Neuropathological Characterization of a Dravet Syndrome Knock-In Mouse Model Useful for Investigating Cannabinoid Treatments

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. 2021 Jan 29;13:602801.

doi: 10.3389/fnmol.2020.602801. eCollection 2020.

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Valentina Satta et al. Front Mol Neurosci. .

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Abstract

Dravet syndrome (DS) is an epileptic syndrome caused by mutations in the Scn1a gene encoding the α1 subunit of the sodium channel Nav1.1, which is associated with febrile seizures that progress to severe tonic-clonic seizures and associated comorbidities. Treatment with cannabidiol has been approved to reduce seizures in DS, but it may also be active against these comorbidities. The aim of this study was to validate a new mouse model of DS having lower mortality than previous models, which may serve to further evaluate therapies for the long-term comorbidities. This new model consists of heterozygous conditional knock-in mice carrying a missense mutation (A1783V) in Scn1a gene expressed exclusively in neurons of the CNS (Syn-Cre/Scn1aWT/A1783V). These mice have been used here to determine the extent and persistence of the behavioral deterioration in different postnatal days (PND), as well as to investigate the alterations that the disease produces in the endocannabinoid system and the contribution of inflammatory events and impaired neurogenesis in the pathology. Syn-Cre/Scn1aWT/A1783V mice showed a strong reduction in hindlimb grasp reflex at PND10, whereas at PND25, they presented spontaneous convulsions and a greater susceptibility to pentylenetetrazole-induced seizures, marked hyperactivity, deficient spatial working memory, lower levels of anxiety, and altered social interaction behavior. These differences disappeared at PND40 and PND60, except the changes in social interaction and anxiety. The analysis of CNS structures associated with these behavioral alterations revealed an elevated glial reactivity in the prefrontal cortex and the dentate gyrus. This was associated in the dentate gyrus with a greater cell proliferation detected with Ki67 immunostaining, whereas double-labeling analyses identified that proliferating cells were GFAP-positive suggesting failed neurogenesis but astrocyte proliferation. The analysis of the endocannabinoid system of Syn-Cre/Scn1aWT/A1783V mice confirmed reductions in CB1 receptors and MAGL and FAAH enzymes, mainly in the cerebellum but also in other areas, whereas CB2 receptors became upregulated in the hippocampus. In conclusion, Syn-Cre/Scn1aWT/A1783V mice showed seizuring susceptibility and several comorbidities (hyperactivity, memory impairment, less anxiety, and altered social behavior), which exhibited a pattern of age expression similar to DS patients. Syn-Cre/Scn1aWT/A1783V mice also exhibited greater glial reactivity and a reactive response in the neurogenic niche, and regional changes in the status of the endocannabinoid signaling, events that could contribute in behavioral impairment.

Keywords: Dravet syndrome; Syn-Cre/Scn1aWT/A1783V mice; cannabinoids; endocannabinoid signaling; infantile epileptic refractory syndromes; inflammation; neurogenesis; neuropathological characterization.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1

Figure 1

Evolution of body weight, rotarod performance, forelimb and hindlimb grasp reflexes, and whisker reflexes in Syn-Cre/Scn1aWT/A1783V mice and their three control groups (Scn1aWT/WT, Syn-Cre/Scn1aWT/WT, and Scn1aWT/A1783V mice) measured at different postnatal days in the range PND10-PND60. Values are means ± SEM of 12–21 animals per group. Data were assessed by using two-way ANOVA for repeated measures followed by the Bonferroni test (***p < 0.005 vs. the other three groups).

Figure 2

Figure 2

Evolution of different behavioral parameters recorded in the computer-aided actimeter in Syn-Cre/Scn1aWT/A1783V mice and their three control groups (Scn1aWT/WT, Syn-Cre/Scn1aWT/WT, and Scn1aWT/A1783V mice) measured at different postnatal days in the range PND25–PND60. Values are means ± SEM of 13–21 animals per group. Data were assessed by using two-way ANOVA for repeated measures followed by the Bonferroni test (*p < 0.05, **p < 0.01, ***p < 0.005 vs. the other three groups).

Figure 3

Figure 3

Evolution of different behavioral parameters recorded in the Y-maze in Syn-Cre/Scn1aWT/A1783V mice and their three control groups (Scn1aWT/WT, Syn-Cre/Scn1aWT/WT, and Scn1aWT/A1783V mice) measured at different postnatal days in the range PND25–PND60. Values are means ± SEM of 13–21 animals per group. Data were assessed by using two-way ANOVA for repeated measures followed by the Bonferroni test.

Figure 4

Figure 4

Evolution of different behavioral parameters recorded in the elevated plus maze (upper panels) and in the social interaction test (lower panels) in Syn-Cre/Scn1aWT/A1783V mice and their three control groups (Scn1aWT/WT, Syn-Cre/Scn1aWT/WT, and Scn1aWT/A1783V mice) measured at different postnatal days in the range PND25–PND60. Values are means ± SEM of 11–21 animals per group. Data were assessed by using two-way ANOVA for repeated measures followed by the Bonferroni test (*p < 0.05, **p < 0.01, ***p < 0.005 vs. control groups; #p < 0.05 vs. Scn1aWT/WT group).

Figure 5

Figure 5

Animal survival in Syn-Cre/Scn1aWT/A1783V mice and their three control groups (Scn1aWT/WT, Syn-Cre/Scn1aWT/WT, and Scn1aWT/A1783V mice), and seizuring activity in Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals after an acute injection of PTZ at PND25 (lower panels). Values are means ± SEM of six to eight animals per group. Data were assessed by Kaplan-Meier analysis (survival data) and by the Student’s t-test (PTZ experiment; *p < 0.05, **p < 0.01 vs. the control group).

Figure 6

Figure 6

Immunoreactivity for the astroglial marker GFAP measured by immunofluorescence in different CNS structures, including representative immunofluorescence images in the prefrontal cortex and the hippocampal dentate gyrus of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are % over controls and correspond to means ± SEM of five to six animals per group. Data were assessed by the Student’s t-test (*p < 0.05, **p < 0.01 vs. the control group). Scale bar = 100 μm.

Figure 7

Figure 7

Immunoreactivity for the microglial marker Iba-1 measured by immunofluorescence in different CNS structures, including representative immunofluorescence images in the prefrontal cortex and the hippocampal dentate gyrus, of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are % over controls and correspond to means ± SEM of five to six animals per group. Data were assessed by the Student’s t-test (*p < 0.05, **p < 0.01 vs. the control group). Scale bar = 100 μm.

Figure 8

Figure 8

Gene expression for TNF-α and BDNF measured by qPCR in different CNS structures of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are fold of change over controls and correspond to means ± SEM of five to six animals per group. Data were assessed by the Student’s t-test.

Figure 9

Figure 9

Double immunofluorescence analysis of Ki67 and GFAP in the hippocampal dentate gyrus, including representative immunofluorescence images, of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are means ± SEM of six animals per group. Data were assessed by the Student’s t-test (*p < 0.05, **p < 0.01 vs. the control group). Scale bar for double labeling = 50 μm (top panels) and 20 μm (bottom panels).

Figure 10

Figure 10

Gene expression for the CB1, CB2, and GPR55 receptors and the NAPE-PLD, DAGL, FAAH, and MAGL enzymes measured by qPCR in different CNS structures of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are fold of change over controls and correspond to means ± SEM of five to six animals per group. Data were assessed by the Student’s t-test (*p < 0.05, **p < 0.01 vs. the control group).

Figure 11

Figure 11

Protein levels for the CB1 and CB2 receptors and the FAAH and MAGL enzymes measured by western blot in the prefrontal cortex and the hippocampus of Syn-Cre/Scn1aWT/A1783V mice compared with Scn1aWT/W animals at PND25. Values are % over controls and correspond to means ± SEM of five to six animals per group. Data were assessed by the Student’s t-test (*p < 0.05 vs. the control group).

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References

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