Epigenomics of Neural Cells: REST-Induced Down- and Upregulation of Gene Expression in a Two-Clone PC12 Cell Model

Cell epigenomics depends on the marks released by transcription factors operating via the assembly of complexes that induce focal changes of DNA and histone structure. Among these factors is REST, a repressor that, via its strong decrease, governs both neuronal and neural cell differentiation and specificity. REST operation on thousands of possible genes can occur directly or via indirect mechanisms including repression of other factors. In previous studies of gene down- and upregulation, processes had been only partially investigated in neural cells. PC12 are well-known neural cells sharing properties with neurons. In the widely used PC12 populations, low-REST cells coexist with few, spontaneous high-REST PC12 cells. High- and low-REST PC12 clones were employed to investigate the role and the mechanisms of the repressor action. Among 15,500 expressed genes we identified 1,770 target and nontarget, REST-dependent genes. Functionally, these genes were found to operate in many pathways, from synaptic function to extracellular matrix. Mechanistically, downregulated genes were predominantly repressed directly by REST; upregulated genes were mostly governed indirectly. Among other factors, Polycomb complexes cooperated with REST for downregulation, and Smad3 and Myod1 participated in upregulation. In conclusion, we have highlighted that PC12 clones are a useful model to investigate REST, opening opportunities to development of epigenomic investigation

Intracerebroventricular delivery of hematopoietic progenitors results in rapid and robust engraftment of microglia-like cells

Recent evidence indicates that hematopoietic stem and progenitor cells (HSPCs) can serve as vehicles for therapeutic molecular delivery to the brain by contributing to the turnover of resident myeloid cell populations. However, such engraftment needs to be fast and efficient to exert its therapeutic potential for diseases affecting the central nervous system. Moreover, the nature of the cells reconstituted after transplantation and whether they could comprise bona fide microglia remain to be assessed. We demonstrate that transplantation of HSPCs in the cerebral lateral ventricles provides rapid engraftment of morphologically, antigenically, and transcriptionally dependable microglia-like cells. We show that the cells comprised within the hematopoietic stem cell compartment and enriched early progenitor fractions generate this microglia-like population when injected in the brain ventricles in the absence of engraftment in the bone marrow. This delivery route has therapeutic relevance because it increases the delivery of therapeutic molecules to the brain, as shown in a humanized animal model of a prototypical lysosomal storage disease affecting the central nervous system

Genome Wide Identification of Aberrant Alternative Splicing Events in Myotonic Dystrophy Type 2

<div><p>Myotonic dystrophy type 2 (DM2) is a genetic, autosomal dominant disease due to expansion of tetraplet (CCTG) repetitions in the first intron of the ZNF9/CNBP gene. DM2 is a multisystemic disorder affecting the skeletal muscle, the heart, the eye and the endocrine system. According to the proposed pathological mechanism, the expanded tetraplets have an RNA toxic effect, disrupting the splicing of many mRNAs. Thus, the identification of aberrantly spliced transcripts is instrumental for our understanding of the molecular mechanisms underpinning the disease. The aim of this study was the identification of new aberrant alternative splicing events in DM2 patients. By genome wide analysis of 10 DM2 patients and 10 controls (CTR), we identified 273 alternative spliced exons in 218 genes. While many aberrant splicing events were already identified in the past, most were new. A subset of these events was validated by qPCR assays in 19 DM2 and 15 CTR subjects. To gain insight into the molecular pathways involving the identified aberrantly spliced genes, we performed a bioinformatics analysis with Ingenuity system. This analysis indicated a deregulation of development, cell survival, metabolism, calcium signaling and contractility. In conclusion, our genome wide analysis provided a database of aberrant splicing events in the skeletal muscle of DM2 patients. The affected genes are involved in numerous pathways and networks important for muscle physio-pathology, suggesting that the identified variants may contribute to DM2 pathogenesis.</p></div

Needle-like instruments for steering through solid organs: A review of the scientific and patent literature

High accuracy and precision in reaching target locations inside the human body is necessary for the success of percutaneous procedures, such as tissue sample removal (biopsy), brachytherapy, and localized drug delivery. Flexible steerable needles may allow the surgeon to reach targets deep inside solid organs while avoiding sensitive structures (e.g. blood vessels). This article provides a systematic classification of possible mechanical solutions for three-dimensional steering through solid organs. A scientific and patent literature search of steerable instrument designs was conducted using Scopus and Web of Science Derwent Innovations Index patent database, respectively. First, we distinguished between mechanisms in which deflection is induced by the pre-defined shape of the instrument versus mechanisms in which an actuator changes the deflection angle of the instrument on demand. Second, we distinguished between mechanisms deflecting in one versus two planes. The combination of deflection method and number of deflection planes led to eight logically derived mechanical solutions for three-dimensional steering, of which one was dismissed because it was considered meaningless. Next, we classified the instrument designs retrieved from the scientific and patent literature into the identified solutions. We found papers and patents describing instrument designs for six of the seven solutions. We did not find papers or patents describing instruments that steer in one-plane on-demand via an actuator and in a perpendicular plane with a pre-defined deflection angle via a bevel tip or a pre-curved configuration.Accepted Author ManuscriptMedical Instruments & Bio-Inspired Technolog

Increased DNMT3L exon 12 skipping in DM2 patients.

<p>A) EAA analysis identified an AS event on exon 12 of DNMT3L mRNA transcript ENST00000418993. The AS area is enlarged and exon 12, more frequently excluded in DM2 patients, is highlighted in solid gray. B) The box plot shows the decreased expression of Affymetrix probe set 3934442 recognizing exon 12, in DM2 patients compared to CTR (n = 10, ** p<0.01). Values are normalized for the levels of the whole transcript. The splice index (SI) is indicated. C) Validation qPCR assays were performed using the specific primer pair indicated as black arrowhead in panel A. Results are shown as fold change (DM2 = 19, CTR = 15; ** p<0.01).</p

Increased PHKA1 exon 19 inclusion in DM2 patients.

<p>A) EAA analysis identified an AS event on exon 19 of PHKA1 mRNA transcript ENST00000339490. The AS area is enlarged and exon 19, more frequently included in DM2 patients, is highlighted in solid gray. B) The box plot shows the increased expression of Affymetrix probe set 4012322 recognizing exon 19, in DM2 patients compared to CTR (n = 10, **** p<0.0001). Values are normalized for the levels of the whole transcript. The splice index (SI) is indicated. C) Validation qPCR assays were performed using the specific primer pair indicated as black arrowhead in panel A. Results are shown as fold change (DM2 = 19, CTR = 15; ** p<0.01).</p

Increased LAMC2 exon 23 inclusion in DM2 patients.

<p>A) EAA analysis identified an AS event on exon 23 of LAMC2 mRNA transcript ENST00000264144. The AS area is enlarged and exon 23, more frequently included in DM2 patients, is highlighted in solid gray. B) The box plot shows the increased expression of Affymetrix probe set 2371184, recognizing exon 23, in DM2 patients compared to CTR (n = 10, * p<0.05). Values are normalized for the levels of the whole transcript. The splice index (SI) is indicated. C) Validation qPCR assays were performed using the specific primer pair indicated as black arrowhead in panel A. Results are shown as fold change (DM2 = 19, CTR = 15; *p<0.05).</p

Increased NDUFV3 exon 3 skipping in DM2 patients.

<p>A) EAA analysis identified an AS event on exon 3 of NDUFV3 mRNA transcript ENST00000354250. The AS area is enlarged and exon 3, less frequently included in DM2 patients, is highlighted in solid gray. B) The box plot shows the decreased expression of Affymetrix probe set 3922937 recognizing exon 3, in DM2 patients compared to CTR (n = 10, **** p<0.0001). Values are normalized for the levels of the whole transcript. The splice index (SI) is indicated. C) Validation qPCR assays were performed using the specific primer pair indicated as black arrowhead in panel A. Results are shown as fold change (DM2 = 19, CTR = 15; ** p<0.01).</p