TMEM106B reduction does not rescue GRN deficiency in iPSC-derived human microglia and mouse models.

Heterozygous mutations in the granulin (GRN) gene are a leading cause of frontotemporal lobar degeneration with TDP-43 aggregates (FTLD-TDP). Polymorphisms in TMEM106B have been associated with disease risk in GRN mutation carriers and protective TMEM106B variants associated with reduced levels of TMEM106B, suggesting that lowering TMEM106B might be therapeutic in the context of FTLD. Here, we tested the impact of full deletion and partial reduction of TMEM106B in mouse and iPSC-derived human cell models of GRN deficiency. TMEM106B deletion did not reverse transcriptomic or proteomic profiles in GRN-deficient microglia, with a few exceptions in immune signaling markers. Neither homozygous nor heterozygous Tmem106b deletion normalized disease-associated phenotypes in Grn -/-mice. Furthermore, Tmem106b reduction by antisense oligonucleotide (ASO) was poorly tolerated in Grn -/-mice. These data provide novel insight into TMEM106B and GRN function in microglia cells but do not support lowering TMEM106B levels as a viable therapeutic strategy for treating FTD-GRN.

Structure and conserved function of iso-branched sphingoid bases from the nematode Caenorhabditis elegans.

Sphingolipids are bio-active metabolites that show structural diversity among eukaryotes. They are essential for growth of all eukaryotic cells but when produced in an uncontrolled manner can lead to cell death and pathologies including auto-immune reactions, cancer, diabetes and neurodegeneration. Caenorhabditis elegans is an important genetic model organism both to find new drug-targets against parasitic nematodes and to study the conserved roles of sphingolipids in animals like their essential functions in very basic cellular processes ranging from maintenance of cell polarity and mitochondrial repair to growth and survival. C. elegans produces sphingoid bases which are structurally distinct from those of other animals as both iso- and anteiso-branched species have been reported. Using metabolic labeling we show that most worm sphingoid bases are iso-branched. We have synthesized the nematode-specific C17 iso-branched sphinganine and its 1-deoxy analogue and could show that both the iso-branch and the 1-hydroxyl group are essential to form functional nematode sphingolipids which are needed to maintain intestinal function. The organism specificity was examined by complementation experiments in Saccharomyces cerevisiae yeast cells lacking sphingoid base synthesis. We found that iso-branched sphingoid base did not support growth of mutant cells and was toxic to wild type yeast. 1-Deoxy sphingolipids have been linked to the hereditary disease HSAN1A and other metabolic disorders including diabetes. We found that in C. elegans the 1-deoxy analogue cannot rescue the intestinal phenotype caused by sphingoid base depletion. In fact, in wild-type animals with normal sphingoid base biosynthesis, exogenous 1-deoxy analogue had a disruptive effect on apical cytoskeletal organization of intestinal cells indicating that atypical bases can interfere with normal sphingolipid function.

Exit-strategies - smart ways to release phospholipid vesicle cargo.

This highlight describes recent trends in fundamental phospholipid research towards possible future drug delivery technology. In particular it focuses on synthetic phospholipids and their vesicular constructs and describes selected "smart" ways to release cargo from liposomes. Various chemical and physical release triggers are discussed such as temperature changes, application of ultrasound, enzyme degradation, changes in pH, redox reactions, photochemical reactions, as well as the effects of shear stress on vesicles.

Synthesis and biological evaluation of non-isomerizable analogues of Ala-tRNA(Ala).

Aminoacyl-tRNAs serve as amino acid donors in many reactions in addition to protein synthesis by the ribosome, including synthesis of the peptidoglycan network in the cell wall of bacterial pathogens. Synthesis of analogs of aminoacylated tRNAs is critical to further improve the mechanism of these reactions. Here we have described the synthesis of two non-isomerizable analogues of Ala-tRNA(Ala) containing an amide bond instead of the isomerizable ester that connects the amino acid with the terminal adenosine in the natural substrate. The non-isomerizable 2' and 3' regioisomers were not used as substrates by FemX(Wv), an alanyl-transferase essential for peptidoglycan synthesis, but inhibited this enzyme with IC50 of 5.8 and 5.5 µM, respectively.

Efficient access to peptidyl-RNA conjugates for picomolar inhibition of non-ribosomal FemX(Wv) aminoacyl transferase.

Peptidyl-RNA conjugates have various applications in studying the ribosome and enzymes participating in tRNA-dependent pathways such as Fem transferases in peptidoglycan synthesis. Herein a convergent synthesis of peptidyl-RNAs based on Huisgen-Sharpless cycloaddition for the final ligation step is developed. Azides and alkynes are introduced into tRNA and UDP-MurNAc-pentapeptide, respectively. Synthesis of 2'-azido RNA helix starts from 2'-azido-2'-deoxyadenosine that is coupled to deoxycytidine by phosphoramidite chemistry. The resulting dinucleotide is deprotected and ligated to a 22-nt RNA helix mimicking the acceptor arm of Ala-tRNA(Ala) by T4 RNA ligase. For alkyne UDP-MurNAc-pentapeptide, meso-cystine is enzymatically incorporated into the peptidoglycan precursor and reduced, and L-Cys is converted to dehydroalanine with O-(mesitylenesulfonyl)hydroxylamine. Reaction of but-3-yne-1-thiol with dehydroalanine affords the alkyne-containing UDP-MurNAc-pentapeptide. The Cu(I)-catalyzed azide alkyne cycloaddition reaction in the presence of tris[(1-hydroxypropyl-1H-1,2,3-triazol-4-yl)methyl]amine provided the peptidyl-RNA conjugate, which was tested as an inhibitor of non-ribosomal FemX(Wv) aminoacyl transferase. The bi-substrate analogue was found to inhibit FemX(Wv) with an IC(50) of (89±9) pM, as both moieties of the peptidyl-RNA conjugate contribute to high-affinity binding.

Synthesis of stable aminoacyl-tRNA analogs.

Aminoacyl-tRNAs have important roles in a variety of biological processes. Here, we describe the synthesis of stable aminoacyl-tRNA analogs containing 1,4-substituted 1,2,3-triazole rings. The procedure involves (i) copper-catalyzed cycloadditions of 3'-or 2'-azido-adenosine and alkynes, (ii) coupling between the resulting triazole-deoxyadenosine derivatives and a deoxycytidine phosphoramidite, and (iii) the enzymatic ligation of the 2'- or 3'-triazole-dinucleotides with a 22-nt RNA microhelix that mimics the acceptor arm of tRNA. Each nucleoside and nucleotide intermediate was characterized by MS spectrometry and (1)H, (31)P, and (13)C NMR spectroscopy, and the tRNA-analogs were assayed for inhibition of FemXWv, an alanyl-transferase essential for the formation of the peptidoglycan network of Gram-positive bacterial pathogens. The low IC(50) values obtained (2 to 4 µM) indicate that the five-membered triazole rings acted as an isosteres of esters and can be used for the design of stable aminoacyl-tRNA analogs.