Effects of CRMP2 DNA Methylation in the Hippocampus on Depressive-Like Behaviors and Cytoskeletal Proteins in Rats

Chronic stress appears to alter DNA methylation and DNA methyltransferases (DNMTs) in brain regions related to emotion. Collapsin response mediator protein-2 (CRMP2) mediates the development of depression by regulating microtubule dynamics. In this study, rats were subjected to chronic unpredictable mild stress (CUMS). At the end of the CUMS procedure, normal saline or fluoxetine was administered to the rats. Moreover, normal saline or the 5-aza-2’-deoxycytidine (5-aza) was administered to the hippocampal CA1 region of the rats. Behavioral tests were performed to evaluate the depressive-like phenotypes. The CRMP2 DNA methylation levels and cytoskeletal microtubular system-related biomarkers were detected by several molecular biology techniques. The results showed that the rat model of depression was successfully established by exposure to CUMS, and fluoxetine treatment exerted an antidepressant-like effect. We observed the upregulation of DNMT1 and DNMT3a in the hippocampus of stressed rats. CUMS induced a decrease in CRMP2 expression and an increase in phosphorylated CRMP2 (pCRMP2) expression in the hippocampus of rats. The rate of DNA methylation in the CpG island of the CRMP2 promoter region in the hippocampus of stressed rats was significantly higher than that in control rats. Moreover, CUMS significantly decreased the interaction between CRMP2 and α-tubulin and decreased the microtubule dynamics. Chronic fluoxetine treatment reversed these changes. Also, hypomethylation induced by 5-aza injection into the hippocampal CA1 region caused antidepressant-like effects and increased CRMP2 expression and microtubule dynamics. These results suggested that CRMP2 DNA methylation may be involved in regulating the cytoskeletal microtubular system and mediating depressive-like behaviors.

Vulnerability of Subcellular Structures to Pathogenesis Induced by Rotenone in SH-SY5Y Cells

Numerous pathological changes of subcellular structures are characteristic hallmarks of neurodegeneration. The main research has focused to mitochondria, endoplasmic reticulum, Golgi apparatus, lysosomal networks as well as microtubular system of the cell. The sequence of specific organelle damage during pathogenesis has not been answered yet. Exposition to rotenone is used for simulation of neurodegenerative changes in SH-SY5Y cells, which are widely used for in vitro modelling of Parkinson´s disease pathogenesis. Intracellular effects were investigated in time points from 0 to 24 h by confocal microscopy and biochemical analyses. Analysis of fluorescent images identified the sensitivity of organelles towards rotenone in this order: microtubular cytoskeleton, mitochondrial network, endoplasmic reticulum, Golgi apparatus and lysosomal network. All observed morphological changes of intracellular compartments were identified before αS protein accumulation. Therefore, their potential as an early diagnostic marker is of interest. Understanding of subcellular sensitivity in initial stages of neurodegeneration is crucial for designing new approaches and a management of neurodegenerative disorders.

The regulatory role of cell mechanics for migration of differentiating myeloid cells

Migration of cells is important for tissue maintenance, immune response, and often altered in disease. While biochemical aspects, including cell adhesion, have been studied in detail, much less is known about the role of the mechanical properties of cells. Previous measurement methods rely on contact with artificial surfaces, which can convolute the results. Here, we used a non-contact, microfluidic optical stretcher to study cell mechanics, isolated from other parameters, in the context of tissue infiltration by acute promyelocytic leukemia (APL) cells, which occurs during differentiation therapy with retinoic acid. Compliance measurements of APL cells reveal a significant softening during differentiation, with the mechanical properties of differentiated cells resembling those of normal neutrophils. To interfere with the migratory ability acquired with the softening, differentiated APL cells were exposed to paclitaxel, which stabilizes microtubules. This treatment does not alter compliance but reduces cell relaxation after cessation of mechanical stress six-fold, congruent with a significant reduction of motility. Our observations imply that the dynamical remodeling of cell shape required for tissue infiltration can be frustrated by stiffening the microtubular system. This link between the cytokeleton, cell mechanics, and motility suggests treatment options for pathologies relying on migration of cells, notably cancer metastasis.