Ers. We next tested in the event the CAMKK2-AMPK pathway is essential for the synaptotoxic effects induced by A42 oligomers in hippocampal neurons in vitro. We initially took advantage 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 handle (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 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 don’t 1 show a significant reduction of spine density following A42 oligomer remedy (Figures 2B and 2D). Second, pharmacological inhibition of CAMKK2 activity working with application on the inhibitor STO-609 in culture prevented the reduce 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 necessary to mediate the synaptotoxic effects of A42 in culture, they did not let to conclude if CAMKK2 acts pre- or postsynaptically, or even indirectly by acting on nonneuronal cells for instance astrocytes, that are critically significant for synapse formation and maintenance (Eroglu and Barres, 2010). As a result, we used a third strategy exactly where CAMKK2 function was inhibited inside a cell-autonomous manner utilizing low transfection efficiency of dominant-negative (kinase-dead, KD) forms 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) form 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 substantial impact on spine density per se (Figures 3CF). These benefits strongly support the notion that the synaptotoxic effects of A42 oligomers need activation with the CAMKK2-AMPK kinase pathway in hippocampal neurons. We subsequent assessed the protective effects of blocking CAMKK2 following A42 oligomer application utilizing a functional strategy.Estriol 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.Dehydroabietic acid As previously shown by Shankar et al.PMID:23558135 (2007) and Wei et al. (2010), application of A42 oligomers (1 .. M for 24 hr) induced a important reduction in mEPSC frequency (manifested as a rise in interevent intervals) compared to manage (INV42) (Figures 3G and 3H). Importantly, overexpression of a KD version of CAMKK2 didn’t impact basal mEPSC frequency but abolished the lower in mEPSC frequency induced by A42 oligomer application (Figures 3G and 3H). None of the remedies had any significant effect on AMPA receptor-mediated mEPSC amplitude (Figure 3I). These final results demonstrate that the CAMKK2-AMPK kinases are important for the early structural and functional effects of A42 oligomers on excitatory synaptic maintenance. The CAMKK2-AMPK Kinase Pathway Is Required for the Dendritic Spine.
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