Ocampal neurons in culture (Figures 1N and 1O). A comparable synaptotoxic
Ocampal neurons in culture (Figures 1N and 1O). A comparable synaptotoxic impact could also be observed upon activation of AMPK working with metformin, which broadly activates AMPK by inducing a metabolic tension involving reduction of ATP level and conversely enhance in ADPAMP level (Hardie, 2006; Hawley et al., 2010) (Figures 1P and 1Q). Ultimately, application of a more particular AMPK activator, A-769662, induced a considerable, dose-dependent lower in spine density inside 24 hr (Figures 1P and 1Q). Taken with each other, these experiments demonstrate that overactivation of CAMKK2 or AMPK is enough to mimic the synaptotoxic effects induced by A42 oligomers. We next tested when the CAMKK2-AMPK pathway is expected for the synaptotoxic effects induced by A42 oligomers in IL-5 Storage & Stability hippocampal neurons in vitro. We very first took benefit of constitutive knockout (KO) mouse lines for CAMKK2 (Ageta-Ishihara et al., 2009) and AMPK (Viollet et al., 2003) and treated dissociated neuronal cultures isolated from 1 manage (CAMKK2 and AMPK , respectively) or KO mice (CAMKK2– and 1 AMPK –) at 21 DIV with INV42 or A42 oligomers (1 .. M for 24 hr) (Figures 2A HDAC6 MedChemExpress andNeuron. Author manuscript; readily available in PMC 2014 April 10.NIH-PA Author Manuscript NIH-PA Author Manuscript NIH-PA Author ManuscriptMairet-Coello et al.Page2C). Quantitative evaluation indicated that CAMKK2 null and AMPK null neurons usually do not 1 show a important reduction of spine density following A42 oligomer treatment (Figures 2B and 2D). Second, pharmacological inhibition of CAMKK2 activity using application on the inhibitor STO-609 in culture prevented the lower of spine density induced by A42 oligomer application in vitro (Figures 3A and 3B). Though the experiments presented above indicated that CAMKK2 and AMPK kinases are needed to mediate the synaptotoxic effects of A42 in culture, they did not permit to conclude if CAMKK2 acts pre- or postsynaptically, and even indirectly by acting on nonneuronal cells such as astrocytes, which are critically essential for synapse formation and maintenance (Eroglu and Barres, 2010). Hence, we applied a third method where CAMKK2 function was inhibited in a cell-autonomous manner making use of low transfection efficiency of dominant-negative (kinase-dead, KD) types of CAMKK2 (CAMKK2 KD) in wild-type (WT) hippocampal neuron cultures. This experiment revealed that cellautonomous inhibition of CAMMK2 function prevents the reduction of spine density induced by A42 oligomer application (Figures 3C and 3D). Similarly, cell-autonomous inhibition of AMPK catalytic activity by expression of a dominant-negative (KD) type of AMPK (AMPK KD) also abolished the reduction of spine density induced by A42 2 oligomers (Figures 3E and 3F). Importantly, neither CAMKK2 KD nor AMPK KD 2 overexpression alone had any significant effect on spine density per se (Figures 3CF). These results strongly support the notion that the synaptotoxic effects of A42 oligomers call for activation of your CAMKK2-AMPK kinase pathway in hippocampal neurons. We next assessed the protective effects of blocking CAMKK2 following A42 oligomer application employing a functional strategy. To perform this, we performed whole-cell patch-clamp recordings of pharmacologically isolated AMPA-type miniature excitatory postsynaptic currents (mEPSCs) in hippocampal cultures at 18 DIV. As previously shown by Shankar et al. (2007) and Wei et al. (2010), application of A42 oligomers (1 .. M for 24 hr) induced a significant reduction in mEPSC f.