Biology subjects

Munoz, E. M.

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

3 recordsLinked to original sources

bioRxiv2025

Seeking rhythmic patterns in microglial cells within the circadian pineal gland from male rats

Microglia, the innate immune cells of the brain, constitute a highly dynamic cell population that displays several functions influenced by the light:dark (L:D) cycle. Within the pineal gland (PG), a key organ of the circadian timing system, microglia actively participate in its development and homeostasis. However, little is known about their rhythmic features in this circadian organ. This study aimed to elucidate morphological and functional phenotypes of pineal microglial cells at two time points of the L:D cycle. We performed immunofluorescence staining and confocal microscopy on paraffin-embedded pineal sections from 3- and 18-month-old Wistar rats, analyzing samples collected at midday (ZT6) and midnight (ZT18). Our results showed that the density and spatial distribution of IBA1+ microglial cells did not vary between ZT6 and ZT18 in the 3-month-old PG. However, these cells exhibited reduced size and amoeboid-like shapes, along with a reduction in the expression of the phagocytosis inhibitor SIRP alpha, at ZT18 compared to ZT6. Moreover, IBA1+ cells were immunoreactive for the lysosomal marker CD68 and the autophagic marker LC3B at both ZTs. Additionally, contacts with PAX6+ cells were detected in the two ZTs analyzed. Interestingly, IBA1+ cells showed attenuated or abolished rhythmicity in morphological parameters and SIRP alpha expression levels in the 18-month-old PG. Our findings suggest that microglia undergo transitions into alerted states at night, characterized by small, rounded shapes and enhanced phagocytic capacity. This adaptation likely prepares them to respond to potential invading agents and other insults in the PG, which could impact the nocturnal melatonin production. Key pointsO_LIMicroglia within the rat pineal gland exhibit daily morphological and functional adaptations, enhancing their effectivity to respond against potential challenges in a time-dependent manner. C_LIO_LIOscillatory features of pineal microglia diminish with aging, suggesting an attenuation of their functions. C_LIO_LINocturnal reactivity of microglia within the pineal organ may influence its physiology, potentially affecting melatonin synthesis. C_LI

neuroscience↗

bioRxiv2024

Impact of aging on the GABAB receptor-mediated connectome

GABA B receptors (GABABRs) are heterodimeric seven-transmembrane receptors that interact with a range of proteins and form large protein complexes on cholesterol-rich membrane microdomains. As the brain ages, membrane cholesterol levels exhibit alterations, although it remains unclear how these changes impact protein-protein interactions and downstream signaling. Herein, we studied the structural bases for the interaction between GABABR and the KCC2 transporter, including their protein expression and distribution, and we compared data between young and aged rat cerebella. Also, we analyzed lipid profiles for both groups, and we used molecular dynamics simulations on three plasma membrane systems with different cholesterol concentrations, to further explore the GABABR-transporter interaction. Based on our results, we report that a significant decrease in GABAB2 subunit expression occurs in the aged rat cerebella. After performing a comparative co-immunoprecipitation analysis, we confirm that GABABR and KCC2 form a protein complex in adult and aged rat cerebella, although their interaction levels are reduced substantially as the cerebellum ages. On the other hand, our lipid analyses reveal a significant increase in cholesterol and sphingomyelin levels of the aged cerebella. Finally, we used the Martini coarse-grained model to conduct molecular dynamics simulations, from which we observed that membrane cholesterol concentrations can dictate whether the GABABR tail domains physically establish G protein-independent contacts with a transporter, and the timing when those associations eventually occur. Taken together, our findings illustrate how age-related alterations in membrane cholesterol levels affect protein-protein interactions, and how they could play a crucial role in regulating GABABRs interactome-mediated signaling. Significance StatementThis study elucidates age-related changes in cerebellar GABAB receptors (GABABRs), KCC2, and plasma membrane lipids, shedding light on mechanisms underlying neurological decline. Molecular dynamics simulations reveal how membrane lipids influence protein-protein interactions, offering insights into age-related neurodegeneration. The findings underscore the broader impact of cerebellar aging on motor functions, cognition, and emotional processing in the elderly. By elucidating plasma membrane regulation and GABAergic dynamics, this research lays the groundwork for understanding aging-related neurological disorders and inspires further investigation into therapeutic interventions.

neuroscience↗

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↗