Percorrer por autor "Torres, Guilherme Machado"
A mostrar 1 - 1 de 1
Resultados por página
Opções de ordenação
- Decoding acylation defects in the nervous system using a novel Zdhhc14 knockout mousePublication . Torres, Guilherme Machado; Brites, Pedro; Ferraz, RicardoThe complexity of biological processes within cells and normal tissue function is, to a great extent, guaranteed by post-translational modifications (PTMs). S-acylation, also known as palmitoylation, is one PTM whose importance in the nervous system has been increasingly gaining attention and comprises a process by which long-chain fatty acids are covalently attached to cysteine residues of proteins. This process can have implications for protein localization, association with lipid membranes, and protein stability and/or function. Palmitoylation is accomplished by a family of zinc-finger DHHC (ZDHHC) motif-containing palmitoyl acyltransferases. ZDHHC14 is abunantly expressed in the nervous system, and its deficiency i sone of the hallmarkers of 6q25 microdeletion syndrome, where patients presente with microencephaly, developmental delay, and cognitive impairements. Despite this, there is a current knowledge gap regarding ZDHHC14 activity, funtion and targets. To address this problema, we generated a knockout (KO) mouse model, representing the first in vivo model for Zdhhc14 deficiency, using CRISPR-Cas9 technology to delete exon 5 of the Zdhhc14 gene. PCR analysis of brain tissue showed that deletion of exon 5 with its ensuing mutation, i.e., R235Lfs1*, leads to nonsense-mediated mRNA decay and virtually undetected Zdhhc14 mRNA in Zdhhc14 KO mice. We also determined that brain regions, including the corpus callosum, hippocampus, and córtex, have the highest expression of Zdhhc14 mRNA. In vitro experiments on cortical neurons revealed that the absence of Zdhhc14 has an impacto n the length and positioning of the axon initial segment (AIS). The AIS of Zdhhc14 KO neurons was shorter and more distally located along the axon. Alternations in synapse formation were also identified in Zdhhc14 KO cortical neurons, with a reduction in density of presynaptic terminals and postsynaptic sites, and a decrease in the number of functionally active synapses. Following preliminar neuropathological data and employing transmission electron microscopy on the optic nerves of wild-type (WT) and Zdhhc14 KO mice, we unraveled a defect in myelination characterized by a reduced thickness of the myelin sheath and an increase in the number and length of myelin outfoldings. Moreover, the measurement of myelin components revealed decreased expression of some myelin markers (e.g., Atp1a1 and Gstp1), which could be related to defective Zdhhc14 activity and may modulate the observed myelin defects. Through confocal imaging and 3D reconstruction softwere, we were able to detect myelin outfoldings in the brains of Zdhhc14 KO mice and accurately depict their three-dimensional structure. In conclusion, our data highlight a crucial role for Zdhhc14 in AIS assembly, synaptogenesis, and myelination of the central nervous system (CNS).