Peer review of: Polyamine regulation of ion channel assembly and implications for nicotinic acetylcholine (2020)

Reviewers' Comments:
Reviewer #1:
Remarks to the Author:
Novel genome-wide screening was used to discover an unlikely candidate for selectively and efficiently
promoting expression of alpha 7 and alpha 4 beta 2 AChRs through a novel mechanism. The small
cytoplasmic protein SAT1 encodes the rate limiting enzyme for polyamine catabolism, and degrading
polyamines turns out to promote assembly of these AChRs through interactions with their large
cytoplasmic domains. The polyamine spermine can block the AChR cation channel, but does not alter
the up regulation of AChRs by SAT1. Upregulation by SAT 1 depends on the presence of the alpha 4 or
alpha 7 AChR large cytoplasmic domain. A cell-permeant inhibitor of SAT1 can block its effects.
Blocking polyamine synthesis with DMFO (a drug that inhibits polyamide synthesis) was shown to up
regulate alpha 4 beta 2 and alpha 7 AChR amounts and thereby nicotine-mediated protection of
postsynaptic neurons from excitotoxicity, thereby suggesting a new approach to developing
neuroprotective drugs. Mutations that reduce SAT1 expression are associated with increased suicide.
Alpha 7 and alpha 4 beta 2 AChR agonists are thought to improve depressive behavior in patients and
animal models.
 These investigations have significant implications for drug development. These investigations also
 reveal previously unknown mechanisms for regulating expression of these AChR subtypes. The data
 presented are extensive, clearly presented, and compelling.
Reviewer #2:
Remarks to the Author:
Dhara et al show for the first time a new function of polyamines, distinct from that previously known
of controlling channel gating.
They demonstrate that the intracellular reduction of polyamine levels, by means of different
approaches, increases the assembly and plasma membrane trafficking of the alpha7 and alpha 4beta2
subtypes. They also clearly demonstrate that the polyamine effects on assembly involve the M3-M4
subunit cytosolic loop.
The effect of polyamine on nAChRs is present in neurons and the polyamine-induced nAChR
upregulation is protective towards glutamate induce excitotoxicity; however, this protective effect is
not present in neurons of NACHO KO mice.
The work is well done, clearly described and discussed.
 The results are very interesting and deserve publication, although the cellular mechanisms acting on
 the different subtypes are not completely elucidated
Minor but important points
1) the title does not reflect properly the content of the paper. In the title there is no mention of
nicotinic receptor subtypes, but in this paper the effect of polyamines is only on some of these
subtypes. Indeed the reference to neuropharmacology is too vague.
2) In the introduction it is written that “polyamine levels selectively up-regulates cerebrocortical α4β2
and α7 levels”. Whereas in neurons it has been shown, by using non permeable Bgtx647, an increase
in alpha7-containing receptors, in the case of alpha4beta2 receptors this has not been demonstrated.
The increase in 3H-epibatidine binding seen in figure 6d may be due to binding to both receptor
subtypes. It will be important to perform the binding of 3H-Epibatidine in presence and absence of 100
nM Cytisine (that inhibits the binding to the alpha4beta2 subtype), in order to quantify in neurons the
increase in alpha7 and alpha4beta2 subtypes, respectively. Moreover by performing in neurons the
Ca+ response in presence of an alpha4beta2 specific allosteric modulator it is also possible to know
whether the functional alpha4beta2 receptors are increased at the cell surface.
3) At page 9 in discussion it is written that “Nicotine and other orthosteric ligands promote assembly
through α7’s extracellular ACh binding domain43”. Please delete “extracellular” because Lester et al
(43) show that the assembly is favoured by the intracellular action of nicotine and orthosteric ligands.