Peer review of: A dietary fatty acid counteracts neuronal mechanical sensitization (2020)

Reviewers' Comments:
 Reviewer #1:
 Remarks to the Author:
 “A dietary fatty acid counteracts neuronal mechanical sensitization”
SUMMARY: This manuscript presents a thoughtful and rigorous characterization of the effects of
margaric acid (MA) enrichment of the plasma membrane on PIEZO2-dependent
mechanotransduction in a variety of mammalian cell types: mouse and rat DRG neurons, N2A
mouse neuroblastoma cell line, Merkel cell caricinoma cells, and human iPSC-derived sensory
neurons. Prior to this study, MA was shown to inhibit mechanically evoked Piezo1 currents. In this
current study, the authors observed that Piezo2 is less sensitive to MA than Piezo1. They also
show that Piezo2 but not Piezo1 is perturbed by pharmacological disruption of the actin
cytoskeleton, echoing earlier findings with Piezo1. Several of the authors’ key experiments, most
notably the testing of Piezo1-Piezo2 chimeric channels, suggest that the beam domain of Piezo1
and Piezo2 ¬¬ confers differential dependence of the channels on cytoskeleton and their different
sensitivities to MA. Using mouse, rat, and iPSC-derived human DRG neurons, the authors show
that MA partially attenuates rapidly adapting currents (attributable to Piezo2) and that MA
increases the apparent threshold for mechanically evoked currents in the cell indentation assay.
They also demonstrate that MA decreases the ability of sensory neurons to recover from 1Hz
indentation stimuli. They propose that MA affects mechanotransduction channels and not voltage-
gated sodium or steady-state potassium channels, and show that resting membrane potential is
not perturbed with MA treatment. Finally, the authors demonstrate that MA attenuates the effect
of bradykinin, an inflammatory endogenous peptide that sensitizes Piezo2 and other cation
channels to mechanical stimuli via G protein signaling, on mouse DRG neurons. Overall, this
manuscript makes substantial strides to advance our understanding how Piezo1 and Piezo2 sense
membrane tension and mechanical force, which is incompletely understood.
The authors’ conclusions are generally supported by the data in its present form. We have several
suggestions for additional analyses and/or experiments to better support the claims in the paper,
in particular with regard to the proposed effects of MA on DRG neurons and the conclusions drawn
from the Piezo2 chimera experiments. However, even in the absence of further revision
experiments we believe this paper is suitable for publication with text revisions and/or analysis of
existing data to address the items detailed below.
Points of concern (note these are all relatively minor)
1. Electrophysiology of mouse DRG neurons (Figure 4 and Supplemental Figure 5):
a. It is difficult to conclude from the data as-is that MA does not affect DRG neuronal excitability
(and hence only affects mechanically-gated ion channels, i.e. Piezo2). For example, not all K+
channels modulate resting potential. It would be good to know if the input resistance of these cells
is changed after MA, which could be calculated from your existing voltage-clamp experiments.
From the methods, it looks like the bath solutions include many cations that could carry currents
aside from stretch-activated ion channels.
b. Furthermore, the AP waveform (4A and Supplemental Figure 5E) does look slightly different in
the traces presented, contrary to the authors’ conclusions regarding MA and neuronal
excitability/AP firing. Specifically, the after hyperpolarization and the shoulder are smaller after
MA. This could be due to cell-to-cell variability (i.e. minor differences in the chosen traces). A
dv/dt plot, or assessment of other AP waveform parameters in your analysis software could reveal
differences due to MA effects on other channels, or convincingly demonstrate no difference. Any
differences should be noted and addressed in the main text.
c. You have some inactivating inward currents after the step from positive potential to
resting/negative in Supplemental Figure 5A. Similar to concern 1a, is this observation reproducible
over multiple cells and if so could this be due to effects of MA on voltage-dependent ion channels?
d. The chosen 1Hz stimulus protocol is nice (Figure 4C and elsewhere). Have you observed
 differences in recovery from inactivation +/- MA using a voltage-clamp paradigm similar to Coste
 et al. PNAS (2013), varying ∆t and comparing the time constant of recovery? This is by no means
 an essential experiment.
 2. Cytoskeleton disruption experiments with Piezo2, Piezo1, and beam chimera in N2A cells (Figure
 2 and Supplemental Figure 3):
 a. The conferral of MA sensitivity in 2E and loss of LatA effect in 2F are striking. More specific
 discussion of how this directly relates to previous experimental findings, such as Cox et al. (2016)
 should be included.
 b. The Eijelkamp et al. (2013) paper on EPAC1 and Piezo2 by no means demonstrates that Piezo2
 requires cytoskeletal elements for normal function (line 60), it suggests cytoskeletal involvement
 but does not prove direct interaction. Treatment with cytoskeletal-disrupting drugs such as
 colchicine and latrunculin A can have many other effects, for example trafficking and recycling of
 other membrane proteins. I would tone down this citation. Furthermore, the citation on line 122
 cites the above article as well as Coste et al. (2010), Moroni et al. (2018), and Wang et al. (2019)
 as evidence that Piezo2 requires cytoskeleton. None of these other papers examined that directly.
 Reviewer #2:
 Remarks to the Author:
 The authors illustrate that Piezo2 currents are inhibited by Margaric Acid (MA) in N2A mouse
 neuroblastoma cells, rodent DRGs and human iPSC-derived DRGs. This study, a follow up to a
 previous study from the same group, finds that compared to Piezo1, greater amounts of MA are
 required for Piezo2 inhibition. However, disruption of the actin cytoskeleton by Latranculin A
 reduces the required amount. MA also inhibited action potentials from mouse and rat DRG
 neurons, and reversed sensitization of mouse DRG currents by bradykinin. The authors suggest
 that MA could be used to attenuate overactive touch responses as seen in tactile allodynia.
 This is a well-executed study, the data are of high quality, and the experiments are appropriately
 controlled and analyzed.
 Major Comments
 The authors suggest that the observations on the relationship between Piezo2 activity and MA
 supplementation suggest a novel method to reduce the effects of overactive sensory receptors in
 tactile allodynia. However, for translation to human DRGs and the proposed clinical applications, it
 is important to show the effect of MA on human iPSC-derived neurons electrophysiology in current-
 clamp mode - does MA treatment inhibit action potentials like in the rodent DRGs? Also, is the
 effect of MA on bradykinin-sensitized currents (as in Fig. 6) also observed in the human iPSC-
 derived neurons? These experiments are necessary to support the conclusions and proposed
 impact of the findings (e.g. “Our findings.....suggest new avenues to treat tactile allodynia”). If the
 authors are unable to perform these experiments, then the claims should be adjusted accordingly
 throughout the manuscript.
 Figure 2 - The authors conclude in this figure that the different beam regions between Piezo1 and
 Piezo2 is the determinant for their different responses to MA supplementation based on a chimeric
 Piezo2 channel that possesses a Piezo1 beam. Their argument would be more robust if they
 included another chimera of Piezo1 with a Piezo2 beam region. If this new chimera under the
 treatment of MA and Latrunculin A behaved more like Piezo2 in response to MA, the authors would
 be able strengthen their conclusion that the beam region in each protein is responsible for the
 individual protein’s response to MA. While this experiment would be highly informative in providing
 a mechanistic basis of the observed effect, in light of the current Covid-19 disruptions and lab
 closures, it may not be feasible to do these experiments. As such, I leave it to the author’s
 discretion.
 Minor Comments
 Line 116: “Thus, as with PIEZO1, PIEZO2 is less active in rigid membranes (>78 pN (ref 24)).”
 It is not clear what >78 pN refers to, nor where this number arises from.
 Supplemental Figure 3A- The colors and layout chosen for this figure make it confusing and hard
 to read. Please consider changing the layout and colors to make it easier to follow.
Line 262 - 264. This speculative statement should be moved to the Discussion section.