Peer review of: Zeta Inhibitory Peptide attenuates learning and memory by inducing NO-mediated (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 
The association between select atypical PKC isoforms, glutamate receptors, and cognitive 
performance is not clear due to conflicting reports. The current manuscript provides interesting 
experimental support that may potentially shed light on discrepancies by identifying a putative 
noncanonical mechanism for one of the most commonly used reagents for the atypical PKC isforms 
PKC/PKMzeta. However, more validation is needed to definitively confirm the authors’ 
interpretations, by inclusion of control experiments to validate mechanism across models, rule out 
other isoforms, provide rigor, and also the association with the provided cognitive task. 
 
• To definitively indicate that ZIP is acting as an arginine donor, other possibilities need to be 
eliminated. Specifically, the authors have not ruled out ZIP effects with PKCzeta (which is 
expressed in examined HEK cells and neuronal tissue based on representative western blots, nor 
has PKC iota/lambda, which has overlapping sequence homology, also been ruled out as a 
contributing factor. 
 
• Throughout the manuscript inferences are made to ‘direct action’ on AMPA Rs by ZIP; however, 
the no supporting data is provided for such conclusion (e.g., colocalization). Either colocalization of 
ZIP-FITC with AMPA receptors should be done, or such conclusions need to be revised. 
 
• The evidence is somewhat convincing for GluA1 AMPA receptor regulation in an immortalized cell 
line, but experimental evidence should be provided that such an even is also occurring in neurons, 
such as using a primary Did not test AMPA redistribution in a more natural model (e.g., neuronal 
culture), given the differences in intracellular signaling cascades between models. 
 
• One of the biggest concerns is with regard to the hypothesis indicating ZIP as an arginine donor 
and provided data. Specifically, evidence is provided that cleavage for arginine as a novel 
noncanonical mechanism; at the same time, data is provided that indicates ZIP-FITC undergoes 
membrane localization. First, nNOS and iNOS (which would expected to be the predominantly 
expressed subtypes) is cytosolic in nature compared to eNOS, which has more membrane 
localization. Second, If indeed ZIP is being subjected to peptidase activity, then it would likely be 
more difficult for FITC to be localized to the membrane. This discrepancy needs to be addressed. 
 
• In Figure 3, a single graph is provided for number of counted cells to potentially rule out cell 
death, but based on the methods, counts were taken from samples ranging 6-24 hrs. Given the 
suggestive decrease following peptide administration, it would be more transparent if the data 
were temporally displayed so as to evaluated whether or not putative cell death events occur at 
later time points. Also, based on the provided methods, it is not clear if cell counts – were based 
only on single or double-labelled nuclei. 
 
• While Peptide B is putatively able to be an arginine donor, evidence is not provide as to whether 
Peptide C, GluA1 with the mutated s-nitrosylation site would prevent S831 phosphorylation. While 
the linkage between nitrosylation and phosphorylation site, has been suggest previously by one 
lab, given the linkage to the hypothesized mechanism, inclusion of such data would increase the 
rigor and reproducibility of this linkage. 
 
• Similarly, Peptide C was not tested as a putative negative control on cocaine CPP. Further, it’s 
not as though CPP is completely absent (it is present, albeit in a weakened state (depending on 
specific statistical analysis – not provided, see below). Related, methodology does not clarivy the 
timing of peptide infusions relative to CPP procedure. Depending on whether peptides are delivered 
during habituation and/or testing will impact interpretation of results (i.e., preventing memory 
acquisition/consolidation, inhibiting recall). 
 

• Statistical methods are provided, but statistical results are not provided in the manuscript All 
statistical information should be provided either in the text or in a table and need to include t- and 
F- values for all t-tests and ANOVAs, respectively, along with main effects where applicable. 
 
• ZIP is not defined in abstract or title 
• Figure 3B – representative image for ZIP localization to neuronal membranes should be replaced 
with a better image, as the current image suggests cytosolic and membrane localization. 
• Figure 4B - legend vs labelling is not consistent. 
• Peptidase cleavage sites should be identified. 
• Number of cells quantified per sample per condition not indicated… 
• Coronal map demonstrating cannula targeting not shown. 
• Stereotaxic microinjections utilized PBS instead of ACSF, the former which is not as suitable for 
site-directed delivery to the brain and should be discussed. 
 
 
 
Reviewer #2: 
Remarks to the Author: 
A complete mechanistic explanation of learning and memory has been one of the great research 
enterprises of neuroscience in our lifetimes. In particular, research of late has adopted a heavy 
molecular biological approach in order to identify biochemical mechanisms, and indeed, particular 
intracellular kinases that may serve to make permanent the changes in neural synaptic weights 
that are thought to underlie learning and memory. 
This particular study examines an alternate mechanism for how the supposedly specific PKMzeta 
inhibitory peptide (ZIP) might induce its LTP- and memory-erasing properties. As promoted by 
previous researchers, PKMzeta has been touted as “The Memory Molecule” since it is activated by 
LTP-producing stimuli (as well as behavioural learning), persists throughout expression (of both 
LTP and memory) and its inactivation by the synthetic peptide ZIP eliminate both LTP and memory 
when applied directly to brain areas that show (or would be presumed to show) synaptic plasticity. 
However, a serious problem with this story is that mice with PKMzeta knock-outs still show both 
LTP memory and, and worse, ZIP still inactivates LTP and memory in these mice (which of course, 
don’t actually express PKMzeta). Despite these contrary findings, there is still continued 
enthusiasm for the strict PKMzeta hypothesis (e.g., Tsokas et al, 2016; ). 
Regardless of the steadfast faith shown by some in PKMzeta, alternate hypotheses exist for the 
memory-abolishing properties of ZIP. In particular, one group has shown that ZIP can promote 
excitotoxic actions on hippocampal neurons (Sadeh et al, 2015). Another has recently shown that 
ZIP impairs ongoing synaptic population activity in the hippocampus (LeBlancq et al, 2016). In the 
present work, Bingor et al show a compelling alternate explanation for the action of ZIP that is 
highly consistent with most of the prior literature. 
Using a cultured cell line that does not express PKMzeta (HEK293), the authors performed live cell 
fluorescence microscopy to show that ZIP alters the localization of the GLuA1 subunit of the AMPA 
receptor (tagged with GFP) to the intracellular compartment, as opposed to the membrane 
surface. Consistent with this, ZIP also depressed AMPA-mediated synaptic currents in neurons 
from the nucleus accumbens (NAc) as assessed using whole cell techniques in brain slices. 
Interestingly, the scrambled version of ZIP also had similar influences to ZIP itself. 
Given the parallel effects of both ZIP and its scrambled version, the authors hypothesized that a 
common component of both might yield its effects. Since both are rich in arginine residues, and 
since arginine is known to activate nitric oxide (shown previously to promote endocytosis of AMPA 
receptors) they tested arginine itself on AMPA-mediated EPSCs, again in NAc. Like ZIP, arginine 
depressed excitatory synaptic currents. Interestingly, an alternate manufactured peptide which 
had the same length as ZIP but had all other non-arginine amino acids replaced with alanine 
(presumably rendering it inactive with respect to PKMzeta) had the same influence as ZIP itself. 
Yet another alternate form of ZIP that was rendered resistant to peptidases yielded no such 
depression, showing that degradation of the peptide with the likely release of arginine was 
necessary for its action. 

Consistent with an effect that would be dependent on NO, the nitric oxide synthase inhibitor L-
NAME occluded the effect of ZIP. Furthermore, in HEK293 cells expressing an altered GluA1 that 
does not allow S-nitrosylation via NO which then leads to increased AMPA receptor endocytosis, 
ZIP no longer promoted the redistribution of receptors from the cell membrane to the intracellular 
compartment, and it also reduced the phosphorylation state of GluA1 in NAc neurons which is a 
necessary step in the endocytosis of AMPA receptors. 
Lastly, and most damningly for the ZIP/PKMzeta story, the authors demonstrate that their alanine 
modified peptide, (like ZIP - as previously shown) reduced a previously acquired cocaine-induced 
conditioned place preference when infused into the NAc. 
 
The authors’ summarize their findings as follows (p8): 
“… ZIP localizes to the cell membrane and is cleaved by peptidases to produce free arginine 
residues. Free arginine induces the production of NO by activation of NOS. NO, in turn, nitrosylates 
cysteine 875 on the C-tail of AMPAR GluA1, which induces phosphorylation of GluA1 on serine 831, 
leading to endocytosis of the GluA1-containing AMPAR and its downregulation.” 
This leads to their major conclusion (p9) that “ZIP acts as a regulator of GluA1 activity, 
independent of PKMzeta.” 
 
Altogether this is a well thought-out and well written piece of work. The authors are to be 
congratulated for a principled and well purposed stand for following up on previous puzzling 
findings regarding the actions of ZIP. Despite my complete enthusiasm for this work, the authors 
may wish to consider some potential suggestions and addendums. 
 
Specific Comments: 
Although the authors assert that many researchers have found that both ZIP and its scrambled 
version promote the same results, I would consider that some prior researches would disagree. 
Some more detailed commentary and comparisons to prior work will likely be necessary here. I 
also note that in the authors’ previous work (Shabashov et al, 2012) that scrambled ZIP was not 
used as a control. Would it, like the manufactured peptide and ZIP itself, be expected to diminish 
CPP as well? 
Perhaps related to the above, one of the striking elements of previous neuro-physiological and 
behavioural studies with ZIP is that testing tends to occur after a period of two hours following 
application. Although the in vivo experiments here appear to have a delay of one day interposed 
between infusions and testing, I would be curious to know if the effects that the authors are 
reporting in both culture and brain slice would be expected to continue past two hours? 
 
 
 
Reviewer #3: 
Remarks to the Author: 
Bingor et al studied the mechanism of synaptic depression and memory erasure by ZIP. The 
authors tested ZIP effect both in vitro and in vivo through confocal imaging and electrophysiology 
as well as behavioral experiment. They reported that the custom-made peptide composed of 
arginine and alanine has effect similar to ZIP. The results suggest that ZIP (and other peptides 
having arginine) provides arginine by being degraded, which is a substrate of NO-synthase, 
thereby activating NO signaling pathway inducing GluA1 endocytosis and memory erasure. 
Although the study tackles an important issue of ZIP specificity, their experimental evidence is not 
enough to support their hypothesis and to justify the publication of the manuscript in Nature 
Communications. Details are as follows. 
 
Major points 
 
1. Direct proof that ZIP is cleaved in the cell is lacking. The myristoyl group of ZIP seems to be 
acting as a membrane anchor, localizing the molecule to lipid membranes. Indeed the authors 
observe FITC conjugated near the myristoyl is still concentrated near the membrane even after an 

hour (Figure 3B). This could be used to prove the point, by use of another ZIP-FITC with a c-
terminal FITC conjugate that should be freed from the myristoyl group and therefore cytosolic 
after cleavage. Showing that the peptidase-resistant Peptide C-FITC does become cytosolic would 
further strengthen the authors' case. 
 
2. GluA1-S831 phosphorylation is not typically linked to AMPAR endocytosis. To prove that S831 
phosphorylation is relevant in the context of ZIP, the authors will need to test cells or mice with a 
phosphomutant GluA1. 
 
3. Throughout the paper, the authors showed the change of GFP signal inside the cell to evaluate 
the GluA1 endocytosis. But without estimating the GFP signal on the membrane, it is hard to 
conclude that there is GluA1 endocytosis with their result. In addition, they seemed to analyze the 
GFP signal in the nucleus rather than cytosol. Indeed, it is not feasible to discriminate the cytosol 
from the plasma membrane in a confocal image. And it is unusual that the endocytosed proteins 
stay in the nucleus. 
 
4. For the behavioral experiment (Figure 5), they microinjected Peptide B instead of ZIP. As 
pointed out above, it is unclear whether Peptide B and ZIP share a common mechanism. In 
addition, it was not tested whether Peptide B exerted its effects via o NO-signaling pathway 
activated by arginines from Peptide B. 
 
 
Minor points 
 
1. If the measurement of membrane potential in Figure 1G corresponds to Vm, then the methods 
section should properly describe how Vm is measured during voltage clamp. If it corresponds to 
holding current, then the label should be corrected and the authors should explain why the holding 
current decreases after 50minutes, whereas Sadeh et al. found that ZIP increased holding current 
over time. 
 
 
2. Figure 4B shows that L-NAME enhances ZIP-induced depression, which is obviously not the 
author's conclusion.