CaMKIIβ knockdown decreases store-operated calcium entry in hippocampal dendritic spines

Abstract

Calcium/calmodulin-dependent protein kinase II (CaMKII) and neuronal store-operated calcium entry (nSOCE) have been implicated in the development of Alzheimer's disease (AD). nSOCE is involved in regulation of dendritic spine shape, particularly in stability of mushroom spines that play role in formation of strong synapses. CaMKII is involved in regulation of induction of long-term potentiation, that is needed for shaping of memory. In the present study, we demonstrated that inhibition of kinase activity of CaMKII by KN-62 decreases nSOCE amplitude in soma of primary hippocampal neurons. We have shown that knockdown of CaMKIIβ leads to the downregulation of nSOCE in dendritic spines. In agreement with previously published data, we have also observed that CaMKIIβ knockdown causes mushroom spine loss in primary hippocampal culture. The effect of CaMKIIβ knockdown on the nSOCE may be associated with a decrease of dendritic spine head size.

New Positive TRPC6 Modulator Penetrates Blood–Brain Barrier, Eliminates Synaptic Deficiency and Restores Memory Deficit in 5xFAD Mice

“…Primary hippocampal neuronal cultures were prepared as previously described [ 19 , 25 , 45 , 46 ]. Briefly, hippocampuses from postnatal day 0–2 FVB/NJ mice were removed and cut into small pieces in ice-cold buffer (1% 10x CMF-HBSS (Gibco, Grand Island, NY, USA; #14185), 1% Pen Strep (Gibco, #15140), 16 m M HEPES (Sigma, St Louis, MO, USA; #H3375), 10 m M NaHCO 3 (Sigma, #S5761); pH = 7.2).…”

“…Neurons were transfected using the calcium phosphate method at DIV 7 as previously described [ 25 ] with alterations that were described earlier [ 46 ]. The transfection kit was obtained from Clontech (TAKARA Biotechnology, Mountain View, CA, USA; #631312).…”

“…For assessment of synapse morphology, hippocampal cultures were transfected with TD-tomato plasmid (pCSCMV:tdTomato was a gift from Gerhart Ryffel (Addgene, Watertown, MA, USA; #30530)) at DIV7 using the calcium phosphate method and treated with Aβ42 working solution and 1 uM, 100 nM, or 10 nM of C20 for 24 h at DIV14 and then fixed. Confocal microscopy parameters and morphological analysis were described previously [ 46 ]. Briefly, a Z-stack of 10–25 optical sections with a 0.2 µm interval was captured using a 100 × lens (UPlanSApo, 100x/1.40 Oil, OLYMPUS, Tokyo, Japan) with a confocal microscope (Thorlabs, Newton, NJ, USA).…”

“…Each image was captured at 1024 × 1024 pixels with a maximum resolution of 0.1 µm/pixel. Morphological analysis of dendritic spines was performed by using the NeuronStudio software package [ 48 ] as previously described [ 25 , 46 ].…”

Piperazine Derivative Stabilizes Actin Filaments in Primary Fibroblasts and Binds G-Actin In Silico

“…One of the TRPC6 agonists, N-(2-chlorophenyl)-2-(4-phenylpiperazine-1-yl) acetamide (51164), restores nSOCE, maintains mushroom spine percentage, and recovers synaptic plasticity in amyloidogenic mouse models of AD under conditions of amyloid toxicity [ 13 ]. We recently revealed that CaMKIIβ plays a role in regulating nSOCE activity in primary hippocampal culture [ 14 ]. In this article, we found that 51164 is unable to upregulate nSOCE under CaMKIIβ knockdown conditions.…”