Recent literature has shown that AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid) receptors lacking the GluR2 subunit (thus calcium permeable) are widely expressed in the CNS, especially in interneurons and glia, where they contribute to synaptic transmission and plasticity. Studies have also indicated that calcium permeable AMPARs (CP-AMPARs) are expressed and participate in synaptic regulation in principal neurons, including hippocampal pyramidal neurons. Furthermore, CP-AMPARs and their resultant calcium influx are implicated in various pathophysiological conditions such as ischemia and seizures. However, the synaptic events activated by calcium influx through CP-AMPARs remain unknown. I took advantage of genetically altered mice without (GluR2-/-) or with reduced GluR2 (GluR2+/-), thus allowing the expression and detailed analysis of synaptic CP-AMPARs in hippocampal pyramidal neurons. Utilizing electrophysiological techniques, I demonstrated that these receptors were capable of inducing numerous forms of long-term potentiation (referred to as CP-AMPAR-dependent LTP) through a number of different induction protocols, including high-frequency stimulation (HFS) and theta-burst stimulation (TBS). This included a previously undemonstrated form of protein-synthesis dependent late-LTP (L-LTP) at CA1 synapses that is NMDA-receptor (NMDAR) independent. This form of plasticity was completely blocked by the selective CP-AMPAR inhibitor IEM-1460. Surprisingly, calcium/calmodulin-dependent kinase II (CaMKII), the key protein kinase that is indispensable for NMDAR-dependent LTP at CA1 synapses appeared to be not required for the induction of CP-AMPAR-dependent LTP due to the lack of effect of two separate pharmacological inhibitors (KN-62 and staurosporine) on this form of potentiation. Both KN-62 and staurosporine strongly inhibited NMDAR dependent LTP in control studies. In contrast, inhibitors for the extracellular signal-regulated kinase/mitogen-activated protein kinase (ERK/MAPK) cascade (PD98059 and U0126) significantly attenuated this CP-AMPAR-dependent LTP. Additional studies with knockout mice revealed that the ERK/MAPK signalling cascade is likely acting through p-21 activated kinase 1 (or PAK1, a Rho-GTPase associated kinase) dependent mechanisms. These results suggest that distinct synaptic signalling underlies GluR2-lacking CP-AMPAR-dependent LTP, and reinforces the recent notions that CP-AMPARs are important facilitators of synaptic plasticity in the brain.

(English) This work is based on five publications studying mostly adenylate cyclase toxin (CyaA) from Bordetella pertussis and its interaction with biological membranes. CyaA permeabilizes cell membranes by forming small cation­selective pores and subverts cellular signaling by delivering an adenylate cyclase (AC) enzyme that converts ATP to cAMP into host cells. First study clarifies the membrane disruption mechanisms of CyaA and another bacterial RTX toxin; α­hemolysin (HlyA) from Escherichia coli. For this purpose, we employed a fluorescence requenching method using liposomes as target membranes. We showed that both toxins induced a graded leakage of liposome content with different ion selectivities (Fišer a Konopásek 2009). Both AC delivery and pore formation were previously shown to involve a predicted amphipathic α­helix(502­522). In the second publication we investigated another predicted transmembrane α­helix(565­591) that comprises a Glu(570) and Glu(581) pair. We examined the roles of these glutamates in the activity of CyaA, mostly on planar lipid membranes end erythrocytes. Negative charge at position 570, but not at position 581, was found to be essential for cation selectivity of the pore, suggesting a role of Glu(570) in...

AMP-activated protein kinase (AMPK) is a key regulator of energy homeostasis in cells. It has been implicated as a therapeutic target for various metabolic diseases like type II diabetes and obesity. However, its role in cancer is context-dependent and therefore warrants further studies to explore its possible use as a therapeutic target. AMPK can either promote or retard the growth of cancer cells depending on other cues and stresses in the milieu of the cancer cells. This study aims to understand AMPK signalling in response to extracellular cues of matrix deprivation and matrix stiffness that are important determinants of metastasis. 1) Calcium-Oxidant Signalling Network Regulates AMPK Activation upon Matrix Deprivation. Recent work from our lab, as well as others, has identified a novel role for the cellular energy sensor AMP-activated protein kinase in epithelial cancer cell survival under matrix deprivation. However, the molecular mechanisms that activate AMPK upon matrix-detachment remain unexplored. In this study, we show that AMPK activation is a rapid and sustained phenomenon upon matrix deprivation, while re-attachment to the matrix leads to its dephosphorylating and inactivation. Since matrix-detachment leads to loss of integrin signalling, we investigate whether integrin signalling negatively regulates AMPK activation. However, modulation of FAK or Src, the major downstream components of integrin signalling, fails to cause a corresponding change in AMPK signalling. Further investigations reveal that the upstream AMPK kinases, LKB1 and CaMKKβ, contribute to AMPK activation upon detachment. Additionally, we show LKB1 phosphorylation and cytosolic translocation upon matrix deprivation, which might also contribute to AMPK activation. In LKB1-deficient cells, we find AMPK activation to be predominantly dependent on Caskβ. We observe no change in ATP levels under detached conditions at early time points suggesting that rapid AMPK activation upon detachment is not triggered by energy stress. We demonstrate that matrix deprivation leads to a spike in intracellular calcium as well as oxidant signalling and both these intracellular messengers contribute to rapid AMPK activation upon detachment. We further show that ER calcium release induced store-operated calcium entry (SOCE) contributes to intracellular calcium increase, leading to ROS production, and AMPK activation. We additionally show that the LKB1/CaMKK-AMPK axis and intracellular calcium levels play a critical role in anchorage-independent cancer sphere formation. We find a significant increase in LKB1 as well as pACC levels in breast tumour tissues in comparison to normal tissues. Further, we observe a significant correlation between LKB1 and pACC levels in breast tumour tissues suggesting that LKB1-AMPK signaling pathway is active in vivo in breast cancers. Thus, the Ca2+/ROS triggered LKB1/CaMKK-AMPK signalling cascade may provide a quick, adaptable switch to promote survival of metastasising cancer cells. 2) Extracellular Matrix Stiffness Regulates Stemless through AMPK. Cancer cells experience changes in extracellular matrix stiffness during cancer progression. However, the signalling pathways utilised in sensing matrix stiffness are poorly understood. In this study, we identify AMPK pathway as a possible mechanosensory pathway in response to matrix stiffness. AMPK activity, as measured by downstream target phosphorylation, is found to be higher in soft matrix conditions. We additionally show that compared to stiff matrices, soft matrices increase stemless properties, as evidenced by the increased expression of stemless markers, which is dependent on AMPK activity. Thus, we elucidate a novel mechanotransduction pathway triggered by matrix stiffness that contributes to stemness of cancer cells by regulating AMPK activity. Taken together, our study identifies a novel calcium-oxidant signaling network in the rapid modulation of AMPK signaling in the context of matrix detachment. This pathway is especially relevant in the context of metastasising cancer cells that may not face energy stress in the blood stream but are matrix-deprived. Inhibition of AMPK might compromise the viability of these circulating cells thereby reducing the metastatic spread of cancer. Our study further suggests that varying stiffnesses experienced by cancer cells can modulate AMPK activity and this, in turn, regulates stem-like properties. Thus our study provides novel insights into various extracellular cues that regulate this kinase and contribute to cell survival and metastasis. This knowledge can be utilised in the stage-specific use of AMPK inhibitors in the treatment of breast cancer patients.