Authors :
Presenting Author: Samantha Thompson, PhD – University of Michigan
Anika Sonig, BS – Cleveland Clinic
Qinglong Miao, PhD – Baylor College of Medicine
Jeffery Noebels, M.D., Ph.D. – Baylor College of Medicine
Rationale:
Childhood absence epilepsy (CAE) is characterized by frequent episodes of spike-wave synchronization driven by phasic bursting of thalamocortical circuitry. CAE originates from loss of function gene mutations in Cacna1a, the alpha subunit for PQ calcium channels, that leads to dysfunctional thalamocortical circuit function driving absence seizures. T-type channels are low voltage-activated calcium channels that regulate thalamocortical circuit burst firing. Gain of function mutations in thalamic big-conductance calcium-activated potassium (BK) channels are also linked to burst firing driving absence seizures. To explore downstream remodeling of thalamic neurons in Cacna1a deficient mice, we performed single cell level transcriptional analysis of the T-channel α-subunit encoding gene Cacna1g and Kcnma1, the α-subunit encoding gene for BK channels. To determine whether transcriptional remodeling is due to loss of intrinsic PQ current in thalamic cells or controlled trans-synaptically, we also examined the Nocor CAE model with targeted Cacna1a deletion in cortical L6 pyramidal cells that eliminates PQ-mediated corticothalamic input while sparing thalamic cell PQ expression.
Methods:
We utilized highly sensitive, fluorescent in situ hybridization RNAscope technology (ACD Bio.) to target mRNA transcripts of Cacna1g, Kcnma1 in wildtype and homozygous tottering or Nocor littermates. To compare expression before and after seizure onset, cohorts were analyzed at two timepoints (P14, P30). High magnification images (40x) of thalamic relay nuclei were quantified using HALO software (Indica Labs). Results:
Cacna1g, Kcnma1 expression was upregulated in thalamic relay cells of Cacna1a mutant mice prior to seizure onset (P14) when compared to wildtype littermates and showed a significant shift in percentage of cells from low expression to high expression. Adult (P30) Cacna1a mice showed a further sustained increase in Cacna1g, Kcnma1 expression. Analysis of these mice revealed a significant increase in the number of NeuN+ cells within an identical size thalamic area in global Cacna1a mutant tottering but not Nocor mice. Further experiments revealed enhanced proliferation during embryonic neurogenesis and stimulation of the b-catenin/Lef1 pathway in tottering mice prior to seizure onset. Conclusions:
We detected downstream upregulation of two pro-epileptic genes (Cacna1g, Kcnma1) in Cacna1a deficient mutants that underlie elevated T-channel current and burst firing driving absence seizures. Transcriptional upregulation occurs prior to onset of seizures and persists in adult mice, suggesting transcriptional dysregulation may play a role in epileptogenesis. This study has also revealed the unexpected finding of increased thalamic neuron number in tottering mice that may be driven by the Lef1/β-catenin pathway. Our study is the first to report the role of PQ channel mutations on early embryonic development of the thalamocortical circuit. Funding:
NINDS F31 NS124345
NINDS R01 NS29709
Blue Bird Circle Foundation