL1 and CTL2 were localized in the plasma membrane and mitochondria, respectively. Functional analysis of choline uptake demonstrated the existence of Na+-independent, pH-dependent, and intermediate-affinity choline transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3), an inhibitor of choline uptake, and increased by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Expression of the mRNA of M1 microglia markers IL-1β and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 (Arg-1) was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. Conclusions: Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases. [ABSTRACT FROM AUTHOR]

Transport of choline via the neuronal high-affinity choline transporter (CHT; SLC5A7) is essential for cholinergic terminals to synthesize and release acetylcholine (ACh). In humans, we previously demonstrated an association between a common CHT coding substitution (rs1013940; Ile89Val) and reduced attentional control as well as attenuated frontal cortex activation. Here, we used a CRISPR/Cas9 approach to generate mice expressing the I89V substitution and assessed, in vivo, CHT-mediated choline transport, and ACh release. Relative to wild-type (WT) mice, CHT-mediated clearance of choline in male and female mice expressing one or two Val89 alleles was reduced by over 80% in cortex and over 50% in striatum. Choline clearance in CHT Val89 mice was further reduced by neuronal inactivation. Deficits in ACh release, 5 and 10min after repeated depolarization at a low, behaviorally relevant frequency, support an attenuated reloading capacity of cholinergic neurons in mutant mice. The density of CHTs in total synaptosomal lysates and neuronal plasma-membrane-enriched fractions was not impacted by the Val89 variant, indicating a selective impact on CHT function. When challenged with a visual disruptor to reveal attentional control mechanisms, Val89 mice failed to adopt a more conservative response bias. Structural modeling revealed that Val89 may attenuate choline transport by altering conformational changes of CHT that support normal transport rates. Our findings support the view that diminished sustained cholinergic signaling capacity underlies perturbed attentional performance in individuals expressing CHT Val89. The CHT Val89 mouse serves as a valuable model to study heritable risk for cognitive disorders arising from cholinergic dysfunction. [ABSTRACT FROM AUTHOR]

Acetylcholine (ACh), an excitatory neurotransmitter, is biosynthesized from choline in cholinergic neurons. Import from the extracellular space to the intracellular environment through the high‐affinity choline transporter is currently regarded to be the only source of choline for ACh synthesis. We recently demonstrated that the P2X2 receptor, through which large cations permeate, functions as an alternative pathway for choline transport in the mouse retina. In the present study, we investigated whether choline entering cells through P2X2 receptors is used for ACh synthesis using a recombinant system. When P2X2 receptors expressed on HEK293 cell lines were stimulated with ATP, intracellular ACh concentrations increased. These results suggest that P2X2 receptors function in a novel pathway that supplies choline for ACh synthesis. [ABSTRACT FROM AUTHOR]

Summary: Maize kernel weight is influenced by the unloading of nutrients from the maternal placenta and their passage through the transfer tissue of the basal endosperm transfer layer (BETL) and the basal intermediate zone (BIZ) to the upper part of the endosperm.Here, we show that Small kernel 10 (Smk10) encodes a choline transporter‐like protein 1 (ZmCTLP1) that facilitates choline uptake and is located in the trans‐Golgi network (TGN). Its loss of function results in reduced choline content, leading to smaller kernels with a lower starch content.Mutation of ZmCTLP1 disrupts membrane lipid homeostasis and the normal development of wall in‐growths. Expression levels of Mn1 and ZmSWEET4c, two kernel filling‐related genes, are downregulated in the smk10, which is likely to be one of the major causes of incompletely differentiated transfer cells. Mutation of ZmCTLP1 also reduces the number of plasmodesmata (PD) in transfer cells, indicating that the smk10 mutant is impaired in PD formation. Intriguingly, we also observed premature cell death in the BETL and BIZ of the smk10 mutant.Together, our results suggest that ZmCTLP1‐mediated choline transport affects kernel development, highlighting its important role in lipid homeostasis, wall in‐growth formation and PD development in transfer cells. [ABSTRACT FROM AUTHOR]

Alzheimer’s disease (AD) is thought to be a series of neuroinflammatory diseases caused by abnormal deposits of amyloid-β (Aβ) and tau protein in the brain as part of its etiology. We focused on Aβ aggregation and M1 and M2 microglial polarity in microglia to search for novel therapeutic agents. It has been reported that the inhibition of choline uptake via choline transporter-like protein 1 (CTL1) in microglia preferentially induces M2 microglial polarity. However, the role of the choline transport system on the regulation of microglial M1/M2 polarity in AD is not fully understood. Licochalcones (Licos) A–E, flavonoids extracted from licorice, have been reported to have immunological anti-inflammatory effects, and Lico A inhibits Aβ aggregation. In this study, we compared the efficacy of five Licos, from Lico A to E, at inhibiting Aβ1-42 aggregation. Among the five Licos, Lico E was selected to investigate the relationship between the inhibition of choline uptake and microglial M1/M2 polarization using the immortalized mouse microglial cell line SIM-A9. We newly found that Lico E inhibited choline uptake and Aβ1-42 aggregation in SIM-A9 cells in a concentration-dependent manner, suggesting that the inhibitory effect of Lico E on choline uptake is mediated by CTL1. The mRNA expression of tumor necrosis factor (TNF-α), a marker of M1 microglia, was increased by Aβ1-42, and its effect was inhibited by choline deprivation and Lico E in a concentration-dependent manner. In contrast, the mRNA expression of arginase-1 (Arg-1), a marker of M2 microglia, was increased by IL-4, and its effect was enhanced by choline deprivation and Lico E. We found that Lico E has an inhibitory effect on Aβ aggregation and promotes polarity from M1 to M2 microglia via inhibition of the CTL1 function in microglia. Thus, Lico E may become a leading compound for a novel treatment of AD.

Background: Microglia are key cells of the immune system in the central nervous system and are suggested to be deeply involved in the development of neurodegenerative diseases. It is well known that microglia have functional plasticity, with an inflammatory M1 phenotype and an anti-inflammatory M2 phenotype. Inhibition of choline transport in macrophages has been reported to suppress the secretion of inflammatory cytokines. However, the role of the choline transport system in regulating microglial M1/M2 polarization has not been fully elucidated to date. In this study, we investigated the mechanism of choline uptake in microglia, and its association with microglial M1/M2 polarization. Methods: The immortalized mouse microglial cell line SIM-A9 was used for [3H]choline uptake and expression analysis of choline transporters. The association between the choline uptake system and the M1/M2 polarization of microglia was also analyzed. Results: Choline transporter-like protein (CTL) 1 and CTL2 were highly expressed in SIM-A9 cells, and CTL1 and CTL2 were localized in the plasma membrane and mitochondria, respectively. Functional analysis of choline uptake demonstrated the existence of Na+-independent, pH-dependent, and intermediate-affinity choline transport systems. Choline uptake was concentration-dependently inhibited by hemicholinium-3 (HC-3), an inhibitor of choline uptake, and increased by lipopolysaccharide (LPS) and interleukin-4 (IL-4). Expression of the mRNA of M1 microglia markers IL-1β and IL-6 was increased by LPS, and their effects were suppressed by choline deprivation and HC-3. In contrast, mRNA expression of the M2 microglial marker arginase-1 (Arg-1) was increased by IL-4, and the effect was enhanced by choline deprivation and HC-3. Conclusions: Our results suggest that inhibition of CTL1-mediated choline uptake in microglia preferentially induces M2 microglia polarization, which is a potential therapeutic approach for inflammatory brain diseases.

Choline is used to synthesize phospholipids and a lack of choline induces a number of liver-related diseases, including non-alcoholic steatohepatitis. The current study characterized the choline uptake system, at molecular and functional levels, in the immortalized human hepatic cell line, Fa2N-4, to identify the specific choline transporter involved in choline uptake. The present study also assesed whether choline deficiency or the inhibited choline uptake affected cell viability and apoptosis. Reverse transcription-quantitative polymerase chain reaction (PCR) revealed choline transporter-like protein 1 (CTL1) and CTL2 mRNA and protein expression in Fa2N-4 cells. [Methyl-3H]choline studies revealed choline uptake was saturable and mediated by a single transport system that functioned in a Na+-independent but pH-dependent manner, which was similar to CTL1. Hemicholinium-3 (HC-3), which is a choline uptake inhibitor, and choline deficiency inhibited cell viability, increased caspase-3 and −7 activities, and increased fluorescein isothiocyanate-Annexin V immunofluorescent staining indicated apoptosis. Immunofluorescent staining also revealed CTL1 and CTL2 localized in plasma and mitochondrial membranes, respectively. [Methyl-3H]choline uptake was enhanced by a protein kinase C (PKC) activator, phorbol-12-myristate 13-acetate (PMA). Immunofluorescence staining and western blot analysis demonstrated increased CTL1 expression on the cell membrane following PMA treatment. The results of current study indicated that extracellular choline is primarily transported via CTL1, relying on a direct H+ gradient that functions as a driving force in Fa2N-4 cells. Furthermore, it was hypothesized that CTL1 and the choline uptake system are strongly associated with cell survival, and that the choline uptake system is modulated by PKC signaling via increased CTL1 expression on the cell surface. These findings provide further insights into the pathogenesis of liver disease involving choline metabolism. [ABSTRACT FROM AUTHOR]

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance. [ABSTRACT FROM AUTHOR]

Diagnosis or exclusion of Hirschsprung disease (HSCR) is a frequent exercise in any pediatric hospital. Although HSCR may present at different ages and with varied clinical findings, the most common presentation is a neonate with severe constipation or signs of intestinal obstruction. A variety of diagnostic tests including contrast enema and anorectal manometry may be used as diagnostic screens, but diagnosis ultimately rests upon histopathological evaluation of a rectal biopsy. For the experienced pathologist, conventional hematoxylin-and-eosin-stained sections often suffice to exclude HSCR or establish the diagnosis. However, ancillary diagnostic tests such as acetylcholinesterase histochemistry or calretinin immunohistochemistry are complementary and extremely helpful in some cases. In this Perspectives article, we review the clinical and pathological features of HSCR, highlight those that are found in most patients, and discuss how to address particularly challenging aspects of the diagnostic workup. [ABSTRACT FROM AUTHOR]

MFSD7b belongs to the Major Facilitator Superfamily of transporters that transport small molecules. Two isoforms of MFSD7b have been identified and they are reported to be heme exporters that play a crucial role in maintaining the cytosolic and mitochondrial heme levels, respectively. Mutations of MFSD7b (also known as FLVCR1) have been linked to retinitis pigmentosa, posterior column ataxia, and hereditary sensory and autonomic neuropathy. Although MFSD7b functions have been linked to heme detoxification by exporting excess heme from erythroid cells, it is ubiquitously expressed with a high level in the kidney, gastrointestinal tract, lungs, liver, and brain. Here, we showed that MFSD7b functions as a facilitative choline transporter. Expression of MFSD7b slightly but significantly increased choline import, while its knockdown reduced choline influx in mammalian cells. The influx of choline transported by MFSD7b is dependent on the expression of choline metabolizing enzymes such as choline kinase (CHKA) and intracellular choline levels, but it is independent of gradient of cations. Additionally, we showed that choline transport function of Mfsd7b is conserved from fly to man. Employing our transport assays, we showed that missense mutations of MFSD7b caused reduced choline transport functions. Our results show that MFSD7b functions as a facilitative choline transporter in mammalian cells., (© 2023. The Author(s), under exclusive licence to Springer Nature Switzerland AG.)

Previous research emphasized the impact of traumatic brain injury on cholinergic systems and associated cognitive functions. Here we addressed the converse question: Because of the available evidence indicating cognitive and neuronal vulnerabilities in humans expressing low-capacity cholinergic systems or with declining cholinergic systems, do injuries cause more severe cognitive decline in such subjects, and what cholinergic mechanisms contribute to such vulnerability? Using mice heterozygous for the choline transporter (CHT+/- mice) as a model for a limited cholinergic capacity, we investigated the cognitive and neuronal consequences of repeated, mild concussion injuries (rmCc). After five rmCc, and compared with wild type (WT) mice, CHT+/- mice exhibited severe and lasting impairments in sustained attention performance, consistent with effects of cholinergic losses on attention. However, rmCc did not affect the integrity of neuronal cell bodies and did not alter the density of cortical synapses. As a cellular mechanism potentially responsible for the attentional impairment in CHT+/- mice, we found that rmCc nearly completely attenuated performance-associated, CHT-mediated choline transport. These results predict that subjects with an already vulnerable cholinergic system will experience severe and lasting cognitive-cholinergic effects after even relatively mild injuries. If confirmed in humans, such subjects may be excluded from, or receive special protection against, activities involving injury risk. Moreover, the treatment and long-term outcome of traumatic brain injuries may benefit from determining the status of cholinergic systems and associated cognitive functions. [ABSTRACT FROM AUTHOR]

Abstract Insect mushroom bodies (MB) have an ensemble of synaptic connections well-studied for their role in experience-dependent learning and several higher cognitive functions. MB requires neurotransmission for an efficient flow of information across synapses with different flexibility to meet the demand of the dynamically changing environment of an insect. Neurotransmitter transporters coordinate appropriate changes for an efficient neurotransmission at the synapse. Till date, there is no transporter reported for any of the previously known neurotransmitters in the intrinsic neurons of MB. In this study, we report a highly enriched expression of Choline Transporter (ChT) in Drosophila MB. We demonstrate that knockdown of ChT in a sub-type of MB neurons called α/β core (α/βc) and ϒ neurons leads to eclosion failure, peristaltic defect in larvae, and altered NMJ phenotype. These defects were neither observed on knockdown of proteins of the cholinergic locus in α/βc and ϒ neurons nor by knockdown of ChT in cholinergic neurons. Thus, our study provides insights into non-canonical roles of ChT in MB. Highlights • Choline transporter is highly enriched in Drosophila mushroom body. • Choline transporter in α/β core and ϒ neurons of mushroom body regulates eclosion. • ChT in α/β core and ϒ neurons of the mushroom body maintains NMJ integrity. • This study provides insights into the non-canonical role of ChT in MB. [ABSTRACT FROM AUTHOR]

Cerebral choline metabolism is crucial for normal brain function, and its homoeostasis depends on carrier-mediated transport. Here, we report on four individuals from three families with neurodegenerative disease and homozygous frameshift mutations (Asp517Metfs*19, Ser126Metfs*8, and Lys90Metfs*18) in the SLC44A1 gene encoding choline transporter-like protein 1. Clinical features included progressive ataxia, tremor, cognitive decline, dysphagia, optic atrophy, dysarthria, as well as urinary and bowel incontinence. Brain MRI demonstrated cerebellar atrophy and leukoencephalopathy. Moreover, low signal intensity in globus pallidus with hyperintensive streaking and low signal intensity in substantia nigra were seen in two individuals. The Asp517Metfs*19 and Ser126Metfs*8 fibroblasts were structurally and functionally indistinguishable. The most prominent ultrastructural changes of the mutant fibroblasts were reduced presence of free ribosomes, the appearance of elongated endoplasmic reticulum and strikingly increased number of mitochondria and small vesicles. When chronically treated with choline, those characteristics disappeared and mutant ultrastructure resembled healthy control cells. Functional analysis revealed diminished choline transport yet the membrane phosphatidylcholine content remained unchanged. As part of the mechanism to preserve choline and phosphatidylcholine, choline transporter deficiency was implicated in impaired membrane homeostasis of other phospholipids. Choline treatments could restore the membrane lipids, repair cellular organelles and protect mutant cells from acute iron overload. In conclusion, we describe a novel childhood-onset neurometabolic disease caused by choline transporter deficiency with autosomal recessive inheritance. [ABSTRACT FROM AUTHOR]

Choline and choline metabolites are essential for all cellular functions. They have also been reported to be crucial for neural development. In this work, we studied the functional characteristics of the choline uptake system in human neural stem cells (hNSCs). Additionally, we investigated the effect of extracellular choline uptake inhibition on the cellular activities in hNSCs. We found that the mRNAs and proteins of choline transporter-like protein 1 (CTL1) and CTL2 were expressed at high levels. Immunostaining showed that CTL1 and CTL2 were localized in the cell membrane and partly in the mitochondria, respectively. The uptake of extracellular choline was saturable and performed by a single uptake mechanism, which was Na+-independent and pH-dependent. We conclude that CTL1 is responsible for extracellular choline uptake, and CTL2 may uptake choline in the mitochondria and be involved in DNA methylation via choline oxidation. Extracellular choline uptake inhibition caused intracellular choline deficiency in hNSCs, which suppressed cell proliferation, cell viability, and neurite outgrowth. Our findings contribute to the understanding of the role of choline in neural development as well as the pathogenesis of various neurological diseases caused by choline deficiency or choline uptake impairment.

Clonorchis sinensis is commonly found in East Asian countries. Clonorchiasis is prevalent in these countries and can lead to various clinical symptoms. In this study, we used overlap extension polymerase chain reaction (PCR) and the Xenopus laevis oocyte expression system to isolate a cDNA encoding the choline transporter of C. sinensis (CsChT). We subsequently characterized recombinant CsChT. Expression of CsChT in X. laevis oocytes enabled efficient transport of radiolabeled choline, with no detectable uptake of arginine, α-ketoglutarate, p-aminohippurate, taurocholate, and estrone sulfate. Influx and efflux experiments showed that CsChT-mediated choline uptake was time- and sodium-dependent, with no exchange properties. Concentration-dependent analyses of revealed saturable kinetics consistent with the Michaelis-Menten equation, while nonlinear regression analyses revealed a Km value of 8.3 μM and a Vmax of 61.0 pmol/oocyte/h. These findings contribute to widen our understanding of CsChT transport properties and the cascade of choline metabolisms within C. sinensis.

Inhibition of the CDP-choline pathway during apoptosis restricts the availability of phosphatidylcholine (PtdCho) for assembly of membranes and synthesis of signaling factors. The N-terminal nuclear localization signal (NLS) in CTP:phosphocholine cytidylyltransferase (CCT)α is removed during apoptosis but the caspase(s) involved and the contribution to suppression of the CDP-choline pathway is unresolved. In this study we utilized siRNA silencing of caspases in HEK293 cells and caspase 3-deficient MCF7 cells to show that caspase 3 is required for CCTα proteolysis and release from the nucleus during apoptosis. CCTα-Δ28 (a caspase-cleaved mimic) expressed in CCTα-deficient Chinese hamster ovary cells was cytosolic and had increased in vitro activity. However, [3H]choline labeling experiments in camptothecin-treated MCF7 cells and MCF7 cells expressing caspase 3 (MCF7-C3) revealed a global suppression of the CDP-choline pathway that was consistent with inhibition of a step prior to CCTα. In camptothecin-treated MCF7 and MCF7-C3 cells, choline kinase activity was unaffected; however, choline transport into cells was reduced by 30 and 60%, respectively. We conclude that caspase 3-mediated removal of the CCTα NLS contributes minimally to the inhibition of PtdCho synthesis during DNA damage-induced apoptosis. Rather, the CDP-choline pathway is inhibited by caspase 3-independent and -dependent suppression of choline transport into cells.

The synaptic uptake of choline via the high-affinity, hemicholinium-3-dependent choline transporter (CHT) strongly influences the capacity of cholinergic neurons to sustain acetylcholine (ACh) synthesis and release. To advance research on the impact of CHT capacity in humans, we established the presence of the neuronal CHT protein in human T lymphocytes. Next, we demonstrated CHT-mediated choline transport in human T cells. To address the validity of T cell-based choline uptake as a proxy for brain CHT capacity, we isolated T cells from the spleen, and synaptosomes from cortex and striatum, of wild type and CHT-overexpressing mice (CHT-OXP). Choline uptake capacity in T cells from CHT-OXP mice was two-fold higher than in wild type mice, mirroring the impact of CHT over-expression on synaptosomal CHT-mediated choline uptake. Monitoring T lymphocyte CHT protein and activity may be useful for estimating human CNS cholinergic capacity and for testing hypotheses concerning the contribution of CHT and, more generally, ACh signaling in cognition, neuroinflammation and disease. [ABSTRACT FROM AUTHOR]

Acetylcholinesterase enzymatic histochemistry (AChE EHC), which highlights abnormal cholinergic nerves in the mucosa of aganglionic bowel, has been used for decades to evaluate rectal biopsies for Hirschsprung disease (HSCR). While useful diagnostically, AChE EHC is not compatible with conventional formalin-fixed and paraffin-embedded (FFPE) tissues and is not widely available. The choline transporter (ChT) is a putative alternative marker of cholinergic nerves. ChT immunohistochemistry (IHC) was investigated using FFPE biopsies and resections from patients with confirmed HSCR, as well as appropriate non-HSCR controls. ChT immunostaining was effective at identifying cases with HSCR and qualitatively similar to AChE EHC on frozen section. Among 3 pathologists, the diagnostic positive and negative predictive values based on ChT IHC ranged from 0.84-0.94 and 0.85-0.89, respectively, with good inter-observer agreement (Cohen kappa=0.70-0.90). ChT IHC was useful in unusual scenarios in which calretinin (CR) IHC failed to correctly identify patients with HSCR. In 10 cases of short-segment HSCR, abnormal ChTþ mucosal innervation was present through the entire aganglionic segment and into portions of the TZ with submucosal nerve hypertrophy. In contrast, mucosal CR IHC was retained in the TZ and adjacent aganglionic bowel, which could lead to misinterpretation of a biopsy as ganglionic bowel. Indeed, 6 such patients were identified with paradoxical CR-positive mucosal innervation in their diagnostic biopsies. ChT IHC was interpreted as unequivocal HSCR in these cases, and HSCR was confirmed on resection. In summary, ChT IHC in FFPE tissue demonstrates high positive and negative predictive values for HSCR, is superior to CR IHC in a subset of cases, and can be incorporated into routine practice without the need for specialized techniques. [ABSTRACT FROM AUTHOR]

Some rats [sign-trackers (STs)] are prone to attribute incentive salience to reward cues, which can manifest as a propensity to approach and contact pavlovian cues, and for addiction-like behavior. STs also exhibit poor attentional performance, relative to goal-trackers (GTs), which is associated with attenuated acetylcholine (ACh) levels in prefrontal cortex (Paolone et al., 2013). Here, we demonstrate a cellular mechanism, linked to ACh synthesis, that accounts for attenuated cholinergic capacity in STs. First, we found that electrical stimulation of the basal forebrain increased cortical choline transporter (CHT)-mediated choline transport in GTs, paralleled by a redistribution of CHTs to the synaptic plasma membrane. Neither increases in choline uptake nor translocation of CHTs occurred in STs. Second, and consistent with uptake/translocation alterations, STs demonstrated a reduced ability to support cortical ACh release in vivo compared with GTs after reverse-dialysis to elevate extracellular potassium levels. Third, rats were significantly more likely to develop sign-tracking behavior if treated systemically before pavlovian conditioned approach training with the CHT inhibitor VU6001221. Consistent with its proposed mechanisms, administration of VU6001221 attenuated potassium-evoked ACh levels in prefrontal cortex measured with in vivo microdialysis. We propose that loss of CHT-dependent activation of cortical cholinergic activity in STs degrades top-down executive control over behavior, producing a bias for bottom-up or stimulus-driven attention. Such an attentional bias contributes to nonadaptive reward processing and thus identifies a novel mechanism that can support psychopathology, including addiction. [ABSTRACT FROM AUTHOR]

Sign-tracking (ST) describes the propensity to approach and contact a Pavlovian reward cue. By contrast, goal-trackers (GTs) respond to such a cue by retrieving the reward. These behaviors index the presence of opponent cognitive-motivational traits, with STs exhibiting attentional control deficits, behavior dominated by incentive motivational processes, and vulnerability for addictive drug taking. Attentional control deficits in STs were previously attributed to attenuated cholinergic signaling, resulting from deficient translocation of intracellular choline transporters (CHTs) into synaptosomal plasma membrane. Here, we investigated a posttranslational modification of CHTs, poly-ubiquitination, and tested the hypothesis that elevated cytokine signaling in STs contributes to CHT modification. We demonstrated that intracellular CHTs, but not plasma membrane CHTs, are highly ubiquitinated in male and female sign-tracking rats when compared with GTs. Moreover, levels of cytokines measured in cortex and striatum, but not spleen, were higher in STs than in GTs. Activation of the innate immune system by systemic administration of the bacterial endotoxin lipopolysaccharide (LPS) elevated ubiquitinated CHT levels in cortex and striatum of GTs only, suggesting ceiling effects in STs. In spleen, LPS increased levels of most cytokines in both phenotypes. In cortex, LPS particularly robustly increased levels of the chemokines CCL2 and CXCL10. Phenotype-specific increases were restricted to GTs, again suggesting ceiling effects in STs. These results indicate that interactions between elevated brain immune modulator signaling and CHT regulation are essential components of the neuronal underpinnings of the addiction vulnerability trait indexed by sign-tracking., Competing Interests: The authors declare no competing financial interests., (Copyright © 2023 Carmon et al.)

The efficient import of choline into cholinergic nerve terminals by the presynaptic, high-affinity choline transporter (CHT, SLC5A7) dictates the capacity for acetylcholine (ACh) synthesis and release. Tissue levels of ACh are significantly reduced in mice heterozygous for a loss of function mutation in Slc5a7 (HET, CHT +/- ), but significantly elevated in overexpressing, Slc5a7 BAC-transgenic mice (BAC). Since the readily-releasable pool of ACh is thought to constitute a small fraction of the total ACh pool, these genotype-dependent changes raised the question as to whether CHT expression or activity might preferentially influence the size of reserve pool ACh vesicles. In the current study, we approached this question by evaluating CHT genotype effects on the release of ACh from suprafused mouse forebrain slices. We treated slices from HET, BAC or wildtype (WT) controls with elevated K + and monitored release of both newly synthesized and storage pools of ACh. Newly synthesized ACh produced following uptake of [ 3 H]choline was quantified by scintillation spectrometry whereas release of endogenous ACh storage pools was quantified by an HPLC-MS approach, from the same samples. Whereas endogenous ACh release scaled with CHT gene dosage, preloaded [ 3 H]ACh release displayed no significant genotype dependence. Our findings suggest that CHT protein levels preferentially impact the capacity for ACh release afforded by mobilization of reserve pool vesicles. [ABSTRACT FROM AUTHOR]

The basal forebrain cholinergic projection system to the cortex mediates essential aspects of visual attention performance, including the detection of cues and the response to performance challenges (top-down control of attention). Higher levels of top-down control are mediated via elevated levels of cholinergic neuromodulation. The neuronal choline transporter (CHT) strongly influences the synthesis and release of acetylcholine (ACh). As the capacity of the CHT to import choline into the neuron is a major, presynaptic determinant of cholinergic neuromodulation, we hypothesize that genetically-imposed CHT capacity variation impacts the balance of bottom-up versus top-down control of visual attention. Following a brief review of the cognitive concepts relevant for this hypothesis, we describe the key results from our research in mice and humans that possess genetically-imposed changes in choline uptake capacity. CHT subcapacity is associated with poor top-down attentional control and attenuated (cholinergic) activation of right frontal regions. Conversely, mice overexpressing the CHT, and humans expressing a CHT variant hypothesized to enhance choline transporter function, are relatively resistant to challenges of visual attention performance. Genetic or environmental modulation of CHT expression and function may be associated with vulnerabilities for cognitive disorders. [ABSTRACT FROM AUTHOR]

Schizophrenia (SZ) is a psychiatric disorder characterized by cognitive dysfunction within the realm of attentional processing. Reduced P3a and P3b event-related potentials (ERPs), indexing involuntary and voluntary attentional processing respectively, have been consistently observed in SZ patients who also express prominent cholinergic deficiencies. The involvement of the brain’s cholinergic system in attention has been examined for several decades; however, further inquiry is required to further comprehend how abnormalities in this system affect neighbouring neurotransmitter systems and contribute to neurocognitive deficits. The objective of this pilot study was to examine the moderating role of the CHRNA4 (rs1044396), CHRNA7 (rs3087454), and SLC5A7 (rs1013940) genes on ERP indices of attentional processing in healthy volunteers ( N = 99; Caucasians and non-Caucasians) stratified by genotype and assessed using the auditory P300 “oddball” paradigm. Results indicated significantly greater P3a and P3b-indexed attentional processing for CT (vs. CC) CHRNA4 carriers and greater P3b for AA (vs. CC) CHRNA7 carriers. SLC5A7 allelic variants did not show significant differences in P3a and P3b processing. These findings expand our knowledge on the moderating effect of cholinergic genes on attention and could help inform targeted drug developments aimed at restoring attention deficits in SZ patients. [ABSTRACT FROM AUTHOR]