TAT-MeCP2 protein variants rescue disease phenotypes in human and mouse models of Rett syndrome.

Rett syndrome (RTT) is a neurodevelopmental disorder caused by pathogenic variants leading to functional impairment of the MeCP2 protein. Here, we used purified recombinant MeCP2e1 and MeCP2e2 protein variants fused to a TAT protein transduction domain (PTD) to evaluate their transduction ability into RTT patient-derived fibroblasts and the ability to carry out their cellular function. We then assessed their transduction ability and therapeutic effects in a RTT mouse model. In vitro, TAT-MeCP2e2-eGFP reversed the pathological hyperacetylation of histones H3K9 and H4K16, a hallmark of abolition of MeCP2 function. In vivo, intraperitoneal administration of TAT-MeCP2e1 and TAT-MeCP2e2 extended the lifespan of Mecp2-/y mice by >50%. This was accompanied by rescue of hippocampal CA2 neuron size in animals treated with TAT-MeCP2e1. Taken together, these findings provide a strong indication that recombinant TAT-MeCP2 can reach mouse brains following peripheral injection and can ameliorate the phenotype of RTT mouse models. Thus, our study serves as a first step in the development of a potentially novel RTT therapy.

TPP2 mutation associated with sterile brain inflammation mimicking MS.

OBJECTIVE: To ascertain the genetic cause of a consanguineous family from Syria suffering from a sterile brain inflammation mimicking a mild nonprogressive form of MS. METHODS: We used homozygosity mapping and next-generation sequencing to detect the disease-causing gene in the affected siblings. In addition, we performed RNA and protein expression studies, enzymatic activity assays, immunohistochemistry, and targeted sequencing of further MS cases from Austria, Germany, Canada and Jordan. RESULTS: In this study, we describe the identification of a homozygous missense mutation (c.82T>G, p.Cys28Gly) in the tripeptidyl peptidase II (TPP2) gene in all 3 affected siblings of the family. Sequencing of all TPP2-coding exons in 826 MS cases identified one further homozygous missense variant (c.2027C>T, p.Thr676Ile) in a Jordanian MS patient. TPP2 protein expression in whole blood was reduced in the affected siblings. In contrast, TPP2 protein expression in postmortem brain tissue from MS patients without TPP2 mutations was highly upregulated. CONCLUSIONS: The homozygous TPP2 mutation (p.Cys28Gly) is likely responsible for the inflammation phenotype in this family. TPP2 is an ubiquitously expressed serine peptidase that removes tripeptides from the N-terminal end of longer peptides. TPP2 is involved in various biological processes including the destruction of major histocompatibility complex Class I epitopes. Recessive loss-of-function mutations in TPP2 were described in patients with Evans syndrome, a rare autoimmune disease affecting the hematopoietic system. Based on the gene expression results in our MS autopsy brain samples, we further suggest that TPP2 may play a broader role in the inflammatory process in MS.

Molecular dissection of dual pseudosymmetric solute translocation pathways in human P-glycoprotein.

The human multispecific drug efflux transporter P-glycoprotein (P-gp) causes drug resistance and modulates the pharmacological profile of systemically administered medicines. It has arisen from a homodimeric ancestor by gene duplication. Crystal structures of mouse MDR1A indicate that P-gp shares the overall architecture with two homodimeric bacterial exporters, Sav1866 and MsbA, which have complete rotational symmetry. For ATP-binding cassette transporters, nucleotide binding occurs in two symmetric positions in the motor domains. Based on the homology with entirely symmetric half-transporters, the present study addressed the key question: can biochemical evidence for the existence of dual drug translocation pathways in the transmembrane domains of P-gp be found? P-gp was photolabeled with propafenone analogs, purified, and digested proteolytically, and peptide fragments were identified by high-resolution mass spectrometry. Labeling was assigned to two regions in the protein by projecting data into homology models. Subsequently, symmetric residue pairs in the putative translocation pathways were identified and replaced by site-directed mutagenesis. Transport assays corroborated the existence of two pseudosymmetric translocation pathways. Although rhodamine123 has a preference to take one path, verapamil, propafenones, and vinblastine preferentially use the other. Two major findings ensued from this study: the existence of two solute translocation pathways in P-gp as a reflection of evolutionary origin from a homodimeric ancestor and selective but not exclusive use of one of these pathways by different P-gp solutes. The pseudosymmetric behavior reconciles earlier kinetic and thermodynamic data, suggesting an alternative concept of drug transport by P-gp that will aid in understanding the off-target quantitative structure activity relationships of P-gp interacting drugs.

The SYBR Green I malaria drug sensitivity assay: performance in low parasitemia samples.

Validation of the sensitivity of the SYBR Green I in vitro test against an enzyme-linked immunosorbent assay (ELISA)-based drug sensitivity assay. Our results suggest that the SYBR Green I assay is a fast and inexpensive malaria drug screening assay for laboratory use. However, because of its lack of sensitivity in whole blood samples its usefulness for testing clinical samples may be limited.