Neuronal TDP-43 depletion affects activity-dependent plasticity.

TAR DNA-binding protein 43 (TDP-43) is a hallmark of some neurodegenerative disorders, such as frontotemporal lobar degeneration and amyotrophic lateral sclerosis. TDP-43-related pathology is characterized by its abnormally phosphorylated and ubiquitinated aggregates. It is involved in many aspects of RNA processing, including mRNA splicing, transport, and translation. However, its exact physiological function and role in mechanisms that lead to neuronal degeneration remain elusive. Transgenic rats that were characterized by TDP-43 depletion in neurons exhibited enhancement of the acquisition of fear memory. At the cellular level, TDP-43-depleted neurons exhibited a decrease in the short-term plasticity of intrinsic neuronal excitability. The induction of long-term potentiation in the CA3-CA1 areas of the hippocampus resulted in more stable synaptic enhancement. At the molecular level, the protein levels of an unedited (R) FLOP variant of α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR) GluR1 and GluR2/3 subunits decreased in the hippocampus. Alterations of FLOP/FLIP subunit composition affected AMPAR kinetics, reflected by cyclothiazide-dependent slowing of the decay time of AMPAR-mediated miniature excitatory postsynaptic currents. These findings suggest that TDP-43 may regulate activity-dependent neuronal plasticity, possibly by regulating the splicing of genes that are responsible for fast synaptic transmission and membrane potential.

[RNA processing TDP-43 protein has a main pathological role in FTLD and ALS]. / TDP-43, wielofunkcyjne bialko regulujace metabolizm RNA, jako patologiczny czynnik chorób neurozwyrodnieniowych FTLD i ALS.

TDP-43 is mainly a nuclear protein belonging to the heterogeneous ribonucleoproteins family. It plays a crucial role in the regulation of gene transcription and RNA processing. In 2006, TDP-43 was characterized as the main component of ubiquitin-positive inclusions observed in Frontotemporal Lobar Degeneration (FTLD) and Amyotrophic Lateral Sclerosis (ALS) cases. Since then, its central role in a number of neurodegenerative diseases was confirmed, originating the TDP-43 proteinopathies term. Pathological TDP-43 redistributes and accumulates in the cytoplasm forming toxic aggregates. Plethora of animal models recapitulating features typical for human TDP-43 proteinopathies has been generated. However, the mechanism involving TDP-43 and causing functional disturbances, like dementia and motoneurons degeneration, remains unknown. Loss and gain of function hypotheses are proposed, but they still need to be verified.

Prototyping in Polymethylpentene to Enable Oxygen-Permeable On-a-Chip Cell Culture and Organ-on-a-Chip Devices Suitable for Microscopy.

With the rapid development and commercial interest in the organ-on-a-chip (OoC) field, there is a need for materials addressing key experimental demands and enabling both prototyping and large-scale production. Here, we utilized the gas-permeable, thermoplastic material polymethylpentene (PMP). Three methods were tested to prototype transparent PMP films suitable for transmission light microscopy: hot-press molding, extrusion, and polishing of a commercial, hazy extruded film. The transparent films (thickness 20, 125, 133, 356, and 653 µm) were assembled as the cell-adhering layer in sealed culture chamber devices, to assess resulting oxygen concentration after 4 days of A549 cell culture (cancerous lung epithelial cells). Oxygen concentrations stabilized between 15.6% and 11.6%, where the thicker the film, the lower the oxygen concentration. Cell adherence, proliferation, and viability were comparable to glass for all PMP films (coated with poly-L-lysine), and transparency was adequate for transmission light microscopy of adherent cells. Hot-press molding was concluded as the preferred film prototyping method, due to excellent and reproducible film transparency, the possibility to easily vary film thickness, and the equipment being commonly available. The molecular orientation in the PMP films was characterized by IR dichroism. As expected, the extruded films showed clear orientation, but a novel result was that hot-press molding may also induce some orientation. It has been reported that orientation affects the permeability, but with the films in this study, we conclude that the orientation is not a critical factor. With the obtained results, we find it likely that OoC models with relevant in vivo oxygen concentrations may be facilitated by PMP. Combined with established large-scale production methods for thermoplastics, we foresee a useful role for PMP within the OoC field.

Generation of Transgenic Rats using a Lentiviral Vector Approach.

Transgenic animal models are fundamentally important for modern biomedical research. The incorporation of foreign genes into early mouse or rat embryos is an invaluable tool for gene function analysis in living organisms. The standard transgenesis method is based on microinjecting foreign DNA fragments into a pronucleus of a fertilized oocyte. This technique is widely used in mice but remains relatively inefficient and technically demanding in other animal species. The transgene can also be introduced into one-cell-stage embryos via lentiviral infection, providing an effective alternative to standard pronuclear injections, especially in species or strains with a more challenging embryo structure. In this approach, a suspension that contains lentiviral vectors is injected into the perivitelline space of a fertilized rat embryo, which is technically less demanding and has a higher success rate. Lentiviral vectors were shown to efficiently incorporate the transgene into the genome to determine the generation of stable transgenic lines. Despite some limitations (e.g., Biosafety Level 2 requirements, DNA fragment size limits), lentiviral transgenesis is a rapid and efficient transgenesis method. Additionally, using female rats that are mated with a fertile male strain with a different dominant fur color is presented as an alternative to generate pseudopregnant foster mothers.

A New Inhibitor of Tubulin Polymerization Kills Multiple Cancer Cell Types and Reveals p21-Mediated Mechanism Determining Cell Death after Mitotic Catastrophe.

Induction of mitotic catastrophe through the disruption of microtubules is an established target in cancer therapy. However, the molecular mechanisms determining the mitotic catastrophe and the following apoptotic or non-apoptotic cell death remain poorly understood. Moreover, many existing drugs targeting tubulin, such as vincristine, have reduced efficacy, resulting from poor solubility in physiological conditions. Here, we introduce a novel small molecule 2-aminoimidazoline derivative-OAT-449, a synthetic water-soluble tubulin inhibitor. OAT-449 in a concentration range from 6 to 30 nM causes cell death of eight different cancer cell lines in vitro, and significantly inhibits tumor development in such xenograft models as HT-29 (colorectal adenocarcinoma) and SK-N-MC (neuroepithelioma) in vivo. Mechanistic studies showed that OAT-449, like vincristine, inhibited tubulin polymerization and induced profound multi-nucleation and mitotic catastrophe in cancer cells. HeLa and HT-29 cells within 24 h of treatment arrested in G2/M cell cycle phase, presenting mitotic catastrophe features, and 24 h later died by non-apoptotic cell death. In HT-29 cells, both agents altered phosphorylation status of Cdk1 and of spindle assembly checkpoint proteins NuMa and Aurora B, while G2/M arrest and apoptosis blocking was consistent with p53-independent accumulation in the nucleus and largely in the cytoplasm of p21/waf1/cip1, a key determinant of cell fate programs. This is the first common mechanism for the two microtubule-dissociating agents, vincristine and OAT-449, determining the cell death pathway following mitotic catastrophe demonstrated in HT-29 cells.

Bafilomycin A1 triggers proliferative potential of senescent cancer cells in vitro and in NOD/SCID mice.

Anticancer therapies that induce DNA damage tend to trigger senescence in cancer cells, a process known as therapy-induced senescence (TIS). Such cells may undergo atypical divisions, thus contributing to tumor re-growth. Accumulation of senescent cancer cells reduces survival of patients after chemotherapy. As senescence interplays with autophagy, a dynamic recycling process, we sought to study whether inhibition of autophagy interferes with divisions of TIS cells. We exposed human colon cancer HCT116 cells to repeated cycles of a chemotherapeutic agent - doxorubicin (doxo) and demonstrated induction of hallmarks of TIS (e.g. growth arrest, hypertrophy, poliploidization and secretory phenotype) and certain properties of cancer stem cells (increased NANOG expression, percentages of CD24+ cells and side population). Colonies of small and highly proliferative progeny appeared shortly after drug removal. Treatment with bafilomycin A1 (BAF A1), an autophagy inhibitor, postponed short term in vitro cell re-population. It was associated with reduction in the number of diploid and increase in the number of poliploid cells. In a long term, a pulse of BAF A1 resulted in reactivation of autophagy in a subpopulation of HCT116 cells and increased proliferation. Accordingly, the senescent HCT116 cells treated with BAF A1 when injected into NOD/SCID mice formed tumors, in contrast to the controls.Our results suggest that senescent cancer cells that appear during therapy, can be considered as dormant cells that contribute to cancer re-growth, when chemotherapeutic treatment is stopped. These data unveil new mechanisms of TIS-related cancer maintenance and re-population, triggered by a single pulse of BAF A1 treatment.

Light-sheet microscopy imaging of a whole cleared rat brain with Thy1-GFP transgene.

Whole-brain imaging with light-sheet fluorescence microscopy and optically cleared tissue is a new, rapidly developing research field. Whereas successful attempts to clear and image mouse brain have been reported, a similar result for rats has proven difficult to achieve. Herein, we report on creating novel transgenic rat harboring fluorescent reporter GFP under control of neuronal gene promoter. We then present data on clearing the rat brain, showing that FluoClearBABB was found superior over passive CLARITY and CUBIC methods. Finally, we demonstrate efficient imaging of the rat brain using light-sheet fluorescence microscopy.