The CNS in inbred transgenic models of 4-repeat Tauopathy develops consistent tau seeding capacity yet focal and diverse patterns of protein deposition

Background

MAPT mutations cause neurodegenerative diseases such as frontotemporal dementia but, strikingly, patients with the same mutation may have different clinical phenotypes.

Methods

Given heterogeneities observed in a transgenic (Tg) mouse line expressing low levels of human (2 N, 4R) P301L Tau, we backcrossed founder stocks of mice to C57BL/6Tac, 129/SvEvTac and FVB/NJ inbred backgrounds to discern the role of genetic versus environmental effects on disease-related phenotypes.

Results

Three inbred derivatives of a TgTau^P301L founder line had similar quality and steady-state quantity of Tau production, accumulation of abnormally phosphorylated 64–68 kDa Tau species from 90 days of age onwards and neuronal loss in aged Tg mice. Variegation was not seen in the pattern of transgene expression and seeding properties in a fluorescence-based cellular assay indicated a single “strain” of misfolded Tau. However, in other regards, the aged Tg mice were heterogeneous; there was incomplete penetrance for Tau deposition despite maintained transgene expression in aged animals and, for animals with Tau deposits, distinctions were noted even within each subline. Three classes of rostral deposition in the cortex, hippocampus and striatum accounted for 75% of pathology-positive mice yet the mean ages of mice scored as class I, II or III were not significantly different and, hence, did not fit with a predictable progression from one class to another defined by chronological age. Two other patterns of Tau deposition designated as classes IV and V, occurred in caudal structures. Other pathology-positive Tg mice of similar age not falling within classes I-V presented with focal accumulations in additional caudal neuroanatomical areas including the locus coeruleus. Electron microscopy revealed that brains of Classes I, II and IV animals all exhibit straight filaments, but with coiled filaments and occasional twisted filaments apparent in Class I. Most strikingly, Class I, II and IV animals presented with distinct western blot signatures after trypsin digestion of sarkosyl-insoluble Tau.

Conclusions

Qualitative variations in the neuroanatomy of Tau deposition in genetically constrained slow models of primary Tauopathy establish that non-synchronous, focal events contribute to the pathogenic process. Phenotypic diversity in these models suggests a potential parallel to the phenotypic variation seen in P301L patients.

     

Synthesis and biological activity of novel tert-amylphenoxyalkyl (homo)piperidine derivatives as histamine H3R ligands

As a continuation of our search for novel histamine H₃ receptor ligands a series of twenty new tert-amyl phenoxyalkylamine derivatives (2–21) was synthesized. Compounds of four to eight carbon atoms spacer alkyl chain were evaluated on their binding properties at human histamine H₃ receptor (hH₃R). The highest affinities were observed for pentyl derivatives 6–8 (K_i = 8.8–23.4 nM range) and among them piperidine derivative 6 with K_i = 8.8 nM. Structures 6, 7 were also classified as antagonists in cAMP accumulation assay (with EC₅₀ = 157 and 164 nM, respectively). Moreover, new compounds were also evaluated for anticonvulsant activity in Antiepileptic Screening Program (ASP) at National Institute of Neurological Disorders and Stroke (USA). Seven compounds (2–4, 9, 11, 12 and 20) showed anticonvulsant activity at maximal electroshock (MES) test in the dose of 30 mg/kg at 0.5 h. In the subcutaneous pentetrazole (scMET) test compound 4 showed protection at 100 and 300 mg/kg dose at mice, however compounds showed high neurotoxicity in rotarod test at used doses. Also, molecular modeling studies were undertaken, to explain affinity of compounds at hH₃R (taking into the consideration X-ray analysis of compound 18). In order to estimate “drug-likeness” of selected compounds in silico and experimental evaluation of lipophilicity, metabolic stability and cytotoxicity was performed.     

The metalloprotease SepA governs processing of accumulation‐associated protein and shapes intercellular adhesive surface properties in Staphylococcus epidermidis

The otherwise harmless skin inhabitant Staphylococcus epidermidis is a major cause of healthcare‐associated medical device infections. The species' selective pathogenic potential depends on its production of surface adherent biofilms. The Cell wall‐anchored protein Aap promotes biofilm formation in S. epidermidis, independently from the polysaccharide intercellular adhesin PIA. Aap requires proteolytic cleavage to act as an intercellular adhesin. Whether and which staphylococcal proteases account for Aap processing is yet unknown. Here, evidence is provided that in PIA‐negative S. epidermidis 1457Δica, the metalloprotease SepA is required for Aap‐dependent S. epidermidis biofilm formation in static and dynamic biofilm models. qRT‐PCR and protease activity assays demonstrated that under standard growth conditions, sepA is repressed by the global regulator SarA. Inactivation of sarA increased SepA production, and in turn augmented biofilm formation. Genetic and biochemical analyses demonstrated that SepA‐related induction of biofilm accumulation resulted from enhanced Aap processing. Studies using recombinant proteins demonstrated that SepA is able to cleave the A domain of Aap at residue 335 and between the A and B domains at residue 601. This study identifies the mechanism behind Aap‐mediated biofilm maturation, and also demonstrates a novel role for a secreted staphylococcal protease as a requirement for the development of a biofilm.     

Poly(ethylenimine-co-L-lactamide-co-succinamide): a biodegradable polyethylenimine derivative with an advantageous pH-dependent hydrolytic degradation for gene delivery

A biodegradable gene transfer vector has been synthesized by linking several low molecular weight (MW) polyethylenimine (PEI, 1200 Da) blocks using an oligo(L-lactic acid-co-succinic acid) (OLSA, 1000 Da). The resulting copolymer P(EI-co-LSA) (8 kDa) is soluble in water and degrades via base-catalyzed hydrolytic cleavage of amide bonds. With regard to its application as a gene transfer agent, the polymer showed an interesting pH dependency of degradation. At pH 5, when DNases are highly active, the degradation proceeds at a slower rate than at a physiological pH of 7.4. PEI and P(EI-co-LSA) spontaneously formed complexes with plasmid DNA. Whereas the complexes formed with PEI were not stable and aggregated, forming particles of up to 1 microm hydrodynamic diameter, P(EI-co-LSA) formed complexes, which were about 150 nm in size and of narrow size distribution. The latter complexes were stable, due to their high surface charge (zeta-potential + 18 mV). Similar to low MW PEI, the copolymer exhibited a low toxicity profile. At the same time, the copolymer showed a significant enhancement of transfection activity in comparison to the low MW PEI. This makes P(EI-co-LSA) a promising candidate for long-term gene therapy where biocompatibility and biodegradability become increasingly important.     

The mechanism of extrachromosomal homologous DNA recombination in plant cells

Summary By cotransfecting plasmids carrying particular mutations in the -glucuronidase (GUS) gene into Nicotiana plumbaginifolia protoplasts and by monitoring the recombination rates using a recently developed transient assay, we were able to obtain insights into the mechanism of extrachromosomal recombination operating in plant cells. An exchange of flanking markers takes place in over 90% of the recombination events. In most of the remaining cases two consecutive, independent single crossover events occur. These events involve the same DNA substrate and lead to two successive exchanges of flanking markers, thus mimicking a presumed double crossover intermediate. A comparison of the outcome of our experiments with the predictions of two recombination models originally proposed for mammalian cells indicates that extrachromosomal recombination in plant cells is best described by the single strand annealing model. According to this model all recombination events result in an exchange of flanking markers. Our results rule out the double strand break repair model which predicts that flanking markers are exchanged in only half of all events.     

Asymmetric-flow field-flow fractionation of prions reveals a strain-specific continuum of quaternary structures with protease resistance developing at a hydrodynamic radius of 15 nm

Prion diseases are transmissible neurodegenerative disorders that affect mammals, including humans. The central molecular event is the conversion of cellular prion glycoprotein, PrP^C, into a plethora of assemblies, PrP^Sc, associated with disease. Distinct phenotypes of disease led to the concept of prion strains, which are associated with distinct PrP^Sc structures. However, the degree to which intra- and inter-strain PrP^Sc heterogeneity contributes to disease pathogenesis remains unclear. Addressing this question requires the precise isolation and characterization of all PrP^Sc subpopulations from the prion-infected brains. Until now, this has been challenging. We used asymmetric-flow field-flow fractionation (AF4) to isolate all PrP^Sc subpopulations from brains of hamsters infected with three prion strains: Hyper (HY) and 263K, which produce almost identical phenotypes, and Drowsy (DY), a strain with a distinct presentation. In-line dynamic and multi-angle light scattering (DLS/MALS) data provided accurate measurements of particle sizes and estimation of the shape and number of PrP^Sc particles. We found that each strain had a continuum of PrP^Sc assemblies, with strong correlation between PrP^Sc quaternary structure and phenotype. HY and 263K were enriched with large, protease-resistant PrP^Sc aggregates, whereas DY consisted primarily of smaller, more protease-sensitive aggregates. For all strains, a transition from protease-sensitive to protease-resistant PrP^Sc took place at a hydrodynamic radius (R_h) of 15 nm and was accompanied by a change in glycosylation and seeding activity. Our results show that the combination of AF4 with in-line MALS/DLS is a powerful tool for analyzing PrP^Sc subpopulations and demonstrate that while PrP^Sc quaternary structure is a major contributor to PrP^Sc structural heterogeneity, a fundamental change, likely in secondary/tertiary structure, prevents PrP^Sc particles from maintaining proteinase K resistance below an R_h of 15 nm, regardless of strain. This results in two biochemically distinctive subpopulations, the proportion, seeding activity, and stability of which correlate with prion strain phenotype.     

Tight linkage between the Beckwith-Wiedemann syndrome and a microsatellite marker for the TH locus

The Beckwith-Wiedemann syndrome (BWS) is characterized by somatic overgrowth, developmental anomalies, and proneness to embryonic tumor development. The majority of cases are sporadic, but several families with an autosomal dominant mode of inheritance with variable expression and reduced penetrance have been described. In three such families, BWS has been linked to DNA markers for the insulin gene (INS) and H-ras on chromosome band 11p15. Two additional families with inherited BWS are described here. Linkage analysis has been performed with a highly informative marker for the tyrosine hydroxylase (TH) locus within the INS-IGF2 (insulin-like growth factor II)-TH gene cluser and confirms the previous observed linkage to this region (lod score 2.16 at = 0). Linkage analysis to TH provides a basis for informed genetic counselling and carrier detection in the hereditary form of the syndrome. Based on the hypothesis that IGF2 may be a candidate gene for BWS, we screened for mutations in the coding exons 7 and 9, but found no abnormalities in 5 unrelated BWS cases.     

In vitro assembly of gap junctions

Gap junction structures were assembled in vitro from octyl-β- -glucopyranoside-solubilized components of lens fiber cell membranes. Individual pore structures (connexons), short double-membrane structures, and other amorphous material were evident in the solubilized mixture. Following the removal of the detergent by dialysis, these connexons associated to form single- and double-layered, two-dimensional hexagonal arrays (unit cell size a = B = 8.5 nm). The formation of larger arrays was dependent on the lipid-to-protein ratio and the presence of Mg²⁺ ions. Crystallographic analysis of electron micrographs revealed that lens junctional connexons consisted of six subunits surrounding a stain-filled channel. Upon further detergent treatment, in vitro assembled gap junctions were insoluble and formed three-dimensional stacks while other components were solubilized. SDS-PAGE and mass data from scanning transmission electron microscopy strongly suggest that a 38-kDa polypeptide, which is a processed form of the lens specific gap junction protein MP70, is a major component of the arrays. The in vitro assembly of gap junctions opens new avenues for the structural analysis of gap junctions and for the study of the intermolecular interactions of connexons during junctional assembly.     

CD8 T-cell induction against vascular endothelial growth factor receptor 2 by Salmonella for vaccination purposes against a murine melanoma

The Salmonella type III secretion system (T3SS) efficiently translocates heterologous proteins into the cytosol of eukaryotic cells. This leads to an antigen-specific CD8 T-cell induction in mice orally immunized with recombinant Salmonella. Recently, we have used Salmonella's T3SS as a prophylactic and therapeutic intervention against a murine fibrosarcoma. In this study, we constructed a recombinant Salmonella strain translocating the immunogenic H-2D(b)-specific CD8 T-cell epitope VILTNPISM (KDR2) from the murine vascular endothelial growth factor receptor 2 (VEGFR2). VEGFR2 is a member of the tyrosine protein kinase family and is upregulated on proliferating endothelial cells of the tumor vasculature. After single orogastric vaccination, we detected significant numbers of KDR2-tetramer-positive CD8 T cells in the spleens of immunized mice. The efficacy of these cytotoxic T cells was evaluated in a prophylactic setting to protect mice from challenges with B16F10 melanoma cells in a flank tumor model, and to reduce dissemination of spontaneous pulmonary melanoma metastases. Vaccinated mice revealed a reduction of angiogenesis by 62% in the solid tumor and consequently a significant decrease of tumor growth as compared to non-immunized mice. Moreover, in the lung metastasis model, immunization with recombinant Salmonella resulted in a reduction of the metastatic melanoma burden by approximately 60%.