Multiple roles of Sonic Hedgehog in the developing human cortex are suggested by its widespread distribution

Sonic Hedgehog (Shh) plays an instrumental role in brain development, fine-tuning processes such as cell proliferation, patterning, and fate specification. Although, mutations in the SHH pathway in humans are associated with various neurodevelopmental disorders, ranging from holoprosencephaly to schizophrenia, its expression pattern in the developing human brain is not well established. We now determined the previously not reported wide expression of SHH in the human fetal cerebral cortex during most of the gestation period (10-40 gestational weeks). This spatiotemporal distribution puts Shh in a position to influence the fundamental processes involved in corticogenesis. SHH expression increased during development, shifting from progenitor cells in the proliferative zones to neurons, both glutamatergic and GABAergic, and astrocytes in upper cortical compartments. Importantly, the expression of its downstream effectors and complementary receptors revealed evolutionary differences in SHH-pathway gene expression between humans and rodents

Potential Immune Biomarkers in Diagnosis and Clinical Management for Systemic Lupus Erythematosus

Background: There is still no reliable, specific biomarker for precision diagnosis and clinical monitoring of systemic lupus erythematosus. The aim of this study was to investigate the importance of the determination of immunofenotypic profiles (T, B lymphocytes and NK cells) and serum cytokine concentrations (IL-17 and IFN-alpha) as potential biomarkers for this disease. Methods: The study included 55 patients with SLE and 25 healthy controls. The proportion of T, B, NK cells were assessed in peripheral blood using flow cytometric assays while the serum cytokine concentration (IL-17 and IFNalpha) was determined by ELISA test. Results: ROC curve analysis showed good accuracy to distinguish between patients and healthy individuals for activated T cells (AUC=0.798; p<0.001), Treg (AUC= 0.651; p=0.036), and memory B cells (AUC=0.285; p=0.002). We found statistically significant difference (p=0.036) in the levels of serum IL-17 between patients with SLE (IL-17=49.27 pg/mL) and controls (IL-17= 28.64 pg/mL). Conclusions: Significant increase in the relative number of Treg lymphocytes, and decrease in memory B cells, as well as decrease level of IL-17, in SLE patients may be implicated in the pathogenesis of the disease. These parameters, as biomarkers, could distinguish SLE patients and no-SLE patients. Monitoring subpopulations of immune cells in peripheral blood using flow cytometry provides insight into abnormal T and B cell function in SLE. Progress in understanding the immunity at SLE, results in concrete benefits for the SLE patients, which include new clinical management and therapeutic strategies

Origins of Cortical GABAergic Neurons in the Cynomolgus Monkey

In human most cortical γ-aminobutyric acidergic (GABAergic) neurons are produced in the proliferative zones of the dorsal telencephalon in contrast to rodents. We report that in cynomolgus monkey fetuses cortical GABAergic neurons are generated in the proliferative zones of the dorsal telencephalon, in addition to the proliferative region of the ventral telencephalon, the ganglionic eminence (GE), however, with a temporal delay. GABAergic neuron progenitors labeled for Mash1 and GAD65 were present mainly in the GE at embryonic days (E) 47–55, and in the entire dorsal telencephalon at E64–75. These progenitors within the dorsal telencephalon are generated locally rather than in the GE. The ventral and dorsal lineages of cortical GABAergic neurons display different laminar distribution. Early generated GABAergic neurons from the GE mostly populate the marginal zone and subplate, whereas cortical plate GABAergic neurons originate from both ventral and dorsal telencephalon. A differential regulation of the two GABA synthesizing enzymes (GAD65 and GAD67) parallels GABAergic neuron differentiation. GAD65 is preferentially expressed in GABAergic progenitors and migrating neurons, GAD67 in morphologically differentiated neurons. Therefore, the dorsal telencephalic origin of cortical GABAergic neurons is not human-specific but appears as a former event in the ascent of evolution that could provide GABAergic neurons to an expending neocortex

Morpholino Gene Knockdown in Adult Fundulus heteroclitus: Role of SGK1 in Seawater Acclimation

The Atlantic killifish (Fundulus heteroclitus) is an environmental sentinel organism used extensively for studies on environmental toxicants and salt (NaCl) homeostasis. Previous research in our laboratory has shown that rapid acclimation of killifish to seawater is mediated by trafficking of CFTR chloride channels from intracellular vesicles to the plasma membrane in the opercular membrane within the first hour in seawater, which enhances chloride secretion into seawater, thereby contributing to salt homeostasis. Acute transition to seawater is also marked by an increase in both mRNA and protein levels of serum glucocorticoid kinase 1 (SGK1) within 15 minutes of transfer. Although the rise in SGK1 in gill and its functional analog, the opercular membrane, after seawater transfer precedes the increase in membrane CFTR, a direct role of SGK1 in elevating membrane CFTR has not been established in vivo. To test the hypothesis that SGK1 mediates the increase in plasma membrane CFTR we designed two functionally different vivo-morpholinos to knock down SGK1 in gill, and developed and validated a vivo-morpholino knock down technique for adult killifish. Injection (intraperitoneal, IP) of the splice blocking SGK1 vivo-morpholino reduced SGK1 mRNA in the gill after transition from fresh to seawater by 66%. The IP injection of the translational blocking and splice blocking vivo-morpholinos reduced gill SGK1 protein abundance in fish transferred from fresh to seawater by 64% and 53%, respectively. Moreover, knock down of SGK1 completely eliminated the seawater induced rise in plasma membrane CFTR, demonstrating that the increase in SGK1 protein is required for the trafficking of CFTR from intracellular vesicles in mitochondrion rich cells to the plasma membrane in the gill during acclimation to seawater. This is the first report of the use of vivo-morpholinos in adult killifish and demonstrates that vivo-morpholinos are a valuable genetic tool for this environmentally relevant model organism

The application of cortical layer markers in the evaluation of cortical dysplasias in epilepsy

The diagnostic criteria for focal cortical dysplasia type I (FCD I) remain to be well and consistently defined. Cortical layer-specific markers (CLM) provide a potential tool for the objective assessment of any dyslamination. We studied expression patterns of recognised CLM using immunohistochemistry for N200, ER81, Otx1, Map1b (subsets of V/VI projection neurones), Pax6, Tbr1, Tbr2 (differentially expressed in cortical neurones from intermediate progenitor cells), Cux 1 (outer cortical layers) and MASH1 (ventricular zone progenitors). Dysplasia subtypes included FCD I and II, dysplasias adjacent to hippocampal sclerosis (HS) or dysembryoplastic neuroepithelial tumours (DNTs); all were compared to neonatal and adult controls. Laminar expression patterns in normal cortex were observed with Tbr1, Map1b, N200 and Otx1. FCDI cases in younger patients were characterised by abnormal expression in layer II for Tbr1 and Otx1. FCDII showed distinct labelling of balloon cells (Pax6, ER81 and Otx1) and dysmorphic neurones (Tbr 1, N200 and Map1b) supporting origins from radial glia and intermediate progenitor cells, respectively. In temporal lobe sclerosis cases with dysplasia adjacent to HS, Tbr1 and Map1b highlighted abnormal orientation of neurones in layer II. Dyslamination was not confirmed in the perilesional cortex of DNT with CLM. Finally, immature cell types (Otx1, Pax6 and Tbr2) were noted in varied pathologies. One possibility is activation of progenitor cell populations which could contribute to the pathophysiology of these lesions

Populations of Radial Glial Cells Respond Differently to Reelin and Neuregulin1 in a Ferret Model of Cortical Dysplasia

Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not

The Werner Syndrome Protein Suppresses Telomeric Instability Caused by Chromium (VI) Induced DNA Replication Stress

Telomeres protect the chromosome ends and consist of guanine-rich repeats coated by specialized proteins. Critically short telomeres are associated with disease, aging and cancer. Defects in telomere replication can lead to telomere loss, which can be prevented by telomerase-mediated telomere elongation or activities of the Werner syndrome helicase/exonuclease protein (WRN). Both telomerase and WRN attenuate cytotoxicity induced by the environmental carcinogen hexavalent chromium (Cr(VI)), which promotes replication stress and DNA polymerase arrest. However, it is not known whether Cr(VI)-induced replication stress impacts telomere integrity. Here we report that Cr(VI) exposure of human fibroblasts induced telomeric damage as indicated by phosphorylated H2AX (γH2AX) at telomeric foci. The induced γH2AX foci occurred in S-phase cells, which is indicative of replication fork stalling or collapse. Telomere fluorescence in situ hybridization (FISH) of metaphase chromosomes revealed that Cr(VI) exposure induced an increase in telomere loss and sister chromatid fusions that were rescued by telomerase activity. Human cells depleted for WRN protein exhibited a delayed reduction in telomeric and non-telomeric damage, indicated by γH2AX foci, during recovery from Cr(VI) exposure, consistent with WRN roles in repairing damaged replication forks. Telomere FISH of chromosome spreads revealed that WRN protects against Cr(VI)-induced telomere loss and downstream chromosome fusions, but does not prevent chromosome fusions that retain telomere sequence at the fusion point. Our studies indicate that environmentally induced replication stress leads to telomere loss and aberrations that are suppressed by telomerase-mediated telomere elongation or WRN functions in replication fork restoration