PhD thesis

The data uploaded here is related to projects incorporated in the PhD thesis titled: "Deciphering the role of the actin cytoskeleton in central nervous system synapses". This thesis arises from work carried out at Dementia Research Centre, Department of Biomedical Sciences, Faculty of Medicine, Health and Human Sciences, Macquarie University, Sydney, Australi

DEHP Decreases Steroidogenesis through the cAMP and ERK1/2 Signaling Pathways in FSH-Stimulated Human Granulosa Cells

DEHP is an endocrine disruptor that interferes with the function of the female reproductive system. Several studies suggested that DEHP affects steroidogenesis in human and rodent granulosa cells (GC). Some studies have shown that DEHP can also affect the FSH-stimulated steroidogenesis in GC; however, the mechanism by which DEHP affects hormone-challenged steroidogenesis in human GC is not understood. Here, we analyzed the mechanism by which DEHP affects steroidogenesis in the primary culture of human cumulus granulosa cells (hCGC) stimulated with FSH. Cells were exposed to DEHP and FSH for 48 h, and steroidogenesis and the activation of cAMP and ERK1/2 were analyzed. The results show that DEHP decreases FSH-stimulated STAR and CYP19A1 expression, which is accompanied by a decrease in progesterone and estradiol production. DEHP lowers cAMP production and CREB phosphorylation in FSH but not cholera toxin- and forskolin-challenged hCGC. DEHP was not able to decrease steroidogenesis in cholera toxin- and forskolin-stimulated hCGC. Furthermore, DEHP decreases FSH-induced ERK1/2 phosphorylation. The addition of EGF rescued ERK1/2 phosphorylation in FSH- and DEHP-treated hCGC and prevented a decrease in steroidogenesis in the FSH- and DEHP-treated hCGC. These results suggest that DEHP inhibits the cAMP and ERK1/2 signaling pathways, leading to the inhibition of steroidogenesis in the FSH-stimulated hCGC

Spermatozoal Mitochondrial Dynamics Markers and Other Functionality-Related Signaling Molecules Exert Circadian-like Response to Repeated Stress of Whole Organism

In the search for the possible role of the mitochondrial dynamics markers in spermatozoa adaptation, an in vivo approach was designed to mimic situations in which human populations are exposed to 3 h of repeated psychological stress (the most common stress in human society) at different time points during the day (24 h). The hormones (stress hormone corticosterone and testosterone), the number and the functionality of spermatozoa (response to acrosome-reaction-inducer progesterone), as well as the transcriptional profiles of 22 mitochondrial dynamics and function markers and 22 signaling molecules regulating both mitochondrial dynamics and spermatozoa number and functionality were followed at three time points (ZT3, ZT11, and ZT23). The results show that repeated stress significantly decreased the number and functionality of spermatozoa at all time points. In the same samples, the transcriptional profiles of 91% (20/22) of mitochondrial dynamics and functionality markers and 86% (19/22) of signaling molecules were disturbed after repeated stress. It is important to point out that similar molecular changes in transcriptional profiles were observed at ZT3 and ZT23, but the opposite was observed at ZT11, suggesting the circadian nature of the adaptive response. The results of PCA analysis show the significant separation of repeated stress effects during the inactive/light and active/dark phases of the day, suggesting the circadian timing of molecular adaptations

Overexpression of Tropomyosin Isoform Tpm3.1 Does Not Alter Synaptic Function in Hippocampal Neurons

Tropomyosin (Tpm) has been regarded as the master regulator of actin dynamics. Tpms regulate the binding of the various proteins involved in restructuring actin. The actin cytoskeleton is the predominant cytoskeletal structure in dendritic spines. Its regulation is critical for spine formation and long-term activity-dependent changes in synaptic strength. The Tpm isoform Tpm3.1 is enriched in dendritic spines, but its role in regulating the synapse structure and function is not known. To determine the role of Tpm3.1, we studied the synapse structure and function of cultured hippocampal neurons from transgenic mice overexpressing Tpm3.1. We recorded hippocampal field excitatory postsynaptic potentials (fEPSPs) from brain slices to examine if Tpm3.1 overexpression alters long-term synaptic plasticity. Tpm3.1-overexpressing cultured neurons did not show a significantly altered dendritic spine morphology or synaptic activity. Similarly, we did not observe altered synaptic transmission or plasticity in brain slices. Furthermore, expression of Tpm3.1 at the postsynaptic compartment does not increase the local F-actin levels. The results suggest that although Tpm3.1 localises to dendritic spines in cultured hippocampal neurons, it does not have any apparent impact on dendritic spine morphology or function. This is contrary to the functional role of Tpm3.1 previously observed at the tip of growing neurites, where it increases the F-actin levels and impacts growth cone dynamics

Syntaxins 6 and 8 facilitate tau into secretory pathways

Tau pathology initiates in defined brain regions and is known to spread along neuronal connections as symptoms progress in Alzheimer's disease (AD) and other tauopathies. This spread requires the release of tau from donor cells, but the underlying molecular mechanisms remained unknown. Here, we established the interactome of the C-terminal tail region of tau and identified syntaxin 8 (STX8) as a mediator of tau release from cells. Similarly, we showed the syntaxin 6 (STX6), part of the same SNARE family as STX8 also facilitated tau release. STX6 was previously genetically linked to progressive supranuclear palsy (PSP), a tauopathy. Finally, we demonstrated that the transmembrane domain of STX6 is required and sufficient to mediate tau secretion. The differential role of STX6 and STX8 in alternative secretory pathways suggests the association of tau with different secretory processes. Taken together, both syntaxins, STX6 and STX8, may contribute to AD and PSP pathogenesis by mediating release of tau from cells and facilitating pathology spreading