Summary: The results reveal the molecular mechanism of acetylcholine in learning and memory.
source: Fujita Health University
Patients with Alzheimer’s disease (AD) have lower levels of the neurotransmitter acetylcholine (ACh) in their brains. Donepezil, an AD medication, increases ACH levels in the brain and improves learning deficits associated with Alzheimer’s disease.
Now, researchers have identified the intracellular signaling cascade through which ACh regulates aversive learning, a major preliminary test for Alzheimer’s drugs.
The researchers also found that donepezil activates this signaling cascade to regulate aversive learning. The results suggest the potential of signaling sequences as drug targets.
Acetylcholine (ACh) is a neuromodulator that has a central role in aversive learning—the rapid conditioning of unpleasant smell, taste, or touch. These learning functions are played by cells called middle spinal neurons that express D2 receptors (D2R-MSNs) located in the striatum/nucleus accumbens (NAc) of the brain. ACh levels in the NAc increase during aversive learning experiences.
Previous studies have shown that ACh acts on D2R-MSNs through a receptor called the M1 muscarinic receptor (M1R), which in turn activates a downstream signaling molecule called protein kinase C (PKC).
However, to date, the precise intracellular signaling mechanism by which ACh influences aversive learning has been unclear, which has limited the development of therapeutic strategies for Alzheimer’s disease that directly target intracellular ACh signaling.
Recently, in a new study published in Molecular PsychiatryIn this study, researchers from Professor Kozu Kaibuchi’s laboratory at Fujita Health University (FHU) have 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 Alzheimer’s disease 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 treatment strategies for Alzheimer’s disease,” he explains. Associate Professor Yuki Yamahashi, lead author of the study.
Molecular signaling cascades are facilitated by a process called phosphorylation, which involves the addition of phosphate groups to specific substrate molecules by intracellular kinases. To study the phosphorylation, the research team used a technique called phosphoproteome-guided kinase analysis, which was developed by Professor Kozo Kaibuchi, corresponding author of the study.
The research team confirmed the role of ACh in PKC stimulation after observing phosphorylation events after ACh binding to M1Rs in striatal/NAc mice slices ex vivo. Next, phosphoproteomic analysis was performed, which yielded 116 candidates of PKC substrate, including ‘β-PIX’, an 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, which is a downstream target of Rac. We next examined the involvement of the identified ACh-M1R-PKC-Rac-β-PIX-PAK cascade. In aversive learning and aversion memory using negative avoidance tests in mice, says Dr. Yamahashi.Finally, the researchers also found that donepezil activates the cascade to enhance aversive learning.
“This study constitutes the first evidence of the intracellular mechanisms of donepezil that regulate learning and memory,” says Dr. Yamahashi.
Their results correlate well with a recent study from Professor Kaibuchi’s laboratory published in Journal of Neurochemistry. The study’s first author, Dr. Muhammad Omar Farooq, was awarded the Mark A. Smith of the International Society of Neurochemistry (ISN).
The study demonstrated the involvement of the “potassium channel KCNQ2 voltage-gated”—which was identified as another candidate PKC substrate in the above phosphoproteome analysis—in aversive learning. Indeed, PKC phosphorylates KCNQ2 directly at threonine 217, a phosphorylation site previously reported for possible involvement in Modulation of channel activity Furthermore, donepezil administration also enhanced the phosphorylation event of the NAc.
The team’s findings directly suggest that the M1R-PKC-β-PIX-PAK signaling cascade is involved in recognition memory and associative learning. This is very important because the series itself provides a platform to screen Alzheimer’s drugs in development.
“While we focused only on β-PIX and elucidating the M1R-PKC-PAK pathway, our phosphoprotein data revealed several other PKC substrates—presynaptic proteins and postsynaptic scaffold proteins to name a few, which were recorded in a database called Kinase- Associated Neural Signals PHOspho (Canphos) (https://kanphos.neuroinf.jp/).
“We are only seeing the tip of the iceberg and we believe that future research could yield new mechanisms of signal transmission in other brain regions,” says Dr. Yamahashi, regarding the future prospects for their research.
About this learning and memory research
original search: open access.
“Acetylcholine pathway phosphorylated proteins enable detection of the PKC-β-PIX-Rac1-PAK cascade as an aversive learning stimulatory signal.Written by Yuki Yamahashi et al. Molecular Psychiatry
Acetylcholine pathway phosphorylated proteins enable detection of the PKC-β-PIX-Rac1-PAK cascade as an aversive learning stimulatory signal.
Acetylcholine is a critical neuromodulator for learning and memory. The cholinesterase inhibitor donepezil increases levels of acetylcholine in the brain and improves learning disabilities associated with Alzheimer’s disease (AD).
Acetylcholine activates striatal dopamine receptors/nucleus accumbens D2 expressing middle spinal neurons (D2R-MSNs), which regulate aversive learning through the M1 muscarinic receptor (M1R). However, the way in which acetylcholine stimulates learning after M1R remains unresolved.
Here, we found that acetylcholine stimulates protein kinase C (PKC) in the mouse nucleus/striatum nucleus/nucleus. Analysis of the original kinase-directed phosphoproteome revealed 116 candidates of a PKC substrate, including the Rac1 β-PIX activator. Acetylcholine induced and activated β-PIX phosphorylation, thereby stimulating Rac1-responsive p21 kinase (PAK).
Aversive stimulation activated the M1R-PKC-PAK pathway in mouse D2R-MSNs. D2R-MSN-specific expression of PAK mutants by the Cre-Flex system that regulates dendritic spine structural plasticity and aversive learning. Donepezil induced activation of PAK in both accumulated D2R-MSNs and in the CA1 region of the hippocampus 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 implicitly suggest the therapeutic potential of the sequence for Alzheimer’s disease as aversive learning is used to screen for Alzheimer’s disease-inducing drugs.
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