Novel fluorescent probe likely to help decode neuronal talking

Numerous cells, including neurons, depend on the intracellular messenger molecule cyclic adenosine monophosphate (cAMP) to function. It does this by encouraging axon growth and maintaining neuronal communication.

Although cAMP’s molecular pathways have been extensively studied and it is known that it plays a significant role in regulating synaptic functions, there are currently no markers that can be used to precisely track its intracellular activity.

Recently, a research team from Doshisha University headed by Associate Professor Naoto Saitoh have developed a novel green fluorescent cAMP indicator called gCarvi to address this issue. Their indicator is easily expressed using an adeno-associated virus vector and is able to measure the intracellular basal level of cAMP (referred to as [cAMP]i) in neurons.

The study was published on July 12th, 2022, in the Proceedings of the National Academy of Sciences journal.

Our new indicator, gCarvi, monitors [cAMP]i at concentrations ranging from 0.2 to 20 μM with a subsecond time resolution and a high specificity over cGMP. It is bright enough to monitor cAMP kinetics in single cells and even at single presynaptic boutons.”

Naoto Saitoh, Study Corresponding Author and Associate Professor, Doshisha University

Seiko Kawata, a PhD candidate at Doshisha University and the study’s lead author, was a member of Saitoh’s team, as was Professor Tomoyuki Takahashi of the Okinawa Institute of Science and Technology.

An ideal cAMP indicator should be able to measure the full intracellular range of cAMP concentration, distinguish cAMP from other nucleotides, like cyclic guanosine monophosphate (cGMP), which is also involved in synaptic modulation, be highly specific for cAMP, not interfere with endogenous cAMP pathways’ physiological functions, and allow stable intracellular imaging.

Regulatory subunits of the mammalian cAMP binding domain (CBD) make up the majority of cAMP indicators currently in use. However, because these molecules are endogenous, their use might interfere with how cAMP pathways work physiologically. The CBD of a bacterial cAMP receptor protein, which does not obstruct mammalian cells’ cAMP pathways, is present in gCarvi, which solves this issue.

The circular permutated green fluorescent protein (cpGFP), which recognizes cAMP concentration by inciting corresponding changes in the intensity of fluorescent light released upon binding to it, was combined with the bacterial CBD by the investigators.

Additionally, it was discovered that gCarvi, despite having a low affinity for cGMP and a high cAMP/cGMP specificity of >100, overcame another drawback of existing cAMP indicators by preventing cGMP detection.

To enhance the ratiometric probe’s capacity to recognize various cAMP concentrations, the team fused gCarvi with the red fluorescent protein mCherry. As a proof of concept, this probe was expressed in hippocampal neurons to track changes in [cAMP]i. Similarly, to identify cAMP and Ca2+, both of which are involved in neuronal regulation, gCarvi was co-expressed with a red fluorescent calcium ion (Ca2+) indicator.

This simultaneous cAMP/Ca2+ detection in hippocampal neurons demonstrated a significant relationship between cAMP and Ca2+ signals. These neurons showed an increase in intracellular cAMP above a specific threshold following treatment with forskolin, a substance that raises cAMP levels, and a phosphodiesterase (PDE) inhibitor, which led to Ca2+ elevation.

Forskolin was also used to evoke irregular cAMP elevations in cerebellar presynaptic boutons, which showed increases in the number of synaptic vesicles being recycled. This suggests that each bouton contains presynaptic cAMP domains, which increase synaptic strength by isolating signaling pathways to prevent cross-talk.

Dr Saitoh elaborates on the potential uses for this innovative indicator in the future.

gCarvi can precisely detect the time of degradation of cAMP signaling in the brain, which results in neuro-degenerative conditions like Alzheimer’s disease. gCarvi can be employed as a screening tool in live-neuron imaging to monitor outcomes of drugs targeting such conditions. Additionally, fluorescent probes help detect invisible molecular dynamics in cells; therefore, the gCarvi cAMP indicator presents invaluable knowledge on neuronal talking.”

Naoto Saitoh, Study Corresponding Author and Associate Professor, Doshisha University

Source:
Journal reference:

Kawata, S., et al. (2022) Green fluorescent cAMP indicator of high speed and specificity suitable for neuronal live-cell imaging. Proceedings of the National Academy of Sciences. doi.org/10.1073/pnas.212261811.

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