AQUAPORIN-4 IN GLIAL CALCIUM HOMEOSTASIS

Astrocytes are active elements of the brain circuitry. They integrate neuronal signals, exhibit Ca++ excitability and process information. Ca++ signaling in activated astrocytes has been proposed to trigger the release of many neuroactive molecules, such as glutamate, ATP and D-serine, which can modulate neuronal excitability, synaptic activity and plasticity. Aquaporin-4 (AQP4) is strongly expressed in astrocyte endfeet and has an important role in brain water flux at the blood-brain and CSF-brain barriers. In the present study we have used WT and AQP4 KO astrocyte primary cultures to show that AQP4 plays a role in glial calcium homeostasis. By Ca++ imaging experiments we demonstrated that under hypotonic stress WT astrocytes exhibited an intracellular Ca++ increase whose amplitude was 6 times higher in the presence of external Ca++, indicating a Ca++ influx from the extracellular and not intracellular stores. The same experiments, performed in parallel on AQP4 KO astrocytes, showed that the amplitude of this phoenomenon was significantly reduced and associated to a delay in calcium influx, suggesting a direct effect of the altered water permeability on the hypotonic shock dependent Ca++ increase. Gadolinium and Ruthenium red were later used to show that this effect was dependent on plasma membrane stretch activated Ca++ channels. Finally, the use of CPA helped us demonstrating that the difference in the influx of calcium was not due to ICrac but mainly related to the magnitude of membrane stretch. All together these findings suggest that AQP4 plays a pivotal role in astrocyte Ca++ homeostasis and is therefore involved in the modulation of neuronal excitability, synaptic activity and plasticity

Epigenetic versus Genetic Deregulation of the KEAP1/NRF2 Axis in Solid Tumors: Focus on Methylation and Noncoding RNAs

Oxidative and electrophilic changes in cells are mainly coordinated by the KEAP1/NRF2 (Kelch-like erythroid-derived cap-n-collar homology- (ECH-) associated protein-1/nuclear factor (erythroid-derived 2)-like 2) axis. The physical interaction between these two proteins promotes the expression of several antioxidant defense genes in response to exogenous and endogenous insults. Recent studies demonstrated that KEAP1/NRF2 axis dysfunction is also strongly related to tumor progression and chemo- and radiotherapy resistance of cancer cells. In solid tumors, the KEAP1/NRF2 system is constitutively activated by the loss of KEAP1 or gain of NFE2L2 functions that leads to its nuclear accumulation and enhances the transcription of many cytoprotective genes. In addition to point mutations, epigenetic abnormalities, as aberrant promoter methylation, and microRNA (miRNA) and long noncoding RNA (lncRNA) deregulation were reported as emerging mechanisms of KEAP1/NRF2 axis modulation. This review will summarize the current knowledge about the epigenetic mechanisms that deregulate the KEAP1/NRF2 cascade in solid tumors and their potential usefulness as prognostic and predictive molecular markers

Inhibition of Aquaporin-1 dependent angiogenesis impairs tumour growth in a mouse model of melanoma

Prohibiting angiogenesis is an important therapeutic approach for fighting cancer and other angiogenic related diseases. Research focused on proteins that regulate abnormal angiogenesis has attracted intense interest in both academia and industry. Such proteins are able to target several angiogenic factors concurrently, thereby increasing the possibility of therapeutic success. Aquaporin-1 (AQP1) is a water channel membrane protein that promotes tumour angiogenesis by allowing faster endothelial cell migration. In this study we test the hypothesis that AQP1 inhibition impairs tumour growth in a mouse model of melanoma. After validating the inhibitor efficacy of two different AQP1 specific siRNAs in cell cultures, RNA interference experiments were performed by intratumoural injections of AQP1 siRNAs in mice. After 6 days of treatment, AQP1 siRNA treated tumours showed a 75% reduction in volume when compared to controls. AQP1 protein level, in AQP1 knockdown tumours, was around 75 % that of the controls and was associated with a significant 40 % reduced expression of the endothelial marker, Factor VIII. Immunofluorescence analysis of AQP1 siRNA treated tumours showed a significantly lower microvessel density. Time course experiments showed that repeated injections of AQP1 siRNA over time are effective in sustaining the inhibition of tumour growth. In conclusion, this study validates AQP1 as a pro-angiogenic protein, relevant for the therapy of cancer and other angiogenic-related diseases such as psoriasis, endometriosis, arthritis and atherosclerosis

Contribution of Aquaporins and TRPV4 to astrocyte cell volume regulation

Regulatory Volume Decrease (RVD) is a process by which cells restore their volume when swollen by hypo-osmotic stress. In this study, we have focused on the role played by two different Aquaporins (AQPs), AQP4 and AQP1, in mediating Ca2+ signaling after hypotonic shock and in triggering RVD, together with the Transient Receptor Potential Vanilloid 4 (TRPV4), a Ca2+-permeable channel activated by the membrane stretching. Using biophysical techniques to measure the water plasma membrane permeability of WT and AQP4 KO astrocytes and of cells transfected with AQP4 or AQP1, we showed that both AQPs play a key role in RVD by affecting the initial kinetics of the swollen phase that is faster and higher in amplitude in the presence of AQPs. By calcium imaging we showed that AQP4 and AQP1-mediated cell swelling significantly increases the amplitude of Ca2+ influx inhibited by the TRPV4 inhibitors, Gadolinium (Gad) and Ruthenium Red (RR). Finally, the effect of Ca2+ influx through TRPV4 on the cell volume regulation was analyzed by measuring RVD in the presence of Gad and RR or removing the external Ca2+. Our results show that the RVD kinetic was unchanged in all these conditions, indicating that the TRPV4 mediated Ca2+ influx does not play a role in RVD. All together these results show that 1) AQPs play a key role in mediating Ca2+ signaling after hypotonic shock together with TRPV4, 2) AQPs are the main trigger for RVD, and 3) Ca2+is not fundamental for RVD to occur