Browsing by Subject "Epilepsy"

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Systematic review registration

Multiple lines of evidence reveal that activation of the tropomyosin related kinase B (TrkB) receptor is a critical molecular mechanism underlying status epilepticus (SE) induced epilepsy development. However, the cellular consequences of such signaling remain unknown. To this point, localization of SE-induced TrkB activation to CA1 apical dendritic spines provides an anatomic clue pointing to Schaffer collateral-CA1 synaptic plasticity as one potential cellular consequence of TrkB activation. Here, we combine two-photon glutamate uncaging with two photon fluorescence lifetime imaging microscopy (2pFLIM) of fluorescence resonance energy transfer (FRET)-based sensors to specifically investigate the roles of TrkB and its canonical ligand brain derived neurotrophic factor (BDNF) in dendritic spine structural plasticity (sLTP) of CA1 pyramidal neurons in cultured hippocampal slices of rodents. To begin, we demonstrate a critical role for post-synaptic TrkB and post-synaptic BDNF in sLTP. Building on these findings, we develop a novel FRET-based sensor for TrkB activation that can report both BDNF and non-BDNF activation in a specific and reversible manner. Using this sensor, we monitor the spatiotemporal dynamics of TrkB activity during single-spine sLTP. In response to glutamate uncaging, we report a rapid (onset less than 1 minute) and sustained (lasting at least 20 minutes) activation of TrkB in the stimulated spine that depends on N-methyl-D-aspartate receptor (NMDAR)-Ca2+/Calmodulin dependent kinase II (CaMKII) signaling as well as post-synaptically synthesized BDNF. Consistent with these findings, we also demonstrate rapid, glutamate uncaging-evoked, time-locked release of BDNF from single dendritic spines using BDNF fused to superecliptic pHluorin (SEP). Finally, to elucidate the molecular mechanisms by which TrkB activation leads to sLTP, we examined the dependence of Rho GTPase activity - known mediators of sLTP - on BDNF-TrkB signaling. Through the use of previously described FRET-based sensors, we find that the activities of ras-related C3 botulinum toxin substrate 1 (Rac1) and cell division control protein 42 (Cdc42) require BDNF-TrkB signaling. Taken together, these findings reveal a spine-autonomous, autocrine signaling mechanism involving NMDAR-CaMKII dependent BDNF release from stimulated dendritic spines leading to TrkB activation and subsequent activation of the downstream molecules Rac1 and Cdc42 in these same spines that proves critical for sLTP. In conclusion, these results highlight structural plasticity as one cellular consequence of CA1 dendritic spine TrkB activation that may potentially contribute to larger, circuit-level changes underlying SE-induced epilepsy.

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Approximately 1 million Americans live with drug-resistant epilepsy. Surgical resection of the brain areas where seizures originate can be curative. However, successful surgical outcomes require delineation of the epileptogenic zone (EZ), the minimum amount of tissue that needs to be resected to eliminate a patient’s seizures. EZ localization is often accomplished using stereo-EEG where 5-30 wires are implanted into the brain through small holes drilled through the skull to map widespread regions of the epileptic network. However, despite the technical advances in surgical planning and epilepsy monitoring, seizure freedom rates following epilepsy surgery have remained at ~60% for decades. In part, seizure freedom rates have not increased because epilepsy neurologists do not have appropriate software tools to optimize stereo-EEG. In this dissertation, we report on the development and analysis of foundational models and software tools to improve the use of stereo-EEG technology and ultimately increase seizure-freedom rates following epilepsy surgery.We developed an automated image-based head-modeling pipeline to generate patient-specific models for stereo-EEG analysis. We assessed the key dipole source model assumption, which assumes that voltages generated by a population of active neurons can be simplified to a single dipole. We found that the dipole source model is appropriate to reproduce the spatial voltage distribution generated by neurons and for source localization applications. Our findings validate a key model parameter for stereo-EEG head-models, which are foundational to all computational tools developed to optimize stereo-EEG. Using the dipole source model, we systematically assessed the origin of recorded brain electrophysiological signals using computational models. We found that, counter to dogma, action potentials contribute appreciably to brain electrophysiological signals. Our findings reshape the cellular interpretation of brain electrophysiological signals and should impact modeling efforts to reproduce neural recordings. We also developed a recording sensitivity metric, which quantifies the cortical areas that are recordable by a set of stereo-EEG electrodes. We used the recording sensitivity metric to develop two software tools to visualize the recording sensitivity on patient-specific brain geometry and to optimize the trajectories of stereo-EEG electrodes. Using the same number of electrodes, our optimization approach identified trajectories that had greater recording sensitivity than clinician-defined trajectories. Using the same target recording sensitivity, our optimization approach found trajectories that mapped the same amount of cortex with fewer electrodes compared to the clinician-defined trajectories. Thus, our optimization approach can improve the outcomes following epilepsy surgery by increasing the chances that an electrode records from the EZ or reduce the risk of surgery by minimizing the number of necessary implanted electrodes. We finally developed a propagating source reconstruction algorithm using a novel TEmporally Dependent Iterative Expansion approach (TEDIE). TEDIE takes as inputs stereo-EEG recordings and patient-specific anatomical images, produces movies of dynamic (moving) neural activity displayed on patient-specific anatomy, and distills the immense intracranial stereo-EEG dataset into an objective reconstruction of the EZ. We validated TEDIE using seizure recordings from 40 patients from two centers. TEDIE consistently localized the EZ closer to the resected regions for patients who are currently seizure-free. Further, TEDIE identified new EZs in 13 of the 23 patients who are currently not seizure-free. Therefore, TEDIE is expected to improve the accuracy of the evaluation of surgical epilepsy candidates, result in increased numbers of patients advancing to surgery, and increase the proportion of patients who achieve seizure freedom through surgery. Together, our suite of software tools constitute important advances to optimize stereo-EEG implantation and analysis, which should lead to more patients achieving seizure freedom following epilepsy surgery.

Epilepsy is the most common acquired neurological disorder and is characterized by spontaneous, recurrent seizures. Of the various forms of epilepsy, Temporal Lobe Epilepsy (TLE) has received intense clinical and research interest. Current therapeutic options for TLE are anti-convulsive and purely symptomatic. Improved treatments are needed that either (1) prevent epileptogenesis or (2) ameliorate existing disease. Studies suggest that TLE may be induced by a preceding episode of prolonged seizure activity (status epilepticus, or SE). Our lab previously utilized a chemical-genetic strategy to establish proof of concept that transient inhibition of the receptor tyrosine kinase TrkB following SE prevented TLE. Subsequent studies identified the downstream effector of TrkB activation by demonstrating that transient administration of a peptide (“pY816”) uncoupling TrkB from the enzyme PLCγ1 also prevented SE-induced TLE.

TLE is analogous to associative memory formation in that both involve activity-determined plasticity. Associative memories can be rendered labile following re-exposure to the inciting stimulus; during this labile period inhibition of molecular mechanisms necessary for initial learning inhibits reconsolidation and results in memory “erasure”. Given the proposed parallels between epileptogenesis and memory formation as well as the central role of TrkB-PLCγ1 signaling in the development of epilepsy, I sought to test whether the occurrence of a seizure introduces a period of lability and whether inhibition of TrkB-PLCγ1 signaling prevents subsequent reconsolidation. I demonstrate in the kindling model of TLE that the combination of an evoked seizure and chemical-genetic inhibition of TrkB kinase, but not inhibition of TrkB kinase alone, reduces the severity of subsequent evoked seizures. Combination of an evoked seizure and pY816 (but not pY816 alone) produces the same effect. These results suggest that seizures induce a period of lability in a model of TLE and perturbation of TrkB-PLCγ1 signaling inhibits reconsolidation of pathologic plasticity.

In specimens from patients who underwent surgical resection for medically refractory TLE there is a striking increase in expression of the ligand for TrkB, BDNF. In a second study, I demonstrate that this increase, as well as an increase in TrkB-PLCγ1 signaling, is also seen in a TLE model exhibiting spontaneous seizures. Given the result that TrkB-PLCγ1 inhibition prevents reconsolidation, I asked what effect treatment with pY816 has in an animal model after spontaneous seizures emerged. I demonstrate that pY816 induced a remission in seizures that persists after treatment termination.

These studies elucidate a signaling pathway (TrkB-PLCγ1) underlying epilepsy progression and persistence, connect TLE to other disorder of pathologic plasticity like PTSD and neuropathic pain, and open the door to a novel therapeutic approach for treating patients with existing epilepsy.

Human genetics has been experiencing a wave of genetic discoveries thanks to the development of several technologies, such as genome-wide association studies (GWAS), whole-exome sequencing, and whole genome sequencing. Despite the massive genetic discoveries of new variants associated with human diseases, several key challenges emerge following the genetic discovery. GWAS is known to be good at identifying the locus associated with the patient phenotype. However, the actually causal variants responsible for the phenotype are often elusive. Another challenge in human genetics is that even the causal mutations are already known, the underlying biological effect might remain largely ambiguous. Functional evaluation plays a key role to solve these key challenges in human genetics both to identify causal variants responsible for the phenotype, and to further develop the biological insights from the disease-causing mutations.

We adopted various methods to characterize the effects of variants identified in human genetic studies, including patient genetic and phenotypic data, RNA chemistry, molecular biology, virology, and multi-electrode array and primary neuronal culture systems. Chapter 1 is a broader introduction for the motivation and challenges for functional evaluation in human genetic studies, and the background of several genetics discoveries, such as hepatitis C treatment response, in which we performed functional characterization.

Chapter 2 focuses on the characterization of causal variants following the GWAS study for hepatitis C treatment response. We characterized a non-coding SNP (rs4803217) of IL28B (IFNL3) in high linkage disequilibrium (LD) with the discovery SNP identified in the GWAS. In this chapter, we used inter-disciplinary approaches to characterize rs4803217 on RNA structure, disease association, and protein translation.

Chapter 3 describes another avenue of functional characterization following GWAS focusing on the novel transcripts and proteins identified near the IL28B (IFNL3) locus. It has been recently speculated that this novel protein, which was named IFNL4, may affect the HCV treatment response and clearance. In this chapter, we used molecular biology, virology, and patient genetic and phenotypic data to further characterize and understand the biology of IFNL4. The efforts in chapter 2 and 3 provided new insights to the candidate causal variant(s) responsible for the GWAS for HCV treatment response, however, more evidence is still required to make claims for the exact causal roles of these variants for the GWAS association.

Chapter 4 aims to characterize a mutation already known to cause a disease (seizure) in a mouse model. We demonstrate the potential use of multi-electrode array (MEA) system for the functional characterization and drug testing on mutations found in neurological diseases, such as seizure. Functional characterization in neurological diseases is relatively challenging and available systematic tools are relatively limited. This chapter shows an exploratory research and example to establish a system for the broader use for functional characterization and translational opportunities for mutations found in neurological diseases.

Overall, this dissertation spans a range of challenges of functional evaluations in human genetics. It is expected that the functional characterization to understand human mutations will become more central in human genetics, because there are still many biological questions remaining to be answered after the explosion of human genetic discoveries. The recent advance in several technologies, including genome editing and pluripotent stem cells, is also expected to make new tools available for functional studies in human diseases.

Background

Epilepsy is a chronic neurological disorder characterized by recurrent seizure activity caused by abnormal electrical activity in the brain. Over 80% of all cases globally occur in Low- and Middle-Income countries. A high treatment gap exists in LMICs, including Uganda, with 80% of people with epilepsy never receiving treatment. Studies have shown that even with existing medical services, a lack of skilled workforce, medication stock-outs, and long distances to health facilities contribute to the high treatment gap. This study describes the capacity, distribution of health facilities, and referral patterns between facilities that care for epilepsy patients in Uganda.

Methods

We conducted a cross-sectional survey adapted from the WHO Tool for Situational Analysis to Assess Emergency and Essential Surgical Care. It was modified to include WHO Mental Health Gap Action Program (mhGAP) resources for epilepsy and the Tool for Situational Analysis to Assess Epilepsy Care. Data collection occurred between July and August 2022. Our sample included all regional and general hospitals and a sample of randomly selected Health Centers in Southwestern Uganda. We used probability proportional to size sampling to determine which Health Centers to include in our sample. We had only public health facilities in our sample. Data were collected through in-person interviews conducted by trained research assistants. A three-tiered categorical score (full, intermediate, and low capacity) was used to describe epilepsy capacity. For scoring, hospitals were stratified into three groups/facility levels: tertiary care (regional referral hospitals), secondary care (district hospitals/health center IVs), and primary care (health center IIIs), as we hypothesized that available resources would differ between the groups. We did geospatial mapping to show the distribution of facilities.

Ethical approval was obtained from the Makerere School of Public Health Research Ethics Committee (Protocol 1104), the Uganda National Council of Science and Technology (Protocol HS2344ES), and Duke University’s Institutional Review Board (Protocol 00110747).

Results63 facilities were surveyed, with 100% completion in all facilities. 63 (100%) facilities provide care for epilepsy patients. None of the facilities surveyed had full capacity to treat epilepsy patients. Most of our sampled facilities had a low capacity to treat epilepsy: 100% of tertiary care facilities, 77.3% of secondary care facilities, and 83.8% of primary care facilities. Overall capacity was weakest in medication, equipment, and human resources and highest in infrastructure and guidelines. Conclusion While epilepsy services are present in Uganda’s southwestern region, a lack of vital medicines, staff shortages, and technology can limit service delivery. Task shifting and sharing have been widely implemented to address workforce shortages. The findings of this study can help inform policy to improve service delivery for epilepsy patients.

Neurodevelopmental disorders develop over time and are characterized by a wide variety of mental, behavioral, and physical phenotypes. The categorization of neurodevelopmental disorders encompasses a broad range of conditions including intellectual disability, autism spectrum disorder, attention deficit hyperactivity disorder, cerebral palsy, schizophrenia, bipolar disorder, and epilepsy, among others. Diagnostic classifications of neurodevelopmental disorders are complicated by comorbidities among these neurodevelopmental disorders, unidentified causal genes, and growing evidence of shared genetic risk factors.

We sought to identify the genetic underpinnings of a variety of neurodevelopmental disorders, with a particular emphasis on the epilepsies, by employing next–generation sequencing to thoroughly interrogate genetic variation in the human genome/exome. First, we investigated four families presenting with a seemingly identical and previously undescribed neurodevelopmental disorder characterized by congenital microcephaly, intellectual disability, progressive cerebral atrophy, and intractable seizures. These families all exhibited an apparent autosomal recessive pattern of inheritance. Second, we investigated a heterogeneous cohort of ∼60 undiagnosed patients, the majority of whom suffered from severe neurodevelopmental disorders with a suspected genetic etiology. Third, we investigated 264 patients with epileptic encephalopathies — severe childhood epilepsy disorders — looking specifically at infantile spasms and Lennox–Gastaut syndrome. Finally, we investigated ∼40 large multiplex epilepsy families with complex phenotypic constellations and unclear modes of inheritance. The studied neurodevelopmental disorders exhibited a range of genetic complexity, from clear Mendelian disorders to common complex disorders, resulting in varying degrees of success in the identification of clearly causal genetic variants.

In the first project, we successfully identified the disease–causing gene. We show that recessive mutations in ASNS (encoding asparagine synthetase) are responsible for this previously undescribed neurodevelopmental disorder. We also characterized the causal mutations in vitro and studied Asns–deficient mice that mimicked aspects of the patient phenotype. This work describes ASNS deficiency as a novel neurodevelopmental disorder, identifies three distinct causal mutations in the ASNS gene, and indicates that asparagine synthesis is essential for the proper development and function of the brain.

In the second project, we exome sequenced 62 undiagnosed patients and their unaffected biological parents (trios). By analyzing all identified variants that were annotated as putatively functional and observed as a novel genotype in the probands (not observed in the unaffected parents or controls), we obtained a genetic diagnosis for 32% (20/62) of these patients. Additionally, we identify strong candidate variants in 31% (13/42) of the undiagnosed cases. We also present additional analysis methods for moving beyond traditional screens, e.g., considering only securely implicated genes, or subjecting qualifying variants from any gene to two unique analysis approaches. This work adds to the growing evidence for the utility of diagnostic exome sequencing, increases patient sizes for rare neurodevelopmental disorders (enabling more detailed analyses of the phenotypic spectrum), and proposes novel analysis approaches which will likely become beneficial as the number of sequenced undiagnosed patients grows.

In the third project, we again employ a trio–based exome sequencing design to investigate the role of de novo mutations in two classical forms of epileptic encephalopathy. We find a significant excess of de novo mutations in the ∼4,000 genes that are the most intolerant to functional genetic variation in the human population (P = 2.9 x 10–3, likelihood analysis). We provide clear statistical evidence for two novel genes associated with epileptic encephalopathy — GABRB3 and ALG13. Together with the 15 well–established epileptic encephalopathy genes, we statistically confirm the association of an additional ten putative epileptic encephalopathy genes. We show that only ∼12% of epileptic encephalopathy patients in our cohort are explained by de novo mutations in one of these 24 genes, highlighting the extreme locus heterogeneity of the epileptic encephalopathies.

Finally, we investigated multiplex epilepsy families to uncover novel epilepsy susceptibility factors. Candidate variants emerging from sequencing within discovery families were further assessed by cosegregation testing, variant association testing in a case–control cohort, and gene–based resequencing in a cohort of additional multiplex epilepsy families. Despite employing multiple approaches, we did not identify any clear genetic associations with epilepsy. This work has, however, identified a set of candidates that may include real risk factors for epilepsy; the most promising of these is the MYCBP2 gene. This work emphasizes the extremely high locus and allelic heterogeneity of the epilepsies and demonstrates that very large sample sizes are needed to uncover novel genetic risk factors.

Collectively, this body of work has securely implicated three novel neurodevelopmental disease genes that inform the underlying pathology of these disorders. Furthermore, in the final three studies, this work has highlighted additional candidate variants and genes that may ultimately be validated as disease–causing as sample sizes increase.

Understanding the mechanisms of limbic epileptogenesis in cellular and molecular terms may provide novel therapeutic targets for its prevention. The neurotrophin receptor tropomyosin-related kinase B (TrkB) is thought to be critical for limbic epileptogenesis. Enhanced activation of TrkB, revealed by immunodetection of enhanced phosphorylated TrkB (pTrkB), a surrogate measure of its activation, has been identified within the hippocampus in multiple animal models. Knowledge of the cellular locale of activated TrkB is necessary to elucidate its functional consequences. Using an antibody selective to pTrkB in conjunction with confocal microscopy and cellular markers, we determined the cellular and subcellular locale of enhanced pTrkB induced by status epilepticus (SE) evoked by infusion of kainic acid into the amygdala of adult mice. SE induced enhanced pTrkB immunoreactivity in two distinct populations of principal neurons within the hippocampus--the dentate granule cells and CA1 pyramidal cells. Enhanced immunoreactivity within granule cells was found within mossy fiber axons and giant synaptic boutons. By contrast, enhanced immunoreactivity was found within apical dendritic shafts and spines of CA1 pyramidal cells. A common feature of this enhanced pTrkB at these cellular locales is its localization to excitatory synapses between excitatory neurons, presynaptically in the granule cells and postsynaptically in CA1 pyramidal cells. Long-term potentiation (LTP) is one cellular consequence of TrkB activation at these excitatory synapses that may promote epileptogenesis.

The importance of TrkB in diverse neuronal processes, as well as its involvement in various disorders of the nervous system, underscores the importance of understanding how it is activated. The canonical neurotrophin ligand which activates TrkB is brain derived neurotrophic factor (BDNF). Zinc, however, has also been demonstrated to activate this receptor through a mechanism whereby it does not directly interact with it, known as transactivation. Presynaptic vesicles of mossy fiber boutons of stratum lucidum are particularly enriched in zinc, where it is co-released with glutamate in an activity dependent fashion, and incorporated into these vesicles by the zinc transporter, ZnT3. Given the presence of large quantities of zinc within stratum lucidum, we hypothesized that this metal may contribute to TrkB transactivation at this locale. To this end, we examined the contributions of both BDNF and synaptic vesicular zinc to TrkB activation in stratum lucidum of mouse hippocampus under physiological conditions. Utilization of mice which are genetic knockouts for BDNF and/or ZnT3 allowed us to examine TrkB activation in the absence of one or both of these ligands. This was done using an antibody for pTrkB in conjunction with confocal microscopy, assaying immunoreactivity at the cellular and synaptic locales within stratum lucidum where pTrkB was previously found to be enriched. Our results suggest that BDNF contributes to TrkB activation within stratum lucidum. Interestingly, ZnT3 mice displayed an increase in BDNF protein and TrkB activation, demonstrating that synaptic zinc regulates BDNF and TrkB signaling at this locale.

Advances in next-generation sequencing (NGS) and the ability to sequence the entire genome of many individuals in a cost-effective manner has led to the revelation of the genetic etiologies of a number of neurological disorders. Parallel advancements in predictive software, for example, have allowed for the annotation of potentially pathogenic variants. However, the development of appropriate systems to functionally interpret variants and identify pathogenic mechanisms has lagged behind. Understanding pathogenic mechanisms is crucial to the development of targeted therapeutics. Therefore, the main challenge to translating genetic findings into targeted therapeutics is functional modeling.

Increased understanding of the genetic architecture of epilepsy and the hyperexcitability that results from many epilepsy-causing variants makes the disease particularly well-suited for the development of model systems for functional interpretation of genetic variation. To capture the effects of genetic variation in neurological diseases, like epilepsy, complex cellular systems are crucial. In my thesis I describe a paradigm that addresses the need for complex cellular systems. The paradigm utilizes cultured neural networks (CNNs) that can be collected either from mouse models or derived from human induced stem cell models (hIPSCs). CNNs retain much of the electrical and network forming capabilities of the intact brain. CNNs plated onto multi-well microelectrode arrays (CNN-MEAs), which capture extracellular activity of electrically active cells, therefore offer a particularly appealing cellular system for the investigation of genetic variants that cause neurological disorders.

In chapter one I review the history of genetics and epilepsy. I discuss how studies of genetic variants that cause epilepsy give insights into the mechanisms of a wide scope of neurological disorders. I suggest that epilepsy is therefore a good place to start in the development of cellular models of disease and targeted therapeutic options. I next introduce the MEA as a platform capable of capturing important electrophysiological data from CNNs, creating the foundations for chapters two and three.

Chapter two describes one application of the CNN-MEA paradigm in which we inhibited microRNA (miRNA) expression in vitro and evaluated the resulting activity profiles. MiRNAs are increasingly linked to epileptogenesis. We show that small differences in miRNA expression can have large effects on network activity. Chapter two offers a proof-of-principle of the utility of the CNN-MEA paradigm in capturing pathogenic hyperexcitability.

Chapter three discusses a second application in which mutations in the ATP1A3 gene were evaluated. Mutations in ATP1A3 cause at least four distinct disorders and it is not yet fully understood how mutations mediate pathophysiologic consequences. We first investigate ATP1A3 mutations in COS7 cells and observe no clear differences. We next evaluate the effect of two mutations that cause the most severe ATP1A3-associated disorder, Alternating Hemiplegia of Childhood (AHC), on network dynamics. We show that mutant cultures demonstrate hypersynchronous activity and distorted bursting properties when compared to wild-type. Using strategic pharmacological manipulation, we illustrate the role of GABA neurotransmission on aberrant network dynamics and further show the partial rescue of activity phenotypes using adenosine triphosphate (ATP) and an anti-epileptic drug. Chapter three illustrates the shortcomings of heterologous cell modeling and provides additional support for the use of CNN-MEAs to study genetic variation.

The CNN-MEA paradigm provides a promising method to evaluate the effect of mutations that cause neurological disorders. Furthermore, with the use of multi-well MEAs, this paradigm provides a scalable option to evaluate multiple parameters simultaneously. Understanding the functional impact of genetic variation using the CNN-MEA paradigm is a crucial step to developing targeted therapeutics.