Dravet in the Dish: Mechanisms of Hyperexcitability

Transcription

1 Current Literature In Basic Science Dravet in the Dish: Mechanisms of Hyperexcitability Purinergic Control of Hippocampal Circuit Hyperexcitability in Dravet Syndrome. Gu F, Hazra A, Aulakh A, Ziburkus J. Epilepsia 2014;55: OBJECTIVE: Severe myoclonic epilepsy in infancy (SMEI) or Dravet syndrome is one of the most devastating childhood epilepsies. Children with SMEI have febrile and afebrile seizures (FS and afs), ataxia, and social and cognitive dysfunctions. SMEI is pharmacologically intractable and can be fatal in 10 20% of patients. It remains to be elucidated how channelopathies that cause SMEI impact synaptic activities in key neural circuits, and there is an ongoing critical need for alternative methods of controlling seizures in SMEI. Using the SCN1A gene knock-in mouse model of SMEI (msmei), we studied hippocampal cell and circuit excitability, particularly during hyperthermia, and tested whether an adenosine A1 receptor (A1R) agonist can reliably control hippocampal circuit hyperexcitability. METHODS: Using a combination of electrophysiology (extracellular and whole-cell voltage clamp) and fast voltage-sensitive dye imaging (VSDI), we quantified synaptic excitation and inhibition, spatiotemporal characteristics of neural circuit activity, and hyperthermia-induced febrile seizure-like events (FSLEs) in juvenile mouse hippocampal slices. We used hyperthermia to elicit FSLEs in hippocampal slices, while making use of adenosine A1R agonist N6-cyclopentyladenosine (CPA) to control abnormally widespread neural activity and FSLEs. RESULTS: We discovered a significant excitation/inhibition (E/I) imbalance in msmei hippocampi, in which inhibition was decreased and excitation increased. This imbalance was associated with an increased spatial extent of evoked neural circuit activation and a lowered FSLE threshold. We found that a low concentration (50 nm) of CPA blocked FSLEs and reduced the spatial extent of abnormal neural activity spread while preserving basal levels of excitatory synaptic transmission. SIGNIFICANCE: Our study reveals significant hippocampal synapse and circuit dysfunctions in msmei and demonstrates that the A1R agonist CPA can reliably control hippocampal hyperexcitability and FSLEs in vitro. These findings may warrant further investigations of purinergic agonists as part of the development of new therapeutic approaches for Dravet syndrome. Commentary Dravet syndrome, formerly known as Severe Myoclonic Epilepsy of Infancy (SMEI), is a devastating neurodevelopmental disorder of intractable epilepsy that begins in early infancy. Tragically, development of affected children is typically on track during the first year of life, whereas progressive developmental decline and prolonged seizures begin to emerge in the second year of life. Individuals with Dravet syndrome are also at higher risk of febrile seizures, prolonged status epilepticus, and of SUDEP (sudden unexplained death in epilepsy) and have a wide range of associated conditions, which all need to be properly treated and managed. Dravet syndrome is now considered a channelopathy with mutations in the gene encoding the α1 subunit (Na v 1.1) of the voltage-gated sodium channel (SCN1A) being one of the most common causes for the condition (1). A genetic change in SCN1A will present according to its severity, the genetic background of the patient and environmental factors; those factors in turn will determine the clinical presentation of Dravet syndrome. While Dravet Epilepsy Currents, Vol. 14, No. 5 (September/October) 2014 pp American Epilepsy Society syndrome is largely refractory to common antiepileptic drugs, ketogenic diet therapy has recently been used for the management of Dravet syndrome and related severe myoclonic epilepsies (2), and in one study, 65 percent of patients with Dravet syndrome treated with the ketogenic diet experienced a greater than 50 percent reduction in seizure frequency (3). To study pathophysiological mechanisms implicated in Dravet syndrome and develop improved treatment options for the condition, several features of the syndrome have been reproduced by creating mice engineered with a SCN1A gene mutation (mscn1a) (4). Animals homozygous for the mutation develop seizures as early as 18 days after birth, and initial mechanistic studies suggested that Nav1.1 plays critical roles in the spike output from parvalbumin-positive interneurons and that altered functions of these inhibitory circuits may contribute to epileptic seizures in the mutant animals (4). The study by Gu et al. was designed to further characterize mechanisms of circuit excitability in mscn1a mice and to test possible therapeutic interventions. The authors used hippocampal slice preparations from juvenile mscn1a mice and employed a combination of extracellular and whole-cell voltage clamp studies with fast voltage-sensitive dye imaging (VSDI). VSDI is a functional imaging technique that uses voltage sensitive dies to study cortical dynamics at high spatial 279

2 Dravet and Hyperexcitability and temporal resolution (5). Using extracellular and whole-cell patch-clamp recordings in area CA1, Gu and colleagues first demonstrated increased field excitatory postsynaptic potentials (fepsps) and decreased spontaneous inhibitory postsynaptic currents (sipscs), indicating an imbalance of excitation and inhibition in this in vitro model of Dravet syndrome. Using VSDI, they further show increased propagation of neural activity in hippocampal circuits from mscna1 mice, suggesting that mechanisms that normally limit seizure spread fail to function in this model. Since Dravet syndrome is characterized by a higher incidence of febrile seizures, Gu and colleagues mimicked febrile seizures in the dish by gradually raising the temperature of their preparations in 1 o C/min increments. The authors were able to reliably create febrile seizure-like events (FSLE) as characterized by repetitive seizures evoked by hyperthermia and simultaneously recorded using extracellular and patch-clamp electrodes. Seizures in slices from heterozygous mscna1 mice were triggered by temperatures under 39 o C, whereas temperatures >40 o C were needed to trigger seizures in hippocampal slices derived from wild-type animals. Further, seizures in the mutant slices were more than twice as frequent and longer in duration. Together, these data suggest that hippocampal slice preparations from mscna1 mice constitute a valid model system of Dravet syndrome that might be a useful tool to screen for effective drug therapies for Dravet syndrome. The purine ribonucleoside adenosine is a potent regulator of brain activity exerting seizure suppression in all studied models of epilepsy through increased activation of adenosine A 1 receptors (A 1 Rs) (6). Based on this rationale, Gu and colleagues added the selective A 1 R agonist N6-cyclopentyladenosine to their preparations. As expected, the A 1 R agonist reliably controlled synaptic, circuit, and hyperthermia-induced hyperexcitability in their in vitro Dravet-model system. These effects could be blocked by an A 1 R antagonist confirming the involvement of the A 1 R. Thus, activation of A 1 R signaling might have therapeutic potential for the treatment of seizures in Dravet syndrome. One limitation of the study from Gu and colleagues is the in vitro approach of therapy assessment. It remains to be determined whether A 1 R stimulation would likewise be effective in suppressing seizures in intact mscna1 mice. The in vivo application of CPA might be problematic because systemic A 1 R activation is associated with major cardiovascular and sedative side effects. Furthermore, adenosine-based therapies for the treatment of seizures in Dravet syndrome need to be considered with caution. Patients with Dravet syndrome are at high risk of SUDEP, which in theory may be precipitated by an excess of adenosine in brainstem (7). Therefore, the therapeutic use of adenosine receptor agonists might not be a safe approach for patients with Dravet syndrome; alternative strategies to harness the therapeutic potential of adenosine might be needed. Therapy with a high-fat low-carbohydrate ketogenic diet might provide such an alternative to harness the therapeutic potential of endogenous adenosine without causing undue side effects (8). Indeed, clinical studies have shown that ketogenic diet therapy is effective in patients with Dravet syndrome, with limited adverse effects (2, 3). To develop more effective treatment options for Dravet syndrome, more research into the underlying mechanisms is needed, and the study from Gu and colleagues provides some interesting insight in particular in regard to the proneness to febrile seizures, which could nicely be mimicked in the in vitro model. However, it still remains to be determined how hyperthermia triggers seizures in this model system, in particular, and in Dravet syndrome in general. by Detlev Boison, PhD References 1. Brunklaus A, Zuberi SM. Dravet syndrome From epileptic encephalopathy to channelopathy [published online ahead of print May 16, 2014]. Epilepsia. doi: /epi McTague A, Cross JH. Treatment of epileptic encephalopathies. CNS Drugs 2013;27: Laux L, Blackford R. The ketogenic diet in Dravet syndrome. J Child Neurol 2013;28: Ogiwara I, Miyamoto H, Morita N, Atapour N, Mazaki E, Inoue I, Takeuchi T, Itohara S, Yanagawa Y, Obata K, Furuichi T, Hensch TK, Yamakawa K. Nav1.1 localizes to axons of parvalbumin-positive inhibitory interneurons: A circuit basis for epileptic seizures in mice carrying an SCN1A gene mutation. J Neurosci 2007;27: Grinvald A, Hildesheim R. VSDI: A new era in functional imaging of cortical dynamics. Nat Rev Neurosci 2004;5: Boison D. Adenosine as a modulator of brain activity. Drug News Persp 2007;20: Shen HY, Li T, Boison D. A novel mouse model for sudden unexpected death in epilepsy (SUDEP): Role of impaired adenosine clearance. Epilepsia 2010;51: Masino SA, Li T, Theofilas P, Sandau US, Ruskin DN, Fredholm BB, Geiger JD, Aronica E, Boison D. A ketogenic diet suppresses seizures in mice through adenosine A1 receptors. J Clin Inv 2011;121:

3 American Epilepsy Society Epilepsy Currents Journal Disclosure of Potential Conflicts of Interest Instructions The purpose of this form is to provide readers of your manuscript with information about your other interests that could influence how they receive and understand your work. Each author should submit a separate form and is responsible for the accuracy and completeness of the submitted information. The form is in four parts. 1. Identifying information. Enter your full name. If you are NOT the main contributing author, please check the box no and enter the name of the main contributing author in the space that appears. Provide the requested manuscript information. 2. The work under consideration for publication. This section asks for information about the work that you have submitted for publication. The time frame for this reporting is that of the work itself, from the initial conception and planning to the present. The requested information is about resources that you received, either directly or indirectly (via your institution), to enable you to complete the work. Checking No means that you did the work without receiving any financial support from any third party that is, the work was supported by funds from the same institution that pays your salary and that institution did not receive third-party funds with which to pay you. If you or your institution received funds from a third party to support the work, such as a government granting agency, charitable foundation or commercial sponsor, check Yes. Then complete the appropriate boxes to indicate the type of support and whether the payment went to you, or to your institution, or both. 3. Relevant financial activities outside the submitted work. This section asks about your financial relationships with entities in the bio-medical arena that could be perceived to influence, or that give the appearance of potentially influencing, what you wrote in the submitted work. For example, if your article is about testing an epidermal growth factor receptor (DGFR) antagonist in lung cancer, you should report all associations with entities pursuing diagnostic or therapeutic strategies in cancer in general, not just in the area of EGFR or lung cancer. Report all sources of revenue paid (or promised to be paid) directly to you or your institution on your behalf over the 36 months prior to submission of the work. This should include all monies from sources with relevance to the submitted work, not just monies from the entity that sponsored the research. Please note that your interactions with the work s sponsor that are outside the submitted work should also be listed here. If there is any question, it is usually better to disclose a relationship than not to do so. For grants you have received for work outside the submitted work, you should disclose support ONLY from entities that could be perceived to be affected financially by the published work, such as drug companies, or foundations supported by entities that could be perceived to have a financial stake in the outcome. Public funding sources, such as government agencies, charitable foundations or academic institutions, need not be disclosed. For example, if a government agency sponsored a study in which you have been involved and drugs were provided by a pharmaceutical company, you need only list the pharmaceutical company. 4. Other relationships Use this section to report other relationships or activities that readers could perceive to have influenced, or that give the appearance of potentially influencing, what you wrote in the submitted work.

4 American Epilepsy Society Epilepsy Currents Journal Disclosure of Potential Conflicts of Interest Section #1 Identifying Information 1. Today s Date: 5/21/13 2. First Name Detlev Last Name Boison Degree PhD 3. Are you the Main Assigned Author? Yes No If no, enter your name as co-author: 4. Manuscript/Article Title: Dravet In The Dish: Mechanisms of Hyperexcitability 5. Journal Issue you are submitting for: 14.5 Section #2 The Work Under Consideration for Publication Did you or your institution at any time receive payment or services from a third party for any aspect of the submitted work (including but not limited to grants, data monitoring board, study design, manuscript preparation, statistical analysis, etc.)? Complete each row by checking No or providing the requested information. If you have more than one relationship just add rows to this table. Type No Money Paid to You Money to Your Institution* Name of Entity Comments** 1. Grant 2. Consulting fee or honorarium 3. Support for travel to meetings for the study or other purposes 4. Fees for participating in review activities such as data monitoring boards, statistical analysis, end point committees, and the like 5. Payment for writing or reviewing the manuscript 6. Provision of writing assistance, medicines, equipment, or administrative support. 7. Other * This means money that your institution received for your efforts on this study. ** Use this section to provide any needed explanation. Page 2 10/17/2014

5 Section #3 Relevant financial activities outside the submitted work. Place a check in the appropriate boxes in the table to indicate whether you have financial relationships (regardless of amount of compensation) with entities as described in the instructions. Use one line for each entity; add as many lines as you need by clicking the Add box. You should report relationships that were present during the 36 months prior to submission. Complete each row by checking No or providing the requested information. If you have more than one relationship just add rows to this table. Type of relationship (in alphabetical order) No Name of Entity Comments** 1. Board membership 2. Consultancy 3. Employment 4. Expert testimony 5. Grants/grants pending 6. Payment for lectures including service on speakers bureaus 7. Payment for manuscript preparation. 8. Patents (planned, pending or issued) 9. Royalties 10. Payment for development of educational presentations 11. Stock/stock options 12. Travel/accommodations/meeti ng expenses unrelated to activities listed.** 13. Other (err on the side of full disclosure) Money Paid to You Money to Your Institution* * This means money that your institution received for your efforts. ** For example, if you report a consultancy above there is no need to report travel related to that consultancy on this line. Section #4 Other relationships Are there other relationships or activities that readers could perceive to have influenced, or that give the appearance of potentially influencing, what you wrote in the submitted work? No other relationships/conditions/circumstances that present a potential conflict of interest. Yes, the following relationships/conditions/circumstances are present: Thank you for your assistance. Epilepsy Currents Editorial Board Page 3 10/17/2014