CDKL5 affects neuronal polarization through its interaction with Shootin1.

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Imbalance of flight-freeze responses and their cellular correlates in the Nlgn3-/y rat model of autism

Abstract Background Mutations in the postsynaptic transmembrane protein neuroligin-3 are highly correlative with autism spectrum disorders (ASDs) and intellectual disabilities (IDs). Fear learning is well studied in models of these disorders, however differences in fear response behaviours are often overlooked. We aim to examine fear behaviour and its cellular underpinnings in a rat model of ASD/ID lacking Nlgn3. Methods This study uses a range of behavioural tests to understand differences in fear response behaviour in Nlgn3 −/y rats. Following this, we examined the physiological underpinnings of this in neurons of the periaqueductal grey (PAG), a midbrain area involved in flight-or-freeze responses. We used whole-cell patch-clamp recordings from ex vivo PAG slices, in addition to in vivo local-field potential recordings and electrical stimulation of the PAG in wildtype and Nlgn3 −/y rats. We analysed behavioural data with two- and three-way ANOVAS and electrophysiological data with generalised linear mixed modelling (GLMM). Results We observed that, unlike the wildtype, Nlgn3 −/y rats are more likely to response with flight rather than freezing in threatening situations. Electrophysiological findings were in agreement with these behavioural outcomes. We found in ex vivo slices from Nlgn3 −/y rats that neurons in dorsal PAG (dPAG) showed intrinsic hyperexcitability compared to wildtype. Similarly, stimulating dPAG in vivo revealed that lower magnitudes sufficed to evoke flight behaviour in Nlgn3 −/y than wildtype rats, indicating the functional impact of the increased cellular excitability. Limitations Our findings do not examine what specific cell type in the PAG is likely responsible for these phenotypes. Furthermore, we have focussed on phenotypes in young adult animals, whilst the human condition associated with NLGN3 mutations appears during the first few years of life. Conclusions We describe altered fear responses in Nlgn3 −/y rats and provide evidence that this is the result of a circuit bias that predisposes flight over freeze responses. Additionally, we demonstrate the first link between PAG dysfunction and ASD/ID. This study provides new insight into potential pathophysiologies leading to anxiety disorders and changes to fear responses in individuals with ASD

Clinical and biochemical characteristics of people experiencing post-coronavirus disease 2019-related symptoms: A prospective follow-up investigation

BackgroundPost-acute coronavirus disease 2019 (COVID-19) syndrome, also known as long COVID, is a prolonged illness after the acute phase of COVID-19. Hospitalized patients were known to have persisting symptoms of fatigue, headache, dyspnea, and anosmia. There is a need to describe the characteristics of individuals with post-COVID-19 symptoms in comparison to the baseline characteristics.PurposeTo investigate the clinical and biochemical characteristics of people who recovered from COVID-19 after 6 months of discharge from the hospital.MethodsThis was a prospective follow-up investigation of hospitalized and discharged COVID-19 patients. Adult patients admitted to King Saud University Medical City, Riyadh, Saudi Arabia, with laboratory-confirmed COVID-19 and discharged were recruited. The baseline demographic information, comorbidities, vital signs and symptoms, laboratory parameters, COVID-19 therapy, and outcomes were collected from the medical records. Blood samples were collected for cytokines estimation. A detailed interview about signs and symptoms was undertaken during the follow-up.ResultsHalf of the followed-up people reported experiencing at least one of the COVID-19-related symptoms. The mean blood pressure was found higher in follow-up. People with the symptoms were characterized by low lymphocyte count, lower serum calcium levels, and hyperglycemia compared to people without any post-COVID-19 symptoms. Cytokines IL-8, VEGF, and MCP-1 were higher in people with the most frequent symptoms.ConclusionPeople with post-COVID-19 symptoms were characterized by lower lymphocyte count, lower serum calcium levels, and hyperglycemia compared to people without symptoms. Individuals with the most frequent post-COVID-19 symptoms had higher baseline pro-inflammatory, chemotactic, and angiogenic cytokines

Plasmonics-based refractive index sensor for detection of hemoglobin concentration

An ultra-compact plasmonics-based sensor is investigated which is excited by Fano resonance. The structure is numerically simulated by the finite-difference time-domain method. The sensor utilizes unique waveguide geometry named as metal-insulator-metal (MIM) waveguide geometry which has an intriguing feature to confine signal far beyond diffraction light. Thus, it is used to devise ultra-compact optical circuits. The MIM waveguide is coupled to a pair of elliptical ring resonators and the interaction between the resonators excites special mode which is known as Fano resonance mode. It is a unique phenomenon which exhibits asymmetrical resonance profile and supports ultra narrow line width. Because of its exciting feature, a large value of sensitivity = 1100 nm/RIU and figure of merit = 224RIU-1 is obtained for the proposed sensor. The sensing performance of the device can be further enhanced by tailoring the geometrical parameters. The applicability of the device is also tested to detect the concentration of hemoglobin in blood. Thus, the device is well suited to design on-chip optical sensors

Angiopoietin-2 level as a tool for cardiovascular risk stratification in hypertensive type 2 diabetic subjects

<p><b>Objectives</b>: This observational cross-sectional study aimed to investigate the relationship between serum Angiopoietin-2 (Ang-2) levels and cardiovascular (CVD) risk factors in drug controlled hypertensive diabetic subjects without cardiovascular complications.</p> <p><b>Methods</b>: All subjects were evaluated for fasting blood glucose (FBG), HbA1c, liver enzymes, lipid profile and serum Ang-2.</p> <p><b>Results</b>: Mean serum Ang-2 level was significantly higher in hypertensive diabetic subjects. In bivariate analysis in diabetic subjects with cardiovascular risk factors, Ang-2 positively correlated with waist circumference, body mass index (BMI), systolic blood pressure (SBP), FBG, HbA1c and triglycerides. In multivariate linear regression analysis, this association remained significant with FBG and triglycerides. Ang-2 levels were independently associated with CVD risk factors in drug controlled Type 2 diabetes (T2D) subjects.</p> <p><b>Conclusions</b>: Further detailed studies in larger population with more attention is needed to consider Ang-2 level as a tool for CVD risk stratification in hypertensive diabetic subjects.</p

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 influences shootin1 phosphorylation in primary cortical neurons.

<p>(A) Total cell extracts of DIV7 cortical neurons were treated with or without lambda phosphatase (λ-PPase) and analyzed by two-dimensional gel electrophoresis and immunoblotting with antibodies against shootin1, β-actin and, as control for the λ-PPase treatment, phopho-ERK1/2. (B) Primary cortical neurons were infected with shLacZ- or shCDKL5#1-expressing viral particles at DIV0 and total cell lysates were prepared at DIV7 and subjected to two-dimensional gel electrophoresis. Shootin1 and NFL were detected by immunoblotting; the single NFL-spot was used as internal control for alignment. Silencing of CDKL5 was confirmed by western blot (right panel). (n = 3).</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