Impaired excitability of fast-spiking neurons in a novel mouse model of KCNC1 epileptic encephalopathy

Reviewer #1 (Public review):

Summary:

The authors have created a new model of KCNC1-related DEE in which a pathogenic patient variant (A421V) is knocked into a mouse in order to better understand the mechanisms through which KCNC1 variants lead to DEE.

Strengths:

(1) The creation of a new DEE model of KCNC1 dysfunction.

(2) InVivo phenotyping demonstrates key features of the model such as early lethality and several types of electrographic seizures.

(3) The ex vivo cellular electrophysiology is very strong and comprehensive including isolated patches to accurately measure K+ currents, paired recording to measure evoked synaptic transmission, and the measurement of membrane excitability at different time points and in two cell types.

Weaknesses:

(1) The assertion that membrane trafficking is impaired by this variant could be bolstered by additional data.

(2) In some experiments details such as the age of the mice or cortical layer are emphasized, but in others, these details are omitted.

(3) The impairments in PV neuron AP firing are quite large. This could be expected to lead to changes in PV neuron activity outside of the hypersynchronous discharges that could be detected in the 2-photon imaging experiments, however, a lack of an effect on PV neuron activity is only loosely alluded to in the text. A more formal analysis is lacking. An important question in trying to understand mechanisms underlying channelopathies like KCNC1 is how changes in membrane excitability recorded at the whole cell level manifest during ongoing activity in vivo. Thus, the significance of this work would be greatly improved if it could address this question.

(4) Myoclonic jerks and other types of more subtle epileptiform activity have been observed in control mice, but there is no mention of littermate control analyzed by EEG.

Reviewer #2 (Public review):

Summary:

Wengert et al. generated and thoroughly characterized the developmental epileptic encephalopathy phenotype of Kcnc1A421V/+ knock-in mice. The Kcnc1 gene encodes the Kv3.1 channel subunit. Analogous to the role of BK channels in excitatory neurons, Kv3 channels are important for the recurrent high-frequency discharge in interneurons by accelerating the downward hyperpolarization of the individual action potential. Various Kcnc1 mutations are associated with developmental epileptic encephalopathy, but the effect of a recurrent A421V mutation was somewhat controversial and its influence on neuronal excitability has not been fully established. In order to determine the neurological deficits and underlying disease mechanisms, the authors generated cre-dependent KI mice and characterized them using neonatal neurological examination, high-quality in vitro electrophysiology, and in vivo imaging/electrophysiology analyses. These analyses revealed excitability defects in the PV+ inhibitory neurons associated with the emergence of epilepsy and premature death. Overall, the experimental data convincingly support the conclusion.

Strengths:

The study is well-designed and conducted at high quality. The use of the Cre-dependent KI mouse is effective for maintaining the mutant mouse line with premature death phenotype, and may also minimize the drift of phenotypes which can occur due to the use of mutant mice with minor phenotype for breeding. The neonatal behavior analysis is thoroughly conducted, and the in vitro electrophysiology studies are of high quality.

Weaknesses:

While not critically influencing the conclusion of the study, there are several concerns.

In some experiments, the age of the animal in each experiment is not clearly stated. For example, the experiments in Figure 2 demonstrate impaired K+ conductance and membrane localization, but it is not clear whether they correlated with the excitability and synaptic defects shown in subsequent figures. Similarly, it is unclear how old mice the authors conducted EEG recordings, and whether non-epileptic mice are younger than those with seizures.

The trafficking defect of mutant Kv3.1 proposed in this study is based only on the fluorescence density analysis which showed a minor change in membrane/cytosol ratio. It is not very clear how the membrane component was determined (any control staining?). In addition to fluorescence imaging, an addition of biochemical analysis will make the conclusion more convincing (while it might be challenging if the Kv3.1 is expressed only in PV+ cells).

While the study focused on the superficial layer because Kv3.1 is the major channel subunit, the PV+ cells in the deeper cortical layer also express Kv3.1 (Chow et al., 1999) and they may also contribute to the hyperexcitable phenotype via negative effect on Kv3.2; the mutant Kv3.1 may also block membrane trafficking of Kv3.1/Kv3.2 heteromers in the deeper layer PV cells and reduce their excitability. Such an additional effect on Kv3.2, if present, may explain why the heterozygous A421V KI mouse shows a more severe phenotype than the Kv3.1 KO mouse (and why they are more similar to Kv3.2 KO). Analyzing the membrane excitability differences in the deep-layer PV cells may address this possibility.

In Table 1, the A421V PV+ cells show a depolarized resting membrane potential than WT by ~5 mV which seems a robust change and would influence the circuit excitability. The authors measured firing frequency after adjusting the membrane voltage to -65mV, but are the excitability differences less significant if the resting potential is not adjusted? It is also interesting that such a membrane potential difference is not detected in young adult mice (Table 2). This loss of potential compensation may be important for developmental changes in the circuit excitability. These issues can be more explicitly discussed.

Reviewer #3 (Public review):

Summary:

Here Wengert et al., establish a rodent model of KCNC1 (Kv3.1) epilepsy by introducing the A421V mutation. The authors perform video-EEG, slice electrophysiology, and in vivo 2P imaging of calcium activity to establish disease mechanisms involving impairment in the excitability of fast-spiking parvalbumin (PV) interneurons in the cortex and thalamic PV cells.

Outside-out nucleated patch recordings were used to evaluate the biophysical consequence of the A421V mutation on potassium currents and showed a clear reduction in potassium currents. Similarly, action potential generation in cortical PV interneurons was severely reduced. Given that both potassium currents and action potential generation were found to be unaffected in excitatory pyramidal cells in the cortex the authors propose that loss of inhibition leads to hyperexcitability and seizure susceptibility in a mechanism similar to that of Dravet Syndrome.

Strengths:

This manuscript establishes a new rodent model of KCNC1-developmental and epileptic encephalopathy. The manuscript provides strong evidence that parvabumin-type interneurons are impaired by the A421V Kv3.1 mutation and that cortical excitatory neurons are not impaired. Together these findings support the conclusion that seizure phenotypes are caused by reduced cortical inhibition.

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of the observed impairments in thalamic neurons in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 it is not clear why these impairments would lead to a more severe disease phenotype than other loss-of-function mutations which have been characterized previously. Lastly, additional analysis of video-EEG data would be helpful for interpreting the extent of the seizure burden and the nature of the seizure types caused by the mutation.

Author response:

Reviewer #1 (Public review):

Weaknesses:

(1) The assertion that membrane trafficking is impaired by this variant could be bolstered by additional data.

We agree with this comment and will perform additional analysis and experiments to support the assertion that membrane trafficking is impaired. As noted by the Reviewers, standard biochemical approaches to obtain such data may be challenging due to the fact that Kv3.1 is expressed in only a subset of cells and that we do not have a Kv3.1-A421V specific antibody.

(2) In some experiments details such as the age of the mice or cortical layer are emphasized, but in others, these details are omitted.

We appreciate that the Reviewer has noted this omission. We will include such details in the resubmission.

(3) The impairments in PV neuron AP firing are quite large. This could be expected to lead to changes in PV neuron activity outside of the hypersynchronous discharges that could be detected in the 2-photon imaging experiments, however, a lack of an effect on PV neuron activity is only loosely alluded to in the text. A more formal analysis is lacking. An important question in trying to understand mechanisms underlying channelopathies like KCNC1 is how changes in membrane excitability recorded at the whole cell level manifest during ongoing activity in vivo. Thus, the significance of this work would be greatly improved if it could address this question.

Yes, the impairments in neocortical PV-IN excitability are more marked than any other PV interneuronopathy that we have studied. We will include a more extensive analysis of the 2-photon imaging data in the resubmission. However, there are limitations to the inferences that can be made as to firing patterns based on 2-photon calcium imaging data, particularly for interneurons.

(4) Myoclonic jerks and other types of more subtle epileptiform activity have been observed in control mice, but there is no mention of littermate control analyzed by EEG.

We did not observe myoclonic jerks in control mice. This data will be included in the resubmission.

Reviewer #2 (Public review):

Weaknesses:

In some experiments, the age of the animal in each experiment is not clearly stated. For example, the experiments in Figure 2 demonstrate impaired K+ conductance and membrane localization, but it is not clear whether they correlated with the excitability and synaptic defects shown in subsequent figures. Similarly, it is unclear how old mice the authors conducted EEG recordings, and whether non-epileptic mice are younger than those with seizures.

We will include explicit information as to the age of the animals used for each experiment in the resubmission.

The trafficking defect of mutant Kv3.1 proposed in this study is based only on the fluorescence density analysis which showed a minor change in membrane/cytosol ratio. It is not very clear how the membrane component was determined (any control staining?). In addition to fluorescence imaging, an addition of biochemical analysis will make the conclusion more convincing (while it might be challenging if the Kv3.1 is expressed only in PV+ cells).

We will include additional information in the Methods section as to how the membrane component was determined in a revised version of the manuscript. We agree with Reviewer #2 regarding the limitations in the ability to further evaluate this.

While the study focused on the superficial layer because Kv3.1 is the major channel subunit, the PV+ cells in the deeper cortical layer also express Kv3.1 (Chow et al., 1999) and they may also contribute to the hyperexcitable phenotype via negative effect on Kv3.2; the mutant Kv3.1 may also block membrane trafficking of Kv3.1/Kv3.2 heteromers in the deeper layer PV cells and reduce their excitability. Such an additional effect on Kv3.2, if present, may explain why the heterozygous A421V KI mouse shows a more severe phenotype than the Kv3.1 KO mouse (and why they are more similar to Kv3.2 KO). Analyzing the membrane excitability differences in the deep-layer PV cells may address this possibility.

We will include recordings from PV-INs in deeper layers of the neocortex in the revised version of the manuscript, as requested.

In Table 1, the A421V PV+ cells show a depolarized resting membrane potential than WT by ~5 mV which seems a robust change and would influence the circuit excitability. The authors measured firing frequency after adjusting the membrane voltage to -65mV, but are the excitability differences less significant if the resting potential is not adjusted? It is also interesting that such a membrane potential difference is not detected in young adult mice (Table 2). This loss of potential compensation may be important for developmental changes in the circuit excitability. These issues can be more explicitly discussed.

We will include a more thorough discussion of this finding in the revised version of the manuscript. However, we do not completely understand this finding. It could be compensatory, as suggested by the Reviewer; however, it is transient and seems to be an isolated finding (i.e., there does not appear to be parallel “compensation” in other properties). Alternatively, it could be that impaired excitability of the Kcnc1-A421V/+ PV-INs may reflect impaired/delayed development, which itself is known to be activity-dependent.

Reviewer #3 (Public review):

Weaknesses:

The manuscript identifies a partial mechanism of disease that leaves several aspects unresolved including the possible role of the observed impairments in thalamic neurons in the seizure mechanism. Similarly, while the authors identify a reduction in potassium currents and a reduction in PV cell surface expression of Kv3.1 it is not clear why these impairments would lead to a more severe disease phenotype than other loss-of-function mutations which have been characterized previously. Lastly, additional analysis of video-EEG data would be helpful for interpreting the extent of the seizure burden and the nature of the seizure types caused by the mutation.

We agree with this comment. We studied neurons in the reticular thalamus as these cells are known to express Kv3.1 and are linked to epilepty pathogenesis. Yet, we focused on neocortical PV-INs over other Kv3.1-expressing neurons such as neurons of the reticular thalamus because we evaluated the impairments of intrinsic excitability to be more profound in neocortical PV-INs. Cross of Kcnc1-Flox(A421V)/+ mice to a cerebral cortex interneuron-specific driver that would avoid recombination in thalamus – such as Ppp1r2-Cre (RRID:IMSR_JAX:012686) – could assist in determining the relative contribution of thalamic reticular nucleus dysfunction to the overall phenotype, as performed by Makinson et al (2017) to address a similar question. There are of course other Kv3.1-expressing neurons in the brain, including in GABAergic interneurons in hippocampus and amygdala. We will include additional discussion in a revised version of the manuscript as to why we think there is more severe impairment in our Kcnc1-Flox(A421V)/+ mice relative to Kv3.1 and Kv3.2 knockout mice. We will include additional data on the epilepsy phenotype in the revised version of the manuscript, as requested.