Current Literature In Basic Science
Neurosteroid Deficiency Associated With Epilepsy
Mutations of Protocadherin 19 in Female Epilepsy (PCDH19-FE) Lead to Allopregnanolone Deficiency. Tan C, Shard C, Ranieri E, Hynes K, Pham DH, Leach D, Buchanan G, Corbett M, Shoubridge C, Kumar R, Douglas E, Nguyen LS, Mcmahon J, Sadleir L, Specchio N, Marini C, Guerrini R, Moller RS, Depienne C, Haan E, Thomas PQ, Berkovic SF, Scheffer IE, and Gecz J. Human Molecular Genetics 2015;24:5250–5259.
Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E−47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.
Commentary Mutations in protocadherin 19 (PCDH19) have been linked to early-onset epilepsy in females, termed protocadherin 19 female associated epilepsy (PCDH19-FE) and formerly EFMR. Patients with PCDH19-FE exhibit early-onset seizures that vary widely in type and severity. In addition, PCDH19-FE patients exhibit a wide range of psychiatric and cognitive comorbidities, including obsessive compulsive behaviors, depression, and autism. The gene responsible, protocadherin 19, has been mapped to the X-chromosome and encodes for a protein thought to be a calcium-dependent cell-adhesion molecule, primarily expressed in the brain. However, the relationship between the function of protocadherin 19 and the pathophysiology of PCDH19-FE remains unclear. The observation that transmitting males are largely unaffected, combined with the broad spectrum of clinical presentation of this disorder, led the authors of the currently highlighted study to explore secondary gene effects that may alter the clinical presentation or penetrance of PCDH19-FE. The highlighted study employs expression profiling to identify gender-biased genes altered in PCDH19-FE. Comparing the expression profiles of both young (mean 8.8 years old) and older (mean 25 years of age) females with PCDH19-FE to healthy female controls identified 140 and 192 dysregulated Epilepsy Currents, Vol. 16, No. 2 (March/April) 2016 pp. 108–109 © American Epilepsy Society
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genes, respectively. The genes associated with PCDH19-FE were then compared against the 223 genes that exhibit gender bias, identified by comparing the expression profiles of healthy males to those of healthy females. This approach identified 43 gender-biased genes that are associated with PCDH19-FE. To gain some insight into the pathways associated with the gene alterations in PCDH19-FE, the authors examined an annotated list of these genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway and Ingenuity Pathway Analysis (IPA). These methods identified cell adhesion and integrin-related signaling pathways, consistent with the role of protocadherin 19 as a cell adhesion molecule. However, none of the pathways identified included gender-biased genes. At this point, the authors had the ingenuity to examine the upstream regulatory elements of the PCDH19-FE associated genes and demonstrated that 22% of them possess progesterone and estrogen regulatory sites. Thus, these steroid hormone regulatory sites may confer gender-specific regulation and be important for PCDH19-FE. Five of these genes (WISP2, OXTR, AKR1C3, APOD, and GRIA1) were validated using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Two of these genes, AKR1C2 and AKR1C3, were of particular interest given their function in neurosteroidogenesis, including the synthesis of allopregnanolone. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone) is a neurosteroid derived from progesterone that acts as a positive allosteric modulator at GABAA receptors. Not only can allopregnanolone directly
Neurosteroid Deficiency in Epilepsy
modulate GABAergic signaling via allosteric modulation, allopregnanolone can also impact GABAergic transmission by altering the expression of specific GABAA receptor subunits. Extrasynaptic GABAA receptors, which mediate tonic GABAergic inhibition, are particularly sensitive to neurosteroid modulation, and actions of allopregnanolone on these receptors are thought to mediate their potent anticonvulsant actions. The steroid hormone precursor to allopregnanolone, progesterone, has been shown to exert anticonvulsant effects (1), mediated at least in part by the production of neurosteroids (2, 3). Consistent with their anticonvulsant actions, exogenous allopregnanolone has been shown to exert anticonvulsant actions in numerous animal models of epilepsy (2, 3). More importantly, allopregnanolone has recently been shown to be beneficial in the treatment of patients with severe status epilepticus (4–6). Withdrawal of steroid hormones has been demonstrated to increase seizure susceptibility, in part owing to the reduction in neurosteroid levels and also attributed to alterations in GABAA receptor subunit expression (7). Interestingly, PCDH19 has been shown to directly interact with at least one GABAA receptor subunit, alpha 1 (8). However, further studies are required to determine whether PCDH19 plays a role in the regulation of GABAA receptors and whether these interactions may contribute to the pathophysiology of PCDH19-FE. Reductions in the levels of allopregnanolone has also been implicated in a subset of epilepsy in which seizures are aggravated by the menstrual cycle, termed catamenial epilepsy (9), which can be ameliorated with progesterone treatment (10). Although the current study focuses on low levels of neurosteroids during early PCDH19-FE development, a similar mechanism for seizure generation has been proposed for neurosteroid withdrawal associated with catamenial epilepsy. These findings suggest that neurosteroids or compounds targeting their site of action—namely, extrasynaptic GABAA receptors—may be useful for treatment of numerous types of epilepsies, including PCDH19-FE. In fact, an open-label study recently demonstrated the effectiveness of allopregnanolone in the treatment of status epilepticus. A clinical trial has also been initiated testing the use of Ganaxolone, a synthetic analog of allopregnanolone, for the treatment of PCDH19 pediatric epilepsy. One of the benefits of the use of neuroste roids for therapy is that they do not exhibit the development
of tolerance (11), in contrast to benzodiazepines. Refinement of compounds related to neurosteroids to improve the halflife, route of administration, efficacy, and bioavailability will likely have significant therapeutic potential particularly for the epilepsies and perhaps, in particular, PCDH19-FE. by Jamie Maguire, PhD References 1. Stevens S, Harden C. Hormonal therapy for epilepsy. Curr Neurol Neurosci Rep 2011;11:435–442. 2. Reddy DS, Rogawski MA. Neurosteroids – Endogenous regulators of seizure susceptibility and role in the treatment of epilepsy. In: Jasper’s Basic Mechanisms of the Epilepsies [Internet]. 4th edition. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) Bethesda, MD: National Center for Biotechnology Information (US); 2012. 3. Reddy DS. Role of anticonvulsant and antiepileptogenic neuroste roids in the pathophysiology and treatment of epilepsy. Frontiers in Endocrinology 2011;2:38. 4. Reddy K, Reife R, Cole AJ. SGE-102: A novel therapy for refractory status epilepticus. Epilepsia 2013;54:81–83. 5. Rogawski MA, Loya CM, Reddy K, Zolkowska D, Lossin C. Neuroactive steroids for the treatment of status epilepticus. Epilepsia 2013;54:93– 98. 6. Broomall E, Natale JE, Grimason M, Goldstein J, Smith CM, Chang C, Kanes S, Rogawski MA, Wainwright MS. Pediatric super-refractory status epilepticus treated with allopregnanolone. Ann Neurol 2014;76:911–915. 7. Smith SS, Shen H, Gong QH, Zhou X. Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits. Pharmacol Ther 2007;116:58–76. 8. Passafaro M. Molecular Characterization of PCDH19 in Rat Hippocampal Neurons. Proceedings of the Third World Conference on PCDH19. Rome, October 24, 2015. Rome: Onlus Insieme per la Ricerca PCDH19 [Together for the Research PCDH19]; 2015. 9. Herzog AG, Klein P, Ransil BJ. Three patterns of catamenial epilepsy. Epilepsia 1997;38:1082–1088. 10. Herzog AG, Frye CA. Seizure exacerbation associated with inhibition of progesterone metabolism. Ann Neurol 2003;53:390–391. 11. Kokate TG, Yamaguchi SI, Pannell LK, Rajamani U, Carroll DM, Grossman AB, Rogawski MA. Lack of anticonvulsant tolerance to the neuroactive steroid pregnanolone in mice. J Pharmacol Exp Ther 1998;287:553–558.
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Neurosteroid Deficiency Associated With Epilepsy
Mutations of Protocadherin 19 in Female Epilepsy (PCDH19-FE) Lead to Allopregnanolone Deficiency. Tan C, Shard C, Ranieri E, Hynes K, Pham DH, Leach D, Buchanan G, Corbett M, Shoubridge C, Kumar R, Douglas E, Nguyen LS, Mcmahon J, Sadleir L, Specchio N, Marini C, Guerrini R, Moller RS, Depienne C, Haan E, Thomas PQ, Berkovic SF, Scheffer IE, and Gecz J. Human Molecular Genetics 2015;24:5250–5259.
Protocadherin 19 (PCDH19) female limited epilepsy (PCDH19-FE; also known as epilepsy and mental retardation limited to females, EFMR; MIM300088) is an infantile onset epilepsy syndrome with or without intellectual disability (ID) and autism. We investigated transcriptomes of PCDH19-FE female and control primary skin fibroblasts, which are endowed to metabolize neurosteroid hormones. We identified a set of 94 significantly dysregulated genes in PCDH19-FE females. Intriguingly, 43 of the 94 genes (45.7%) showed gender-biased expression; enrichment of such genes was highly significant (P = 2.51E−47, two-tailed Fisher exact test). We further investigated the AKR1C1-3 genes, which encode crucial steroid hormone-metabolizing enzymes whose key products include allopregnanolone and estradiol. Both mRNA and protein levels of AKR1C3 were significantly decreased in PCDH19-FE patients. In agreement with this, the blood levels of allopregnanolone were also (P < 0.01) reduced. In conclusion, we show that the deficiency of neurosteroid allopregnanolone, one of the most potent GABA receptor modulators, may contribute to PCDH19-FE. Overall our findings provide evidence for a role of neurosteroids in epilepsy, ID and autism and create realistic opportunities for targeted therapeutic interventions.
Commentary Mutations in protocadherin 19 (PCDH19) have been linked to early-onset epilepsy in females, termed protocadherin 19 female associated epilepsy (PCDH19-FE) and formerly EFMR. Patients with PCDH19-FE exhibit early-onset seizures that vary widely in type and severity. In addition, PCDH19-FE patients exhibit a wide range of psychiatric and cognitive comorbidities, including obsessive compulsive behaviors, depression, and autism. The gene responsible, protocadherin 19, has been mapped to the X-chromosome and encodes for a protein thought to be a calcium-dependent cell-adhesion molecule, primarily expressed in the brain. However, the relationship between the function of protocadherin 19 and the pathophysiology of PCDH19-FE remains unclear. The observation that transmitting males are largely unaffected, combined with the broad spectrum of clinical presentation of this disorder, led the authors of the currently highlighted study to explore secondary gene effects that may alter the clinical presentation or penetrance of PCDH19-FE. The highlighted study employs expression profiling to identify gender-biased genes altered in PCDH19-FE. Comparing the expression profiles of both young (mean 8.8 years old) and older (mean 25 years of age) females with PCDH19-FE to healthy female controls identified 140 and 192 dysregulated Epilepsy Currents, Vol. 16, No. 2 (March/April) 2016 pp. 108–109 © American Epilepsy Society
108
genes, respectively. The genes associated with PCDH19-FE were then compared against the 223 genes that exhibit gender bias, identified by comparing the expression profiles of healthy males to those of healthy females. This approach identified 43 gender-biased genes that are associated with PCDH19-FE. To gain some insight into the pathways associated with the gene alterations in PCDH19-FE, the authors examined an annotated list of these genes using the Database for Annotation, Visualization and Integrated Discovery (DAVID) pathway and Ingenuity Pathway Analysis (IPA). These methods identified cell adhesion and integrin-related signaling pathways, consistent with the role of protocadherin 19 as a cell adhesion molecule. However, none of the pathways identified included gender-biased genes. At this point, the authors had the ingenuity to examine the upstream regulatory elements of the PCDH19-FE associated genes and demonstrated that 22% of them possess progesterone and estrogen regulatory sites. Thus, these steroid hormone regulatory sites may confer gender-specific regulation and be important for PCDH19-FE. Five of these genes (WISP2, OXTR, AKR1C3, APOD, and GRIA1) were validated using reverse transcriptase-quantitative polymerase chain reaction (RT-qPCR). Two of these genes, AKR1C2 and AKR1C3, were of particular interest given their function in neurosteroidogenesis, including the synthesis of allopregnanolone. Allopregnanolone (3α-hydroxy-5α-pregnan-20-one or 3α,5α-tetrahydroprogesterone) is a neurosteroid derived from progesterone that acts as a positive allosteric modulator at GABAA receptors. Not only can allopregnanolone directly
Neurosteroid Deficiency in Epilepsy
modulate GABAergic signaling via allosteric modulation, allopregnanolone can also impact GABAergic transmission by altering the expression of specific GABAA receptor subunits. Extrasynaptic GABAA receptors, which mediate tonic GABAergic inhibition, are particularly sensitive to neurosteroid modulation, and actions of allopregnanolone on these receptors are thought to mediate their potent anticonvulsant actions. The steroid hormone precursor to allopregnanolone, progesterone, has been shown to exert anticonvulsant effects (1), mediated at least in part by the production of neurosteroids (2, 3). Consistent with their anticonvulsant actions, exogenous allopregnanolone has been shown to exert anticonvulsant actions in numerous animal models of epilepsy (2, 3). More importantly, allopregnanolone has recently been shown to be beneficial in the treatment of patients with severe status epilepticus (4–6). Withdrawal of steroid hormones has been demonstrated to increase seizure susceptibility, in part owing to the reduction in neurosteroid levels and also attributed to alterations in GABAA receptor subunit expression (7). Interestingly, PCDH19 has been shown to directly interact with at least one GABAA receptor subunit, alpha 1 (8). However, further studies are required to determine whether PCDH19 plays a role in the regulation of GABAA receptors and whether these interactions may contribute to the pathophysiology of PCDH19-FE. Reductions in the levels of allopregnanolone has also been implicated in a subset of epilepsy in which seizures are aggravated by the menstrual cycle, termed catamenial epilepsy (9), which can be ameliorated with progesterone treatment (10). Although the current study focuses on low levels of neurosteroids during early PCDH19-FE development, a similar mechanism for seizure generation has been proposed for neurosteroid withdrawal associated with catamenial epilepsy. These findings suggest that neurosteroids or compounds targeting their site of action—namely, extrasynaptic GABAA receptors—may be useful for treatment of numerous types of epilepsies, including PCDH19-FE. In fact, an open-label study recently demonstrated the effectiveness of allopregnanolone in the treatment of status epilepticus. A clinical trial has also been initiated testing the use of Ganaxolone, a synthetic analog of allopregnanolone, for the treatment of PCDH19 pediatric epilepsy. One of the benefits of the use of neuroste roids for therapy is that they do not exhibit the development
of tolerance (11), in contrast to benzodiazepines. Refinement of compounds related to neurosteroids to improve the halflife, route of administration, efficacy, and bioavailability will likely have significant therapeutic potential particularly for the epilepsies and perhaps, in particular, PCDH19-FE. by Jamie Maguire, PhD References 1. Stevens S, Harden C. Hormonal therapy for epilepsy. Curr Neurol Neurosci Rep 2011;11:435–442. 2. Reddy DS, Rogawski MA. Neurosteroids – Endogenous regulators of seizure susceptibility and role in the treatment of epilepsy. In: Jasper’s Basic Mechanisms of the Epilepsies [Internet]. 4th edition. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) Bethesda, MD: National Center for Biotechnology Information (US); 2012. 3. Reddy DS. Role of anticonvulsant and antiepileptogenic neuroste roids in the pathophysiology and treatment of epilepsy. Frontiers in Endocrinology 2011;2:38. 4. Reddy K, Reife R, Cole AJ. SGE-102: A novel therapy for refractory status epilepticus. Epilepsia 2013;54:81–83. 5. Rogawski MA, Loya CM, Reddy K, Zolkowska D, Lossin C. Neuroactive steroids for the treatment of status epilepticus. Epilepsia 2013;54:93– 98. 6. Broomall E, Natale JE, Grimason M, Goldstein J, Smith CM, Chang C, Kanes S, Rogawski MA, Wainwright MS. Pediatric super-refractory status epilepticus treated with allopregnanolone. Ann Neurol 2014;76:911–915. 7. Smith SS, Shen H, Gong QH, Zhou X. Neurosteroid regulation of GABA(A) receptors: Focus on the alpha4 and delta subunits. Pharmacol Ther 2007;116:58–76. 8. Passafaro M. Molecular Characterization of PCDH19 in Rat Hippocampal Neurons. Proceedings of the Third World Conference on PCDH19. Rome, October 24, 2015. Rome: Onlus Insieme per la Ricerca PCDH19 [Together for the Research PCDH19]; 2015. 9. Herzog AG, Klein P, Ransil BJ. Three patterns of catamenial epilepsy. Epilepsia 1997;38:1082–1088. 10. Herzog AG, Frye CA. Seizure exacerbation associated with inhibition of progesterone metabolism. Ann Neurol 2003;53:390–391. 11. Kokate TG, Yamaguchi SI, Pannell LK, Rajamani U, Carroll DM, Grossman AB, Rogawski MA. Lack of anticonvulsant tolerance to the neuroactive steroid pregnanolone in mice. J Pharmacol Exp Ther 1998;287:553–558.
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