Lipid‐associated aggregate formation of superoxide dismutase‐1 is initiated by membrane‐targeting loops

Copper‐Zinc superoxide dismutase 1 (SOD1) is a homodimeric enzyme that protects cells from oxidative damage. Hereditary and sporadic amyotrophic lateral sclerosis may be linked to SOD1 when the enzyme is destabilized through mutation or environmental stress. The cytotoxicity of demetallated or apo‐SOD1 aggregates may be due to their ability to cause defects within cell membranes by co‐aggregating with phospholipids. SOD1 monomers may associate with the inner cell membrane to receive copper ions from membrane‐bound copper chaperones. But how apo‐SOD1 interacts with lipids is unclear. We have used atomistic molecular dynamics simulations to reveal that flexible electrostatic and zinc‐binding loops in apo‐SOD1 dimers play a critical role in the binding of 1‐octanol clusters and phospholipid bilayer, without any significant unfolding of the protein. The apo‐SOD1 monomer also associates with phospholipid bilayer via its zinc‐binding loop rather than its exposed hydrophobic dimerization interface. Our observed orientation of the monomer on the bilayer would facilitate its association with a membrane‐bound copper chaperone. The orientation also suggests how membrane‐bound monomers could act as seeds for membrane‐associated SOD1 aggregation. Proteins 2014; 82:3194–3209. © 2014 Wiley Periodicals, Inc.     

PI3K‐p110‐alpha‐subtype signalling mediates survival,proliferation and neurogenesis of cortical progenitor cells via activation of mTORC2

Development of the cerebral cortex is controlled by growth factors among which transforming growth factor beta (TGFβ) and insulin‐like growth factor 1 (IGF1) have a central role. The TGFβ‐ and IGF1‐pathways cross‐talk and share signalling molecules, but in the central nervous system putative points of intersection remain unknown. We studied the biological effects and down‐stream molecules of TGFβ and IGF1 in cells derived from the mouse cerebral cortex at two developmental time points, E13.5 and E16.5. IGF1 induces PI3K, AKT and the mammalian target of rapamycin complexes (mTORC1/mTORC2) primarily in E13.5‐derived cells, resulting in proliferation, survival and neuronal differentiation, but has small impact on E16.5‐derived cells. TGFβ has little effect at E13.5. It does not activate the PI3K‐ and mTOR‐signalling network directly, but requires its activity to mediate neuronal differentiation specifically at E16.5. Our data indicate a central role of mTORC2 in survival, proliferation as well as neuronal differentiation of E16.5‐derived cortical cells. mTORC2 promotes these cellular processes and is under control of PI3K‐p110‐alpha signalling. PI3K‐p110‐beta signalling activates mTORC2 in E16.5‐derived cells but it does not influence cell survival, proliferation and differentiation. This finding indicates that different mTORC2 subtypes may be implicated in cortical development and that these subtypes are under control of different PI3K isoforms.

     

Bioenergetic adaptation in response to autophagy regulators during rotenone exposure

Parkinson's disease is the second most common neurodegenerative disorder with both mitochondrial dysfunction and insufficient autophagy playing a key role in its pathogenesis. Among the risk factors, exposure to the environmental neurotoxin rotenone increases the probability of developing Parkinson's disease. We previously reported that in differentiated SH‐SY5Y cells, rotenone‐induced cell death is directly related to inhibition of mitochondrial function. How rotenone at nM concentrations inhibits mitochondrial function, and whether it can engage the autophagy pathway necessary to remove damaged proteins and organelles, is unknown. We tested the hypothesis that autophagy plays a protective role against rotenone toxicity in primary neurons. We found that rotenone (10–100 nM) immediately inhibited cellular bioenergetics. Concentrations that decreased mitochondrial function at 2 h, caused cell death at 24 h with an LD50 of 10 nM. Overall, autophagic flux was decreased by 10 nM rotenone at both 2 and 24 h, but surprisingly mitophagy, or autophagy of the mitochondria, was increased at 24 h, suggesting that a mitochondrial‐specific lysosomal degradation pathway may be activated. Up‐regulation of autophagy by rapamycin protected against cell death while inhibition of autophagy by 3‐methyladenine exacerbated cell death. Interestingly, while 3‐methyladenine exacerbated the rotenone‐dependent effects on bioenergetics, rapamycin did not prevent rotenone‐induced mitochondrial dysfunction, but caused reprogramming of mitochondrial substrate usage associated with both complex I and complex II activities. Taken together, these data demonstrate that autophagy can play a protective role in primary neuron survival in response to rotenone; moreover, surviving neurons exhibit bioenergetic adaptations to this metabolic stressor. Exposure to the neurotoxin rotenone is a risk factor for Parkinson's disease. We tested the hypothesis that autophagy is protective against rotenone toxicity in primary neurons. Exposure to nanomolar concentrations of rotenone caused immediate mitochondrial dysfunction, associated with a suppression of macroautophagy. However, mitophagy occurred that was independent of LC3II accumulation, and the surviving neurons exhibited adaptations to their cellular bioenergetic profiles. Cotreatment with the autophagy enhancer rapamycin was protective, whereas further inhibition of autophagy with 3‐methyladenine (3‐MA) exacerbated cell death, resulting in additional bioenergetic adaptations in the surviving neurons.

     

Contribution of cysteine aminotransferase and mercaptopyruvate sulfurtransferase to hydrogen sulfide production in peripheral neurons

Hydrogen sulfide (H₂S) is a gaseous neuromodulator produced from L‐cysteine. H₂S is generated by three distinct enzymatic pathways mediated by cystathionine γ‐lyase (CSE), cystathionine β‐synthase (CBS), and mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT). This study investigated the relative contributions of these three pathways to H₂S production in PC12 cells (rat pheochromocytoma‐derived cells) and the rat dorsal root ganglion. CBS, CAT, and MPST, but not CSE, were expressed in the cells and tissues, and appreciable amounts of H₂S were produced from L‐cysteine in the presence of α‐ketoglutarate, together with dithiothreitol. The production of H₂S was inhibited by a CAT inhibitor (aminooxyacetic acid), competitive CAT substrates (L‐aspartate and oxaloacetate), and RNA interference (RNAi) against MPST. Immunocytochemistry revealed a mitochondrial localization of MPST in PC12 cells and dorsal root ganglion neurons, and the amount of H₂S produced by CAT/MPST at pH 8.0, a physiological mitochondrial matrix pH, was comparable to that produced by CSE and CBS in the liver and the brain, respectively. Furthermore, H₂S production was markedly increased by alkalization. These results indicate that CAT and MPST are primarily responsible for H₂S production in peripheral neurons, and that the regulation of mitochondrial metabolism may influence neuronal H₂S generation.

     

Morphology of antennal sensilla of the brown spruce longhorn beetle,Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae)

The antennal sensilla of the brown spruce longhorn beetle, Tetropium fuscum (Fabr.) (Coleoptera: Cerambycidae) were examined with particular focus on the sensilla present on the apical flagellomere. T. fuscum antennae are composed of 11 segments, namely the scape, pedicel, and nine flagellomeres. Nine types of sensilla were observed: three types of sensilla chaetica, sensilla trichodea, two types of sensilla basiconica, grooved peg sensilla, thick-walled sensilla, and Böhm bristles. Seven of these types were present on the apical flagellomere, the exceptions were sensilla chaetica type 3 and Böhm bristles. There were no significant differences in the distribution or density of sensilla present on the ninth flagellomere of males and females, except that males had significantly more sensilla chaetica type 1, which are put forward as the putative contact chemoreceptors for T. fuscum.     

OdoriFy: A conglomerate of artificial intelligence–driven prediction engines for olfactory decoding

The molecular mechanisms of olfaction, or the sense of smell, are relatively underexplored compared with other sensory systems, primarily because of its underlying molecular complexity and the limited availability of dedicated predictive computational tools. Odorant receptors (ORs) allow the detection and discrimination of a myriad of odorant molecules and therefore mediate the first step of the olfactory signaling cascade. To date, odorant (or agonist) information for the majority of these receptors is still unknown, limiting our understanding of their functional relevance in odor-induced behavioral responses. In this study, we introduce OdoriFy, a Web server featuring powerful deep neural network–based prediction engines. OdoriFy enables (1) identification of odorant molecules for wildtype or mutant human ORs (Odor Finder); (2) classification of user-provided chemicals as odorants/nonodorants (Odorant Predictor); (3) identification of responsive ORs for a query odorant (OR Finder); and (4) interaction validation using Odorant–OR Pair Analysis. In addition, OdoriFy provides the rationale behind every prediction it makes by leveraging explainable artificial intelligence. This module highlights the basis of the prediction of odorants/nonodorants at atomic resolution and for the ORs at amino acid levels. A key distinguishing feature of OdoriFy is that it is built on a comprehensive repertoire of manually curated information of human ORs with their known agonists and nonagonists, making it a highly interactive and resource-enriched Web server. Moreover, comparative analysis of OdoriFy predictions with an alternative structure-based ligand interaction method revealed comparable results. OdoriFy is available freely as a web service at https://odorify.ahujalab.iiitd.edu.in/olfy/.     

Activity-dependent regulation of HCN1 protein in cortical neurons

Homeostasis of neuronal activity is crucial to neuronal physiology. In dendrites, hyperpolarization-activated cyclic nucleotide-gated channel (HCN) 1 is considered to play critical roles in this process. While electrophysiological studies have demonstrated the dynamic modulation of I_h current mediated by HCN1 proteins, little is known about the underlying molecular and cellular mechanisms. In this study, we utilized cortical cultured neurons and biochemical methods to identify molecular and cellular mechanisms that mediate the physiological regulation of HCN1 channel functions in cortical neurons. Pharmacological manipulations of neuronal activity resulted in changes in the expression level of HCN1. In addition, the surface expression of HCN1 was dynamically regulated by neuronal activity. Both of these changes led to functional modulations of HCN1 channels. Our study suggests that coordinated changes in protein expression and surface expression of HCN1 serve as the key regulatory mechanisms controlling the function of endogenous HCN1 protein in cortical neurons.     

Amphibian larvae and zinc sulphate: a suitable model to study the role of brain-derived neurotrophic factor (BDNF) in the neuronal turnover of the olfactory epithelium

The vertebrate olfactory system has fascinated neurobiologists over the last six decades because of its ability to replace its neurons and synaptic connections continuously throughout adult life, under both physiological and pathological conditions. Among the factors that are proposed to be involved in this regenerative potential, brain-derived neurotrophic factor (BDNF) is a candidate for having an important role in the neuronal turnover in the olfactory epithelium (OE) because of its well-documented neurogenic and trophic effects throughout the nervous system. The aim of the present study was to generate a suitable model to study the participation of BDNF in the recovery of the OE after injury in vivo. We developed an experimental design in which the OE of Rhinella arenarum tadpoles could be easily and selectively damaged by immersing the animals in ZnSO₄ solutions of various concentrations for differing time periods. Image analysis of histological sections showed that different combinations of each of these conditions produced statistically different degrees of injury to the olfactory tissue. We also observed that the morphology of the OE was restored within a few days of recovery after ZnSO₄ treatment. Immunohistochemical analysis of BDNF was performed with an antiserum whose specificity was confirmed by Western blotting, and which showed drastic changes in the abundance and distribution pattern of this neurotrophin in the damaged olfactory system. Our results thus suggest that BDNF is involved in the regeneration of the OE of amphibian larvae, and that our approach is suitable for further investigations of this topic. This work was supported by grants from CONICET (PIP 5842), Universidad de Buenos Aires (UBACYT X131) and ANPCYT (PICT 32219).     

Calcium oscillations in the olfactory nonsensory cells of the goldfish, Carassius auratus

Background

The olfactory nonsensory cells contribute to the maintenance of normal functions of the olfactory epithelium (OE). Specifically, the ciliated nonsensory cells of teleosts play important roles in the odorant detection by OE in aqueous environment. Their cilia show strong beating activities and cause water flow at the OE surface, making the detection of odorants by OE more efficient. Because intracellular Ca²⁺ level has been reported to play an important role in ciliary beating, the ciliary beating activity may be regulated by intracellular Ca²⁺ dynamics of these ciliated nonsensory cells.

Methods

We performed Ca²⁺ imaging experiments to analyze the Ca²⁺ dynamics in acutely dissociated OE cells of the goldfish. Furthermore, we examined the contribution of the Ca²⁺ dynamics to the ciliary beating frequency (CBF) at the surface of the intact OE.

Results

Olfactory nonsensory cells showed both spontaneous intracellular Ca²⁺ oscillations and propagating intercellular Ca²⁺ waves. Application of 2-aminoethoxydiphenylborate (2-APB), which antagonizes IP₃-induced Ca²⁺ release from intracellular stores suppressed these Ca²⁺ oscillations. Furthermore, 2-APB application to the intact OE lamellae resulted in the decrease of CBF at the surface of the OE.

Conclusions

These results indicate that spontaneous intracellular calcium oscillations persistently up-regulate the ciliary beating at the surface of the OE in teleosts.

General significance

Ciliary beating activity at the surface of OE can be regulated by the Ca²⁺ dynamics of olfactory nonsensory cells. Because this ciliary movement causes inflow of external fluid into the nostril, this regulation is suggested to influence the efficiency of odorant detection by OE.     

向蟾蜍脊髓灰质腹角注射谷氨酸钠对腓肠肌收缩的影响

首先绘制了蟾蜍脊髓灰质腹角运动神经元池的精确定位图谱,然后分别将浓度为1、0.5、0.1、0.01 mol/L的谷氨酸钠溶液及生理盐水（0.65% NaCl,对照组）微量（0.1 µL）注射到蟾蜍脊髓灰质腹角运动神经元池,采用BL-420F 生物机能实验系统记录腓肠肌收缩曲线,以收缩波的上升相持续时间、最大张力、张力变化速率、下降相持续时间为指标对各组腓肠肌的收缩曲线进行比较。结果：4组实验组腓肠肌的收缩形式是不同程度的强直收缩;各组收缩波的上升相持续时间、最大张力、张力变化速率在一定程度上存在谷氨酸钠量效依赖关系,且1 mol/L组腓肠肌强直收缩的张力、张力变化速率显著大于其他各组,这可能是由于谷氨酸钠与运动神经元上谷氨酸受体结合的数量不同造成的。