In the last grant cycle, we characterized, validated and published a unique in vitro tool for the study of epileptogenesis - hippocampal organotypic slice cultures that develop spontaneous seizures after 1 week in vitro. We used this preparation to blindly screen over 500 drug-concentration combinations for activity in chronic, post-traumatic epilepsy at speeds that are orders of magnitude faster than any other therapeutic testing strategy for chronic epilepsy. We then used double-blind in vivo EEG testing in the kainate model of chronic epilepsy to confirm the anticonvulsant effect of a lead compound, celecoxib. A key innovation that made these speeds possible was the use of lactate and lactate dehydrogenase (LDH) levels in spent media as assays for seizure burden and cell death, respectively. In searching for the cause of the increase in lactate, we found a persistent neuronal membrane leak that increases cytoplasmic sodium and calcium (? Nai & Cai) days before histochemical evidence of cell death. New data indicate that COX2 induction leads to translocation of Bax, a canonical mitochondrial permeabilizing protein, to the cytoplasmic membrane, where it forms pores that admit Na+ and Ca2+. We propose to test the following pathophysiology: traumatic or ictal injury induces Ca2+-dependent Bax translocation to the cytosolic membrane, where it creates a progressive Ca2+ and Na+ leak that should kill the neuron. However, neurons survive for some time due to their uniquely high ion transport capacity. The ion transport consumes a lot of ATP, and lactate production is a consequence of ATP generation. Progression of the leak eventually leads to membrane depolarization, which may contribute to ictogenesis, and cell death. We will test these ideas by correlating seizures, lactate, and ATP production in Aim 1.
In Aim 2 we will establish the nature of the membrane leak.
In Aim 3 we will evaluate the consequences of the membrane leak on ATP production, membrane potential, ictogenesis, and cell death. We propose to use cell-type specific expression of ratiometric, fluorescent reporters of Na+, Ca2+, ATP, NADH, lactate, caspase and membrane potential in the organotypic slice model, together with multiphoton and custom- built low-light, wide-field microscopes to address these questions at temporal and spatial resolutions that have not previously been feasible.

Public Health Relevance

These investigations will 1) provide estimates regarding the amount of seizure activity that is injurious to neurons 2) identify a progressive membrane leak that may contribute to the persistence of epilepsy 3) establish anticonvulsant mechanisms and therapies that are unique to chronic epilepsy 4) provide the capacity to rapidly screen promising drugs for activity in chronic epilepsy.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
4R37NS077908-09
Application #
9981860
Study Section
Clinical Neuroplasticity and Neurotransmitters Study Section (CNNT)
Program Officer
Churn, Severn Borden
Project Start
2011-08-01
Project End
2022-07-31
Budget Start
2019-08-01
Budget End
2020-07-31
Support Year
9
Fiscal Year
2019
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
073130411
City
Boston
State
MA
Country
United States
Zip Code
02114
Lillis, K P; Staley, K J (2018) Optogenetic dissection of ictogenesis: in search of a targeted anti-epileptic therapy. J Neural Eng 15:041001
Glykys, Joseph; Dzhala, Volodymyr; Egawa, Kiyoshi et al. (2017) Chloride Dysregulation, Seizures, and Cerebral Edema: A Relationship with Therapeutic Potential. Trends Neurosci 40:276-294
Liu, Jing; Saponjian, Yero; Mahoney, Mark M et al. (2017) Epileptogenesis in organotypic hippocampal cultures has limited dependence on culture medium composition. PLoS One 12:e0172677
Berdichevsky, Yevgeny; Saponjian, Yero; Park, Kyung-Il et al. (2016) Staged anticonvulsant screening for chronic epilepsy. Ann Clin Transl Neurol 3:908-923
Song, Yu; Pimentel, Corrin; Walters, Katherine et al. (2016) Neuroprotective levels of IGF-1 exacerbate epileptogenesis after brain injury. Sci Rep 6:32095
Lillis, Kyle P; Wang, Zemin; Mail, Michelle et al. (2015) Evolution of Network Synchronization during Early Epileptogenesis Parallels Synaptic Circuit Alterations. J Neurosci 35:9920-34