Authors :
Presenting Author: Yukun Yuan, PhD – University of Michigan Medical School
Chunling Chen, M.D. – University of Michigan Medical School; Yosuke Niibori, Ph.D. – University of Toronto; Robert Duba-Kiss, Ph.D. – University of Toronto; Samantha Hodges, Ph.D. – University of Michigan Medical School; Heather O’Malley, Ph.D. – University of Michigan Medical School; Yan Chen, B.S. – University of Michigan Medical School; Karl Habig, B.S. – University of Michigan Medical School; David Hampson, Ph.D. – Professor, University of Toronto; Lori Isom, Ph.D. – Chair and Professor, Pharmacology, University of Michigan Medical School
Rationale:
Homozygous recessive loss-of-function variants in SCN1B, the gene encoding voltage-gated sodium channel β1/β1B subunits, are linked to severe developmental and epileptic encephalopathy (DEE), including Dravet Syndrome. We showed previously that Scn1b-/- mice have spontaneous generalized seizures and SUDEP in 100% of mice within the first three weeks of life. Scn1b-/- cortical parvalbumin-positive (PV+) fast-spiking interneurons are hypoexcitable. In addition, Scn1b-/- somatosensory cortex is haploinsufficient for Scn1a. Here, we asked whether administration of an AAV vector driving expression of myc-tagged b1 subunits to Scn1b-/- brain at postnatal day (P2) impacted their disease phenotype.
Methods:
Male and female Scn1b+/+ or Scn1b-/- pups received a single intracerebroventricular (ICV) injection of AAV9-GADv1-Naβ1 or empty vector at P2. Survival was monitored by continuous infrared video recording. A subset of animals was euthanized for immunohistochemical (IHC) analysis of exogenous myc-tagged b1 expression in brain. In a separate cohort of animals, cortical slices were prepared from the brains of mice at P16-21. Individual pyramidal neurons or PV+ interneurons in layers two through six were visually identified with IR-DIC optics based on their size, shape, and location or by td-tomato epifluorescence labeling, respectively. Action potentials (APs) and firing patterns of neurons were recorded using the whole cell patch-clamp recording technique. Scn1a mRNA abundance was measured in somatosensory cortex in both genotypes at P 17-18.Results:
Consistent with previous data, untreated and empty vector treated Scn1b-/- mice seized and died by P21. In contrast, AAV-Navb1 injected mice lived up to 160 days. IHC analysis showed exogenous b1 expression in cortex and hippocampus in both excitatory and inhibitory neurons. Whole cell current-clamp recordings showed Scn1b-/- PV+ interneurons had a significantly more depolarized resting membrane potential, higher membrane input resistance, smaller peak amplitude and slower maximal rise rate compared to those of WT PV+ interneurons. There were no significant differences in half-amplitude duration, maximal decay rate or threshold potential for the initiation of APs between genotypes. Notably, Scn1b-/- PV+ interneurons fired more APs at lower intensity ranges of depolarizing current injections and were sensitive to depolarization-induced block at higher intensity ranges of depolarizing current injection compared to Scn1b+/+ interneurons. A single ICV injection of AAV9-GADv1-Naβ1 at P2 restored all observed defects in passive and active membrane electric properties, and excitability of PV+ neurons recorded at P16-21. Finally, AAV-Navb1 administration restored Scn1a mRNA abundance in Scn1b-/- somatosensory cortex.
Conclusions:
These data show that AAV9-GADv1-Naβ1 administration prolongs survival, restores Scn1a mRNA abundance, and restores neuronal excitability of PV+ interneurons in the Scn1b-/- model of DEE. This work provides preclinical evidence for the future development of gene therapies to treat SCN1B-linked DEEs in human patients.Funding:
Supported by NIH grant R37-NS-076752 to LLI & PJT-183963 from the Canadian Institutes of Health Research to DRH and LLI.