LIS1 RNA-binding orchestrates the mechanosensitive properties of embryonic stem cells in AGO2-dependent and independent ways.

Lissencephaly-1 (LIS1) is associated with neurodevelopmental diseases and is known to regulate the molecular motor cytoplasmic dynein activity. Here we show that LIS1 is essential for the viability of mouse embryonic stem cells (mESCs), and it governs the physical properties of these cells. LIS1 dosage substantially affects gene expression, and we uncovered an unexpected interaction of LIS1 with RNA and RNA-binding proteins, most prominently the Argonaute complex. We demonstrate that LIS1 overexpression partially rescued the extracellular matrix (ECM) expression and mechanosensitive genes conferring stiffness to Argonaute null mESCs. Collectively, our data transforms the current perspective on the roles of LIS1 in post-transcriptional regulation underlying development and mechanosensitive processes.

Heterogeneous nuclear ribonucleoprotein U (HNRNPU) safeguards the developing mouse cortex.

HNRNPU encodes the heterogeneous nuclear ribonucleoprotein U, which participates in RNA splicing and chromatin organization. Microdeletions in the 1q44 locus encompassing HNRNPU and other genes and point mutations in HNRNPU cause brain disorders, including early-onset seizures and severe intellectual disability. We aimed to understand HNRNPU's roles in the developing brain. Our work revealed that HNRNPU loss of function leads to rapid cell death of both postmitotic neurons and neural progenitors, with an apparent higher sensitivity of the latter. Further, expression and alternative splicing of multiple genes involved in cell survival, cell motility, and synapse formation are affected following Hnrnpu's conditional truncation. Finally, we identified pharmaceutical and genetic agents that can partially reverse the loss of cortical structures in Hnrnpu mutated embryonic brains, ameliorate radial neuronal migration defects and rescue cultured neural progenitors' cell death.

Perception of palliative medicine by health care professionals at a teaching community hospital: what is the key to a "palliative attitude"?

Background: With growing expense in chronic illness and end-of-life (EOL) care, population-based interventions are needed to reduce the health care cost and improve patients' quality of life. The authors believe that promotion of palliative medicine is one such intervention and this promotion depends on the acceptance of palliative medicine concepts by health care professionals. Aims of the studies: Perception of palliative medicine in chronic illness and in EOL care by health care professionals was learned in two studies carried out at a teaching community hospital 14 years apart. Participants and methods: Voluntary and anonymous surveys were randomly distributed among physicians, nurses, and social workers/case managers. Participants in the two studies presented two different groups of health care providers. Results of the studies: Results of the two studies were essentially similar. On most of the issues, respondents' perceptions were consistent with palliative medicine concepts and confidence in palliation grew over the 14-year period. The authors call this approach a "palliative attitude." Physicians with greater experience performed better in care planning. Younger physicians were more perceptive to withdrawal of care in futile cases. Participants' religion had no influence on perception of palliative medicine. Attendance of educational activities did not influence attitudes of health care professionals. Health care providers who favored involvement of palliative care teams in patients' management were better in care planning, interpretation of the DNR consent, use of opioids at the EOL, use of intensive care, and evaluation of the disease trajectory. Conclusion: The authors conclude that direct interaction between palliative and interdisciplinary teams in clinical practice is the key factor in the education of health care professionals, in the development of a "palliative attitude," and in the promotion of palliative medicine.

Complement C3 Affects Rac1 Activity in the Developing Brain.

The complement system, which is part of the innate immune response system, has been recently shown to participate in multiple key processes in the developing brain. Here we aimed to elucidate downstream signaling responses linking complement C3, a key molecule of the pathway, to small GTPases, known to affect the cytoskeleton. The expression pattern of the activated small GTPase Rac1 resembled that of complement C3. C3-deficient mice exhibited reduced Rac1 and elevated RhoA activity in comparison with control mice. The most pronounced reduction of Rac1 activity occurred at embryonic day 14. Rac1 has been implicated in neuronal migration as well as neuronal stem cell proliferation and differentiation. Consistent with the reduction in Rac1 activity, the expression of phospho-cofilin, decreased in migrating neurons. Reduced Rac1-GTP was also correlated with a decrease in the expression of progenitor markers (Nestin, Pax6 and Tbr2) and conversely the expression of neuronal markers (Dcx and NeuN) increased in C3 knockout (KO) cortices in comparison with wild-type (WT) cortices. More specifically, C3 deficiency resulted in a reduction in the number of the cells in S-phase and an elevation in the number of cells that precociously exited the cell cycle. Collectively, our findings suggest that C3 impacts the activity of small GTPases resulting in cell cycle defects and premature neuronal differentiation.

Serping1/C1 Inhibitor Affects Cortical Development in a Cell Autonomous and Non-cell Autonomous Manner.

Current knowledge regarding regulation of radial neuronal migration is mainly focused on intracellular molecules. Our unbiased screen aimed at identification of non-cell autonomous mechanisms involved in this process detected differential expression of Serping1 or C1 inhibitor, which is known to inhibit the initiation of the complement cascade. The complement cascade is composed of three pathways; the classical, lectin, and the alternative pathway; the first two are inhibited by C1 inhibitor, and all three converge at the level of C3. Knockdown or knockout of Serping1 affected neuronal stem cell proliferation and impaired neuronal migration in mice. Knockdown of Serping1 by in utero electroporation resulted in a migration delay of the electroporated cells as well as their neighboring cells demonstrating a non-cell autonomous effect. Cellular polarity was also affected. Most importantly, expression of protein components mimicking cleaved C3 rescued the knockdown of Serping1, indicating complement pathway functionality. Furthermore, we propose that this activity is mediated mainly via the complement peptide C5a receptors. Whereas addition of a selective C3a receptor agonist was minimally effective, the addition of a dual C3aR/C5a receptor agonist significantly rescued Serping1 knockdown-mediated neuronal migration defects. Our findings suggest that modulating Serping1 levels in the developing brain may affect the complement pathway in a complex way. Collectively, our findings demonstrate an unorthodox activity for the complement pathway during brain development.

Developmental activities of the complement pathway in migrating neurons.

In recent years the notion that malfunctioning of the immune system may result in developmental brain diseases has emerged. However, the role of immune molecules in the developing brain has not been well explored. The complement pathway converges to cleave C3. Here we show that key proteins in the lectin arm of this pathway, MASP1, MASP2 and C3, are expressed in the developing cortex and that neuronal migration is impaired in knockout and knockdown mice. Molecular mimics of C3 cleavage products rescue the migration defects that have been seen following knockdown of C3 or Masp2. Pharmacological activation of the downstream receptors rescue Masp2 and C3 knockdown as well as C3 knockout. Therefore, we propose that the complement pathway is functionally important in migrating neurons of the developing cortex.

Non-cell autonomous and non-catalytic activities of ATX in the developing brain.

The intricate formation of the cerebral cortex requires a well-coordinated series of events, which are regulated at the level of cell-autonomous and non-cell autonomous mechanisms. Whereas cell-autonomous mechanisms that regulate cortical development are well-studied, the non-cell autonomous mechanisms remain poorly understood. A non-biased screen allowed us to identify Autotaxin (ATX) as a non-cell autonomous regulator of neural stem cells. ATX (also known as ENPP2) is best known to catalyze lysophosphatidic acid (LPA) production. Our results demonstrate that ATX affects the localization and adhesion of neuronal progenitors in a cell autonomous and non-cell autonomous manner, and strikingly, this activity is independent from its catalytic activity in producing LPA.

Use of RNA interference by in utero electroporation to study cortical development: the example of the doublecortin superfamily.

The way we study cortical development has undergone a revolution in the last few years following the ability to use shRNA in the developing brain of the rodent embryo. The first gene to be knocked-down in the developing brain was doublecortin (Dcx). Here we will review knockdown experiments in the developing brain and compare them with knockout experiments, thus highlighting the advantages and disadvantages using the different systems. Our review will focus on experiments relating to the doublecortin superfamily of proteins.

Gene trapping: an antibody-dependent approach for verifying integration in your favorite gene.

Gene trapping is used to introduce genome-wide insertional mutations in embryonic stem cells. Determining the integration site is based on highthroughput PCR, which has inevitable possibilities for mistakes, thus necessitating clone verification prior to the generation of mutant mice. Here, we propose a rapid method to validate gene identity based on the fact that many high throughput gene-trapping integrations result in fusion proteins encompassing the N-terminal portion of the gene of interest and LacZ being expressed in embryonic stem cells. Our method utilizes an immunoprecipitation assay using a specific N-terminal-directed antibody to the protein product of the gene of interest followed by a color LacZ assay of the immunoprecipitate, strongly supporting the formation of a fusion protein when the color develops.

Spontaneous atherothrombosis and medial degradation in Apoe-/-, Npc1-/- mice.

BACKGROUND: The formation of an occluding thrombus on a ruptured or eroded atherosclerotic plaque is the hallmark event leading to acute coronary syndromes, myocardial infarction, and sudden death in humans. However, other species are highly resistant to plaque complications, and the specific processes predisposing to plaque destabilization and thrombosis are poorly understood. METHODS AND RESULTS: Mice carrying a null mutation of a gene regulating intracellular cholesterol transport (the Niemann-Pick C1 [Npc1] gene) were crossed with apolipoprotein E (Apoe) knockout mice to examine the effect of Npc1 on atherosclerotic lesion formation. Double-mutant mice showed greater lesion area compared with Apoe-/- littermates. Remarkably, the double mutants also developed large, protruding thrombi associated with the plaques and prominent medial degradation with inflammatory cell infiltration into the adventitia. Genetic studies suggested that the BALB background was permissive for plaque complications compared with C57BL/6J, and a BALB susceptibility locus was mapped by linkage analysis to chromosome 6. Examination of clotting parameters in double-knockout mice revealed that native clotting times were shortened and thrombin-antithrombin complex and soluble CD40 ligand levels were elevated compared with wild-type controls. In addition, cathepsin K was induced in Npc1-/- macrophages, and cathepsin K immunostaining and elastase activity were increased in proximal aortas of double-mutant mice compared with controls. CONCLUSIONS: A defect in intracellular cholesterol trafficking caused by the Npc1 null mutation predisposes to increased lesion formation, atherothrombosis, and medial degradation. Plaque complications may require a procoagulant state and an increased protease activity, leading to plaque destabilization.