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  1. Author Response

    Reviewer #1 (Public Review):

    a) A "hidden gem" in the work is an exploration of whether lamotrigine directly enhances HCN function and finding it did not. While an important negative result, this was not demonstrated in native tissue, leaving the question open regarding direct effects on the native channel in neurons.

    The point is well taken, and we have added this caveat in the relevant section (page 17).

    b) One weakness of the study is the data from the set of experiments exploring impact of overexpression of the variants in neurons. This technique can be highly variable and the data interpretation in this case would benefit from more rigor.

    It is indeed very difficult to rigorously compare expression patterns obtained using different viruses. To address the reviewer’s concerns, we carried out the following additional experiments and analyses:

    i. We repeated the viral injection experiments using two different AAV serotypes for each series (HA-WT, HA-GD, and HA-MI in AAV2/8; HA-WT, HA-GD, and HA-MI in AAV2/9) to ensure that our results are reproducible and independent of virus preparation.

    ii. We evaluated multiple independent injection sites in each series, ensuring that an adequate number of repetitions was executed under the same conditions (equal virus titer, injection volume, time before animal perfusion, tissue processing, and imaging).

    iii. We presented our results in a series of new figures (Figure 7 and Figure 7 – figure supplements 1 and 2) with added panels showing equivalent vs. boosted laser intensities and gain conditions, where necessary, and parvalbumin protein counter-labeling for reference.

    c) There are minor questions about statistical methods for comparing and concluding about the significance of differences between some experimental groups.

    We have now added statistical analysis supporting all our comparisons and conclusions regarding differences between groups (please see the detailed response to Reviewer #1, Recommendations for the Authors, points g,j,l, and q).

    d) An important conceptual gap remains unanswered by the study. Given the phenotypic similarities between patients with sequence variation in Na+ channel and HCN genes, as well as evidence of reduction of other channels or pumps in this case and the strong co-localization of Na+ channels and HCN channels in the PV+ neurons thought critical in the epilepsy of the HCN sequence variants, is it possible that Na+ channels are impacted as a secondary effect of HCN channel dysfunction here?

    This is certainly a possibility, and indeed one that we very much favor. We have added a new analysis of AP morphology (Figure 5 – figure supplement 1) and performed a microarray-based experiment to screen for changes in Na+ channel expression (Source Data 1). While these experiments yielded negative results, they do not definitively rule out potential cell-type specific alterations in the function of Na+ channels or other conductances. A more thorough experimental examination of this important question will have to await future studies. We have added text to underscore how changes in other conductances may indeed impact neurons’ intrinsic properties in our mice (pages 10-11).

    Reviewer #2 (Public Review):

    a) It is not clear whether the mouse equivalent of the severe developmental disability seen in humans was present in mice.

    We have added new behavioral experiments, which show impairment in some cognitive abilities in Hcn1GD/+ mice but not in Hcn1MI/+ mice, consistent with the more severe development disability observed in patients carrying the p.G391D variant compared to patients carrying the p.M153I variant (new Figure 3 and text on page 6 and 7).

    b) (…) there is no demonstration of hyperexcitability at a cellular or network level, so we do not know how HCN1 mutation predisposes to seizures. In fact, hippocampal pyramidal neurons were shown to be hypoexcitable, at least to one method of action potential generation. There is a suggestion that parvalbumin-positive interneurons may be affected, but there is no evaluation of their excitability. It is possible that HCN1 mutation is directly causing neuronal hyperexcitability, but this would only be uncovered by studying HCN1 channel effects on pyramidal neuron dendrite excitability (where they are mostly localized); synaptic function; or on interneuron excitability. There is also no direct demonstration of the effects of channel mutation on HCN1-mediated current (Ih) in native neurons, so we cannot assess how channel biophysics is altered.

    We agree with the Reviewer that there are indeed limitations to the interpretation of our study. Each of these important questions will need additional experimentation before they can be answered definitively. We have added text to underscore such limitations in the Results (pages 10 and 17) and Discussion (pages 20-21) sections. In future studies, we plan to evaluate both the excitability of interneurons through genetic labeling of PV+ cells and patch-clamp recordings, as well as evaluate their synaptic function. Voltage-clamp recordings in pyramidal neurons and possibly dendritic recordings may also be attempted. However, each of these lines of experimentation will require considerable time to complete, particularly because of the difficulty in obtaining patch-clamp recordings from hippocampal slices from the mouse mutants. So we ask that we be allowed to leave them to a future study.

    Reviewer #3 (Public Review):

    a) The authors characterize cerebellum-dependent functional deficits in the mutant mice, basing their studies on the high expression levels of HCN1 in cerebellum, citing Notomi & Shigemoto, They do not present phenotypic deficits in function ascribed to hippocampus or cortex. (…) Therefore, it should be excellent if the authors presented functional tests of hippocampus or cortex dependent behaviors, regardless of the outcome in Fig.2. At a minimum, they should modify the text and downplay the cerebellar emphasis.

    Following the Reviewer’s helpful recommendations, we have added new behavioral experiments testing short-term and long-term memory (see new Figure 3) and modified the panels in Fig 2. The manuscript text has been revised accordingly (pages 6 and 7).

    b) The authors base their proposed mechanism for the pro-epileptic effects of the mutation on the notion that HCN1 Channels are localized to axons only of PV interneurons. Whereas this fact may be true for the adult, during development, axonal targeting is not unique to basket-type interneurons. It is observed in the developing hippocampal circuit, in medial entorhinal cortex neurons innervating dentate gyrus granule cells, i.e., the perforant path. Have the authors looked at axonal targeting in this region in the mutant mice during appropriate developmental stages? Its absence might modulate the firing of GCs, specifically during development (Bender et al., J Neurosci 2007). At a minimum this point merits discussion, particularly in view of the developmental nature of the epilepsies described.

    The Reviewer correctly points out that HCN1 channels are present not only in the axons of PV+ interneurons but also in the axons of certain subclasses of excitatory neurons (see Huang et al., 2011, 2012, and 2019). Regarding axons from medial entorhinal cortex neurons innervating dentate gyrus granule cells, i.e., the perforant path, there is an interesting difference between mice and rats. While HCN1 channel subunits at this site are downregulated in adult rats, they persist in adult mice. This can be seen in the immunostainings shown in Figure 5A (formerly 4A) of the manuscript. Similar to hippocampal PV+ axons in CA3 (Figure 7A, formerly 6A), it can be noted that HCN1 expression in the perforant path is considerably decreased in Hcn1GD/+ mice compared to wildtype and Hcn1MI/+ mice.

    c) In this context, there are distinct developmental profiles for the 4 HCN subunits, including HCN1, and these profiles might contribute to age-specific defects leading to seizures. This point merits discussion.

    We thank the reviewer for raising this important point and have added text underscoring the potential contribution of altered HCN1 channel function to brain development (page 19) to address this issue, and in accord with the comments raised by Reviewer #1 above (see point p).

    d) Whereas the focus of this paper is on the role of genetic mutations in HCN1 in epilepsy, the paper may be enriched by being placed in the context of the overall contributions of HCN1 channels to human epilepsy, including "acquired epilepsy"" via potential epigenetic changes in the expression of normal HCN channels (Bender et al., 2003 and others).

    We agree with the Reviewer and now refer to these datasets in the Introduction, citing the excellent review by Brennan et al., 2016 (page 4).

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  2. Evaluation Summary:

    This is an innovative and important paper with interest to basic and translational neuroscientists that demonstrates the power of experimental models to advance our understanding of human disease. The authors focus on early-life epilepsy, a devastating and common disorder, and specifically on genetic epilepsies generated via pathological sequence variations in the hyperpolarization-activated nonspecific cation (HCN) channel subtype 1. They delineate the epileptic phenotype and demonstrate some of the potential mechanisms leading to the generation of spontaneous seizures in genetically engineered mice.

    (This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 and Reviewer #3 agreed to share their names with the authors.)

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  3. Reviewer #1 (Public Review):

    The authors employ CRISPR/Cas9 technology to create 2 knock-in mouse lines harboring separate pathological variations in the HCN1 gene, p.G391D and p.M153I, that have been identified in human epilepsy. The goal was to develop preclinical models that recapitulate key aspects of human disease and provide a novel resource for studying mechanisms and evaluating new therapies for epilepsy.

    The authors largely achieved their aims. The key strengths of this work were the expert team assembled to execute the project, the successful use of novel CRISPR/Cas9 technology to generate new mouse lines for the study, and the use of cutting-edge techniques to study the molecular and physiological consequences of the genetic variants in the brain. A "hidden gem" in the work is an exploration of whether lamotrigine directly enhances HCN function and finding it did not. While an important negative result, this was not demonstrated in native tissue, leaving the question open regarding direct effects on the native channel in neurons.

    One weakness of the study is the data from the set of experiments exploring impact of overexpression of the variants in neurons. This technique can be highly variable and the data interpretation in this case would benefit from more rigor. There are minor questions about statistical methods for comparing and concluding about the significance of differences between some experimental groups.

    An important conceptual gap remains unanswered by the study. Given the phenotypic similarities between patients with sequence variation in Na+ channel and HCN genes, as well as evidence of reduction of other channels or pumps in this case and the strong co-localization of Na+ channels and HCN channels in the PV+ neurons thought critical in the epilepsy of the HCN sequence variants, is it possible that Na+ channels are impacted as a secondary effect of HCN channel dysfunction here?

    Overall, the impact of this work to the field will be important. The personalized medicine framework here is a good example of how the field will in the future address diseases caused by distinct sequence variation. The choice to generate mice in which phenotypes analogous human variations were replicated in multiple patients and the phenotype was severe was logical and a good example to others about increasing odds of success in approaches like this....

    The work is also important in connecting prior in vitro work, in which changes in channel function in divergent directions predict certain in vivo consequences, to the context of the animal in vivo, where predictions can be tested, and in this case, leading to surprising results. Indeed, these results point in new directions for understanding new aspects of channel "function" such as trafficking and targeting to subcellular domains in neurons.

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  4. Reviewer #2 (Public Review):

    The authors have created transgenic mouse models incorporating single-nucleotide sequence variations of the HCN1 gene known to cause severe early infantile epileptic encephalopathy in early life in humans. The human syndrome includes severe, drug resistant epilepsy as well as developmental regression and delay.

    The study makes some important contributions to our understanding of HCN1 EIEE. First, the two genetically-altered mouse lines harbor an epileptic condition as well as excess mortality similar to that seen in human patients with similar genetic changes. Thus, these mice will be a useful platform for future discovery in understanding the causes of epilepsy. It is not clear whether the mouse equivalent of the severe developmental disability seen in humans was present in mice.

    Second, the authors perform some key antiepileptic drug treatment trials in vivo demonstrating that phenytoin and lamotrigine exacerbate seizure frequency whereas valproic acid does not. In this response the mouse models resemble the differential drug responses seen in another human EIEE, Dravet syndrome. Although not a clinical trial, this information will be useful to clinicians encountering children with HCN1-related EIEE.

    The authors performed a substantial amount of experimentation to show the consequences of HCN1 mutation on the ion channel's subcellular localization and on neuronal excitability. The results are complicated. It does appear that the subcellular localization of HCN1 channels is disrupted in both pyramidal neurons and interneurons, presumably causing loss of function. Also, current clamp recordings of hippocampal pyramidal neurons suggest some loss of HCN1-mediated contributions to their passive membrane properties, but these effects are not entirely consistent with sole mediation by HCN1 channels. This suggests that there are changes to other ionic conductances as well.

    There are some significant limitations to interpretation of this study. First, there is no demonstration of hyperexcitability at a cellular or network level, so we do not know how HCN1 mutation predisposes to seizures. In fact, hippocampal pyramidal neurons were shown to be hypoexcitable, at least to one method of action potential generation. There is a suggestion that parvalbumin-positive interneurons may be affected, but there is no evaluation of their excitability. It is possible that HCN1 mutation is directly causing neuronal hyperexcitability, but this would only be uncovered by studying HCN1 channel effects on pyramidal neuron dendrite excitability (where they are mostly localized); synaptic function; or on interneuron excitability.

    There is also no direct demonstration of the effects of channel mutation on HCN1-mediated current (Ih) in native neurons, so we cannot assess how channel biophysics is altered.

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  5. Reviewer #3 (Public Review):

    This is an innovative and important paper that demonstrates the power of experimental models to advance our understanding of human disease. The authors focus on early-life epilepsy, a devastating and common disorder, and specifically on genetic epilepsies generated via de novo mutations in the hyperpolarization-activated nonspecific cation (HCN) channels subtype 1. They delineate the epileptic phenotype and demonstrate some of the potential mechanisms leading to the generation of spontaneous seizures in genetically engineered mice.

    The key strengths of the paper are:

    a. Starting off with human mutations known to cause disease, generating knock-in mice, and recapitulating the human phenotype, thus creating a powerful platform to study mechanisms which, in turn, will inform human therapies

    b. Identifying plausible mechanisms, including pronounced alterations in the levels and distribution of HCN1 protein, including disrupted targeting to the axon terminals of basket cell interneurons.

    c. Probing the functional consequences of the mutation on the properties of the HCN1 channels, discovering a clinically relevant paradoxical seizure induction by anticonvulsant drugs targeting sodium channels (lamotrigine and phenytoin).

    d. Identifying a novel pharmacology of the mutated channels, which are unresponsive to classic antagonists. This supports the need to screen mutated channels to identify novel compounds that might overcome the mutation-induced functional deficits of the channels. In other words, the authors make a strong case for employing tailored therapies for specific channel gene variants.

    While overall, the work is rigorous and the majority of the conclusions are robust, there are several issues that require addressing:

    a. The authors characterize cerebellum-dependent functional deficits in the mutant mice, basing their studies on the high expression levels of HCN1 in cerebellum, citing Notomi & Shigemoto, They do not present phenotypic deficits in function ascribed to hippocampus or cortex. However
    1. Notomi and Shigemoto state: "Immunoreactivity for HCN1 showed predominantly cortical distribution, being intense in the neocortex, hippocampus, superior colliculus, and cerebellum,"
    2. Importantly, the seizures of the HCN1 mutant mice are unlikely to arise from the cerebellum, and the encephalopathies elements of HCN1-related neonatal epileptic encephalopathies clearly derive from cortex and hippocampus. Therefore, it should be excellent if the authors presented functional tests of hippocampus or cortex dependent behaviors, regardless of the outcome in Fig.2. At a minimum, they should modify the text and downplay the cerebellar emphasis.

    b. The authors base their proposed mechanism for the pro-epileptic effects of the mutation on the notion that HCN1 Channels are localized to axons only of PV interneurons. Whereas this fact may be true for the adult, during development, axonal targeting is not unique to basket-type interneurons. It is observed in the developing hippocampal circuit, in medial entorhinal cortex neurons innervating dentate gyrus granule cells, i.e., the perforant path. Have the authors looked at axonal targeting in this region in the mutant mice during appropriate developmental stages? Its absence might modulate the firing of GCs, specifically during development (Bender et al., J Neurosci 2007). At a minimum this point merits discussion, particularly in view of the developmental nature of the epilepsies described.

    c. In this context, there are distinct developmental profiles for the 4 HCN subunits, including HCN1, and these profiles might contribute to age-specific defects leading to seizures. This point merits discussion.

    d. Whereas the focus of this paper is on the role of genetic mutations in HCN1 in epilepsy, the paper may be enriched by being placed in the context of the overall contributions of HCN1 channels to human epilepsy, including "acquired epilepsy"" via potential epigenetic changes in the expression of normal HCN channels (Bender et al., 2003 and others).

    Was this evaluation helpful?