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Zeta inhibitory peptide, so what is it doing after all?
Submit responseIn their comment, Fenton and Sacktor propose that our results on ZIP and ScrZIP in cultured hippocampal neurons (Sadeh et al. 2015), contradict numerous published observations regarding specific effects of ZIP on plasticity and PKMzeta. However, their conclusion seems to overlook some published data. There are two issues that need to be dealt with separately: is PKMzeta a ???memory molecule', and is ZIP a selective blocker of PKMzeta. Our paper (Sadeh et al. 2015) does not deal with the first issue. We did not study memory, plasticity (e.g., LTP), or PKMzeta in that paper. What we did do, was test the effects of ZIP on neuronal activity in tissue culture and brain slices, effects that may or may not prove relevant to the previously published data on the postulated role of ZIP in plasticity or memory.
Fenton and Sacktor posit that our results are irrelevant to previously published data on the physiological effects of ZIP because of the high concentrations used. However, we did report an effect on network activity already at 1uM ZIP, and significant toxicity at 5uM in culture. These concentrations are 3-4 orders of magnitude below those injected into the brain in previous experiments. It has been reported (Bogard and Tavalin, 2015) that the KD of ZIP for PKMzeta is near 0.5uM, and of ScrZIP about 5uM, so one should wonder why ScrZIP does not work in vivo at 10mM if its target is indeed PKMzeta.
The timing/duration of effects of ZIP in the literature is rather confusing. In their comment, Fenton and Sacktor mention that they did not see an effect of ZIP on membrane conductance in their recent eLife paper. Yet in their supp Fig1, they record the exposure to ZIP for only 10 minutes. This might have been too short a time to see an effect, as can be seen in their extracellular recording (Figure 1A) and in our slice data.
Fenton and Sacktor further argue that with one exception (Volk et al, 2013), nobody has reported an effect of ZIP on baseline synaptic responses. Yet in their own original publication (Pastalkova et al, 2006, Figure 1E) they show a distance-dependent immediate silencing of population spikes in the hippocampus, lasting for at least 2 hours. This in fact much preceded the recent report (Leblancq et al. 2016) that ZIP can silence hippocampal activity. Notably, Leblancq et al. (2016) report that most of their ZIP-treated rats underwent an epileptic seizure, which is what could be predicted based on our in vitro data. Furthermore, in their support of a lack of effect of ZIP on hippocampal activity Fenton and Sacktor cite Barry et al, (2012). But in that study the authors recorded activity only 2 hours after ZIP, not sooner, and the activity was quite erratic.
Finally, Shema et al (2009) do not find an effect of ZIP on memory if applied right after learning, hinting at the possibility that it might not affect memory by blocking an immediate LTP-like mechanism.
Given that the ability of ZIP to block or erase long-term memory was replicated by several groups in multiple systems, it is of interest to clarify how this peptide acts. Ignoring multiple effects of ZIP that might not necessarily reinforce the hypothesis that its only target is PKMzeta, is in our view not particularly constructive in approaching the goal that we all share.
References
Barry JM, Rivard B, Fox SE, Fenton AA, Sacktor TC, Muller RU (2012). Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32(40):13753-62.
Bogard AS, Tavalin SJ (2015) Protein Kinase C (PKC)zeta Pseudosubstrate Inhibitor Peptide Promiscuously Binds PKC Family Isoforms and Disrupts Conventional PKC Targeting and Translocation. Mol Pharmacol 88(4):728-35.
LeBlancq MJ, McKinney TL, Dickson CT. (2016) ZIP It: Neural Silencing Is an Additional Effect of the PKM-Zeta Inhibitor Zeta-Inhibitory Peptide. J Neurosci. 36:6193-8.
Pastalkova E, Serrano P, Pinkhasova D, Wallace E, Fenton AA, Sacktor TC. (2006) Storage of spatial information by the maintenance mechanism of LTP. Science 313(5790):1141-4.
Sadeh N, Verbitsky S, Dudai Y, Segal M (2015) Zeta Inhibitory Peptide, a Candidate Inhibitor of Protein Kinase Mzeta, Is Excitotoxic to Cultured Hippocampal Neurons. J Neurosci 35(36):12404-11.
Shema R, Hazvi S, Sacktor TC, Dudai Y. (2009). Boundary conditions for the maintenance of memory by PKMzeta in neocortex. Learn Mem 16(2):122-8.
Volk LJ, Bachman JL, Johnson R, Yu Y, Huganir RL. (2013) PKM-zeta is not required for hippocampal synaptic plasticity, learning and memory. Nature 493(7432):420-3.
Conflict of Interest:
None declared
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On ZIP, cellular toxicity, and the differences between the results of Sadeh et al. and other reports examining effects of ZIP on LTP and memory
Submit responseSadeh et al. highlight the need for appropriate control experiments to test for non-specific toxic effects of ZIP. Studies using ZIP have controlled for toxic effects: 1) by demonstrating the lack of effect of the equivalent dose of the scrambled peptide, which is less effective on PKMzeta than ZIP (Pastalkova et al., 2006; Lee et al., 2013; Yao et al 2013); 2) by rescuing the effect of ZIP, in which the reversal of LTP maintenance and disruption of established memory by ZIP are prevented by blocking the reversal of PKMzeta's action on AMPAR potentiation (Migues et al., 2010; Pauli et al., 2012; Evuarherhe et al., 2014); 3) by directly measuring cellular integrity using electrophysiological assays. Consequently, it is difficult to reconcile the observations of Sadeh et al. with numerous published observations.
1) Using whole-cell recordings from hippocampal slices, Tsokas et al. (2016) demonstrate no effect of 5 microM ZIP on baseline synaptic transmission (see also Wang et al., 2012), nor on PKMzeta-independent LTP induced by phorbol esters, nor on CA1 pyramidal cell membrane conductance despite the concentration of ZIP being effective at reversing PKMzeta- mediated LTP (see Tsokas et al., 2016 Fig. 1A, B, Fig. 1--figure supplement 1; https://elifesciences.org/content/5/e14846/figure1/figure- supp1).
2) Most hippocampus slice studies studying late-LTP use interface chambers for optimal slice health and maximal ZIP concentrations of 5 microM, and report no effects in baseline pathways and only effects in tetanized or facilitated pathways by measuring fEPSPs (Ling et al., 2002; Sajikumar et al., 2005; Serrano et al., 2005; Navakkode et al., 2010; Lin et al., 2012; Panaccione et al., 2013; Chen et al., 2014), and by measuring EPSCs (Li et al., 2010; Velez-Hernandez et al., 2013; Yao et al., 2013; Li et al., 2014). The only report of effects of ZIP on baseline synaptic transmission come from studies of late-LTP in submerged slices (Volk et al., 2013).
3) Pastalkova et al. (2006) report no effects of the scrambled peptide on memory (although Sadeh et al. reports toxic effects in cultured neurons of both scrambled peptide and ZIP), and demonstrate no effect of the in vivo intracranial delivery of 10 nmol/microl ZIP on baseline synaptic transmission.
4) Barry et al. (2012) demonstrate no effect of the same intracranial delivery and dose of ZIP on action potential waveforms of hippocampal pyramidal cells (place cells). The action potentials were recorded as close as 1 mm from the ZIP injection site in freely-behaving rats, demonstrating that the dose of ZIP and the method of delivery that is effective for memory disruption causes undetectable electrophysiological changes. These observations are incompatible with ZIP-induced cellular toxicity. Additional in vivo studies also provide no evidence that the effects of ZIP on memory are due to cellular toxicity (Cooke and Bear, 2010; Madronal et al., 2010; Dong et al., 2015).
It is therefore unlikely that the effects of ZIP on memory are due to cellular toxicity. The contrast with the findings of Sadeh et al. may be due to greater sensitivity of the culture system they have used as well as their use of a higher (10 microM) concentration of ZIP than has been used in prior studies to assess the role of PKMzeta in LTP.
References
Barry et al. 2012. Inhibition of protein kinase Mzeta disrupts the stable spatial discharge of hippocampal place cells in a familiar environment. J Neurosci 32: 13753-13762.
Chen, S., et al. (2014). "Reinstatement of long-term memory following erasure of its behavioral and synaptic expression in Aplysia." Elife 3: e03896.
Cooke, S. F. and M. F. Bear (2010). "Visual experience induces long- term potentiation in the primary visual cortex." J Neurosci 30(48): 16304- 16313.
Dong, Z., et al. (2015). "Long-term potentiation decay and memory loss are mediated by AMPAR endocytosis." J Clin Invest 125(1): 234-247.
Evuarherhe O, Barker GR, Savalli G, Warburton EC, Brown MW. 2014. Early memory formation disrupted by atypical PKC inhibitor ZIP in the medial prefrontal cortex but not hippocampus. Hippocampus, 24, 934-42.
Lee, A. M., et al. (2013). "Prkcz null mice show normal learning and memory." Nature 493(7432): 416-419.
Li, W., et al. (2014). "Upregulation of glutamatergic transmission in anterior cingulate cortex in the diabetic rats with neuropathic pain." Neurosci Lett 568: 29-34.
Li, X. Y., et al. (2010). "Alleviating neuropathic pain hypersensitivity by inhibiting PKMzeta in the anterior cingulate cortex." Science 330: 1400-1404.
Lin, Y. T., et al. (2012). "Oxytocin promotes long-term potentiation by enhancing epidermal growth factor receptor-mediated local translation of protein kinase Mzeta." J Neurosci 32: 15476-15488.
Ling, D. S., et al. (2002). "Protein kinase Mzeta is necessary and sufficient for LTP maintenance." Nat Neurosci 5: 295-296.
Madronal, N., et al. (2010). "PKMzeta inhibition reverses learning- induced increases in hippocampal synaptic strength and memory during trace eyeblink conditioning." PLoS One 5: e10400.
Migues PV, Hardt O, Wu DC, Gamache K, Sacktor TC, Wang YT, Nader K. 2010. PKM? maintains memories by regulating GluR2-dependent AMPA receptor trafficking. Nat Neurosci, 13, 630-4.
Navakkode, S., et al. (2010). "Protein kinase Mzeta is essential for the induction and maintenance of dopamine-induced long-term potentiation in apical CA1 dendrites." Learn Mem 17: 605-611.
Panaccione, I., et al. (2013). "Constitutively active group I mGlu receptors and PKMzeta regulate synaptic transmission in developing perirhinal cortex." Neuropharmacology 66: 143-150.
Pastalkova, E., et al. (2006). "Storage of spatial information by the maintenance mechanism of LTP." Science 313(5790): 1141-1144.
Pauli WM, Clark AD, Guenther HJ, O'reilly RC, Rudy JW. 2012. Inhibiting PKMzeta reveals dorsal lateral and dorsal medial striatum store the different memories needed to support adaptive behavior. Learn Mem, 19, 307-14.
Sajikumar, S., et al. (2005). "Synaptic tagging and cross-tagging: the role of protein kinase Mzeta in maintaining long-term potentiation but not long-term depression." J Neurosci 25: 5750-5756.
Serrano, P., et al. (2005). "Persistent phosphorylation by protein kinase Mzeta maintains late-phase long-term potentiation." J Neurosci 25: 1979-1984.
Tsokas et. al. 2016. Compensation for PKMzeta in long-term potentiation and spatial long-term memory in mutant mice. eLife 5:e14846.
Velez-Hernandez, M. E., et al. (2013). "Inhibition of Protein kinase Mzeta (PKMzeta) in the mesolimbic system alters cocaine sensitization in rats." J Drug Alcohol Res 2: 235669.
Wang, J., et al. (2012). "Metabotropic actions of the volatile anaesthetic sevoflurane increase protein kinase M synthesis and induce immediate preconditioning protection of rat hippocampal slices." J Physiol 590: 4093-4107.
Yao, Y., et al. (2013). "Matching biochemical and functional efficacies confirm ZIP as a potent competitive inhibitor of PKMzeta in neurons." Neuropharmacology 64: 37-44.
Conflict of Interest:
None declared
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