“Time windows” for switching kinase-phosphatase balance during inhibition of long-term potentialization of hippocampal synapses by amyloid aggregates
Authors: Maltsev A. V. , Balaban P. M. 
Published: May 2024

Phosphorylation and dephosphorylation of target proteins are crucial mechanisms for regulating cellular functions. These processes are mediated by protein kinases and phosphatases, respectively. Changes in the kinase-phosphatase balance, known as K-P balance, are well documented during the progression of amyloidosis. The overactivation of kinases or the failed activity of phosphatases is the root cause of hyperphosphorylation of the structural tau protein leading to the formation of fibrillar tangles. The data suggests bidirectional regulation of phosphorylation/dephosphorylation processes during amyloidosis-related states is consistent across different animal models and many protocols. Therefore, it is crucial to identify the “time windows” during which the K-P balance can be altered.
Field excitatory postsynaptic potentials (fEPSPs) were recorded from the stratum radiatum located in area CA1. Baseline synaptic responses were generated by paired-pulse stimulation of the Schaffer collaterals at a frequency of 0.033 Hz using a bipolar electrode. Four 100 Hz trains were delivered 5 minutes apart to induce long-term potentiation (LTP). Serine and threonine phosphatase activity was assessed using a non-radioactive molybdate dye-based assay kit (Promega, USA) based on the manufacturer’s instructions. The procedure relies on the creation of a colored complex of phosphates with a dye containing molybdenum, followed by the measurement of optical densities of supernatants that were incubated against a control at 640 nm. The activity of protein kinase C (PKC) will be measured directly via an enzyme immunoassay (ELISA) on a plate reader, using a commercial Abcam kit (Abcam, USA). The technique relies on the binding of engineered antibodies to activated PKC, and optical densities of incubated supernatants are measured against a control at 450 nm.
In this study, we identified two important time periods during the application of amyloid Aβ25-35 aggregates that affect plasticity in the CA3-CA1 synapses. Specifically, the PKC isoforms are activated during early neurochemical events (0–20 min of incubation of slices in the presence of Aβ25–35 aggregates). If the hippocampal CA3-CA1 synapses are tetanized during this period, we did not observe any inhibition of synaptic plasticity either in the early or late phase development of LTP. Biochemical analysis indicates that hippocampal slices pretreated with Aβ25–35 aggregates for 20–30 minutes exhibit increased PKC activity, whereas no such effect is observed after one hour of incubation. Simultaneously, the hour-long incubation of hippocampal slices in the presence of Aβ25–35 aggregates resulted in the disappearance of long-term potentiation and the return of responses to the pre-tetanic level of synaptic transmission during the late-LTP phase (3 hours after tetanus induction). Inhibitory analysis revealed that an abrupt rise in the activity of stress-induced phosphatase 1α (PP1α) interrupts kinase-mediated signals after LTP induction, leading to the suppression of fEPSPs in the CA3-CA1 synapses. These findings suggest that exposing hippocampal slices to Aβ25–35 aggregates for 20–30 minutes may alter the K-P balance and enhance kinase activity through PKC isoform induction. The majority of PKC isoforms exhibit a tendency to desensitize following activation. Probably, the depletion of the pool of PKCs, which occurs within the first 20–30 minutes after incubation of hippocampal slices with Aβ25–35 aggregates, along with a notable elevation in the activity of stress-induced phosphatase PP1α, is accountable for the second time window for switching the kinase-phosphatase balance, stabilized after an hour of incubation of hippocampal slices in the presence of Aβ25–35 aggregates.
Incubating hippocampal slices with Aβ25–35 aggregates (for 0–30 minutes) leads to a shift in the kinase-phosphatase balance towards the activity of kinases due to the activation of PKC isoforms. After this stage, there is a significant rise in phosphatase activity, resulting from the induction of PP1α. This induces a shift in the balance between kinase and phosphatase towards dephosphorylation processes.