Probing PAC1 receptor activation across species with an engineered sensor

Reviewer #1 (Public Review):

Summary:

The manuscript "Engineering of PAClight1P78A: A High-Performance Class-B1 GPCR-Based Sensor for PACAP1-38" by Cola et al. presents the development of a novel genetically encoded sensor, PAClight1P78A, based on the human PAC1 receptor. The authors provide a thorough in vitro and in vivo characterization of this sensor, demonstrating its potential utility across various applications in life sciences, including drug development and basic research.

The diverse methods to validate PAClight1P78A demonstrate a comprehensive approach to sensor engineering by combining biochemical characterization with in vivo studies in rodent brains and zebrafish. This establishes the sensor's biophysical properties (e.g., sensitivity, specificity, kinetics, and spectral properties) and demonstrates its functionality in physiologically relevant settings. Importantly, the inclusion of control sensors and the testing of potential intracellular downstream effects such as G-protein activation underscore a careful consideration of specificity and biological impact.

Strengths:

The fundamental development of PAClight1P78A addresses a significant gap in sensors for Class-B1 GPCRs. The iterative design process -starting from PAClight0.1 to the final PAClight1P78A variant - demonstrates compelling optimization. The innovative engineering results in a sensor with a high apparent dynamic range and excellent ligand selectivity, representing a significant advancement in the field. The rigorous in vitro characterization, including dynamic range, ligand specificity, and activation kinetics, provides a critical understanding of the sensor's utility. Including in vivo experiments in mice and zebrafish larvae demonstrates the sensor's applicability in complex biological systems.

Weaknesses:

The manuscript shows that the sensor fundamentally works in vivo, albeit in a limited capacity. The titration curves show sensitivity in the nmol range at which endogenous detection might be possible. However, perhaps the sensor is not sensitive enough or there are not any known robust paradigms for PACAP release. A more detailed discussion of the sensors's limitations, particularly regarding in vivo applications and the potential for detecting endogenous PACAP release, would be helpful.

There are several experiments with an n=1 and other low single-digit numbers. I assume that refers to biological replicates such as mice or culture wells, but it is not well defined. n=1 in experimental contexts, particularly in Figure 1, raises significant concerns about the exact dynamic range of the sensor, data reproducibility, and the robustness of conclusions drawn from these experiments. Also, ROI for cell cultures, like in Figure 1, is not well defined. The methods mentioned ROIs were manually selected, which appears very selective, and the values in Figure 1c become unnecessarily questionable. The lack of definition for "ROI" is confusing. Do ROIs refer to cells, specific locations on the cell membrane, or groups of cells? It would be best if the authors could use unbiased methods for image analysis that include the majority of responsive areas or an explanation of why certain ROIs are included or excluded.

Reviewer #2 (Public Review):

Summary:

The PAClight1 sensor was developed using an approach successful for the development of other fluorescence-based GPCR sensors, which is the complete replacement of the third intracellular loop of the receptor with a circularly-permuted green fluorescent protein. When expressed in HEK cells, this sensor showed good expression and a weak but measurable response to the extracellular presence of PACAP1-38 (a F/Fo of 43%). Additional mutation near the site of insertion of the linearized GPF, at the C-terminus of the receptor, and within the second intracellular loop produced a final optimized sensor with F/Fo of >1000%. Finally, screening of mutational libraries that also included alterations in the extracellular ligand-binding domain of the receptor yielded a molecule, PAClight1P78A, that exhibited a high ligand-dependent fluorescence response combined with a high differential sensitivity to PACAP (EC50 30 nM based on cytometric sorting of stably transfected HEK293 cells) compared to its congener VIP, (with which PACAP shares two highly related receptors, VPAC1 and VPAC2) as well as several unrelated neuropeptides, and significantly slowed activation kinetics by PACAP in the presence of a 10-fold molar excess of the PAC1 antagonist PACAP6-38. A structurally highly similar control construct, PAClight1P78Actl, showed correspondingly similar basal expression in HEK293 cells, but no PACAP-dependent enhancement in fluorescent properties.

PAClight1P78A was expressed in neurons of the mouse cortex via AAV9.hSyn-mediated gene transduction. Slices taken from PAClight1P78A-transfected cortex, but not slices taken from PAClight1P78Actl-transfected cortex exhibited prompt and persistent elevation of F/Fo after 2 minutes of perfusion with PACAP1-38 which persisted for up to 14 minutes and was statistically significant after perfusion with 3000, but not 300 or 30 nM, of peptide. Likewise, microinfusion of 200 nL of 300 uM PACAP1-38 into the cortex of optical fiber-implanted freely moving mice elicited a F/Fo (%) of greater than 15, and significantly higher than that elicited by application of similar concentrations of VIP, CRF, or enkephalin, or vehicle alone. In vivo experiments were carried out in zebrafish larvae by the introduction of PAClight1P78A into single-cell stage Danio rerio embryos using a Tol2 transposase-based plasmid with a UAS promoter via injection (of plasmid and transposase mRNA), and sorting of post-fertilization embryos using a marker for transgenesis carried in the UAS : PAClight1P78A construct. Expression of PAClight1P78A was directed to cells in the olfactory bulb which express the fish paralog of the human PAC1 receptor by using the Tg(GnRH3:gal4ff) line, and fluorescent signals were elicited by intracerebroventricular administration of PACAP1-38 at a single concentration (1 mM), which were specific to PACAP and to the presence of PAClight1P78A per se, as controlled by parallel experiments in which PAClight1P78Actl instead of PAClight1P78A was contained in the transgenic plasmid.

Major strengths and weaknesses of the methods and results:

The report represents a rigorous demonstration of the elicitation of fluorescent signals upon pharmacological exposure to PACAP in nervous system tissue expressing PAClight1P78A in both mammals (mice) and fish (zebrafish larvae). Figure 4d shows a change in GFP fluorescence activation by PACAP occurring several seconds after the cessation of PACAP perfusion over a two-minute period, and its persistence for several minutes following. One wonders if one is apprehending the graphical presentation of the data incorrectly, or if the activation of fluorescence efficiency by ligand presentation is irreversible in this context, in which case the utility of the probe as a real-time indicator, in vivo, of released peptide might be diminished.

Appraisal of achievement of aims, and data support of conclusions:

Small cavils with controls are omitted for clarity; the larger issue of appraisal of results based on the scope of the designed experiments is discussed in the section below. An interesting question related to the time dependence of the PACAP-elicited activation of PAClight1P87A is its onset and reversibility, and additional data related to this would be welcome.

Discussion of the impact of the work, and utility of the methods and data:

Increasingly, neurotransmitter function may be observed in vivo, rather than by inferring in vivo function from in vitro, in cellular, or ex vivo experimentation. This very valuable report discloses the invention of a genetically encoded sensor for the class B1 GPCR PAC1. PAC1 is the major receptor for the neuropeptide PACAP, which in turn is a major neurotransmitter involved in brain response to psychogenic stress, or threat, in vertebrates as diverse as mammals and fishes. If this sensor possesses the sensitivity to detect endogenously released PACAP in vivo it will indeed be an impactful tool for understanding PACAP neurotransmission (and indeed PACAP action in general, in immune and endocrine compartments as well) in future experiments.

However, the sensor has not yet been used to detect endogenously released PACAP. Until this has been done, one cannot answer the question as to whether the levels of exogenously perfused/administered PACAP used here merely to calibrate the sensor's sensitivity are indeed unphysiologically high. If endogenous PACAP levels don't get that high, then the sensor will not be useful for its intended purpose. The authors should address this issue and allude to what kind of experiments would need to be done in order to detect endogenous PACAP release in living tissue in intact animals. The authors could comment upon the success of other GPCR sensors that have been used to observe endogenous ligand release, and where along the pathway to becoming a truly useful reagent this particular sensor is.

Reviewer #3 (Public Review):

Summary:

The manuscript introduces PAClight1P78A, a novel genetically encoded sensor designed to facilitate the study of class-B1 G protein-coupled receptors (GPCRs), focusing on the human PAC1 receptor. Addressing the significant challenge of investigating these clinically relevant drug targets, the sensor demonstrates a high dynamic range, excellent ligand selectivity, and rapid activation kinetics. It is validated across a variety of experimental contexts including in vitro, ex vivo, and in vivo models in mice and zebrafish, showcasing its utility for high-throughput screening, basic research, and drug development efforts related to GPCR dynamics and pharmacology.

Strengths:

The innovative design of PAClight1P78A successfully bridges a crucial gap in GPCR research by enabling real-time monitoring of receptor activation with high specificity and sensitivity. The extensive validation across multiple models emphasizes the sensor's reliability and versatility, promising significant contributions to both the scientific understanding of GPCR mechanisms and the development of novel therapeutics. Furthermore, by providing the research community with detailed methodologies and access to the necessary viral vectors and plasmids, the authors ensure the sensor's broad applicability and ease of adoption for a wide range of studies focused on GPCR biology and drug targeting.

Weaknesses
To further strengthen the manuscript and validate the efficacy of PAClight1P78A as a selective PACAP sensor, it is crucial to demonstrate the sensor's ability to detect endogenous PACAP release in vivo under physiological conditions. While the current data from artificial PACAP application in mouse brain slices and microinfusion in behaving mice provide foundational insights into the sensor's functionality, these approaches predominantly simulate conditions with potentially higher concentrations of PACAP than naturally occurring levels.

Although the sensor's specificity for the PAC1 receptor and its primary ligand is a pivotal achievement, exploring its potential application to other GPCRs within the class-B1 family or broader categories could enhance the manuscript's impact, suggesting ways to adapt this technology for a wider array of receptor studies. Additionally, while the sensor's performance is convincingly demonstrated in short-term experiments, insights into its long-term stability and reusability in more prolonged or repeated measures scenarios would be valuable for researchers interested in chronic studies or longitudinal behavioral analyses. Addressing these aspects could broaden the understanding of the sensor's practical utility over extended research timelines.

Furthermore, the current in vivo experiments involving microinfusion of PACAP near sensor-expressing areas in behaving mice are based on a relatively small sample size (n=2), which might limit the generalizability of the findings. Increasing the number of subjects in these experimental groups would enhance the statistical power of the results and provide a more robust assessment of the sensor's in vivo functionality. Expanding the sample size will not only validate the findings but also address potential variability within the population, thereby reinforcing the conclusions drawn from these crucial experiments.