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L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodrugs Can Improve the Delivery of Drugs into Neurons, Astrocytes and Microglia.

ABSTRACT: L-Type Amino Acid Transporter 1 (LAT1/Lat1) is responsible for carrying large, neutral L-amino acids as well as several drugs and prodrugs across the blood-brain barrier (BBB). However, the BBB is not the only barrier that hinders drugs acting effectively within the brain; the brain parenchymal cell membranes represent a secondary barrier for the drugs with intracellular target sites. In this study, expression and function of Lat1 was quantified in mouse primary neuron, astrocyte and immortalized microglia (BV2) cultures. Moreover, ability of Lat1 to carry prodrugs inside these brain cells was evaluated. The results showed that Lat1 was localized at the similar level in all studied cells (3.07?±?0.92-3.77?±?0.91 fmol/µg protein). The transporter was also functional in all three cell types, astrocytes having the highest transport capacity and affinity for the LAT1/Lat1-substrate, [¹⁴C]-L-leucine, followed by neurons and microglia. The designed prodrugs (1-6) were able to utilize Lat1 for their cellular uptake and it was mainly much higher than the one of their parent drugs. Interestingly, improved cellular uptake was also achieved in cells representing Alzheimer's Disease phenotype. Therefore, improved delivery and intra-brain targeting of drugs can be attained by utilizing LAT1/Lat1 and prodrug approach.

SUBMITTER: Huttunen J

PROVIDER: S-EPMC6731241 | biostudies-literature | 2019 Sep

REPOSITORIES: biostudies-literature

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L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodrugs Can Improve the Delivery of Drugs into Neurons, Astrocytes and Microglia.

Huttunen Johanna J Peltokangas Soile S Gynther Mikko M Natunen Teemu T Hiltunen Mikko M Auriola Seppo S Ruponen Marika M Vellonen Kati-Sisko KS Huttunen Kristiina M KM

Scientific reports 20190906 1

L-Type Amino Acid Transporter 1 (LAT1/Lat1) is responsible for carrying large, neutral L-amino acids as well as several drugs and prodrugs across the blood-brain barrier (BBB). However, the BBB is not the only barrier that hinders drugs acting effectively within the brain; the brain parenchymal cell membranes represent a secondary barrier for the drugs with intracellular target sites. In this study, expression and function of Lat1 was quantified in mouse primary neuron, astrocyte and immortalize ...[more]

PMID: 31492955

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Project description:BackgroundCellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. In this study, we developed an in vivo tagging mouse model (Camk2a-NuTRAP) for paired isolation of neuronal DNA and RNA without cell sorting and then used this model to assess epigenomic regulation, DNA modifications in particular, of gene expression between neurons and glia.ResultsAfter validating the cell-specificity of the Camk2a-NuTRAP model, we performed TRAP-RNA-Seq and INTACT-whole genome oxidative bisulfite sequencing (WGoxBS) to assess the neuronal translatome and epigenome in the hippocampus of young mice (4 months old). WGoxBS findings were validated with enzymatic methyl-Seq (EM-Seq) and nanopore sequencing. Comparing neuronal data to microglial and astrocytic data from NuTRAP models, microglia had the highest global mCG levels followed by astrocytes and then neurons, with the opposite pattern observed for hmCG and mCH. Differentially modified regions between cell types were predominantly found within gene bodies and distal intergenic regions, rather than proximal promoters. Across cell types there was a negative correlation between DNA modifications (mCG, mCH, hmCG) and gene expression at proximal promoters. In contrast, a negative correlation of gene body mCG and a positive relationship between distal promoter and gene body hmCG with gene expression was observed. Furthermore, we identified a neuron-specific inverse relationship between mCH and gene expression across promoter and gene body regions.ConclusionsNeurons, astrocytes, and microglia demonstrate different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.

Dataset Information

L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodrugs Can Improve the Delivery of Drugs into Neurons, Astrocytes and Microglia.

Publications

L-Type Amino Acid Transporter 1 (LAT1/Lat1)-Utilizing Prodrugs Can Improve the Delivery of Drugs into Neurons, Astrocytes and Microglia.

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