Molecular mechanisms behind learning and memory identified

Summary: The results reveal the molecular mechanism for acetylcholine in learning and memory.

Source: Fujita Health University

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

Now researchers have identified the intracellular signaling cascade through which ACh regulates aversive learning, an important pre-test for AD drugs.

Researchers also found that donepezil activates this signaling cascade to regulate aversive learning. The results indicate the potential of the signaling cascade as drug targets.

Acetylcholine (ACh) is a neuromodulator with a central role in aversive learning—rapid conditioning to unpleasant smell, taste, or touch. These learning functions take place in cells called D2 receptor-expressing medium spiny neurons (D2R-MSNs), 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 MSNs through a receptor called muscarinic receptor M1 (M1R), which in turn activates the downstream signaling molecule called protein kinase C (PKC).

However, until now, the precise intracellular signaling mechanism by which ACh affects aversive learning has been unclear, which has limited the development of AD therapy strategies that directly target intracellular ACh signaling.

Recently in a new study published in Molecular PsychiatryResearchers from Prof. Kozo Kaibuchi’s laboratory at Fujita Health University (FHU) have elucidated the molecular mechanisms of ACh for learning and memory.

“This is the first time in 45 years since the cholinergic hypothesis of AD was put forward. 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 Prof. Yukie Yamahashi, one of the lead authors of the study.

Molecular signaling cascades are facilitated by a process called phosphorylation, in which phosphate groups are attached to specific substrate molecules by kinases in cells. To study phosphorylation, the research team used a technique called kinase-oriented phosphoproteomics analysis, 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, phosphoproteomic analysis was performed, which revealed 116 PKC substrate candidates, including “β-PIX”, the activator of a protein called “small GTPase Rac”.

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

“This study represents the first evidence of donepezil’s intracellular mechanisms that regulate learning and memory,” says Dr. Yamahashi.

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

The study demonstrated the involvement of the “voltage-gated potassium channel KCNQ2”, which was identified as another PKC substrate candidate in the above phosphoproteomic analysis, in aversive learning. Indeed, PKC phosphorylates KCNQ2 directly at threonine 217, the phosphorylation site previously reported to possibly be involved in the modulation of its channel activity. In addition, administration of donepezil also enhanced the phosphorylation event in the NAc.

This shows a chart from the study
Upon an aversive stimulus (electric footshock), 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 subsequently increases neuronal activity in response to glutamatergic input. Activation of both PAK-mediated and KCNQ2-mediated signaling pathways results in aversive behavior. Photo credits: Kozo Kaibuchi and Yukie Yamahashi from Fujita Health University

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

“While we only focused on β-PIX and elucidating the M1R-PKC-PAK signaling pathway, our phosphoproteomic data revealed many other PKC substrates – presynaptic proteins and postsynaptic scaffolding proteins, to name a few, that are in a database.” called Kinase-Associated Neural PHOspho signaling (KANPHOS) (

“We are only seeing the tip of the iceberg and believe that future research could reveal new mechanisms of signaling in other areas of the brain,” says Dr. Yamahashi on the future prospects of their research.

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About these news from learning and memory research

Author: press office
Source: Fujita Health University
Contact: Press Office – Fujita Health University
Picture: The image is credited to Kozo Kaibuchi and Yukie Yamahashi of Fujita Health University

Original research: Open access.
“Phosphoproteomics of acetylcholine signaling allows discovery of PKC-β-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning” by Yukie Yamahashi et al. Molecular Psychiatry


Phosphoproteomics of acetylcholine signaling allows the discovery of the PKC-β-PIX-Rac1-PAK cascade as a stimulatory signal for aversive learning

Acetylcholine is a neuromodulator critical to learning and memory. The cholinesterase inhibitor donepezil increases levels of acetylcholine in the brain and improves the learning disabilities associated with Alzheimer’s disease (AD).

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

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

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

These results demonstrate that acetylcholine stimulates M1R-PKC-β-PIX-Rac1-PAK signaling in D2R-MSNs for aversive learning, and implicate the therapeutic potential of the cascade for AD since aversive learning is used to preempt AD drugs to screen.