Aristea S. Galanopoulou

Neuroprotective Effects of Estrogens on Hippocampal Cells in Adult Female Rats After Status Epilepticus

Summary: Purpose: Estrogens have neuroprotective effects in ischemia, stroke, and other conditions leading to neuronal cell death (e.g., Alzheimers disease). The present study examined whether estrogens may have neuroprotective effects after seizures.

Identification of new epilepsy treatments: issues in preclinical methodology.

Preclinical research has facilitated the discovery of valuable drugs for the symptomatic treatment of epilepsy. Yet, despite these therapies, seizures are not adequately controlled in a third of all affected individuals, and comorbidities still impose a major burden on quality of life. The introduction of multiple new therapies into clinical use over the past two decades has done little to change this. There is an urgent demand to address the unmet clinical needs for: (1) new symptomatic antiseizure treatments for drug‐resistant seizures with improved efficacy/tolerability profiles, (2) disease‐modifying treatments that prevent or ameliorate the process of epileptogenesis, and (3) treatments for the common comorbidities that contribute to disability in people with epilepsy. New therapies also need to address the special needs of certain subpopulations, that is, age‐ or gender‐specific treatments. Preclinical development in these treatment areas is complex due to heterogeneity in presentation and etiology, and may need to be formulated with a specific seizure, epilepsy syndrome, or comorbidity in mind. The aim of this report is to provide a framework that will help define future guidelines that improve and standardize the design, reporting, and validation of data across preclinical antiepilepsy therapy development studies targeting drug‐resistant seizures, epileptogenesis, and comorbidities.

A Pulse Rapamycin Therapy for Infantile Spasms and Associated Cognitive Decline

Infantile spasms are seizures manifesting within a spectrum of epileptic encephalopathies of infancy that often lead to cognitive impairment. Their current therapies, including adrenocorticotropic hormone (ACTH), high dose steroids, or vigabatrin, are not always effective and may be associated with serious side effects. Overactivation of the TORC1 complex of the mTOR pathway is implicated in the pathogenesis of certain genetic and acquired disorders that are linked with infantile spasms, like tuberous sclerosis. Here, we tested the therapeutic potential of rapamycin, a TORC1 inhibitor, as a potential treatment for infantile spasms in the multiple-hit rat model of ACTH-refractory symptomatic infantile spasms, which is not linked to tuberous sclerosis. Rapamycin or vehicle was given after spasms appeared. Their effects on spasms, other seizures, performance in Barnes maze, and expression of the phosphorylated S6 ribosomal protein (pS6: a TORC1 target) in the cortex, using immunofluorescence, were compared. Rapamycin suppressed spasms dose-dependently and improved visuospatial learning, although it did not reduce the frequency of other emerging seizures. High-dose pulse rapamycin effected acute and sustained suppression of spasms and improved cognitive outcome, without significant side effects. Therapeutically effective rapamycin doses normalized the pS6 expression, which was increased in perilesional cortical regions of pups with spasms. These findings support that pathological overactivation of TORC1 may be implicated in the pathogenesis of infantile spasms, including those that are not linked to tuberous sclerosis. Furthermore, a high-dose, pulse rapamycin treatment is a promising, well tolerated and disease-modifying new therapy for infantile spasms, including those refractory to ACTH.

Finding a better drug for epilepsy: the mTOR pathway as an antiepileptogenic target.

The mammalian target of rapamycin (mTOR) signaling pathway regulates cell growth, differentiation, proliferation, and metabolism. Loss‐of‐function mutations in upstream regulators of mTOR have been highly associated with dysplasias, epilepsy, and neurodevelopmental disorders. These include tuberous sclerosis, which is due to mutations in TSC1 or TSC2 genes; mutations in phosphatase and tensin homolog (PTEN) as in Cowden syndrome, polyhydramnios, megalencephaly, symptomatic epilepsy syndrome (PMSE) due to mutations in the STE20‐related kinase adaptor alpha (STRADalpha); and neurofibromatosis type 1 attributed to neurofibromin 1 mutations. Inhibition of the mTOR pathway with rapamycin may prevent epilepsy and improve the underlying pathology in mouse models with disrupted mTOR signaling, due to PTEN or TSC mutations. However the timing and duration of its administration appear critical in defining the seizure and pathology‐related outcomes. Rapamycin application in human cortical slices from patients with cortical dysplasias reduces the 4‐aminopyridine–induced oscillations. In the multiple‐hit model of infantile spasms, pulse high‐dose rapamycin administration can reduce the cortical overactivation of the mTOR pathway, suppresses spasms, and has disease‐modifying effects by partially improving cognitive deficits. In post–status epilepticus models of temporal lobe epilepsy, rapamycin may ameliorate the development of epilepsy‐related pathology and reduce the expression of spontaneous seizures, but its effects depend on the timing and duration of administration, and possibly the model used. The observed recurrence of seizures and epilepsy‐related pathology after rapamycin discontinuation suggests the need for continuous administration to maintain the benefit. However, the use of pulse administration protocols may be useful in certain age‐specific epilepsy syndromes, like infantile spasms, whereas repetitive‐pulse rapamycin protocols may suffice to sustain a long‐term benefit in genetic disorders of the mTOR pathway. In summary, mTOR dysregulation has been implicated in several genetic and acquired forms of epileptogenesis. The use of mTOR inhibitors can reverse some of these epileptogenic processes, although their effects depend upon the timing and dose of administration as well as the model used.

Issues related to symptomatic and disease‐modifying treatments affecting cognitive and neuropsychiatric comorbidities of epilepsy

Many symptoms of neurologic or psychiatric illness—such as cognitive impairment, depression, anxiety, attention deficits, and migraine—occur more frequently in people with epilepsy than in the general population. These diverse comorbidities present an underappreciated problem for people with epilepsy and their caregivers because they decrease quality of life, complicate treatment, and increase mortality. In fact, it has been suggested that comorbidities can have a greater effect on quality of life in people with epilepsy than the seizures themselves. There is increasing recognition of the frequency and impact of cognitive and behavioral comorbidities of epilepsy, highlighted in the 2012 Institute of Medicine report on epilepsy. Comorbidities have also been acknowledged, as a National Institutes of Health (NIH) Benchmark area for research in epilepsy. However, relatively little progress has been made in developing new therapies directed specifically at comorbidities. On the other hand, there have been many advances in understanding underlying mechanisms. These advances have made it possible to identify novel targets for therapy and prevention. As part of the International League Against Epilepsy/American Epilepsy Society workshop on preclinical therapy development for epilepsy, our working group considered the current state of understanding related to terminology, models, and strategies for therapy development for the comorbidities of epilepsy. Herein we summarize our findings and suggest ways to accelerate development of new therapies. We also consider important issues to improve research including those related to methodology, nonpharmacologic therapies, biomarkers, and infrastructure.

A model of symptomatic infantile spasms syndrome.

Infantile spasms are characterized by age-specific expression of epileptic spasms and hypsarrhythmia and often result in significant cognitive impairment. Other epilepsies or autism often ensue especially in symptomatic IS (SIS). Cortical or subcortical damage, including white matter, have been implicated in the pathogenesis of SIS. To generate a model of SIS, we recreated this pathology by injecting rats with lipopolysaccharide and doxorubicin intracerebrally at postnatal day (P) 3 and with p-chlorophenylalanine intraperitoneally at P5. Spasms occurred between P4 and 13 and were associated with ictal EEG correlates, interictal EEG abnormalities and neurodevelopmental decline. After P9 other seizures, deficits in learning and memory, and autistic-like behaviors (indifference to other rats, increased grooming) were observed. Adrenocorticotropic hormone (ACTH) did not affect spasms. Vigabatrin transiently suppressed spasms at P5. This new model of SIS will be useful to study the neurobiology and treatment of SIS, including those that are refractory to ACTH.

Sexually dimorphic expression of KCC2 and GABA function

GABA(A) receptors have an age-adapted function in the brain. During early development, they mediate depolarizing effects, which result in activation of calcium-sensitive signaling processes that are important for the differentiation of the brain. In more mature stages of development and in adults, GABA(A) receptors acquire their classical hyperpolarizing signaling. The switch from depolarizing to hyperpolarizing GABA(A)-ergic signaling is triggered through the developmental shift in the balance of chloride cotransporters that either increase (i.e. NKCC1) or decrease (i.e. KCC2) intracellular chloride. The maturation of GABA(A) signaling follows sex-specific patterns, which correlate with the developmental expression profiles of chloride cotransporters. This has first been demonstrated in the substantia nigra, where the switch occurs earlier in females than in males. As a result, there are sensitive periods during development when drugs or conditions that activate GABA(A) receptors mediate different transcriptional effects in males and females. Furthermore, neurons with depolarizing or hyperpolarizing GABA(A)-ergic signaling respond differently to neurotrophic factors like estrogens. Consequently, during sensitive developmental periods, GABA(A) receptors may act as broadcasters of sexually differentiating signals, promoting gender-appropriate brain development. This has particular implications in epilepsy, where both the pathophysiology and treatment of epileptic seizures involve GABA(A) receptor activation. It is important therefore to study separately the effects of these factors not only on the course of epilepsy but also design new treatments that may not necessarily disturb the gender-appropriate brain development.

Sex-specific KCC2 expression and GABAA receptor function in rat substantia nigra

GABA(A) receptor activation by muscimol has sex and age specific effects on substantia nigra reticulata (SNR)-mediated control of generalized seizures. GABA(A) receptor agonists depolarize or hyperpolarize neurons depending upon the level of expression of the neuronal specific potassium chloride contransporter KCC2. We studied KCC2 mRNA expression in the SNR as a function of sex and age and correlated KCC2 expression with the in vivo and in vitro effects of muscimol. Methods included in situ hybridization, gramicidin-perforated patch clamp and fura-2 AM imaging of acute SNR slices. KCC2 mRNA expression increased between postnatal days (PN) 15 and 30 in both sexes, and reached adult levels in males by PN30. Female PN15 and PN30 SNR neurons contained more KCC2 mRNA compared with age-matched males. In male PN14-17 rats, bath application of the GABA(A) receptor agonist muscimol in acute SNR slices depolarized neurons and increased intracellular calcium concentration ([Ca(2+)](i)). Furthermore, acute in vivo administration of muscimol upregulated, whereas blockade of L-type voltage sensitive calcium channels with nifedipine downregulated KCC2 mRNA. In contrast, in female PN14-17 rats, bath application of muscimol hyperpolarized SNR neurons and did not alter [Ca(2+)](i). In vivo muscimol administration acutely downregulated KCC2 mRNA expression whereas nifedipine had no effect. The lower expression of KCC2 mRNA in infantile male SNR neurons may explain why muscimol-induced depolarization and [Ca(2+)](i) increases occur only in males. Consequently, GABA(A) receptor activation selectively upregulates the expression of calcium-regulated genes, such as KCC2, in male SNR, promoting the sexual differentiation of the SNR.

The challenge and promise of anti-epileptic therapy development in animal models

Translation of successful target and compound validation studies into clinically effective therapies is a major challenge, with potential for costly clinical trial failures. This situation holds true for the epilepsies-complex diseases with different causes and symptoms. Although the availability of predictive animal models has led to the development of effective antiseizure therapies that are routinely used in clinical practice, showing that translation can be successful, several important unmet therapeutic needs still exist. Available treatments do not fully control seizures in a third of patients with epilepsy, and produce substantial side-effects. No treatment can prevent the development of epilepsy in at-risk patients or cure patients with epilepsy. And no specific treatment for epilepsy-associated comorbidities exists. To meet these demands, a redesign of translational approaches is urgently needed.

Issues related to development of antiepileptogenic therapies.

Several preclinical proof‐of‐concept studies have provided evidence for positive treatment effects on epileptogenesis. However, none of these hypothetical treatments has advanced to the clinic. The experience in other fields of neurology such as stroke, Alzheimers disease, or amyotrophic lateral sclerosis has indicated several problems in the design of preclinical studies, which likely contribute to failures in translating the positive preclinical data to the clinic. The Working Group on “Issues related to development of antiepileptogenic therapies” of the International League Against Epilepsy (ILAE) and the American Epilepsy Society (AES) has considered the possible problems that arise when moving from proof‐of‐concept antiepileptogenesis (AEG) studies to preclinical AEG trials, and eventually to clinical AEG trials. This article summarizes the discussions and provides recommendations on how to design a preclinical AEG monotherapy trial in adult animals. We specifically address study design, animal and model selection, number of studies needed, issues related to administration of the treatment, outcome measures, statistics, and reporting. In addition, we give recommendations for future actions to advance the preclinical AEG testing.