The proto-oncogene c-myc in terminal B lymphocyte differentiation: its role in plasma cell and memory B cell generation

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultal de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 01-07-201

Dystonia Type 6 Gene Product Thap1: Identification Of a 50 kDa DNA-binding Species In Neuronal Nuclear Fractions

Mutations in THAP1 result in dystonia type 6, with partial penetrance and variable phenotype. The goal of this study was to examine the nature and expression pattern of the protein product(s) of the Thap1 transcription factor (DYT6 gene) in mouse neurons, and to study the regional and developmental distribution, and subcellular localization of Thap1 protein. The goal was accomplished via overexpression and knock-down of Thap1 in the HEK293T cell line and in mouse striatal primary cultures and western blotting of embryonic Thap1-null tissue. The endogenous and transduced Thap1 isoforms were characterized using three different commercially available anti-Thap1 antibodies and validated by immunoprecipitation and DNA oligonucleotide affinity chromatography. We identified multiple, novel Thap1 species of apparent Mr 32 kDa, 47 kDa, and 50–52 kDa in vitro and in vivo, and verified the previously identified species at 29–30 kDa in neurons. The Thap1 species at the 50 kDa size range was exclusively detected in murine brain and testes and were located in the nuclear compartment. Thus, in addition to the predicted 25 kDa apparent Mr, we identified Thap1 species with greater apparent Mr that we speculate may be a result of posttranslational modifications. The neural localization of the 50 kDa species and its nuclear compartmentalization suggests that these may be key Thap1 species controlling neuronal gene transcription. Dysfunction of the neuronal 50 kDa species may therefore be implicated in the pathogenesis of DYT6

VPA and TSA do not inhibit induction of Egr-1 by BDNF, but do inhibit induction of Nab2 by BDNF.

<p>(<b>a</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 15, 30, and 60 min. The level of Egr-1 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>b</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), VPA (3 mM), or BDNF (10 ng/ml) plus VPA (3 mM) for 3 hours and the level of Nab2 was determined by western blotting. Results representative of at least 2 separate platings, performed in triplicate. Error bars indicate SEM (*p<0.05). (<b>c</b>) Primary striatal neurons were treated with BDNF (10 ng/ml), TSA (10 nM), or BDNF (10 ng/ml) plus TSA (10 nM) for 3 hours. Levels of Egr-1 and Nab2 were determined by Western blotting. Results representative of 3 separate platings performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001). (<b>d</b>) Primary striatal cultures were treated as in (b) followed by gel shift analysis. Anti-Nab2, 2 µg, was added before addition of DNA except in Lane 5, where it was added after the labeled oligonucleotide. Representative of 2 experiments performed in duplicate. The band representing binding of Egr-1 <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076842#pone.0076842-Keilani1" target="_blank">[7]</a> is represented by the filled arrow, and the super-shift band generated by addition of α-Nab2 is indicated by the unfilled arrow. (<b>e</b>) Primary striatal cultures were treated with vehicle (NI:non-infected), Ad-GFP (Adenovirus – GFP), or Ad-Nab2 (Adenovirus – Nab2) with and without BDNF, as described in Methods. Representative of 3 platings, performed in duplicate. Error bars indicate SEM (*p<0.05, **p<0.01, ***p<0.001).</p

Membrane Particles Derived From Adipose Tissue Mesenchymal Stromal Cells Improve Endothelial Cell Barrier Integrity

Proinflammatory stimuli lead to endothelial injury, which results in pathologies such as cardiovascular diseases, autoimmune diseases, and contributes to alloimmune responses after organ transplantation. Both mesenchymal stromal cells (MSC) and the extracellular vesicles (EV) released by them are widely studied as regenerative therapy for the endothelium. However, for therapeutic application, the manipulation of living MSC and large-scale production of EV are major challenges. Membrane particles (MP) generated from MSC may be an alternative to the use of whole MSC or EV. MP are nanovesicles artificially generated from the membranes of MSC and possess some of the therapeutic properties of MSC. In the present study we investigated whether MP conserve the beneficial MSC effects on endothelial cell repair processes under inflammatory conditions. MP were generated by hypotonic shock and extrusion of MSC membranes. The average size of MP was 120 nm, and they showed a spherical shape. The effects of two ratios of MP (50,000; 100,000 MP per target cell) on human umbilical vein endothelial cells (HUVEC) were tested in a model of inflammation induced by TNFα. Confocal microscopy and flow cytometry showed that within 24 hours >90% of HUVEC had taken up MP. Moreover, MP ended up in the lysosomes of the HUVEC. In a co-culture system of monocytes and TNFα activated HUVEC, MP did not affect monocyte adherence to HUVEC, but reduced the transmigration of monocytes across the endothelial layer from 138 ± 61 monocytes per microscopic field in TNFα activated HUVEC to 61 ± 45 monocytes. TNFα stimulation induced a 2-fold increase in the permeability of the HUVEC monolayer measured by the translocation of FITC-dextran to the lower compartment of a transwell system. At a dose of 1:100,000 MP significantly decreased endothelial permeability (1.5-fold) respect to TNFα Stimulated HUVEC. Finally, MP enhanced the angiogenic potential of HUVEC in an in vitro Matrigel assay by stimulating the formation of angiogenic structures, such as percentage of covered area, total tube length, total branching points, total loops. In conclusion, MP show regenerative effects on endothelial cells, opening a new avenue for treatment of vascular diseases where inflammatory processes damage the endothelium

AcH3 is increased in striatal neurons by HDAC inhibitors and is enriched downstream of the transcription start site in <i>Ppp1r1b</i> striatal chromatin.

<p>(<b>a</b>) Cells were treated with BDNF (10 ng/mL), (a) trichostatin A (10 nM), (b) valproic acid (3 mM) or (c) HDACi 4b (5 µM) for 3 h. N = 3−5, +/− SEM (*p<0.05, ***p<0.001). (<b>b</b>) Chromatin was purified from adult mouse striatal tissue (STR) (DARPP-32-positive) and NIH-3T3 cells (DARPP-32-negative). Chromatin immunoprecipitation coupled with real-time PCR showed an increased association of acetylated histone H3 within a 1 kb region downstream of the DARPP-32 transcriptional start site (TSS) in striata relative to NIH 3T3 cells. Results are representative of two separate experiments performed in triplicates. Values for IgG are set at 100%. Error bars indicate SEM (*p<0.05).</p

Trichostatin A, valproic acid and HDACi 4b up-regulate markers of the differentiated MSN but do not increase survival of striatal neurons <i>in vitro</i>.

<p>(<b>a</b>) Over 2,000 neurons were counted for each condition, derived from 3 separate platings. Untreated control, BDNF (10 ng/ml), HDACi 4b (5 µM), trichostatin A (10 nM) and valproic acid (3 mM), p = 0.94, one-way ANOVA. (<b>b–d</b>) Primary striatal neurons were treated with (b) TSA (10 nM), (c) VPA (3 mM), or (d) HDACi 4b (5 µM) for 24 h, each of which increased the level of DARPP-32 protein. Representative of 3 platings, each performed in duplicate. (<b>e</b>) Calbindin is increased after 24 h of treatment with VPA, (<b>f</b>) Transcript levels of DARPP-32, ARPP-21, D1 type receptors (D1R), D2 type receptors (D2R), encephalin (ENK) and dynorphin (DYN) were measured by RT-qPCR after 24 h treatment with BDNF or TSA. N = 5–8; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p

Bcl11b protein level is down-regulated in the presence of HDAC inhibitors.

<p>(<b>a</b>) Ontogeny of Bcl11b in striatum in Swiss-Webster mice. Bcl11b levels decrease markedly post-natally, particularly between the first and second week of life. (<b>b</b>) Primary striatal neurons were treated with VPA (3 mM) or TSA (10 nM) with or without BDNF (10 ng/ml) for 24 h. Similar results were observed after treatment with compound 4 b (not shown). N = 3–5; +/− SEM (*p<0.05, **p<0.01, ***p<0.001).</p

iPSC-derived familial Alzheimer’s PSEN2 N141I cholinergic neurons exhibit mutation-dependent molecular pathology corrected by insulin signaling

Abstract Background Type 2 diabetes (T2D) is a recognized risk factor for the development of cognitive impairment (CI) and/or dementia, although the exact nature of the molecular pathology of T2D-associated CI remains obscure. One link between T2D and CI might involve decreased insulin signaling in brain and/or neurons in either animal or postmortem human brains as has been reported as a feature of Alzheimer’s disease (AD). Here we asked if neuronal insulin resistance is a cell autonomous phenomenon in a familial form of AD. Methods We have applied a newly developed protocol for deriving human basal forebrain cholinergic neurons (BFCN) from skin fibroblasts via induced pluripotent stem cell (iPSC) technology. We generated wildtype and familial AD mutant PSEN2 N141I (presenilin 2) BFCNs and assessed if insulin signaling, insulin regulation of the major AD proteins Aβ and/or tau, and/or calcium fluxes is altered by the PSEN2 N141I mutation. Results We report herein that wildtype, PSEN2 N141I and CRISPR/Cas9-corrected iPSC-derived BFCNs (and their precursors) show indistinguishable insulin signaling profiles as determined by the phosphorylation of canonical insulin signaling pathway molecules. Chronic insulin treatment of BFCNs of all genotypes led to a reduction in the Aβ42/40 ratio. Unexpectedly, we found a CRISPR/Cas9-correctable effect of PSEN2 N141I on calcium flux, which could be prevented by chronic exposure of BFCNs to insulin. Conclusions Our studies indicate that the familial AD mutation PSEN2 N141I does not induce neuronal insulin resistance in a cell autonomous fashion. The ability of insulin to correct calcium fluxes and to lower Aβ42/40 ratio suggests that insulin acts to oppose an AD-pathophysiology. Hence, our results are consistent with a potential physiological role for insulin as a mediator of resilience by counteracting specific metabolic and molecular features of AD

CRISPR/Cas9-Correctable mutation-related molecular and physiological phenotypes in iPSC-derived Alzheimer’s PSEN2 N141I neurons

Abstract Basal forebrain cholinergic neurons (BFCNs) are believed to be one of the first cell types to be affected in all forms of AD, and their dysfunction is clinically correlated with impaired short-term memory formation and retrieval. We present an optimized in vitro protocol to generate human BFCNs from iPSCs, using cell lines from presenilin 2 (PSEN2) mutation carriers and controls. As expected, cell lines harboring the PSEN2 N141I mutation displayed an increase in the Aβ42/40 in iPSC-derived BFCNs. Neurons derived from PSEN2 N141I lines generated fewer maximum number of spikes in response to a square depolarizing current injection. The height of the first action potential at rheobase current injection was also significantly decreased in PSEN2 N141I BFCNs. CRISPR/Cas9 correction of the PSEN2 point mutation abolished the electrophysiological deficit, restoring both the maximal number of spikes and spike height to the levels recorded in controls. Increased Aβ42/40 was also normalized following CRISPR/Cas-mediated correction of the PSEN2 N141I mutation. The genome editing data confirms the robust consistency of mutation-related changes in Aβ42/40 ratio while also showing a PSEN2-mutation-related alteration in electrophysiology

Additional file 1: of iPSC-derived familial Alzheimer’s PSEN2 N141I cholinergic neurons exhibit mutation-dependent molecular pathology corrected by insulin signaling

Figure S1. Western blots of iPSC-derived basal cholinergic neuroprecursors cell lines. Cells were insulin deprived overnight before the addition 1000 ng/ml of insulin (insulin was not added to time 0′). Lysates were collected at 0, 10, or 30-min exposure. Quantified western blot data was normalized over Gapdh and expressed as fold change of 0 ng/ml dose. An effect on concentration response was detected by 2-way ANOVA if indicated. These data correspond to results of three independent experiments. ** P < 0.01; *** P < .001. (H) Representative blots from a single experiment showing three of the lines. Figure S2. (A) Western blot quantification of iPSC derived basal cholinergic neurons (two independent experiments). Cells (DIV 34) were insulin deprived for 3 days or given 1000 ng/mL every 24 h for 3 days. (B) Representative blots from a single experiment. (DOCX 262 kb