CURE Epilepsy Discovery: Funding Basic Mechanisms Research Drives Momentum Toward a CURE

Key Points:

  • For 25 years, CURE Epilepsy has been funding breakthrough research to advance science to find a cure for epilepsy. A key focus of our research grants has been understanding the basic biological mechanisms that result in epilepsy, which provides foundational knowledge that will ultimately lead to a cure for epilepsy.
  • One initiative funded by CURE Epilepsy, the Infantile Spasms (IS) Initiative, brought together a diverse team of medical and scientific experts to rapidly advance IS research and was the first initiative of its kind in the field of epilepsy.
  • John Swann of Baylor College of Medicine, whose work is discussed herein, was one of the grantees involved in the IS Initiative. He has progressed his initial discoveries, demonstrating the importance of funding basic mechanisms research to put us one step closer to a cure.
  • Basic research provides hope for a cure for the epilepsies; by better understanding the mechanisms that cause seizures, we can develop curative treatments for the epilepsies.


Deep dive

Twenty-five years ago, CURE Epilepsy was founded finding a cure for epilepsy and related seizure disorders, which now impacts 3.4 million Americans and 65 million people worldwide. The founders saw a need to push the research community to think differently about epilepsy research. This resulted in a paradigm shift for the community, moving from seeking treatments and therapies that would control seizures to focusing on innovative approaches that would advance science and find a cure for epilepsy. Achieving this goal would provide freedom from seizures and the negative side effects of medications. The organization determined that it could have the largest impact by focusing on understanding the basic biological mechanisms underlying the causes of epilepsy. Understanding is the first step in the scientific process, where researchers study the brain to gain a better understanding of why and how seizures are caused. These findings create foundational knowledge that may translate to new ideas to treat epilepsy and eventually preclinical and clinical trials. Clinical trials may ultimately lead to improved and potential cures. Hence, while the benefits of basic epilepsy research are not immediate, the rewards that basic research provides in terms of our understanding epilepsy are unparalleled.

Since 1998, CURE Epilepsy has funded over 280 research grants, and many have addressed the need to learn more about the basic biological mechanisms that underlie epilepsy. Many of these grants have formed the basis for further study, learning, and advancements that may lead us to a cure. One example of this is within infantile spasms (IS), a rare and particularly severe form of epilepsy, with approximately 90% of cases diagnosed in the first year of life. Infantile spasms manifest as sudden, jerking movements of the arms and legs, and are often accompanied by an irregular brainwave pattern on the electroencephalogram (EEG) called hypsarrhythmia.[1]These seizures are also often accompanied by significant cognitive and physical deterioration.[2] Current therapies for IS are effective in only half of the children with IS [3] and are associated with negative side effects, highlighting the need to find better and more effective treatments.

In 2013, CURE Epilepsy launched the Infantile Spasms Initiative, with $4 million in funding. The IS Initiative employed a multi-disciplinary and multi-location team science approach to study the basic biological mechanisms underlying IS, search for biomarkers and novel drug targets, and develop improved treatments. Work done as part of the IS Initiative proved successful across multiple dimensions and led to more than 19 publications. More about the IS Initiative can be found here.[4]

Understanding the basic biological mechanisms underlying IS was a key focus of the IS Initiative. One example of the IS Initiative’s success in understanding a key underlying basic mechanism is from the team led by Dr. John Swann of Baylor College of Medicine.[1] Dr. Swann and his team discovered that treatment with a derivative of the growth hormone insulin-like growth factor 1 (IGF-1) called (1-3) IGF-1 reduced spasms and irregular brain wave patterns on the EEG in an animal model. Adding this compound to vigabatrin, an FDA-approved treatment for IS, reduced the dose of vigabatrin required to eliminate the spasms. Adding this compound to vigabatrin, an FDA-approved treatment for IS reduced the dose of vigabatrin required to eliminate the spasms. Reducing the dosage also decreased the risk of serious side effects, including the potential for irreversible peripheral vision loss. The Swann lab patented this combination treatment and used the discovery to obtain two National Institutes of Health (NIH) grants. The NIH grants enabled Dr. Swann to build on the discoveries from the CURE Epilepsy-funded IS Initiative. In a subsequent study, data from Dr. Swann’s team revealed that the levels IGF-1 itself were lower in brain tissue from both a rat model and from infants with IS. Data also indicated that reduced expression of IGF-1 in the rat model affected the biological pathways critical for neurodevelopmental processes.[6]

Using the learnings from the IS Initiative as a foundation, through a series of additional experiments, the team confirmed that the (1-3) IGF-1 could also cross the blood-brain barrier with much higher efficiency than the full-length IGF-1 and activate the same biological pathways as full-length IGF-1.[7] The researchers administered it to the rats in their experiment, and successfully eliminated both the spasms and the hypsarrhythmia in most rodents. This exciting finding suggests that this smaller (1-3) IGF-1 or perhaps an IGF-1-like drug may one day be used to treat IS patients immediately after the condition is diagnosed. You can read more about this study here.

Dr. Swann and his team have continued to build on the learnings initially funded through their CURE Epilepsy grant; recently, the team studied seizure progression in IS, and the impact of spasms on learning and memory.[8] Infants with IS show developmental delay and behavioral abnormalities, with only 16% of patients with IS exhibiting normal intellectual development.[9] The reasons for a delay in intellectual development could be many, though they have not yet been determined.[10,11] Additionally, the trajectory of the decline in intellectual and behavioral abilities has not yet been documented due to the variability of the condition, and limitations in assessing intellectual abilities in infants. Spasms can be subtle, making an accurate diagnosis of the exact start of the spasms challenging.[12] Hence, whether the behavioral decline is caused by or simply associated in time with seizures in IS is an area where more research is necessary. Given the difficulties of understanding this relationship between seizures and cognitive decline in infants with IS, Dr. Swann’s team used rats with a history of spasms and assessed them in a series of tests to gauge their ability to learn and remember. Swann’s team used rats with a history of spasms and assessed them in a series of memory tests to gauge their ability to learn and remember. The team also studied their brainwaves using EEG.

Previous work by Dr. Swann’s team had developed a model to simulate IS in animal models.[13] In this model, a substance known as tetrodotoxin (TTX) is infused into the brains of infant rats 10-12 days after birth which causes many of the characteristics of IS, including spasms, seen in humans.[13] The research team then used tests to examine spatial and working memory. Spatial memory helps us remember locations and the relationship between locations, and working memory helps us remember a small part of the information in our minds temporarily. To better understand the brainwaves in rats that had spasms, Dr. Swann’s team performed continuous EEG recordings for a total of seven weeks after infusion of TTX.[8] Rats with spasms were compared with rats that did not have spasms. After seven weeks of EEG recording, behavioral tests were done to test learning and memory. The study showed that rats experiencing spasms showed impairment on the behavioral tests, pointing to issues in learning and memory, which are also seen in infants with IS.[8] EEG analysis showed that there was an increase in spasms for two weeks, and after the two weeks, spasms stabilized.[8] Seizure progression in epilepsy has long been a topic of intense research. The current study suggests that like other seizure disorders[14,15], there may be a critical period in IS when there is a gradual increase in spasm intensity over time. A better understanding of seizure progression patterns in IS could lead to clues about therapies, management, and prognosis. This work from Dr. Swann’s lab is unique as the team did rigorous EEG monitoring and behavioral analysis; these techniques are time and labor-intensive, and seizures in IS have not been studied this deeply before.[8] The neurological mechanisms that underlie memory disturbances and seizure progression in IS are not fully known. So, seizures could be correlated with the learning deficits, but exact details are not clear. Additional research using EEG monitoring coupled with behavioral analysis in the same animals could provide clarity into the relationship.

In conclusion, basic research provides a foundational understanding of underlying biology of a disease process from which cures for the epilepsies will be found. CURE Epilepsy has been funding basic research for 25 years with the sole mission of finding a cure for epilepsy. Dr. Swann’s work as part of the IS Initiative is one example of how strategic, long-term investment in basic research can advance our knowledge by leaps and bounds.



Literature Cited:

  1. Gibbs EL, Fleming MM, Gibbs FA. Diagnosis and prognosis of hypsarhythmia and infantile spasms Pediatrics. 1954 Jan;13:66-73.
  2. Cowan LD, Hudson LS. The epidemiology and natural history of infantile spasms J Child Neurol. 1991 Oct;6:355-364.
  3. Knupp KG, Coryell J, Nickels KC, Ryan N, Leister E, Loddenkemper T, et al. Response to treatment in a prospective national infantile spasms cohort Ann Neurol. 2016 Mar;79:475-484.
  4. Lubbers L, Iyengar SS. A team science approach to discover novel targets for infantile spasms (IS). Epilepsia Open. 2021;6:49-61.
  5. Swann J, Lee, CL., Le, JT. and Frost Jr, JD. , inventor; Combination therapies for treating infantile spasms and other treatment resistant epilepsies 2022.
  6. Ballester-Rosado CJ, Le JT, Lam TT, Mohila CA, Lam S, Anderson AE, et al. A Role for Insulin-like Growth Factor 1 in the Generation of Epileptic Spasms in a murine model Ann Neurol. 2022 Jul;92:45-60.
  7. Yamamoto H, Murphy LJ. Enzymatic conversion of IGF-I to des(1-3)IGF-I in rat serum and tissues: a further potential site of growth hormone regulation of IGF-I action J Endocrinol. 1995 Jul;146:141-148.
  8. Le JT, Ballester-Rosado CJ, Frost JD, Jr., Swann JW. Neurobehavioral deficits and a progressive ictogenesis in the tetrodotoxin model of epileptic spasms Epilepsia. 2022 Dec;63:3078-3089.
  9. Hrachovy RA, Frost JD, Jr. Infantile epileptic encephalopathy with hypsarrhythmia (infantile spasms/West syndrome) J Clin Neurophysiol. 2003 Nov-Dec;20:408-425.
  10. Wirrell EC, Shellhaas RA, Joshi C, Keator C, Kumar S, Mitchell WG. How should children with West syndrome be efficiently and accurately investigated? Results from the National Infantile Spasms Consortium Epilepsia. 2015 Apr;56:617-625.
  11. Osborne JP, Lux AL, Edwards SW, Hancock E, Johnson AL, Kennedy CR, et al. The underlying etiology of infantile spasms (West syndrome): information from the United Kingdom Infantile Spasms Study (UKISS) on contemporary causes and their classification Epilepsia. 2010 Oct;51:2168-2174.
  12. Lux AL, Osborne JP. A proposal for case definitions and outcome measures in studies of infantile spasms and West syndrome: consensus statement of the West Delphi group Epilepsia. 2004 Nov;45:1416-1428.
  13. Lee CL, Frost JD, Jr., Swann JW, Hrachovy RA. A new animal model of infantile spasms with unprovoked persistent seizures Epilepsia. 2008 Feb;49:298-307.
  14. Jeavons PM, Bower BD. The natural history of infantile spasms Arch Dis Child. 1961 Feb;36:17-22.
  15. Golomb MR, Garg BP, Williams LS. Outcomes of children with infantile spasms after perinatal stroke Pediatr Neurol. 2006 Apr;34:291-295.

CURE Epilepsy Discovery: A Look Into the Journeys of CURE Epilepsy Taking Flight Grantees

Key Points:

  • CURE Epilepsy is the leading non-profit organization focused on funding research to find cures for epilepsy.
  • To achieve our mission, CURE Epilepsy created grant mechanisms to support research to understand the basic biological mechanisms or foundations of what causes seizures that become epilepsy as well as awards for pre-clinical research and more.
  • The Taking Flight Award was created to support epilepsy investigators early in their careers to develop a research focus separate from their mentor’s.
  • This Discovery highlights three Taking Flight awardees who received grants for diverse projects, ranging from work on sudden unexpected death in epilepsy (SUDEP) to mapping epileptic brain networks, to an exploration of circadian function as a potential mechanism and a therapeutic target for epilepsy.
  • The three awardees are Dr. William Nobis from Vanderbilt University Medical Center, Dr. Flavia Vitale from The University of Pennsylvania, and Dr. Cristina Reschke at the Royal College of Surgeons in Ireland. These awardees share their motivations for pursuing epilepsy research as a career, the importance of the CURE Epilepsy Taking Flight in their careers, and the impact they hope to have in the epilepsy community.

Deep dive

Epilepsy is one of the most common neurological disorders and affects 65 million people worldwide [1] and 3.4 million Americans [2]. Epilepsy can impact a person at any point in their lifetime, regardless of age, demographics, race, or socioeconomics. Those who live with epilepsy can face a lifetime of challenges. CURE Epilepsy’s mission is to find a cure for epilepsy, by promoting and funding patient-focused research, CURE Epilepsy has developed a variety of granting mechanisms [3], including the Taking Flight Award.[4] This one-year, $100,000 grant is meant to fund studies that will provide new insights into epilepsy, its prevention, and cures. An additional goal of the Taking Flight Award is to foster scientific independence of early career investigators and provide them with the means to collect the necessary data to apply for subsequent funding, thus further advancing their career in epilepsy research.[4]

Through 2021, CURE Epilepsy has funded 48 early career scientists through the Taking Flight Award, supporting them on their path to research independence. Recently, we spoke with three Taking Flight awardees about their research, the impact that receiving the Taking Flight Award had on their research and their vision for the future of their research. The three awardees and their projects were: Dr. William Nobis at Vanderbilt University Medical Center who studied a part of the brain called the amygdala and its role in sudden unexpected death in epilepsy (SUDEP); Dr. Flavia Vitale at The University of Pennsylvania who explored a technique to accurately map epileptic brain networks; and Dr. Cristina Reschke at the Royal College of Surgeons in Ireland, who examined the role of the circadian function in epilepsy.

The awardees noted varying inspirations to pursue the field of epilepsy research. For example, Dr. Nobis mentioned, “I have always been interested in neuroscience research, my initial motivation for training to be a physician-scientist was my experience with my grandmother and Alzheimer’s disease. During my residency training in neurology, I had exposure to epilepsy patients and experience with epileptologists, and I liked the impact you could have on patients’ lives by controlling seizures.” Dr. Nobis continues to be inspired by the patients he aims to serve. He says, “I want to do research that has the opportunity to make an impact on my patients’ lives – this is something that is within reach for epilepsy researchers. We have come far with controlling epilepsy but there are still so many people impacted by seizures.

Controlling epilepsy and preventing SUDEP are both interesting problems that will involve cutting edge circuit-based science and are also of immense importance and impact for the lives of our patients.” Previous research on SUDEP had shown that difficulties in breathing (respiratory depression) could lead to deficits in heart function, ultimately leading to death.[3] To understand this process better, Dr. Nobis’ team analyzed intracranial electroencephalograph (EEG), i.e., electrical signals from deep within the brain, and combined this with the measurement of breathing.[4] They found a specific role of the amygdala and the brainstem in respiratory depression during seizures. The discovery that this circuit may play a role in SUDEP means that in the future, we may be able to target it with interventions to prevent SUDEP.

According to Dr. Nobis, the Taking Flight Award gave him the capability to commit full-time to the SUDEP research, which had been a side project that he was working on in his mentor’s lab. He was able to publish his studies and take the next step in his career by accepting a position at Vanderbilt University Medical Center.[5] Subsequent mentored career-development awards helped him launch his research program centered on the physiological mechanisms of SUDEP. Looking into the future, Dr. Nobis commended the epilepsy research community with these words, “I love the epilepsy research community; I have not been around a group of more earnest and supportive group of scientists that are dedicated to improving the treatment of epilepsy patients. To do something that will improve the lives of epilepsy patients and work towards the goal of no SUDEP, no seizures, and no side effects.”

The basis for Dr. Vitale’s research is the premise that epilepsy arises from disrupted brain networks. To properly treat seizure-related conditions, it is important to map these networks as precisely as possible, but current methods of mapping epileptic neuronal networks do not provide the fine level of detail that is necessary. As part of Dr. Vitale’s Taking Flight research project, she developed miniature, flexible electrodes that could be controlled independently after they were implanted in the brain.[10] This technology has already shown success in detecting seizures in mice [6,7], and it is envisioned that this work of detecting seizure activity in experimental animals could ultimately lead to technologies to map epileptic networks in patients in a highly precise manner.

Speaking to her motivation, Dr. Vitale, was interested in joining a multidisciplinary team that “brings together engineers, clinicians, and neuroscientists to develop novel technological and therapeutic approaches to understand, diagnose, and treat neurological disorders, and in particular epilepsy.” Dr. Vitale found that working closely with clinicians and surgeons whose mission is to fight epilepsy, made her realize how engineers developing new devices, algorithms, modeling, and analysis tools can play a key role in supporting clinicians in their decision-making and provide them with more accurate and effective tools to diagnose and treat their patients. She also mentioned that the personal stories from patients and families reinforced her decision to start a career in neuro-engineering for epilepsy research.

Dr. Vitale describes the Taking Flight Award as an “inflection point” in her career, as the award enabled her to start her lab at the University of Pennsylvania.[8] In her words, “this award jumpstarted my research program in epilepsy research and helped me to gather key preliminary data that later supported successful grant applications and publications. Finally, the award provided recognition among the epilepsy research community and helped me establish new fruitful collaborations.” She summarizes her views on epilepsy research as, “Our mission is to ultimately translate our findings and technological innovations to patients. With more precise and safer electrodes, it will be easier to find and remove the areas of the brain where seizures originate, which has been shown to improve seizure-free rates. We are also developing easier-to-use, portable, and low-cost non-invasive brain monitoring systems to improve access and quality epilepsy care in low-resource settings and for at-home monitoring. Such technologies will offer more accurate data for diagnosis and therapy and reduce the burden of travel to patients in remote areas.”

Dr. Reschke’s work focuses on how circadian rhythms may impact epilepsy.[9,11] Previous studies have hypothesized a link between epilepsy and the 24-hour biological rhythms present in humans that are called “circadian rhythms.”[12] Dr. Reschke’s award, funded by The Cameron Boyce Foundation, aims to explore whether the mechanisms that control circadian rhythms are involved in the process of epileptogenesis (the process by which a non-epileptic circuit is transformed into an epileptic circuit). She will also develop a gene therapy approach in mice to see whether restoring a gene involved in circadian rhythms can halt epileptogenesis.[9,11] 

Dr. Reschke emphasized the excitement of discovery and the patient-centeredness of research as two main reasons she was attracted to working in epilepsy. “If we understand a bit more about the brain, we can develop focused and tailored approaches to epilepsy and accompanying comorbidities.” To her, while the science and research are extremely interesting, the possibility of impacting people with epilepsy, “makes the long hours worthwhile.”

Dr. Reschke also found her independence as a researcher thanks to the Taking Flight Award. The award allowed her to obtain, first, a temporary, and later, a permanent position at the Royal College of Surgeons in Ireland. She was able to secure lab space and personnel to do the work. The Taking Flight Award, in her words, “was extremely important and instrumental in establishing my career in epilepsy in academia.” In addition to the support for research, the award also gave her international recognition and helped her enhance her professional networks. Leveraging her research, recognition, and networks, Dr. Reschke is heavily involved in advocacy and education as well. She feels that “communicating research to patients’ families and caregivers is crucial,” and she feels that it is critical to put her clinical work into practice. She is also passionate about educating the next generation of epilepsy researchers by promoting training and fellowships to give them, “the opportunities that I have; to give them a chance to work on research that is deeply meaningful.”

The Taking Flight Awards have deeply impacted the grantees, and through them, numerous people that are affected by epilepsy every day. Awardees cited the capacity to contribute to scientific excellence while having a positive impact on patients with epilepsy and their caregivers as the most rewarding aspect of the award. The money granted by CURE Epilepsy to the researchers to gain scientific independence, develop new techniques, and discover novel mechanisms of epileptogenesis will help the organization achieve our goal of a world without epilepsy.


Literature Cited:

  1. Ngugi AK, Bottomley C, Kleinschmidt I, Sander JW, Newton CR. Estimation of the burden of active and lifetime epilepsy: a meta-analytic approach Epilepsia. 2010 May;51:883-890.
  2. National and State Estimates of the Numbers of Adults and Children with Active Epilepsy — United States, 2015.
  3. CURE Epilepsy: Grant Opportunities Available at: Accessed November 11
  4. CURE Epilepsy Taking Flight award Available at: Accessed November 11
  5. Ryvlin P, Nashef L, Lhatoo SD, Bateman LM, Bird J, Bleasel A, et al. Incidence and mechanisms of cardiorespiratory arrests in epilepsy monitoring units (MORTEMUS): a retrospective study Lancet Neurol. 2013 Oct;12:966-977.
  6. Nobis WP, González Otárula KA, Templer JW, Gerard EE, VanHaerents S, Lane G, et al. The effect of seizure spread to the amygdala on respiration and onset of ictal central apnea J Neurosurg. 2019 Apr 5;132:1313-1323.
  7. Department of Neurology; William P. Nobis, MD, Ph.D. Available at: Accessed November 11
  8. Mulcahey PJ, Chen Y, Driscoll N, Murphy BB, Dickens OO, Johnson ATC, et al. Multimodal, Multiscale Insights into Hippocampal Seizures Enabled by Transparent, Graphene-Based Microelectrode Arrays eNeuro. 2022 May-Jun;9.
  9. Driscoll N, Rosch RE, Murphy BB, Ashourvan A, Vishnubhotla R, Dickens OO, et al. Multimodal in vivo recording using transparent graphene microelectrodes illuminates spatiotemporal seizure dynamics at the microscale Commun Biol. 2021 Jan 29;4:136.
  10. Flavia Vitale, Ph.D.; Penn Medicine Available at: Accessed November 11
  11. Restoration of Circadian Function as a Novel Therapy for Epilepsy. Available at: Accessed November 11.
  12. Jin B, Aung T, Geng Y, Wang S. Epilepsy and Its Interaction With Sleep and Circadian Rhythm Front Neurol. 2020;11:327.

CURE Epilepsy Discovery: CURE Epilepsy Grantees Explore Genetic Determinants of Sudden Unexpected Death in Pediatrics (SUDP)

Key Points:

  • Sudden infant death syndrome (SIDS), sudden unexpected infant death (SUID), and sudden unexplained death in childhood (SUDC) are tragic conditions referring to the unexplained death of an infant or child. While previously thought of as separate entities, evidence suggests that there is an overlap between these conditions and that they may be considered under the overarching umbrella of sudden unexpected death in pediatrics (SUDP).
  • A previous study awarded by CURE Epilepsy and made possible by funding from the Isaiah Stone Foundation to Dr. Annapurna Poduri and colleagues suggested that genes associated with epilepsy may be involved in SUDP.[1]
  • A new study*, which was an outgrowth of the earlier CURE Epilepsy funded research, included 352 SUDP cases and employed state-of-the-art genetic techniques, in-depth analysis of family history and circumstances of death, and analysis of parental genetic information as well.[2]
  • The study showed evidence for genetic factors that may play a role in SUDP; while some genes were already potentially associated with sudden death in children, several variants in genes previously not associated with SUDP were identified.
  • In addition to providing genetic information about SUDP, the group’s work is proof of concept that a multidisciplinary lens to study SUDP is not only feasible, but necessary to advance the field.


Deep dive

The sudden death of a child is a tragic occurrence. Of all the child and infant deaths in the United States, more than 10% occur without any apparent cause, compounding the grief of families who have lost their children.[3] These sudden, unexpected deaths typically impact seemingly healthy children and are classified as sudden infant death syndrome (SIDS), sudden unexpected infant death (SUID), or sudden unexplained death in childhood (SUDC). Together, these three entities are thought of as sudden unexpected death in pediatrics (SUDP).[4] Through the generous support of the Isaiah Stone Foundation, CURE Epilepsy funded Dr. Annapurna Poduri and colleagues Rick Goldstein, Hannah Kinney, and Ingrid Holm in Robert’s Program** at Boston Children’s Hospital to explore the genetic basis of SUDP; the hypothesis for this work is that there are common, underlying, genetic mechanisms behind the three entities of sudden childhood death, epilepsy and sudden unexpected death in epilepsy (SUDEP).

Earlier work from these researchers had highlighted a link between SIDS and variants in a gene called SCN1A, which is associated with epilepsy and SUDEP.[1, 5, 6] What was notable is that while this gene is traditionally thought to be related to epilepsy, the children who died suddenly and unexpectedly had no history of seizures or epilepsy. This and other studies have suggested that SUDP is an overarching disorder consisting of rare and yet-undiagnosed diseases with potentially overlapping genetic mechanisms.[1, 7, 8]

To build on the previous work supported by CURE Epilepsy, Dr. Poduri and colleagues conducted a larger, more extensive study* to explore genetic risk factors for SUDP.[2] The ultimate goal was to find more accurate ways of diagnosing children at risk of sudden death and eventually prevent such incidences. The current study led by Dr. Poduri and her colleagues Drs. Hyunyong Koh and Alireza Haghighi included 352 SUDP cases.[2] This study looked at “trio-based” cohorts; meaning they studied the child and the child’s parents. This is a stronger approach to studying genetic contributions to a disease process, especially for SUDP, where a genetic link is suspected.[9, 10] Additionally, since the mechanisms underlying SUDP are complex and not yet fully known, the team took a “multidisciplinary undiagnosed diseases approach”[11] that combined genetic analysis, autopsy data, and in-depth study of the child’s phenotype (or observable characteristics). Parents agreed to all analyses performed. 

The genetic analysis included a candidate-gene approach where scientists have some information to suggest that a certain gene may be associated with a certain disease. In this case, 294 genes plausibly related to SUDP, called SUDP genes were studied, many of which were associated with neurologic disorders, cardiac disorders, or systemic/syndromic conditions. [12, 13] Systemic conditions and associated genes affect the entire body, and syndromic conditions include genes for metabolism and those responsible for the functioning of multiple body systems. The team performed a genetic technique called exome sequencing, where the parts of genes that eventually become functional proteins were analyzed for variants that may have caused or contributed to sudden death.[2]

The multidisciplinary team at Robert’s Program on SUDP, directed by Dr. Rick Goldstein, characterized fully the conditions surrounding the death of the child, performed an in-depth analysis of the child’s medical and family history, and conducted exome sequencing. The study showed that SUDP was associated with specific genetic factors, some of which were previously known, but many of which were novel. Detailed analysis showed that the majority of the children were between two and six months old, and 57% were male. Out of the 352 cases, death was associated with sleep in an overwhelming majority (346 children).[2] Genetic analysis revealed variants in genes related to cardiac disease, neurologic diseases, and systemic/syndromic diseases. Most variants were de novo, or new, while some were inherited. Burden analysis, in which the trios were compared to controls, showed that there were more SUDP trio cases with rare, damaging de novo variants as compared to controls. There were also clues as to the presence of febrile seizures, a family history of SIDS or SUDC, and a family history of epilepsy and cardiac disease in several cases. When the genes implicated in SUDP were classified by the age of death of the child, a pattern emerged. Specifically, variants in neurological and syndromic genes appeared in the age ranges associated with SIDS (the child being less than one year old) and SUDC (the child is greater than one year old), and variants in cardiac genes were preferentially seen only in the earlier age range, i.e., associated with SIDS.[2]

Overall, the study found that there was a genetic contribution to SUDP in 11% of the cases and suggests that these genetic variants may increase susceptibility to sudden death. Many genes that were previously not linked with SUDP were able to be reclassified as being associated with SUDP. The power of this study is that the genes for SUDP that were investigated were not the ones previously examined. A few specific genes found to be implicated in SUDP are SCN1A and DEPDC5, which have also been shown to be relevant in SUDEP. Other genes were associated with cardiac issues such as arrhythmia and cardiomyopathy (a condition that makes it harder for the heart to pump blood).[2]

The study also shows the importance of looking at trio data, as in this case, it led to the reclassification of several genetic variants. SUDP is a particularly difficult condition to study because unfortunately, a genetic condition may never have been diagnosed or suspected. Hence, the trio approach was instrumental in the success of the current study. The study also adds to evidence [7, 8] that conditions such as stillbirth, SIDS, and SUDC are not separate entities, but represent a continuum of events associated with unexplained death from fetal life to childhood.[14] It is possible and even likely that there are overlapping genetic variants (SCN1A being one) common to these conditions. Notably, another study from a different group also performed genetic analysis from trios and found de novo mutations associated with sudden unexplained death in childhood.[15]

Future studies will look at more trio data and an even more detailed genetic analysis. While many genetic risk factors were found in the current study, it does not mean that they were necessarily the cause of death; more work will be needed to look at specific neurologic mechanisms. In addition to the scientific findings, the study also sets the scene for a model where a multidisciplinary team engages with parents who have lost their children to unexplained death. In addition to providing scientific answers, a multidisciplinary team also can help provide counseling for family members at a much-needed time.



*Supported by funds from the Robert’s Program on Sudden Unexpected Death in Pediatrics, the Cooper Trewin Memorial SUDC Research Fund, CURE Epilepsy through the Isaiah Stone Foundation Award, Three Butterflies SIDS Foundation, The Florida SIDS Alliance, Borrowed Time 151, and The Eunice Kennedy Shriver National Institute of Child Health and Human Development under grant numbers R21 HD096355 and R01 HD090064.

**Robert’s Program at Boston Children‘s Hospital provides comprehensive clinical care to those that have lost a child suddenly and unexpectedly and performs research to better understand SUDP.


Literature Cited:

  1.      Brownstein CA, Goldstein RD, Thompson CH, Haynes RL, Giles E, Sheidley B, et al. SCN1A variants associated with sudden infant death syndrome Epilepsia. 2018 Apr;59:e56-e62.
  2.      Koh HY, Haghighi A, Keywan C, Alexandrescu S, Plews-Ogan E, Haas EA, et al. Genetic Determinants of Sudden Unexpected Death in Pediatrics Genet Med. 2022 Apr;24:839-850.
  3.      About Underlying Cause of Death, 1999-2020. Available at: Accessed October 4.
  4.     Goldstein RD, Nields HM, Kinney HC. A New Approach to the Investigation of Sudden Unexpected Death Pediatrics. 2017 Aug;140.  
  5.     Escayg A, Goldin AL. Sodium channel SCN1A and epilepsy: mutations and mechanisms Epilepsia. 2010 Sep;51:1650-1658.
  6.    Goldman AM. Mechanisms of sudden unexplained death in epilepsy Curr Opin Neurol. 2015 Apr;28:166-174.
  7.    Perrone S, Lembo C, Moretti S, Prezioso G, Buonocore G, Toscani G, et al. Sudden Infant Death Syndrome: Beyond Risk Factors Life (Basel). 2021 Feb 26;11.
  8.    Weese-Mayer DE, Ackerman MJ, Marazita ML, Berry-Kravis EM. Sudden Infant Death Syndrome: review of implicated genetic factors Am J Med Genet A. 2007 Apr 15;143a:771-788.
  9.    Richards S, Aziz N, Bale S, Bick D, Das S, Gastier-Foster J, et al. Standards and guidelines for the interpretation of sequence variants: a joint consensus recommendation of the American College of Medical Genetics and Genomics and the Association for Molecular Pathology Genet Med. 2015 May;17:405-424.
  10.   Rehm HL. A new era in the interpretation of human genomic variation Genet Med. 2017 Oct;19:1092-1095.
  11.   MacNamara EF, D’Souza P, Tifft CJ. The undiagnosed diseases program: Approach to diagnosis Transl Sci Rare Dis. 2020 Apr 13;4:179-188.
  12.   An Online Catalog of Human Genes and Genetic Disorders (OMIM). Available at: Accessed October 4.
  13.   The Human Gene Mutation Database (HGMD®). Available at: Accessed October 4.
  14.   Goldstein RD, Kinney HC, Willinger M. Sudden Unexpected Death in Fetal Life Through Early Childhood Pediatrics. 2016 Jun;137.
  15.   Halvorsen M, Gould L, Wang X, Grant G, Moya R, Rabin R, et al. De novo mutations in childhood cases of sudden unexplained death that disrupt intracellular Ca(2+) regulation Proc Natl Acad Sci U S A. 2021 Dec 28;118.

CURE Epilepsy Discovery: Brain Aging in Childhood-Onset Epilepsy: A Long-Term, Population-Based Study

Key Points:

  • Little is known about how the brain and brain function change with aging in older people with epilepsy, particularly among those with childhood-onset epilepsy (COE).
  • CURE Epilepsy funded Dr. Bruce Hermann (University of Wisconsin, United States) and Dr. Matti Sillanpää (University of Turku, Finland) to study the intersection of epilepsy and aging in a unique population-based group of people with epilepsy [1]. This group of people, whom Dr. Sillanpää had been following over decades, consists of persons diagnosed with COE when they were less than 16 years old (4.6 years of age on average).
  • The current study examined these individuals 55 years after the initial epilepsy diagnosis. Individuals with COE showed signs of brain aging that in several respects were more accelerated than those without epilepsy. Additionally, specific risk factors predictive of problematic brain aging outcomes were identified.  
  •  If confirmed with a larger group of people, these data could impact epilepsy treatment and our understanding of how epilepsy and aging overlap.


Deep Dive:

Studies in the general population have shown that the aging process changes brain functioning and cognition [2] and there is now much interest in discovering ways to protect the brain and cognition with aging. Through multiple grants funded by CURE Epilepsy including a CURE Innovator Award, a CURE  Epilepsy Award, and an Epilepsy Research Continuity Fund Award, Drs. Bruce Hermann at the University of Wisconsin and Matti Sillanpää at the University of Turku studied a unique patient population of individuals with childhood-onset epilepsy (COE) to investigate neurological and cognitive outcomes 55 years after the initial diagnosis [1].

Earlier studies with this same group (at 50 years after the initial diagnosis) showed that while seizure outcomes in this population were excellent, subsets of people with COE showed some signs of possible neurological and cognitive decline [3, 4]. However, the apparent declines could have been due to multiple factors including epilepsy, aging that was intensified by epilepsy, or simply longstanding abnormalities independent of aging. To get further clarity, Drs. Hermann and Sillanpää recruited many of the same individuals five years after the previous study (i.e., 55 years after the initial diagnosis) [1] to describe the seizure and neurological status of this population and identify the prospective changes at 55 years post-initial diagnosis by comparison to their status at 50 years post-initial diagnosis [4].

The study population, developed by Dr. Sillanpää, consisted of people in Turku, Finland who were diagnosed with active epilepsy between 1961 – 1964 when they were less than 16 years old. The initial population consisted of 245 people of which approximately 100 had epilepsy without other initial neurologic impairments, who were the focus of this investigation [3, 5]. The study performed 50 years after the initial diagnosis consisted of 51 subjects with COE and 52 non-epilepsy controls [4], and the current study at 55 years had complete data sets for 37 subjects with COE and 39 controls [1]. The following outcomes were examined in people with COE and controls:

  1.     clinical neurological signs including but not limited to consciousness, behavior, orientation, reflexes, and sensory functions.
  2.     magnetic resonance imaging (MRI) abnormalities in the brain identified on structured clinical review.
  3.     non-neurologic disorders such as high blood pressure, hypercholesteremia, and obesity.

The rationale for examining non-neurologic outcomes was based on previous studies that have shown a link between epilepsy and obesity [6], and between elevated cholesterol and triglycerides and epilepsy [7], and their roles in the brain aging process remained to be determined. As part of neurologic outcomes, an area of the brain called the cerebellum was also examined since abnormalities in this area have long been documented in epilepsy but are poorly understood [8]. The presence of seizures and their types were also assessed. Additionally, the dose of four commonly prescribed antiseizure medications (ASMs) was assessed to calculate lifelong ASM doses, and associations between ASMs and biological parameters were assessed.

In the current cohort, the average age of the participants was 63.2 years for the COE group and 63.0 years for the control group. A quarter of individuals with COE had active epilepsy and the rest were seizure-free for 10 years or more. Subjects with COE that had active epilepsy had more severe neurologic changes in general, and cerebellar abnormalities specifically. This is important because while the role of the cerebellum in epilepsy has been documented [8], it remains unclear whether abnormalities in the cerebellum are a cause for seizures, a consequence of seizures or their treatment, or purely coincidental. In this study, subjects with COE did not originally have cerebellar abnormalities, suggesting that changes are related to ongoing epilepsy and/or medical treatment [9]. Data analyses also revealed other interesting associations including that a high lifetime dose of ASMs was correlated with an increase in neurologic changes, and there was a link between peripheral neuropathy (damage of nerves outside the central nervous system) and use of ASMs. Another significant association that was noted was a decreased volume of a part of the brain called the hippocampus as seen on MRI and high arterial hypertension (vs. normal blood pressure) in people with COE. In addition, high cholesterol was more prevalent in people with focal epilepsy as compared to generalized epilepsy [4]. When the entire cohort (those with COE and controls) was compared to earlier results at 50 years post-initial diagnoses, the authors found that overall, more people exhibited neurologic changes at the 55-year follow-up [4].

In conclusion, when examined decades after diagnosis, adults with COE who continue to have active epilepsy appear to have an accelerated tendency towards brain aging as compared to controls. The study provides a unique and exceptionally long-term perspective on the intersection of aging and epilepsy. This is also the first study to explore changes in the brain through MRI and neurologic functioning in individuals with COE as compared to controls. Additional findings to follow from this investigation will address prospective quantitative MRI changes, biomarkers of abnormal aging, and prospective cognitive trajectories over the five years. While results from the current study will need to be confirmed, it has the potential to impact the treatment of epilepsy and change the way we think of the intersection of epilepsy and aging.


Literature Cited:

  1.       Sillanpää M, Hermann B, Rinne JO, Parkkola R, Saarinen MM, Karrasch M, et al. Differences in brain changes between adults with childhood-onset epilepsy and controls: A prospective population-based study Acta Neurol Scand. 2022 Mar;145:322-331.
  2.       How the aging brain affects thinking Available at: Accessed September 5.
  3.       Sillanpää M. Medico-social prognosis of children with epilepsy. Epidemiological study and analysis of 245 patients Acta Paediatr Scand Suppl. 1973;237:3-104.
  4.       Sillanpää M, Anttinen A, Rinne JO, Joutsa J, Sonninen P, Erkinjuntti M, et al. Childhood-onset epilepsy five decades later. A prospective population-based cohort study Epilepsia. 2015 Nov;56:1774-1783.
  5.       Sillanpää M, Jalava M, Kaleva O, Shinnar S. Long-Term Prognosis of Seizures with Onset in Childhood New England Journal of Medicine. 1998;338:1715-1722.
  6.       Ladino LD, Téllez-Zenteno JF. Chapter 7 – Epilepsy and obesity: A complex interaction. In: Mula M, editor. The Comorbidities of Epilepsy: Academic Press; 2019. p. 131-158.
  7.       Harnod T, Chen HJ, Li TC, Sung FC, Kao CH. A high risk of hyperlipidemia in epilepsy patients: a nationwide population-based cohort study Ann Epidemiol. 2014 Dec;24:910-914.
  8.       Streng ML, Krook-Magnuson E. The cerebellum and epilepsy Epilepsy & Behavior. 2021 2021/08/01/;121:106909.
  9.       Hagemann G, Lemieux L, Free SL, Krakow K, Everitt AD, Kendall BE, et al. Cerebellar volumes in newly diagnosed and chronic epilepsy J Neurol. 2002 Dec;249:1651-1658.

CURE Epilepsy Discovery: Developing Precision Medicine Treatments for Genetic Epilepsies: Present Challenges, Recent Scientific Advances, and Future Prospects

Key Points:

  • Epilepsy is a serious neurological disorder with many possible causes, and those directly linked to genetic abnormalities have undergone significant scientific breakthroughs in recent years.
  • Precision medicine is “an emerging approach for disease treatment and prevention that takes into account individual variability in genes, environment, and lifestyle for each person”. This concept is being applied to genetic epilepsies, but significant challenges have limited the rate at which basic science has translated into new treatments.
  • New strategies and scientific techniques may hasten the process. A recent publication in Epilepsia highlights some of them, along with the basic science that has fostered the hope for the eventual realization of precision medicine [1]. The authors suggest that greater coordination of efforts by scientists, physicians, patient advocates, and the federal government will accelerate effective, ethical, and equitable precision medicine for genetic epilepsy.
  • This publication stems from discussions at the Epilepsy Precision Medicine conference, funded in part by CURE Epilepsy and held in Washington, DC in 2019. This conference brought together the many stakeholders involved in developing precision therapies for epilepsy including researchers, physicians, funding agencies, and people with lived experience to share their experiences of epilepsy. The publication’s writing team was led by recent CURE Epilepsy Taking Flight grantee Juliet Knowles, MD, PhD.


Deep Dive:

Epilepsy is a debilitating but surprisingly common neurological disorder, with 1 in 26 people in the United States developing it over the course of their lives [2]. Despite the availability of numerous antiseizure medications (ASMs), one-third of people with epilepsy have seizures that remain treatment-resistant [3]. There are many possible causes of epilepsy, ranging from traumatic brain injuries to specific genetic mutations. Regardless of the cause, treatment remains primarily empirical or based on observation, with patients and their epileptologists often trying different and multiple ASMs in an attempt to eliminate the seizures while managing unwanted side effects. Ideally, treatments for epilepsy would precisely target the underlying biological mechanism, control seizures, and reduce the occurrence of negative side effects.

Optimism for this approach of “precision medicine” for epilepsy grew following the complete sequencing of the human genome and fueled the hope that individual genetic information could be used to develop more specific ways to treat epilepsy. Precision medicine, also known as personalized medicine, is the “tailoring of medical treatment to the individual characteristics of each patient. It does not literally mean the creation of drugs or medical devices that are unique to a patient, but rather the ability to classify individuals into subpopulations that differ in their susceptibility to a particular disease or their response to a specific treatment.” Unfortunately, for most types of genetic epilepsy, the individual genetic makeup of a patient has not yet translated to clinical application of precision medicines for epilepsy. This has been due, in part, to the complexity of the underlying biological mechanisms as well as limitations in the technologies needed to advance genetic discovery to appropriate treatments.

However, the authors describe how epilepsy research is entering an exciting new phase that may enable new precision therapies for many more types of genetic epilepsy. Over the last decade, significant progress in advancing precision medicine approaches has been achieved for epilepsies caused by discrete mutation(s) in a single gene. This work has involved 1) acceleration and efficiency of gene sequencing technology and identification of epilepsy-causing, including the location and type of specific mutations in the DNA sequence of these genes, and 2) clarification of the neuronal function(s)/dysfunction of the corresponding protein and underlying biochemical pathways. In addition, the development of specific laboratory methods such as cell-based models that replicate aspects of the structure and function of the human brain and the use of zebrafish that are sensitive to ASMs have accelerated the testing of novel epilepsy treatments. Finally, new epilepsy gene-targeted technologies, for example, antisense oligonucleotides, are being tested in clinical trials, and there is active discussion about changes in clinical trial design that could enable smaller clinical trials needed for rare genetic epilepsies.

Despite these successes, multiple challenges remain for the future development and accessibility of precision therapies for epilepsy. First, genetic testing and counseling remain inaccessible to many groups, including the elderly and the poor, across the world. Second, nearly 70% of epilepsy cases involve more than one gene and thus require an improved understanding of disease risk in the context of multiple genetic mutations, overall genetic background, and environmental exposure. Third, although gene therapy is conceptually encouraging there are challenges related to large-scale development of safe, ethical, and equitable delivery of gene-based therapies to overcome. It will be critical for the research community to work together to overcome these challenges to ensure the delivery of new precision therapies for genetic epilepsies.

An important driver for the advancements that have been made toward the development of precision therapies are the many new stakeholders calling for action. Numerous patient advocacy groups, professional societies such as the American Epilepsy Society, government and non-profit funding agencies such as the National Institute of Neurological Disorders and Stroke and CURE Epilepsy, respectively, have collectively called for a coordinated and systematic approach to developing new epilepsy treatments. Progress stemming from this call to action could bring a new age of treatments for those with epilepsy, shifting from observational experience to data-driven and patient-centered precision therapy.


Literature Cited:

  1. Knowles JK, Helbig I, Metcalf CS, Lubbers LS, Isom LL, Demarest S, Goldberg EM, George AL, Lerche H, Weckhuysen S, Whittemore V, Berkovic SF, Lowenstein DH. Precision medicine for genetic epilepsy on the horizon: Recent advances, present challenges, and suggestions for continued progress. Epilepsia 2022
  2. Hesdorffer D, Logroscino G, Benn E, Katri N. Cascino G, Hauser W. Estimating risk for developing epilepsy. A population-based study in Rochester, Minnesota. Neurology 2011; 76:23–27
  3. Chen Z, Brodie MJ, Liew D, Kwan P. Treatment outcomes in patients with newly diagnosed epilepsy treated with established and new antiepileptic drugs. A 30-Year Longitudinal Cohort Study. JAMA Neurology 2018 75:279-286.

CURE Epilepsy Discovery: Identifying a Promising Novel Treatment for Infantile Spasms

Key Points:

  •  John Swann, PhD, and his team explored an underlying cause of infantile spasms (IS), a devastating epileptic encephalopathy (an epilepsy syndrome that can lead to deterioration of the brain) that typically begins within the first year of life. This new research, funded by the National Institutes of Health (NIH), was a direct result of Dr. Swann’s findings from his work as a member of the CURE Epilepsy Infantile Spasms Initiative, conducted from 2013-2017.
  • Standard treatments for IS work in only approximately 50% of patients and can have severe side effects. The need for additional effective therapies drove Dr. Swann and his team to explore a more effective treatment with fewer or, ideally, no side effects.
  • Through extensive experimentation with an established rat model of IS and parallel studies in human tissue removed during epilepsy surgery, Dr. Swann observed very low levels of an important growth factor in the brain which has the potential to be a promising new treatment for this severe form of epilepsy.

Deep Dive:

Infantile spasms (IS) is a rare catastrophic form of epilepsy with approximately 90% of the cases beginning within the first year of life [1,2]. The condition is characterized by seizures with sudden brief jerking movements of the arms and legs or head bobs and often, though not always, an atypical, chaotic pattern of brain waves on the electroencephalogram (EEG) known as hypsarrhythmia [3]. The seizures are accompanied by significant development delays as well as cognitive and physical deterioration [2]. Standard treatments include adrenocorticotropic hormone (ACTH) or prednisone, and the antiseizure medication vigabatrin [4]. Unfortunately, only approximately 50% of children with IS respond to these treatments and there remains no reliable way of predicting who will respond favorably [4]. Even if these treatments diminish IS symptoms for a specific patient, they can have serious side effects. Therefore, scientists have been searching for other drug targets with the ultimate goal of developing alternative therapies.

One of these scientists is Dr. John Swann, Professor of Pediatrics at the Baylor College of Medicine, Director of the Gordon and Mary Cain Pediatric Neurology Research Foundation, and Principal investigator at the Jan and Dan Duncan Neurological Research Institute at Texas Children’s Hospital, who leveraged findings from his work as part of the CURE Epilepsy Infantile Spasms Initiative (2013-2017). With additional funding from NIH, Dr. Swann and his team used a previously developed rat model of IS [5] that mirrors many of this disorder’s symptoms, to investigate spasms that result from pediatric brain injuries, such as those suffered during a traumatic birth.

He and the team wanted to determine if the level of a substance known as insulin-like growth factor-1 (IGF-1) was altered in the injured brains of both their rat model and in IS patients, the latter using brain tissue from IS patients who had undergone neurosurgery to stop their seizures. The rationale behind this experiment was based on two observations. The first is that the level of IGF-1 in the cerebrospinal fluid of IS patients with preexisting brain damage is low [6], and second is that IGF-1 activates a biological pathway crucial for proper brain development and neuronal function [7]. As hypothesized, data revealed that IGF-1 levels were lower in brain tissue from both the rat model and from infants with IS. Data also showed that reduced expression of IGF-1 in the rat model affected the biological pathways critical for neurodevelopmental processes [8].

These promising findings suggested that increasing the amount of IGF-1 in the brains of the rat model might alleviate at least some of the symptoms of IS. To test this idea, the researchers employed a shorter version of IGF-1 called (1-3)IGF-1 which is a natural breakdown product of IGF-1 that can cross the blood-brain barrier with much higher efficiency than the full-length IGF-1 [9].

After confirming that (1-3)IGF-1 could activate the same biological pathways responsible for regulating the processes involved in early brain development as full-length IGF-1, the researchers administered it to their rat model and successfully eliminated both the spasms as well as the hypsarrhythmia in most rodents. This exciting finding suggests that this smaller (1-3)IGF-1 or perhaps an IGF-1-like drug may one day be used to treat IS patients immediately after the condition is diagnosed. This new approach could potentially reduce or even eliminate the associated neurodevelopmental and cognitive effects of this devastating disorder without the side effects of the currently available treatments. Dr. Swann states that this research and subsequent additional funding from NIH to continue the work would not have been possible without his participation in the CURE Epilepsy Infantile Spasms Initiative.


Literature Cited:

  1. Pellock, JM et al. Infantile spasms: a US consensus report. Epilepsia 2010; 51: 2175-2189
  2. Cowan, L.D. & Hudson, L.S. The epidemiology and natural history of infantile spasms. Child Neurol. 1991; 6(4): 355-364.
  3. Gibbs, E.L., Fleming, N.M, & Gibbs, F.A. Diagnosis and prognosis of hypsarrhythmia and infantile spasms. Pediatrics 1954; 13(1): 66-73.
  4. Knupp, K.G. et al. Response to treatment in a prospective national infantile spasms cohort. Neurol. 2016; 79(3): 475-484.
  5. Lee, C.L. et al. A new animal model of infantile spasms with unprovoked persistent seizures. Epilepsia 2008; 49(2): 298-307.
  6. Riikonen, R.S. et al. Insulin-like growth factor-1 is associated with cognitive outcome in infantile spasms. Epilepsia 2010; 51(7): 1283-1289.
  7. O’Kusky, J. & Ye, P. Neurodevelopmental effects of insulin-like growth factor signaling. Neuroendrocrincrinol. 2012; 33(3): 230-251.
  8. Ballester-Rosado, C.J. et al. A role for insulin-like growth factor 1 in the generation of epileptic spasms in a murine model. Neurol. 2022; 92(1): 45-60.
  9. Yamamoto, H. & Murphy, L.J. Enzymatic conversion of IGF-1 to des(1-3)IGF-1 in rat serum and tissues: a further potential site of growth hormone regulation of IGF-1 action. Endocrinol. 1995; 146(1): 141-148.

CURE Epilepsy Discovery: Investigating Mechanism of the Progression of Epilepsy

Key Points:

  • The development of seizures is associated with many changes in the brain; one of these changes is alterations in the white matter (the deep part of the brain) composed of axons covered in myelin. Myelin is a substance that acts as a nerve insulator and is critical for communication between neurons.
  • Dr. Juliet Knowles at Stanford University was granted both a CURE Epilepsy Taking Flight and a CURE Epilepsy Research Continuity Fund award to investigate whether changes in myelin might play a role in the development of epilepsy. Through her research, the team discovered that abnormal neuronal activity during absence seizures may lead to changes in myelination. The changes in myelin, in turn, lead to seizure progression.
  • This research paves the way for future studies that may identify ways to prevent harmful changes in myelination to treat some forms of epilepsy.

Deep Dive:

Some types of epilepsy are progressive, and the disease progression may manifest in the form of more frequent seizures, worsened control of seizures, or decline of cognition.[1] Progression of seizures has a direct correlation to the severity of epilepsy as time progresses.[2] Multiple factors may contribute to how seizures progress, but one factor that had not been investigated is a change, (known as plasticity), in the white matter or myelin of the brain. Myelin is a substance that acts as a form of insulation around the nerve cells of the brain and is essential for the conduction of electrical impulses between neurons and for the proper functioning of the brain. In fact, this white coating of myelin is what gives the collection of axons deep inside the brain the name “white matter”. Plasticity or changes in myelination can occur in response to neuronal activity and are important in the non-epileptic brain for functions such as learning, memory, and attention.[3,4] The team found that seizures in newborn infants with genetic forms of epilepsy may be associated with abnormalities in myelination.[5] These findings suggested that myelin plasticity might also occur in response to seizures. 

With her CURE Epilepsy Taking Flight Award, generously funded by the Ravichandran Foundation, Dr. Knowles and her colleagues sought to determine whether changes in myelination caused by a type of seizure called an absence seizure (formerly known as a petit mal seizure) contribute to the progression of epilepsy.[6] And when COVID-19 forced this study to be put on a temporary hold, the CURE Epilepsy Research Continuity Fund award, generously funded by the Cotton Family in memory of Vivian Cotton, helped Dr. Knowles’ team to finish these experiments when laboratories reopened. For her study, Dr. Knowles hypothesized that changes in myelin might influence seizure severity in absence seizure spreading.

Absence seizures are characterized by the abrupt stopping of behavior (“behavioral arrest”) and a specific pattern of brain activity. Even though absence seizures are typically brief, an individual can have an extremely high number of seizures in a day.[7, 8] Absence seizures originate in connections between two parts of the brain called the thalamus and cortex, after which seizure activity is transmitted through a large brain network by myelin-coated axons in white matter.[9, 10] 

The team used rodent models which had specific genetic mutations in their neurons that produce absence seizures. The pattern of seizure progression in these animals is similar to what is found in children with progressive forms of epilepsy.[2, 8] Electroencephalogram (EEG) was used to study electrical activity in the brain and the structure of neurons was examined using sophisticated microscopy techniques.[6] Additional techniques used to investigate the role of seizures in myelination included genetic interventions to block myelination (the process by which layers of myelin are produced) and drugs that decreased seizures.[6]

By using these methods, Dr. Knowles’ team first observed that there was an increase in myelination within the seizure network in seizure-prone animals, but this was seen only once seizures had started. This suggests that there is something unique and specific about the seizure activity that impacts myelination. Additionally, when Dr. Knowles’ team blocked seizures either genetically or using drugs, the increase in myelination was prevented. When the team blocked this abnormal myelination, the number of seizures decreased, and neuronal hypersynchrony decreased as well.[6] Neuronal hypersynchrony is the process by which networks of neurons fire together in an extremely organized and coordinated but abnormal way and is thought to underlie some aspects of seizure onset and spreading.[11]

The current study by Dr. Knowles’ group is the first that clearly shows that abnormal neuronal activity (in this case, due to absence seizures) can lead to harmful changes in myelination, which contribute to the continued progression of epilepsy.[6] Future work in the field will look at the exact molecules and neurotransmitters involved to better characterize this change in myelination. Although more studies are necessary, the current work suggests that developing treatments that address both abnormal neuronal activity and the associated abnormal myelination could more effectively prevent seizures and cognitive difficulties.[12] 


Literature Cited:

  1. Coan AC, Cendes F. Epilepsy as progressive disorders: what is the evidence that can guide our clinical decisions and how can neuroimaging help? Epilepsy Behav. 2013 Mar;26:313-321.
  2. Brigo F, Trinka E, Lattanzi S, Bragazzi NL, Nardone R, Martini M. A brief history of typical absence seizures – Petit mal revisited Epilepsy Behav. 2018 Mar;80:346-353.
  3. McKenzie IA, Ohayon D, Li H, de Faria JP, Emery B, Tohyama K, et al. Motor skill learning requires active central myelination Science. 2014 Oct 17;346:318-322.
  4. Pan S, Mayoral SR, Choi HS, Chan JR, Kheirbek MA. Preservation of a remote fear memory requires new myelin formation Nature Neuroscience. 2020 2020/04/01;23:487-499.
  5. Sandoval Karamian AG, Wusthoff CJ, Boothroyd D, Yeom KW, Knowles JK. Neonatal genetic epilepsies display convergent white matter microstructural abnormalities Epilepsia. 2020 Dec;61:e192-e197.
  6. Knowles JK, Xu H, Soane C, Batra A, Saucedo T, Frost E, et al. Maladaptive myelination promotes generalized epilepsy progression Nature Neuroscience. 2022 2022/05/01;25:596-606.
  7. Albuja A.C. KGQ. Absence Seizure. [Updated 2021 Dec 13]. StatPearls [Internet]. Treasure Island (FL): StatPearls Publishing 2022.
  8. Guerrini R, Marini C, Barba C. Generalized epilepsies Handb Clin Neurol. 2019;161:3-15.
  9. Musgrave J, Gloor P. The role of the corpus callosum in bilateral interhemispheric synchrony of spike and wave discharge in feline generalized penicillin epilepsy Epilepsia. 1980 Aug;21:369-378.
  10. Fogerson PM, Huguenard JR. Tapping the Brakes: Cellular and Synaptic Mechanisms that Regulate Thalamic Oscillations Neuron. 2016 Nov 23;92:687-704.
  11. Jiruska P, de Curtis M, Jefferys JGR, Schevon CA, Schiff SJ, Schindler K. Synchronization and desynchronization in epilepsy: controversies and hypotheses J Physiol. 2013;591:787-797.
  12. Li MCH, Cook MJ. Deep brain stimulation for drug-resistant epilepsy Epilepsia. 2018 Feb;59:273-290.

CURE Epilepsy Discovery: Identifying Human Brain Regions that Regulate Breathing as Eventual Targets for Direct SUDEP Intervention

Key Points:

  • CURE Epilepsy Award grantee Dr. Nuria Lacuey and her team sought to identify specific parts of the brain essential for regulating breathing, a fundamental function whose failure following a seizure is primarily responsible for Sudden Unexpected Death in Epilepsy (SUDEP).
  • The team recruited patients who were being evaluated for epilepsy surgery and who formally agreed to enroll in a study that had them perform various breathing exercises while having different areas of their brain electrically stimulated with varying intensities.
  • Quantitative analyses of the data revealed that four specific areas of the cortex of the brain affected the patients’ breathing responses, depending on the strength and frequency of the electrical stimulation. Two of these areas resulted in enhanced respiratory activity.
  • Additional data from more patients is needed, but Dr. Lacuey hopes to use these valuable results to develop a device that will stimulate critical areas of the brain following seizures to enhance breathing and avoid its cessation, thereby preventing SUDEP.

Deep Dive:

SUDEP is the most frequent cause of death among people with drug-resistant epilepsy [1,2]. Although different biological processes may contribute to SUDEP, the most prominent appears to be a phenomenon known as central apnea, a condition in which breathing repeatedly stops and starts, usually while sleeping, during or immediately after a severe seizure [3,4]. There is compelling evidence that breathing irregularities are an underlying cause of SUDEP. Research to date has almost exclusively concentrated on the role of an area of the brain called the brainstem [5], which ultimately connects higher cortical regions of the brain to the spinal cord. Although the brainstem plays a crucial role in maintaining respiratory activity, it may not be the only contributing area. Indeed, areas of the cortex have also been implicated [6], but the specific roles different areas of the cortex play in modulating breathing is unclear. Most importantly, there are currently no strategies for directly improving respiratory function during the dangerous period between seizure-induced central apnea and death.

One possible approach would be to electrically stimulate specific areas of the brain to maintain respiratory function during this critical period. As a first step, it is vital to assess the role of specific areas of the brain and what intensity and frequency of the electrical current might have beneficial or detrimental effects on breathing. Developing such an innovative method requires a detailed understanding of the relationship between brain electrical activity and breathing responses, specifically how brain regions are structurally and functionally linked through their neuronal connections, collectively known as the connectome [7,8].

Research on this approach was conducted by Dr. Lacuey and her team in the Department of Neurology at the University of Texas Health Science Center  in Houston, TX. Nineteen patients who suffered from drug-resistant epilepsy and who were being evaluated for epilepsy surgery consented to be enrolled in the study [9]. Pinpointing the exact seizure focus without damaging surrounding healthy tissue necessitated placing electrodes directly on the brains of these patients and then, electrically stimulating various brain regions for clinical mapping.

Electrodes were implanted in seven brain regions common to all 19 participants, and thus, these were the regions selected for comprehensive investigation. The goal was to ascertain whether electrical stimulation of each of these seven regions would affect breathing responses and, if so, whether the resulting respiratory activity would be enhanced or inhibited. Equally important was to determine the stimulation intensity as well as frequency necessary to elicit such responses. Electrical stimulation was carried out at a current of 1-10 milliamps (mA) and a frequency of 50 Hertz for 0.2 milliseconds [9].

Quantitative analyses of the data showed that electrical stimulation affected breathing responses in four of the seven different brain regions tested. Stimulation of two of these regions, specifically within the frontal portions of areas called the temporal lobe and cingulate gyrus, promoted breathing enhancement at a relatively low current (less than 3 mA) but not at the higher electrical current conditions tested [9]. Future experiments will require a larger group of patients, finer mapping of the identified brain regions, and exploration of brain regions other than the seven examined in the current study. Subsequent experiments will also include an evaluation of electric current and frequency as well as how any observed changes in respiratory control interact with the breathing mechanisms of the brainstem.

The fact that electrical stimulation of four cortical regions affected respiration, and that two of these areas enhanced breathing is an exciting finding. Significantly, this CURE Epilepsy funded research supports the idea that an implantable device capable of electrically stimulating pre-identified cortical regions of the brain to enhance breathing at critical times to prevent SUDEP may eventually be possible.


Literature Cited:

  1. Jones, L.A. & Thomas, R.H. Sudden unexpected death in epilepsy: insights from the last 25 years. Seizure 2017; 44: 232-236.
  2.   Devinsky, O. et al. Sudden unexpected death in epilepsy: epidemiology, mechanisms, and prevention. Lancet Neurol. 2016; 15(10): 1075-1088.
  3.   Vilella, L. et al. Postconvulsive central apnea as a biomarker for sudden unexpected death in epilepsy (SUDEP). Neurology 2019; 92(3): e171-e182.
  4.   So, E.L., Sam, M.C., & Lagerlund, T.L. Postictal central apnea as a cause of SUDEP: evidence from near-SUDEP incident. Epilepsia 2000; 41(11): 1494-1497.
  5.   Patodia, S. et al. The ventrolateral medulla and medulla raphe in sudden unexpected death in epilepsy. Brain 2018; 141(6): 1719-1733.
  6.   Herrero, J.L. Breathing above the brain stem: volitional control and attentional modulation in humans. J. Neurophysiol. 2018; 119(1): 145-159.
  7.   Bethlehem, R.A.J. et al. Brain charts for the human lifespan. Nature 2022; 604(7906): 525-533.
  8.   Fan, Q. et al. Mapping the human connectome using diffusion MRI at 300 mT/m gradient strength: Methodological advances and scientific impact. Neuroimage 2022; 254:118958.
  9.   Ganne, C. et al. Limbic and paralimbic respiratory modulation: from inhibition to enhancement. Epilepsia 2022; Epub

CURE Epilepsy Discovery: Preventing Post-Traumatic Epilepsy May be Possible by Inhibiting Two Inflammation-Based Signaling Pathways

Key Points:

  • For his CURE Epilepsy “Prevention of Acquired Epilepsies” grant, Dr. Xiaoming Jin and his team sought to understand the role of two related signaling pathways called TLR4 and RAGE, in the development of post-traumatic epilepsy (PTE) following brain injury in mice [1-3].
  • The team found that inhibiting either of these two inflammatory pathways soon after injury decreased seizure susceptibility as well as frequency.
  • In addition, inhibiting these pathways changed the levels of three types of brain cells, improving neuron survival and reducing brain tissue scarring.
  • These results suggest that inhibiting either of these inflammatory pathways may impede the development of PTE. 


Deep Dive:

Post-traumatic epilepsy (PTE) is one of the most devasting consequences of a traumatic brain injury (TBI). Depending on the severity of the injury, anywhere from 5% to 53% of people with TBI may develop PTE [1,4,5], and, unfortunately, PTE is often resistant to currently available antiseizure medications. Importantly, there is often a span of time between the injury and the onset of epilepsy, known as the “latent period,” during which treatments could be initiated to either reduce the chance of or completely prevent PTE [1,6].

One potential cause of PTE is inflammation in the brain. In hopes of preventing PTE or decreasing the  probability of it developing, researchers are working to understand the role of inflammation in the brain as a means to prevent PTE. The inflammatory process is regulated by “parent” proteins that, when activated, bind to target receptors to subsequently activate downstream signaling pathways. One of these “parent” proteins is known as HMGB1, and two of its receptor partner systems are TLR4 and RAGE [7]. All three of these proteins have been implicated in development of seizures, a process called epileptogenesis [8,9]. With funding from CURE Epilepsy, Dr. Jin and his team at the Stark Neurosciences Institute of the Indiana University School of Medicine sought to determine if these proteins also played a role in PTE and whether inhibiting these pathways could represent an approach for reducing the likelihood of epileptogenesis and PTE following TBI [10].

To test their hypothesis, the researchers first confirmed that the expression of HMGB1, RAGE, and TLR4 increased in three types of brain cells (neurons [“regular” nerve cells], astrocytes, and microglia) in their PTE mouse model [3,10] soon after injury. After completing this initial experiment, the team evaluated the ability of inhibitors of TLR4 or RAGE to lower seizure susceptibility and frequency in their PTE mice.  

For TLR4, they used the drug TAK242 (also known as resatorvid), a substance that has previously been employed by other researchers to prevent epileptogenesis in a different rodent model of PTE [11]. For RAGE, the researchers used an antibody (mAb) specifically designed in the laboratory to bind to the RAGE protein (RAGE mAb) and inhibit its signaling pathway. The team found that when either substance (TAK242 or RAGE mAb) was administered to mice one week after injury, the treated PTE mice were less prone to having tonic-clonic seizures and remained seizure-free for a longer period of time [10]. 

To provide additional evidence for the role of RAGE signaling in PTE, the researchers used mice in which RAGE had been genetically deleted (“RAGE knockout mice”). Data revealed that the RAGE knockout mice exhibited a higher threshold of seizure susceptibility and a longer period of seizure freedom after a TBI than their control counterparts.

The fact that similar results were obtained from two different, but complementary, types of experiments (pharmacological and genetic) provide corroboration for the critical roles that RAGE and TLR4 play in the onset of PTE. 

Once Dr. Jin’s team demonstrated that inhibiting TLR4 or RAGE seemed to have therapeutic value, they sought to understand what would happen at the cellular level when these two pathways were pharmacologically inhibited in their PTE model. Mice that were treated with either TAK242 or RAGE mAb one week after injury lost fewer neurons compared to those that were not treated with one of the two substances. Along with neurons, the team examined astrocytes and microglia. These cells mediate a process known as gliosis, a type of nonspecific scarring of brain tissue generated in response to brain damage that can lead to drug-resistant seizures [12]. Analogous to the results with neurons, there was much less gliosis in treated versus untreated PTE mice.  

Research to assess what happens in the brain after TBI is crucial to discovering possible therapeutic options to prevent epilepsy from developing. Data from Dr. Jin’s lab further validate the roles of HMGB1, its receptors TLR4 and RAGE, and their downstream inflammatory pathways in the PTE process itself, including cellular level changes, and how blocking either of these pathways may one day prevent PTE.


Literature Cited:

  1. Golub, V.M. & Reddy, D.S. Post-traumatic epilepsy and comorbidities: advanced models, molecular mechanisms, biomarkers, and novel therapeutic interventions. Pharmacol. Rev. 2022; 74(2): 387-438.
  2. Pitkänen, A. & McIntosh, T.K. Animal models of post-traumatic epilepsy. J. Neurotrauma 2006; 23(2): 241-261.
  3. Ping, X. & Jin, X. Chronic posttraumatic epilepsy following neocortical undercut lesion in mice. PLoS One 2016; 11(6): e0158231.
  4. Frey, L.C. Epidemiology of posttraumatic epilepsy: a critical review. Epilepsia 2003; 44(s10):11-17.
  5. Lowenstein, D.H. Epilepsy after head injury: an overview. Epilepsia 2009; 50(Suppl. 2): 4-9.
  6. Dulla, C.G. & Pitkänen, A. Novel approaches to prevent epileptogenesis after traumatic brain injury. Neurotherapeutics 2021; 18(3): 1582-1601.
  7. Yang, H. et al. Targeting inflammation driven by HMGB1. Front. Immunol. 2020; 11: 484.
  8. Friedman, A. and Dingledine, R. Molecular cascades that mediate the influence of inflammation on epilepsy. Epilepsia 2011; 52(Suppl 3): 33-39.
  9. Maroso, M. et al. Toll-like receptor 4 and high-mobility group box-1 are involved in ictogenesis and can be targeted to reduce seizures. Nat. Med. 2010; 16(4): 413-419.
  10. Ping, X. et al. Blocking receptor for advanced glycation end products (RAGE) or toll-like receptor 4 (TLR4) prevents posttraumatic epileptogenesis in mice. Epilepsia 2021; 62(12): 3105-3116.
  11. Zhang, D. et al. TLR4 inhibitor resatorvid provides neuroprotection in experimental traumatic brain injury: implication in the treatment of human brain injury. Neurochem. Int. 2014; 75: 11-18.
  12. Losi, G., Cammarota, M. & Carmignoto, G. The role of astroglia in the epileptic brain. Front. Pharmacol. 2012; 3(132): 1-13.

CURE Epilepsy Discovery: Investigating Spreading Depolarization in SUDEP

Key Points:

  • Dr. Stuart Cain’s CURE Epilepsy Taking Flight Award received while at the University of British Columbia, explored the mechanisms underlying Sudden Unexpected Death in Epilepsy (SUDEP).
  • SUDEP occurs when the heart and respiration both stop in a process called “cardiorespiratory arrest.” An area of the brain called the brainstem is critical in maintaining both heart function and respiration and is, therefore, an important brain area for researchers to study in order to understand the biological process through which SUDEP occurs. The brainstem is also connected to areas called the superior and inferior colliculus.
  • A phenomenon known as “spreading depolarization is known to contribute to respiratory arrest. Dr. Cain’s team investigated mechanisms underlying spreading depolarization in the areas of the brain connected to the brain stem called the superior and inferior colliculus. Their research showed the specific role of the superior colliculus in spreading depolarization to the brainstem, causing SUDEP.
  • This study establishes a foundation for continued study on the superior colliculus to ultimately develop preventative approaches for SUDEP.


Deep Dive:

Sudden Unexpected Death in Epilepsy (SUDEP) is one of the most tragic consequences of epilepsy. SUDEP occurs when a seemingly healthy person with epilepsy dies unexpectedly for no known reason. The biological causes of SUDEP are still not fully understood [1, 2]. Research suggests that SUDEP occurs because of the effects of seizures on the cardiovascular and respiratory systems resulting in “cardiorespiratory arrest”. Given that the brainstem is the part of the brain that controls heart rate and respiration, scientists have investigated the role of this region of the brain in causing SUDEP [3]. One phenomenon that has emerged as a possible explanation for SUDEP is called “spreading depolarization.” Spreading depolarization can be described as a wave of abnormal brain activity that travels through the layers of the brain in an organized fashion. Earlier work done by Dr. Cain and other researchers showed that spreading depolarization that engages the brainstem can be fatal. They also observed that additional areas in the brain called the superior and inferior colliculus are susceptible to spreading depolarization, but only during seizures, and that spreading depolarization that traveled into the brainstem during seizures was fatal [4]. 

Past research studies have shown that the superior and inferior colliculus may be involved in epilepsy [5]. Dr. Cain’s research, funded by the CURE Epilepsy Taking Flight Award, sought to investigate if the superior and inferior colliculus may play a role in spreading depolarization to the brainstem [6]. In this study, Dr. Cain’s team used a genetic mouse model that is susceptible to seizures and SUDEP and has been previously shown to be a good model to understand activity in the brainstem during fatal seizures [4]. The team did several experiments using these mice, called Cacna1aS218L mice, and state-of-the-art techniques. Using these techniques, Dr. Cain’s team first showed that when they stimulated the superior or the inferior colliculus, the mice experienced severe seizures, interrupted breathing (respiratory depression), and ultimately, death. Stimulation of the superior or the inferior colliculus started a wave of spreading depolarization that reached the brainstem in Cacna1aS218L mice, but not in normal mice. This wave of spreading depolarization that started in the superior and inferior colliculus traveled to several other brain regions, and then finally to the brainstem. Previous work done by Dr. Cain and other researchers also suggested the potential role of an additional brain structure called the thalamus in spreading depolarization that reaches the brainstem [4, 7]. By performing an additional experiment, the team observed that while stimulation of the thalamus initiated spreading depolarization in the Cacna1aS218L mice, the wave of activity did not reach the brainstem, and hence, was not associated with arresting breathing. The thalamus, therefore, does not appear to be involved in  spreading depolarization that leads to SUDEP.

To dive deeper into whether the superior or the inferior colliculus is important in spreading depolarization in the brainstem, Dr. Cain’s team used electrophysiology to measure electrical signals in these regions. They found that brain cells (neurons) of the superior colliculus of the Cacna1aS218L mice were inherently more excitable compared to superior colliculus neurons from normal mice. This finding suggests that the superior colliculus is what is critical for the brainstem spreading depolarization to occur which may in turn lead to SUDEP.  

Taken together, these novel results suggest the critical role of the superior colliculus in seizures that may lead to SUDEP. The results also strengthen the understanding of the sequence of events that may cause SUDEP. Spreading depolarization in the superior and inferior colliculus reaching the brainstem was associated with respiratory arrest, followed by cardiac arrest that is seen in SUDEP (as seen in the chart below). While more studies are necessary to understand the role of these brain structures in SUDEP, these data help envision methods to target and address brainstem spreading depolarization as a way to prevent SUDEP.

Literature Cited:

  1. Buchanan, G.F., Impaired CO(2)-Induced Arousal in SIDS and SUDEP. Trends Neurosci, 2019. 42(4): p. 242-250.
  2. Massey, C.A., et al., Mechanisms of sudden unexpected death in epilepsy: the pathway to prevention. Nat Rev Neurol, 2014. 10(5): p. 271-82.
  3. Ryvlin, P., et al., Incidence and mechanisms of cardiorespiratory arrests in epilepsy monitoring units (MORTEMUS): a retrospective study. Lancet Neurol, 2013. 12(10): p. 966-77.
  4. Loonen, I.C.M., et al., Brainstem spreading depolarization and cortical dynamics during fatal seizures in Cacna1aS218L mice. Brain, 2019. 142(2): p. 412-425.
  5. Faingold, C.L., Neuronal networks in the genetically epilepsy-prone rat. Adv Neurol, 1999. 79: p. 311-21.
  6. Cain, S.M., et al., Hyperexcitable superior colliculus and fatal brainstem spreading depolarization in a model of sudden unexpected death in epilepsy. Brain Communications, 2022: p. fcac006.
  7. Cain, S.M., et al., In vivo imaging reveals that pregabalin inhibits cortical spreading depression and propagation to subcortical brain structures. Proc Natl Acad Sci U S A, 2017. 114(9): p. 2401-2406.