Biology subjects

Wyneken, U.

Publications and source records attributed to Wyneken, U..

4 recordsLinked to original sources

bioRxiv2024

Local Synthesis of Reticulon-1C Lessens the Outgrowth of Injured Axons by Controlling Spastin Activity

The regenerative potential of developing cortical axons depends on intrinsic mechanisms, such as axon-autonomous protein synthesis, that are still not fully understood. An emerging factor in this regenerative response is the bi-directional interplay between microtubule dynamics and the axonal endoplasmic reticulum (ER). We hypothesize that locally synthesized ER proteins regulate microtubule dynamics and the regeneration of cortical axons. RNA data-mining identified the ER-shaping protein Reticulon-1 as a relevant candidate across eight axonal transcriptomes. Using microfluidic chambers, we demonstrate that local knockdown of Reticulon-1 synthesis increases the outgrowth of injured cortical axons while reducing their distal tubulin levels. Furthermore, we show by live-cell imaging that axonal Reticulon-1 knockdown restores the microtubule growth rate and track length modified by injury. Interestingly, local inhibition of the microtubule- severing protein Spastin prevents the axonal knockdown-mediated effects over outgrowth and tubulin levels. We demonstrate that the Reticulon-1C isoform is locally synthesized within axons and support its isoform-specific role in attenuating Spastin-mediated microtubule severing. These findings uncover a mechanism by which axonal protein synthesis finely controls microtubule dynamics and outgrowth upon injury.

neuroscience↗

bioRxiv2023

Ketogenic diet administration later in life improves memory and regulates the synaptic cortical proteome through the cAMP/PKA signaling pathway in aging mice.

Aging is a complex biological process that compromises brain function and neuronal network activity, leading to cognitive decline and synaptic dysregulation. In recent years, a cyclic Ketogenic Diet (KD) has emerged as a potential treatment to ameliorate cognitive decline by improving memory in aged mice after long-term administration. However, the molecular mechanisms that govern such changes remains unclear. Additionally, whether short-term cyclic KD administration later in life preserves memory has not been addressed in detail. Accordingly, here we investigated how a short-term cyclic KD starting at 20-23 months-old regulates brain function of aged mice. Behavioral testing and long-term potentiation (LTP) recordings revealed that a cyclic KD improves working memory and hippocampal LTP in 24-27 months-old mice after 16 weeks of treatment. Moreover, the diet also promotes higher dendritic arborization complexity and dendritic spine density in the prefrontal cortex. Furthermore, to elucidate the molecular mechanisms underlying the memory improvements elicited by a cyclic KD, cortical synaptosomes of aged mice fed with this diet for 1 year were analyzed by mass spectrometry. Bioinformatics analysis revealed that long-term cyclic KD administration predominantly modulates the presynaptic compartment by inducing changes in the cAMP/PKA signaling pathway, the synaptic vesicle cycle, and the actin/microtubule cytoskeleton. To test these findings in vivo, synaptic proteins from cortices of 24-27-month-old mice fed with control or cyclic KD for 16 weeks were analyzed by western blot. Interestingly, increased Brain Derived Neurotrophic Factor abundance, MAP2 phosphorylation and PKA activity were observed. Overall, we show that a cyclic KD regulates brain function and memory even when it is administered at late mid-life and significantly triggers several molecular features of long-term administration, including the PKA signaling pathway and cytoskeleton dynamics, thus promoting synaptic plasticity at advanced age.

neuroscience↗

bioRxiv2020

SUMOylation regulates protein cargo in Astrocyte-derived small extracellular vesicles

Recent studies have described a new mechanism of intercellular communication mediated by various types of extracellular vesicles (EVs). In particular, exosomes are small EVs (sEVs) released to the extracellular environment by the fusion of the endosomal pathway-related multivesicular bodies (containing intraluminal vesicles) with the plasma membrane. sEVs contain a molecular cargo consisting of lipids, proteins, and nucleic acids. However, the loading mechanisms for this complex molecular cargo have not yet been completely elucidated. In that line, the post translational modification SUMO (Small Ubiquitin-like Modifier) has been shown to impact the incorporation of select proteins into sEVs. We therefore decided to investigate whether SUMOylation is a mechanism that defines protein loading to sEVs. In order to investigate the role of SUMOylation in cargo loading into sEVs, we utilized astrocytes, an essential cell type of the central nervous system with homeostatic functions, to study the impact of SUMOylation on the protein cargo of sEVs. Following SUMO overexpression, achieved by transfection of SUMO plasmids or experimental conditions that modulate SUMOylation in primary astrocyte cultures, we detected proteins related to cell division, translation, and transcription by mass-spectrometry. In astrocyte cultures treated with the general SUMOylation inhibitor 2-D08 (2',3',4'-trihydroxy-flavone, 2-(2,3,4-Trihydroxyphenyl)-4H-1-Benzopyran-4-one) we observed an increase in the number of sEVs and a decreased amount of protein cargo within them. In turn, in astrocytes treated with the stress hormone corticosterone, we found an increase of SUMO-2 conjugated proteins and sEVs from these cells contained an augmented protein cargo. In this case, the proteins detected with mass-spectrometry were mostly proteins related to protein translation. To test whether astrocyte-derived sEVs obtained in these experimental conditions could modulate protein synthesis in target cells, we incubated primary neurons with astrocyte-derived sEVs. sEVs from corticosterone-treated astrocytes stimulated protein synthesis while no difference was found with sEVs derived from 2-D08-treated astrocytes. Our results show that SUMO conjugation plays a fundamental role in defining the protein cargo of sEVs impacting the physiological function of target cells.

cell biology↗

bioRxiv2020

The eNOS-dependent S-nitrosylation of the NF-κB subunit p65 has neuroprotective consequences in excitotoxicity

Cell death by glutamate excitotoxicity, mediated by N-methyl-D-aspartate (NMDA) receptors, negatively impacts brain function, including but not limited to hippocampal neurons. The NF-{kappa}B transcription factor (composed mainly of p65/p50 subunits) contributes to neuronal death in excitotoxicity, while its inhibition should improve cell survival. Using the biotin switch method, subcellular fractionation, immunofluorescence and luciferase reporter assays, we found that NMDA stimulated NF-{kappa}B activity selectively in hippocampal neurons, while endothelial nitric oxide synthase (eNOS), an enzyme expressed in neurons, is involved in the S-nitrosylation of p65 and consequent NF-{kappa}B inhibition in cerebrocortical, i.e., resistant neurons. The S-nitro proteomes of cortical and hippocampal neurons revealed that different biological processes are regulated by S-nitrosylation in susceptible and resistant neurons, bringing to light that protein S-nitrosylation is a ubiquitous post-translational modification, able to influence a variety of biological processes including the homeostatic inhibition of the NF-{kappa}B transcriptional activity in cortical neurons exposed to NMDA receptor overstimulation.

neuroscience↗