CDKL5 regulates neuronal polarization of hippocampal neurons through interaction with shootin1.

missin

17,β-estradiol inhibits hepatitis C virus mainly by interference with the release phase of its life cycle

Rationale & Aim: Estrogen and estrogen-mediated signalling protect from hepatitis C virus through incompletely understood mechanisms. We aimed to ascertain which phase(s) of HCV life cycle is/are affected by estrogens. Methods: Huh7 cells infected with the JFH1 virus (genotype 2a) were exposed to dehydroepiandrosterone, testosterone, progesterone and 17β-estradiol (tested with/without its receptor antagonist fulvestrant). Dose-response curves were established to calculate IC50 values. To dissect how 17β-estradiol interferes with phases of HCV life cycle, its effects were measured on the HCV pseudo-particle system (viral entry), the sub-genomic replicon N17/JFH1 and the replicon cell line Huh7-J17 (viral replication). Finally, in a dual-step infection model, infectious supernatants, collected from infected cells exposed to hormones, were used to infect naïve cells. Results: Progesterone and testosterone showed no inhibitory effect on HCV; dehydroepiandrosterone was only mildly inhibitory. In contrast, 17β-estradiol inhibited infection by 64-67% (IC50 values 140 to 160 nM). Fulvestrant reverted the inhibition by 17β-estradiol in a dose-dependent manner. 17β-estradiol exerted only a slight inhibition (<20%) on HCV pseudo-particles, and had no effect on cells either transiently or stably (Huh7-J17 cells) expressing the N17/JFH1 replicon. In the dual-step infection model, a significant IC50 decline occurred between primary (134 nM) and secondary (100 nM) infections (p=0.02), with extracellular HCV RNA and infectivity being reduced to a higher degree in comparison to its intracellular counterpart. Conclusions: 17β-estradiol inhibits HCV acting through its intracellular receptors, mainly interfering with late phases (assembly/release) of the HCV life cycle

CDKL5 regulates neuronal polarization of hippocampal neurons through interaction with shootin1.

missin

The MeCP2/YY1 interaction regulates ANT1 expression at 4q35: novel hints for Rett syndrome pathogenesis

Rett syndrome is a severe neurodevelopmental disorder mainly caused by mutations in the transcriptional regulator MeCP2. Although there is no effective therapy for Rett syndrome, the recently discovered disease reversibility in mice suggests that there are therapeutic possibilities. Identification of MeCP2 targets or modifiers of the phenotype can facilitate the design of curative strategies. To identify possible novel MeCP2 interactors, we exploited a bioinformatic approach and selected Ying Yang 1 (YY1) as an interesting candidate. We demonstrate that MeCP2 interacts in vitro and in vivo with YY1, a ubiquitous zinc-finger epigenetic factor regulating the expression of several genes. We show that MeCP2 cooperates with YY1 in repressing the ANT1 gene encoding a mitochondrial adenine nucleotide translocase. Importantly, ANT1 mRNA levels are increased in human and mouse cell lines devoid of MeCP2, in Rett patient fibroblasts and in the brain of Mecp2-null mice. We further demonstrate that ANT1 protein levels are upregulated in Mecp2-null mice. Finally, the identified MeCP2-YY1 interaction, together with the well-known involvement of YY1 in the regulation of D4Z4-associated genes at 4q35, led us to discover the anomalous depression of FRG2, a subtelomeric gene of unknown function, in Rett fibroblasts. Collectively, our data indicate that mutations in MeCP2 might cause the aberrant overexpression of genes located at a specific locus, thus providing new candidates for the pathogenesis of Rett syndrome. As both ANT1 mutations and overexpression have been associated with human diseases, we consider it highly relevant to address the consequences of ANT1 deregulation in Rett syndrome

CDKL5 and shootin1 interact and concur in regulating neuronal polarization

In the last years, the X-linked cyclin-dependent kinase-like 5 (CDKL5) gene has been associated with epileptic encephalopathies characterized by the early onset of intractable epilepsy, severe developmental delay, autistic features, and often the development of Rett syndrome-like features. Still, the role of CDKL5 in neuronal functions is not fully understood. By way of a yeast two hybrid screening we identified the interaction of CDKL5 with shootin1, a brain specific protein acting as a determinant of axon formation during neuronal polarization. We found evidence that CDKL5 is involved, at least in part, in regulating neuronal polarization through its interaction with shootin1. Indeed, the two proteins interact in vivo and both are localized in the distal tip of outgrowing axons. By using primary hippocampal neurons as model system we find that adequate CDKL5 levels are required for axon specification. In fact, a significant number of neurons overexpressing CDKL5 is characterized by supernumerary axons, while the silencing of CDKL5 disrupts neuronal polarization. Interestingly, shootin1 phosphorylation is reduced in neurons silenced for CDKL5 suggesting that the kinase affects, directly or indirectly, the post-translational modification of shootin1. Finally, we find that the capacity of CDKL5 to generate surplus axons is attenuated in neurons with reduced shootin1 levels, in agreement with the notion that two proteins act in a common pathway. Altogether, these results point to a role of CDKL5 in the early steps of neuronal differentiation that can be explained, at least in part, by its association with shootin1

CDKL5 interacts with shootin1 in vivo.

<p>(A) A yeast two-hybrid screening identified shootin1 as a CDKL5 interacting protein. The C-terminal region of hCDKL5, spanning amino acids 299–1030, was used as bait (upper, thick bar). The diagram below shows shootin1 with its coiled coil domains in black. The clones identified in the screen are indicated as black bars and the minimum CDKL5 interacting region as a black bar. (B) Coimmunoprecipitation of P5-7 brain lysates with anti-CDKL5 (upper, n = 3) or anti-shootin1 (lower, n = 3) antibodies (both rabbit). IgGs were used as negative control. The immunoprecipitates and inputs (5% of the brain lysates) were analyzed by immunoblotting for CDKL5 and shootin1 (using a goat anti-shootin1 antibody). Asterisks indicate the immunoglobulin heavy chains and the open circle an unspecific band detected with anti-CDKL5. (C) Coimmunoprecipitation of HeLa cells overexpressing either Flag-CDKL5 or shootin1 or both proteins together. Whole cell lysates were immunoprecipiated with an anti-Flag resin and inputs (5%) and immunocomplexes analyzed by western blotting as indicated. Asterisk shows an anti-shootin1 reactive protein that copurifies with CDKL5. (n = 3).</p

CDKL5 regulates axon outgrowth through shootin1.

<p>(A) Immunofluorescence of neurons nucleofected before plating with a bicistronic vector expressing GFP alone or together with CDKL5 and subsequently infected with lentiviral particles expressing shRNAs against shootin1 or LacZ. At DIV5 neurons were stained for GFP, Tau1, and CDKL5 (green, blue, and red, respectively). (B) Quantification of neuronal polarization of GFP-positive neurons with increased CDKL5 expression. Data are expressed as means ±SEM. **p<0,01, *p<0,05. (n≥24 neurons/condition in 3 independent experiments; ANOVA two-way). Scale bar: 20 μm.</p

CDKL5 promotes axon formation.

<p>(A) Western blot showing CDKL5 levels in primary hippocampal neurons nucleofected before plating with bicistronic vectors expressing GFP alone or together with CDKL5 or CDKL5-K42R. Cell lysates were prepared at DIV5 and analyzed for CDKL5 levels using Tuj1 as loading control. (B) Representative images showing the localization of endogenous and exogenous CDKL5 in the soma of nucleofected GFP-positive neurons at DIV5. The exposure time of the GFP-expressing neuron (left) was increased to reveal the staining of endogenous CDKL5. (C) Representative images showing hippocampal neurons at DIV5 transfected with vectors expressing GFP together with CDKL5 or the K42R derivative. GFP and CDKL5 signals are in green and red, respectively. The arrows indicate neurons with increased CDKL5 levels. Scale bar: 20 μm. (D) Quantitative analysis of neuronal polarization. Axon specification was analyzed at DIV5 by determining the number of neurons with a single axon (polarized, black bars), multiple axons (dark grey bars) and neurons with no axon (light grey bars). Data are expressed as mean of 4 independent experiments ±SEM; ***p<0,001, **p<0,01, *p<0,05 (n≥100 neurons/condition, Student’s <i>t</i> test). (E) Graph showing the length of the longest axon and dendrite of transfected neurons. Data present neurite length as means ±SEM (n>28 neurons/condition, 4 independent experiments); **p<0,01. (Student’s <i>t</i> test).</p

CDKL5 and shootin1 are coexpressed in brains and neurons.

<p>(A) Western blot analysis showing CDKL5 and shootin1 levels in mouse brain at the indicated developmental stages using Tuj1 as loading control. (n = 2) (B) <i>Shootin1</i> is expressed in the cortex, as early as E13, in the cortical plate (cp) and its levels increase ongoing with development (a,b,c,d); <i>Cdkl5</i> (b’,c’,d’) follow the same pattern. Low but detectable levels of <i>shootin1</i> and <i>Cdkl5</i> mRNAs are present in cells migrating out of the ventricular and sub-ventricular zone (vz-svz) towards their final destination in the cortical plate (b,b’). At E18 <i>shootin1</i> and <i>Cdkl5</i> are strongly expressed throughout the whole thickness of the cortex (d,d’). Scale bars: 50 μm: b,b’,c,c’; 100 μm: d,d’; 200 μm: a. (C) Western blot showing CDKL5 and shootin1 levels in cultured primary hippocampal neurons at the indicated stages. A longer exposure of the 18 h time point is shown to the right. (n = 2). (D) Immunofluorescence analysis (left) of hippocampal neurons at stages 2–3 with antibodies against CDKL5 (green) and shootin1 (red). The small panels show the magnification of the minor processes/axons indicated with asterisks. Quantitative profiles showing the fluorescence intensities of shootin1 (red) and CDKL5 (green) from the soma to the distal tip of the neurites/axons indicated with asterisks are shown to the right. Scale bar: 10 μm.</p