Dysregulation of microtubule stability impairs morphofunctional connectivity in primary neuronal networks

Functionally related neurons assemble into connected networks that process and transmit electrochemical information. To do this in a coordinated manner, the number and strength of synaptic connections is tightly regulated. Synapse function relies on the microtubule (MT) cytoskeleton, the dynamics of which are in turn controlled by a plethora of MT-associated proteins, including the MT-stabilizing protein Tau. Although mutations in the Tau-encodingMAPT gene underlie a set of neurodegenerative disorders, termed tauopathies, the exact contribution of MT dynamics and the perturbation thereof to neuronal network connectivity has not yet been scrutinized. Therefore, we investigated the impact of targeted perturbations of MT stability on morphological (e.g., neurite- and synapse density) and functional (e.g., synchronous calcium bursting) correlates of connectivity in networks of primary hippocampal neurons. We found that treatment with MT-stabilizing or -destabilizing compounds impaired morphofunctional connectivity in a reversible manner. We also discovered that overexpression of MAPT induced significant connectivity defects, which were accompanied by alterations in MT dynamics and increased resistance to pharmacological MT depolymerization. Overexpression of a MAPT variant harboring the P301L point mutation in the MT-binding domain did far less, directly linking neuronal connectivity with Tau's MT binding affinity. Our results show that MT stability is a vulnerable node in tauopathies and that its precise pharmacological tuning may positively affect neuronal network connectivity. However, a critical balance in MT turnover causes it to be a difficult therapeutic target with a narrow operating window

Statins and histone deacetylase inhibitors affect lamin A/C - histone deacetylase 2 interaction in human cells

We recently identified lamin A/C as a docking molecule for human histone deacetylase 2 (HDAC2) and showed involvement of HDAC2-lamin NC complexes in the DNA damage response. We further showed that lamin NC-HDAC2 interaction is altered in Hutchinson-Gilford Progeria syndrome and other progeroid laminopathies. Here, we show that both inhibitors of lamin A maturation and small molecules inhibiting HDAC activity affect lamin NC interaction with HDAC2. While statins, which inhibit prelamin A processing, reduce protein interaction, HDAC inhibitors strengthen protein binding. Moreover, treatment with HDAC inhibitors restored the enfeebled lamin NC-HDAC2 interaction observed in HGPS cells. Based on these results, we propose that prelamin A levels as well as HDAC2 activation status might influence the extent of HDAC2 recruitment to the lamin A/C-containing platform and contribute to modulate HDAC2 activity. Our study links prelamin A processing to HDAC2 regulation and provides new insights into the effect of statins and histone deacetylase inhibitors on lamin NC functionality in normal and progeroid cells

Single cell epigenetic visualization assay

Abstract Characterization of the epigenetic status of individual cells remains a challenge. Current sequencing approaches have limited coverage, and it is difficult to assign an epigenetic status to the transcription state of individual gene alleles in the same cell. To address these limitations, a targeted microscopy-based epigenetic visualization assay (EVA) was developed for detection and quantification of epigenetic marks at genes of interest in single cells. The assay is based on an in situ biochemical reaction between an antibody-conjugated alkaline phosphatase bound to the epigenetic mark of interest, and a 5′-phosphorylated fluorophore-labeled DNA oligo tethered to a target gene by gene-specific oligonucleotides. When the epigenetic mark is present at the gene, phosphate group removal by the phosphatase protects the oligo from λ-exonuclease activity providing a quantitative fluorescent readout. We applied EVA to measure 5-methylcytosine (5mC) and H3K9Ac levels at different genes and the HIV-1 provirus in human cell lines. To link epigenetic marks to gene transcription, EVA was combined with RNA-FISH. Higher 5mC levels at the silenced compared to transcribed XIST gene alleles in female somatic cells validated this approach and demonstrated that EVA can be used to relate epigenetic marks to the transcription status of individual gene alleles.</jats:p

Microbial community of predatory bugs of the genus Macrolophus (Hemiptera: Miridae)

<p>Abstract</p> <p>Background</p> <p>The predatory mirids of the genus <it>Macrolophus</it> are key natural enemies of various economically important agricultural pests. Both <it>M. caliginosus</it> and <it>M. pygmaeus</it> are commercially available for the augmentative biological control of arthropod pests in European greenhouses. The latter species is known to be infected with <it>Wolbachia</it> -inducing cytoplasmic incompatibility in its host- but the presence of other endosymbionts has not been demonstrated. In the present study, the microbial diversity was examined in various populations of <it>M. caliginosus</it> and <it>M. pygmaeus</it> by 16S rRNA sequencing and denaturing gradient gel electrophoresis.</p> <p>Results</p> <p>Besides <it>Wolbachia</it>, a co-infection of 2 <it>Rickettsia</it> species was detected in all <it>M. pygmaeus</it> populations. Based on a concatenated alignment of the <it>16S rRNA</it> gene, the <it>gltA</it> gene and the <it>coxA</it> gene, the first is phylogenetically related to <it>Rickettsia bellii</it>, whereas the other is closely related to <it>Rickettsia limoniae</it>. All <it>M. caliginosus</it> populations were infected with the same <it>Wolbachia</it> and <it>limoniae</it>-like <it>Rickettsia</it> strain as <it>M. pygmaeus</it>, but did not harbour the <it>bellii</it>-like <it>Rickettsia</it> strain. Interestingly, individuals with a single infection were not found. A PCR assay on the ovaries of <it>M. pygmaeus</it> and <it>M. caliginosus</it> indicated that all endosymbionts are vertically transmitted. The presence of <it>Wolbachia</it> and <it>Rickettsia</it> in oocytes was confirmed by a fluorescence <it>in situ</it> hybridisation. A bio-assay comparing an infected and an uninfected <it>M. pygmaeus</it> population suggested that the endosymbionts had minor effects on nymphal development of their insect host and did not influence its fecundity.</p> <p>Conclusion</p> <p>Two species of the palaearctic mirid genus <it>Macrolophus</it> are infected with multiple endosymbionts, including <it>Wolbachia</it> and <it>Rickettsia</it>. Independent of the origin, all tested populations of both <it>M. pygmaeus</it> and <it>M. caliginosus</it> were infected with three and two endosymbionts, respectively. There was no indication that infection with endosymbiotic bacteria had a fitness cost in terms of development and fecundity of the predators.</p

Vapor nanobubble is the more reliable photothermal mechanism for inducing endosomal escape of siRNA without disturbing cell homeostasis

Strategies for controlled delivery of therapeutic siRNA into living cells are in high demand as endosomal escape remains the most prominent bottleneck at the intracellular level. Photothermal properties of gold nanoparticles (AuNP) can be used to overcome the endosomal membrane barrier upon laser irradiation by two mechanisms: endosomal rupture by mechanical energy from water vapor nanobubbles (VNBs), or permeabilization of the endosomal membrane by heat diffusion. Here we evaluated how both mechanisms influence cargo release, transfection efficiency, acute cytotoxicity and cell homeostasis. Using a siRNA/AuNP drug delivery system we found that the in vitro release of siRNA from the AuNP carrier occurs equally efficiently by VNB formation or heat generation. Heat-mediated endosomal escape happened more efficiently in cells that had more particles per endosome, resulting in variable siRNA-induced downregulation (20-50%). VNB-mediated endosomal escape did not dependent on the number of AuNP per endosome, yielding high downregulations (50-60%) independent of the cell type. Effects on cell homeostasis by whole transcriptome analysis, showed a quick recover after 24 h or 48 h for either of both photothermal mechanisms. We conclude that VNBs are more consistent to induce efficient endosomal escape and gene silencing independent of the cell type without long lasting effects on cell homeostasis

Identification of healthspan-promoting genes in Caenorhabditis elegans based on a human GWAS study

To find drivers of healthy ageing, a genome-wide association study (GWAS) was performed in healthy and unhealthy older individuals. Healthy individuals were defined as free from cardiovascular disease, stroke, heart failure, major adverse cardiovascular event, diabetes, dementia, cancer, chronic obstructive pulmonary disease (COPD), asthma, rheumatism, Crohn’s disease, malabsorption or kidney disease. Six single nucleotide polymorphisms (SNPs) with unknown function associated with ten human genes were identified as candidate healthspan markers. Thirteen homologous or closely related genes were selected in the model organism C. elegans for evaluating healthspan after targeted RNAi-mediated knockdown using pathogen resistance, muscle integrity, chemotaxis index and the activity of known longevity and stress response pathways as healthspan reporters. In addition, lifespan was monitored in the RNAi-treated nematodes. RNAi knockdown of yap-1, wwp-1, paxt-1 and several acdh genes resulted in heterogeneous phenotypes regarding muscle integrity, pathogen resistance, chemotactic behaviour, and lifespan. Based on these observations, we hypothesize that their human homologues WWC2, CDKN2AIP and ACADS may play a role in health maintenance in the elderly

Cardiomiocitos derivados de células pluripotentes inducidas (hiPSC-CM) con progerina inducible para estudiar envejecimiento cardiaco

Molecular bases of cardiac aging still remain in the dark. Though hiPSC-CM are a usefull tool to study mechanisms, they present an immature phenotype. here, we present a maturation strategy and defind the progerin induction contitions for further aging analyisis&nbsp;in vitro.Las bases moleculares del envejecimiento cardiaco no se conocen en profundidad. Los hiPSC-CM son una herramienta útil para este tipo de estudios, pero presentan un fenotipo inmaduro. En este estudio, proponemos una estrategia de maduración y definimos las condiciones de inducción de progerina para el posterior estudio del envejecimiento in vitro

Measurement of S-phase duration of adult stem cells in the flatworm Macrostomum lignano by double replication labelling and quantitative colocalization analysis

Platyhelminthes are highly attractive models for addressing fundamental aspects of stem cell biology in vivo. These organisms possess a unique stem cell system comprised of neoblasts that are the only proliferating cells during adulthood. We have investigated T-s (S-phase duration) of neoblasts during homoeostasis and regeneration in the flatworm, Macrostomum lignano. A double immunohistochemical technique was used, performing sequential pulses with the thymidine analogues CldU (chlorodeoxyuridine) and IdU (iododeoxyuridine), separated by variable chase times in the presence of colchicine. Owing to the localized nature of the fluorescent signals (cell nuclei) and variable levels of autofluorescence, standard intensity-based colocalization analyses could not be applied to accurately determine the colocalization. Therefore, an object-based colocalization approach was devised to score the relative number of double-positive cells. Using this approach, T-s (S-phase duration) in the main population of neoblasts was similar to 13 h. During early regeneration, no significant change in T-s was observed