SUMOylation of NaV1.2 channels regulates the velocity of backpropagating action potentials in cortical pyramidal neurons

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    This important work will be of interest to neuroscientists working on synaptic transmission and modulation of ion channel activity. This work provides solid evidence of how modulation of Nav1.2 channels by SUMOYLation alters the function of layer 5 pyramidal neurons, using convincing methodology that includes the use of a mouse engineered to eliminate the SUMOYLation site on Nav1.2. Some aspects need to be revised to strengthen data analysis and interpretation.

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Voltage-gated sodium channels located in axon initial segments (AIS) trigger action potentials (AP) and play pivotal roles in the excitability of cortical pyramidal neurons. The differential electrophysiological properties and distributions of Na V 1.2 and Na V 1.6 channels lead to distinct contributions to AP initiation and propagation. While Na V 1.6 at the distal AIS promotes AP initiation and forward propagation, Na V 1.2 at the proximal AIS promotes the backpropagation of APs to the soma. Here, we show the small ubiquitin-like modifier (SUMO) pathway modulates Na + channels at the AIS to increase neuronal gain and the speed of backpropagation. Since SUMO does not affect Na V 1.6, these effects were attributed to SUMOylation of Na V 1.2. Moreover, SUMO effects were absent in a mouse engineered to express Na V 1.2-Lys38Gln channels that lack the site for SUMO linkage. Thus, SUMOylation of Na V 1.2 exclusively controls I NaP generation and AP backpropagation, thereby playing a prominent role in synaptic integration and plasticity.

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  1. eLife assessment

    This important work will be of interest to neuroscientists working on synaptic transmission and modulation of ion channel activity. This work provides solid evidence of how modulation of Nav1.2 channels by SUMOYLation alters the function of layer 5 pyramidal neurons, using convincing methodology that includes the use of a mouse engineered to eliminate the SUMOYLation site on Nav1.2. Some aspects need to be revised to strengthen data analysis and interpretation.

  2. Reviewer #1 (Public Review):

    Previous work, much of it by some of the authors, has characterized modification of various ion channels by SUMOYLation. However, there has been relatively little work exploring the effects of such modulation on function of neurons. This manuscript begins by showing quite large reciprocal effects of either enhancing or reducing SUMOYLation in layer 5 pyramidal neurons. One of the effects found is a leftward shift of the voltage-dependence of persistent sodium current. The authors then test the hypothesis that these changes result in part from SUMOYLation of Nav1.2 channels, and using a mouse engineered to eliminate Nav1.2 SUMOYLation, nicely show that modulation of Nav1.2 shifts the voltage-dependence of Nav1.2 and speeds back-propagation of action potentials from the AIS into the soma.

    The results in the manuscript are interesting, important, and convincing. Besides being important for describing effects of SUMOYLation on overall neuronal function, the selectivity of SUMOYLation for modifying Nav1.2 but not Nav1.6 channels - and the observation of slowing of back-propagation but not forward propagation from the AIS - adds to the previous data on the distinct functional roles of Nav1.2 and Nav1.6 channels in the AIS.

    It is puzzling that the authors focused so much on the effects of SUMOYLation on back-propagation and so little on the large effect on the frequency of firing, which seemed quite dramatic and potentially equally or more important for overall neuronal function than speeding back-propagation. In fact, after introducing the idea that the change in the f-I slope might reflect modulation of Na channels, there is little further discussion of the significance of the changes in firing frequency. Evidently, selective modulation of Nav1.2 channels but not Nav1.6 channels greatly affects firing. Is this explained only by the leftward shift of persistent sodium current from Nav1.2 channels? Or does the leftward shift of Nav1.2 channel gating affect spike threshold? Does modeling of selective modulation of Nav1.2 channels capture these changes in firing frequency and the slope of the f-I curve?

  3. Reviewer #2 (Public Review):

    In this work, Kotler et al. examine the consequences of SUMOylation in the regulation of cortical pyramidal neurons excitability. The authors take advantage of previous articles from their groups in which they have shown that ion channel activity is regulated by the SUMO pathway. Most of the experiments are whole-cell recordings including purified SUMO1 or SENP peptides in the pipette solution, in order to show the effects of SUMOylation and deSUMOylation, respectively. They study repetitive firing and passive membrane properties of cortical neurons, finding that SUMO1 increases the neuronal excitability and the neuronal gain. Interestingly, deSUMOylation with SENP1 dialysis produces opposite effects, indicating that an endogenous basal level of ion channel SUMOylation is present. Importantly, they generate (using CRISPR/Cas9 technology) a mouse model with a point mutation that renders Nav1.2 channels insensible to SUMOylation (Nav1.2-K38Q). In cortical neurons from this mouse model, SUMOylation affects mainly passive neuronal properties; so, modification of neuronal gain by SUMO1 seems to be mediated by changes in Nav1.2 activity. Next, they make use of voltage-clamp recordings and simultaneous fluorescence imaging of Na+ fluxes to assess changes in the persistent sodium current, one of the main factors that can modify neuronal gain. SUMOylation causes a leftward shift in the activation kinetics of INaP, and that change is not observed in Nav1.2-K38Q mice. Finally, the authors show that SUMOylation of Nav1.2 channels affects EPSPs and, of great relevance, the speed of back-propagating action potentials, without modifying the speed of forward-propagating action potentials.

    Overall, the conclusions of this paper are mostly well supported by data, the manuscript is well-written, nicely organized, and breaks new ground in the role of SUMOylation modulating neuronal activity and action potential backpropagation, a key aspect for synaptic plasticity, and should be of broad interest.

    However, some aspects need to be clarified.

    1. The statistical analysis for the first two figures (lines 156 to 215 in the main text) seems to contain some errors, that could change the interpretation of the results. Or, by the contrary, there are some errors in the data provided (mean +/- S.E., number of replicates). Details on this subject are indicated in "recommendations for the authors". Figure 5b has not included statistical analysis. Mean values plus S.E. and "n" are not indicated in Figure 3 - Figure Supplement 1 and 2, and in Figure 4.

    2. The authors mention that the recordings are done in L5 pyramidal neurons, but there are two main classes of these neurons: "thick-tufted" and "slender tufted". These two classes have different morphological and electrophysiological profiles. For example, spiking properties and input resistance can be quite different in both types, as showed by van Aerde & Feldmeyer, 2015 ("Morphological and Physiological Characterization of Pyramidal Neuron Subtypes in Rat Medial Prefrontal Cortex", Cerebral Cortex, 25:788-805). The number of neurons recorded for each condition is around 5-7 for most of the experiments in the article. This number should be increased to avoid bias that could provide differences between different treatments, if the neurons are chosen randomly and not selected by type. The values for input resistance, time constant, etc., should be comparable between conditions just after break-in (before SUMO1 or SENP1 dialysis, and similar to control internal solution). For example, in the case immediately after the break-in, the F-I curves showed in Figure 1 - Figure Supplement 1 should be similar for SUMO1, SENP1 and Control, but they are not.

    3. The neurons in the mutant (Nav1.2-Lys38Gln mouse model) are provided with Nav1.2 channels that are constantly deSUMOylated. This condition could likely drive compensatory changes in the expression/activity of different ion channels in the neurons from mutant mice. A more complete characterization of neuronal properties from this mouse model, in control internal solution, should be desirable, and also a comparison with parameters obtained from wild type neurons with SENP1 internal solution (after dialysis).

  4. Reviewer #3 (Public Review):

    SUMOylation of sodium channels has been implicated as a substantial modulator of current properties. However, prior studies have been limited as they have not examined the impact of SUMOylation in developed neurons. Here the investigators made a mouse with the key SUMOylation site (K38) in Nav1.2 mutated to prevent SUMOylation (K38Q). They characterize modulation of cortical pyramidal neuron firing while manipulating SUMOylation using recombinant proteins in wild-type and SCN2A-K38Q mouse neurons. SUMOylation modulates sodium currents elicited with ramp depolarizations and alters back-propagation of action potentials and thus impacts excitatory post-synaptic potentials. The K38Q mutation prevents these effects on neuronal sodium currents. The work does indeed suggest that SUMOylation modulates specific ionic currents in neurons and that SUMOylation of Nav1.2 may play a role in synaptic integration.

    While the work is interesting, it is limited in several aspects. First, previous studies have reported that SUMOylation modulates the voltage-dependence of Nav1.2 activation and steady-state inactivation. Perhaps because of the difficulties associated with voltage clamping neurons in slice, the current work focuses on ramp currents. While the study states that SUMOylation "exclusively controls InaP generation", this can be misleading as other sodium current properties were not examined in the neurons. Alterations in the voltage-dependence of activation could contribute to the observed changes in ramp currents which are characterized as persistent currents in this study. Second, the study does not examine the impact of the K38Q mutation on behavior. It will be very interesting to see how this mutation impacts learning and memory in the mice.