Biology subjects

Guido, M. E.

Publications and source records attributed to Guido, M. E..

3 recordsLinked to original sources

bioRxiv2023

Pharmacological modulation of the cytosolic oscillator affects glioblastoma cell biology

The circadian system is a conserved time-keeping machinery that regulates a wide range of processes such as sleep/wake, feeding/fasting, and activity/rest cycles to coordinate behavior and physiology. Circadian disruption can be a contributing factor in the development of metabolic diseases, inflammatory disorders, and higher risk of cancer. Glioblastoma (GBM) is a highly aggressive grade 4 brain tumor that is resistant to conventional therapies and has a poor prognosis after diagnosis, with a median survival of only 12-15 months. GBM cells kept in culture were shown to contain a functional circadian oscillator. In seeking more efficient therapies with lower side effects, we evaluated the pharmacological modulation of the circadian clock by targeting the cytosolic kinases glycogen synthase kinase-3 (GSK-3) and casein kinase {varepsilon}/{delta} (CK1{varepsilon}/{delta}) with specific inhibitors (CHIR99022 and PF670462, respectively), the cryptochrome protein stabilizer (KL001), or circadian disruption after Per2 knockdown expression in GBM-derived cells. CHIR99022-treated cells had a significant effect on cell viability, clock protein expression, migration, and cell cycle distribution. Moreover, cultures exhibited higher levels of reactive oxygen species and alterations in lipid droplet content after GSK-3 inhibition as compared with control cells. The combined treatment of CHIR99022 with temozolomide was found to improve the effect on cell viability compared to temozolomide therapy alone. Per2 disruption affected both GBM migration and cell cycle progression. Overall, our results suggest that pharmacological modulation or molecular clock disruption severely affects glioblastoma cell biology.

cancer biology↗

bioRxiv2022

Spatio-temporal dynamics of nuclear CREB1: what does it mean?

In the mammalian pineal gland (PG), cyclic AMP responsive element-binding protein 1 (CREB1) participates in the nocturnal melatonin synthesis that rhythmically modulates physiology and behavior. Phosphorylation of CREB1 present in pinealocyte nuclei is one of the key regulatory steps that drives pineal transcription. The spatio-temporal dynamics of CREB1 itself within PG cell types have not yet been documented. In this study we analyzed total CREB1 via Western blot, and the dynamism of CREB1 nuclear distribution in individual rat pinealocytes using fluorescence immunohistochemistry followed by confocal laser-scanning microscopy and quantitative analysis. Total CREB1 levels remained constant in the PG throughout the light:dark cycle. The distribution pattern of nuclear CREB1 did vary, however, among different PG cells. Pinealocytes emerged as having discrete CREB1 domains within their nucleoplasm that were especially distinct. The number, size, and location of CREB1 foci fluctuated among pinealocytes, within the same PG and among Zeitgeber times. A significantly larger dispersion of CREB1-immunoreactive nuclear sites was found at night. This was not accompanied by changes in the overall transcription activity, which was mostly conserved between the light and dark phases, as shown by the expression of a particular phosphorylated form of the RNA polymerase II (RNAPII-pSer5CTD). Suppression of the nocturnal norepinephrine pulse by chronic bilateral superior cervical ganglionectomy increased CREB1 dispersion in pinealocyte nuclei, as compared to sham-derived cells. In addition, differences in CREB1 distribution were found between sham-operated and non-operated rats at early night. Together, these data suggest that in mature pinealocytes nuclear CREB1 is subjected to a dynamic spatio-temporal distribution. Further studies are necessary to elucidate the underlying mechanisms, including the role of chromatin and interchromatin elements, and to understand the impact of CREB1 reorganization in the pineal transcriptome.

neuroscience↗

bioRxiv2021

Glial Cell Responses to constant low Light exposure in Rat Retina.

Retinal damage promoted by constant illumination of low intensity resulted in a diminution in classical photoreceptors cells. Glial cells exert profound effects on neurons, vasculature and other glial cells. Macroglia and microglia with specific morphological, physiological, and antigenic characteristics may play an essential role in both the maintenance and control of retinal homeostasis, or to exert mechanisms that promote cell death. The role of glial cells and immune function in the pathogenesis promoted by low light is poorly understood. We performed glial cells characterization along the time-course of retinal degeneration induced by chronic exposure to low intensity of light in Wistar rats. We exposed the animals at constant light from 2 to 8 days and assessed the retinal glia. After 6 days of light exposure, retinas presented increased levels of GFAP, a macroglia marker and microglia markers Iba1 and CD68 displayed increased mRNA levels after 6 days. The number of Iba1 positive cells increased in the outer nuclear layer, showing ameboid morphology with thicker processes characteristic of microglial activated cells. The expression levels of immune mediators TNF-{square} and IL-6 were also significantly increased after 6 days. Finally, chemokines analysis showed that CX3CR1 and CCL2 expression levels were significantly elevated after 6 days. Hence, all the events of glial activation occurred after 5-6 days of constant light exposure, when the number of cells of the outer nuclear layer has already decreased significantly. Herein we demonstrated that glial and immune activation are secondary to neurodegeneration; in this scenario, our results suggest that photoreceptor death is an early event that may be induced by phototransduction-dependent mechanisms.

neuroscience↗