Identified molecular mechanisms behind learning and memory

Summary: Findings reveal the molecular mechanism of acetylcholine in learning and memory.

Source: Fujita University of Health

Patients with Alzheimer’s disease (AD) have lower levels of the neuromodulator acetylcholine (ACh) in the brain. Donepezil, an AD drug, increases the brain’s ACh levels and improves AD-associated learning deficits.

Researchers have now identified the intracellular signaling cascade through which ACh regulates aversive learning, an important preliminary test for AD medications.

Researchers also found that donepezil activates this signal cascade to regulate aversive learning. The findings indicate the potential of the signal cascade as a medical target.

Acetylcholine (ACh) is a neuromodulator with a central role in aversive learning – rapid conditioning to unpleasant odor, taste or touch. These learning functions take place in cells called D2-receptor-expressing medium spiny neurons (D2R-MSNs) which are located in the striatum / nucleus accumbens (NAc) of the brain. ACh levels increase in NAc during aversive learning experiences.

Previous studies have shown that ACh acts on D2R-MSN through a receptor called muscarinic receptor M1 (M1R), which in turn activates the downstream signaling molecule called protein kinase C (PKC).

So far, however, the exact intracellular signaling mechanism by which ACh affects aversive learning has been unclear, which has limited the development of AD therapeutic strategies that directly target ACh intracellular signaling.

Recently, in a new study published in Molecular psychiatryresearchers from Prof. The Kozo Kaibuchi Laboratory at Fujita Health University (FHU) has elucidated the molecular mechanisms of ACh for learning and memory.

“This is the first time this has been achieved in the 45 years since the cholinergic hypothesis of AD was established. Our study also led us to understand the intracellular mechanism of donepezil and its effect on learning and memory. This exciting discovery opens doors to new therapeutic strategies for AD, ”explains Assistant Professor Yukie Yamahashi, a lead author of the study.

Molecular signal cascades are simplified by a process called phosphorylation, which involves the addition of phosphate groups to certain substrate molecules of kinases in cells. To study phosphorylation, the research team used a technique called kinase-oriented phosphoproteomic analysis, which was developed by Prof. Kozo Kaibuchi, the corresponding author of the study.

The research team confirmed the role of ACh in stimulating PKC after monitoring phosphorylation events after ACh binding to M1Rs in mouse striatal / NAc slices ex vivo. Then, the phosphoproteomic assay was performed, which yielded 116 PKC substrate candidates, including “β-PIX”, the activator of a protein called “small GTPase Rac.”

“We discovered that PKC phosphorylated and activated β-PIX downstream of ACh, which in turn activated a kinase called PAK, a downstream target of Rac. We then investigated the involvement of the identified ACh-M1R-PKC-Rac-β-PIX-PAK cascade. in aversive learning and aversion memory using passive avoidance tests on mice, says Dr. Yamahashi. Finally, the researchers also found that donepezil activates the cascade to improve aversive learning.

“This study provides the first evidence for the intracellular mechanisms of donepezil that regulate learning and memory,” said Dr. Yamahashi.

Their findings agree well with a recent study from Prof. Kaibuchi’s laboratory published in Journal of Neurochemistry. The first author of the study, Dr. Md. Omar Faruk, has been awarded the Mark A. Smith Prize by the International Society for Neurochemistry (ISN).

The study showed the involvement of the “voltage-gated potassium channel KCNQ2” – which was identified as another PKC substrate candidate in the above phosphoproteomic assay – in aversive learning. In fact, PKC directly phosphorylates KCNQ2 at threonine 217, the phosphorylation site previously reported for possible involvement of modulation of channel activity. Furthermore, the administration of donepezil also enhanced the phosphorylation event in NAc.

In aversive stimulus (electric foot shock), acetylcholine activates PAK kinase through the M1R-PKC cascade to facilitate synaptic plasticity. It also enhances PKC-mediated KCNQ2 phosphorylation to stimulate neuronal excitability, which in turn increases neural firing in response to glutamatergic input. Activation of both PAK-mediated and KCNQ2-mediated pathways results in aversive behavior. Credit: Kozo Kaibuchi and Yukie Yamahashi of Fujita Health University

The team’s findings directly suggest that the signal cascade, M1R-PKC-β-PIX-PAK, is involved in recognition memory and associative learning. This is very important as the cascade itself offers a platform for screening AD drugs that are under development.

While focusing only on β-PIX and elucidating the M1R-PKC-PAK pathway, our phosphoproteomic data revealed many other PKC substrates – presynaptic proteins and postsynaptic scaffold proteins to name a few, which are recorded in a database called Kinase-Associated Neural PHOspho-Signaling (KANPHOS) (

“We only see the tip of the iceberg and believe that future research could provide new mechanisms for signal transmission in other areas of the brain,” says Dr. Yamahashi, regarding the future prospects of their research.

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About this learning and memory research news

Author: Press office
Source: Fujita University of Health
Consult: Press Office – Fujita Health University
Picture: The photo is credited to Kozo Kaibuchi and Yukie Yamahashi from Fujita Health University

Original research: Open access.
“Phosphoproteomic of the acetylcholine pathway allows the detection of the PKC-β-PIX-Rac1-PAK cascade as a stimulating signal for aversive learning” by Yukie Yamahashi et al. Molecular psychiatry


Phosphoproteomic of the acetylcholine pathway allows the discovery of the PKC-β-PIX-Rac1-PAK cascade as a stimulating signal for aversive learning

Acetylcholine is a neuromodulator that is critical for learning and memory. The cholinesterase inhibitor donepezil increases the brain’s acetylcholine level and improves Alzheimer’s disease (AD) -associated learning difficulties.

Acetylcholine activates striatal / nucleus accumbens dopamine receptor D2-expressing medium spiny neurons (D2R-MSNs), which regulate aversive learning through muscarinic receptor M1 (M1R). However, how acetylcholine stimulates learning beyond M1Rs remains unresolved.

Here we found that acetylcholine stimulated protein kinase C (PKC) in mouse striatal / nucleus accumbens. Our original kinase-oriented phosphoproteomic analysis revealed 116 PKC substrate candidates, including Rac1 activator β-PIX. Acetylcholine induced β-PIX phosphorylation and activation, thereby stimulating Rac1 effector p21-activated kinase (PAK).

Aversive stimulus activated the M1R-PKC-PAK pathway in mouse D2R-MSN. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system regulated structural plasticity in dendritic spine and aversive learning. Donepezil induced PAH activation in both the accumulative D2R-MSN and in the CA1 region of the hippocampus and enhanced D2R-MSN-mediated aversive learning.

These findings show that acetylcholine stimulates M1R-PKC-β-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning and suggests the therapeutic potential of the cascade for AD as aversive learning is used to screen AD medications.