Optogenetics for controlling seizure circuits for translational approaches.

The advent of optogenetic tools has had a profound impact on modern neuroscience research, revolutionizing our understanding of the brain. These tools offer a remarkable ability to precisely manipulate specific groups of neurons with an unprecedented level of temporal precision, on the order of milliseconds. This breakthrough has significantly advanced our knowledge of various physiological and pathophysiological processes in the brain. Within the realm of epilepsy research, optogenetic tools have played a crucial role in investigating the contributions of different neuronal populations to the generation of seizures and hyperexcitability. By selectively activating or inhibiting specific neurons using optogenetics, researchers have been able to elucidate the underlying mechanisms and identify key players involved in epileptic activity. Moreover, optogenetic techniques have also been explored as innovative therapeutic strategies for treating epilepsy. These strategies aim to halt seizure progression and alleviate symptoms by utilizing the precise control offered by optogenetics. The application of optogenetic tools has provided valuable insights into the intricate workings of the brain during epileptic episodes. For instance, researchers have discovered how distinct interneuron populations contribute to the initiation of seizures (ictogenesis). They have also revealed how remote circuits in regions such as the cerebellum, septum, or raphe nuclei can interact with hyperexcitable networks in the hippocampus. Additionally, studies have demonstrated the potential of closed-loop systems, where optogenetics is combined with real-time monitoring, to enable precise, on-demand control of seizure activity. Despite the immense promise demonstrated by optogenetic approaches, it is important to acknowledge that many of these techniques are still in the early stages of development and have yet to reach potential clinical applications. The transition from experimental research to practical clinical use poses numerous challenges. In this review, we aim to introduce optogenetic tools, provide a comprehensive survey of their application in epilepsy research, and critically discuss their current potential and limitations in achieving successful clinical implementation for the treatment of human epilepsy. By addressing these crucial aspects, we hope to foster a deeper understanding of the current state and future prospects of optogenetics in epilepsy treatment.

Combinatorial gene therapy for epilepsy: Gene sequence positioning and AAV serotype influence expression and inhibitory effect on seizures.

Gene therapy with AAV vectors carrying genes for neuropeptide Y and its receptor Y2 has been shown to inhibit seizures in multiple animal models of epilepsy. It is however unknown how the AAV serotype or the sequence order of these two transgenes in the expression cassette affects the actual parenchymal gene expression levels and the seizure-suppressant efficacy. To address these questions, we compared three viral vector serotypes (AAV1, AAV2 and AAV8) and two transgene sequence orders (NPY-IRES-Y2 and Y2-IRES-NPY) in a rat model of acutely induced seizures. Wistar male rats were injected bilaterally with viral vectors and 3 weeks later acute seizures were induced by a subcutaneous injection of kainate. The latency until 1st motor seizure, time spent in motor seizure and latency to status epilepticus were measured to evaluate the seizure-suppressing efficacy of these vectors compared to an empty cassette control vector. Based on the results, the effect of the AAV1-NPY-IRES-Y2 vector was further investigated by in vitro electrophysiology, and its ability to achieve transgene overexpression in resected human hippocampal tissue was evaluated. The AAV1-NPY-IRES-Y2 proved to be better to any other serotype or gene sequence considering both transgene expression and ability to suppress induced seizures in rats. The vector also demonstrated transgene-induced decrease of glutamate release from excitatory neuron terminals and significantly increased both NPY and Y2 expression in resected human hippocampal tissue from patients with drug-resistant temporal lobe epilepsy. These results validate the feasibility of NPY/Y2 receptor gene therapy as a therapeutic opportunity in focal epilepsies.

Shaping the future of European epilepsy research: Final meeting report from EPICLUSTER.

Collaboration is essential to the conduct of basic, applied and clinical research and its translation into the technologies and treatments urgently needed to improve the lives of people living with brain diseases and the health professionals who care for them. EPICLUSTER was formed in 2019 by the European Brain Research Area (EBRA) to support the coordination of epilepsy research in Europe. A key objective was to provide a platform to discuss shared research priorities by bringing together scientists and clinicians with multiple stakeholders including patient organisations and industry and the networks and infrastructures that provide healthcare and support research. Additional objectives were to facilitate access and sharing of data and biosamples, working together to ensure epilepsy is a priority for research funding, and embedding a culture of public and patient involvement (PPI) among epilepsy researchers. In this meeting report, we summarise the shared research priorities discussed by the leadership of EPICLUSTER at the recent final meeting. We also briefly review the discussion on patient and industry priorities, guidance on starting PPI for epilepsy researchers, and the sustainability of funding and infrastructures needed to ensure a comprehensive stakeholder-embedded community for epilepsy research.

Transplantation of Human Stem Cell-Derived GABAergic Neurons into the Early Postnatal Mouse Hippocampus to Mitigate Neurodevelopmental Disorders.

A reduced number or dysfunction of inhibitory interneurons is a common contributor to neurodevelopmental disorders. Therefore, cell therapy using interneurons to replace or mitigate the effects of altered neuronal circuits is an attractive therapeutic avenue. To this end, more knowledge is needed about how human stem cell-derived GABAergic interneuron-like cells (hdINs) mature, integrate, and function over time in the host circuitry. Of particular importance in neurodevelopmental disorders is a better understanding of whether these processes in transplanted cells are affected by an evolving and maturing host brain. The present protocol describes a fast and highly efficient generation of hdINs from human embryonic stem cells based on the transgenic expression of the transcription factors Ascl1 and Dlx2. These neuronal precursors are transplanted unilaterally, after 7 days in vitro, to the hippocampus of neonatal 2-day-old mice. The transplanted neurons disperse in the ipsi- and contralateral hippocampus of a mouse model of cortical dysplasia-focal epilepsy syndrome and survive for up to 9 months after transplantation. This approach allows for investigating the cellular identity, integration, functionality, and therapeutic potential of transplanted interneurons over an extended time in developing healthy and diseased brains.

GDNF Increases Inhibitory Synaptic Drive on Principal Neurons in the Hippocampus via Activation of the Ret Pathway.

Glial cell line-derived neurotrophic factor (GDNF) has been shown to counteract seizures when overexpressed or delivered into the brain in various animal models of epileptogenesis or chronic epilepsy. The mechanisms underlying this effect have not been investigated. We here demonstrate for the first time that GDNF enhances GABAergic inhibitory drive onto mouse pyramidal neurons by modulating postsynaptic GABAA receptors, particularly in perisomatic inhibitory synapses, by GFRα1 mediated activation of the Ret receptor pathway. Other GDNF receptors, such as NCAM or Syndecan3, are not contributing to this effect. We observed similar alterations by GDNF in human hippocampal slices resected from epilepsy patients. These data indicate that GDNF may exert its seizure-suppressant action by enhancing GABAergic inhibitory transmission in the hippocampal network, thus counteracting the increased excitability of the epileptic brain. This new knowledge can contribute to the development of novel, more precise treatment strategies based on a GDNF gene therapy approach.

Transcription factor-based direct conversion of human fibroblasts to functional astrocytes.

Astrocytes are emerging key players in neurological disorders. However, their role in disease etiology is poorly understood owing to inaccessibility of primary human astrocytes. Pluripotent stem cell-derived cells fail to mimic age and due to their clonal origin do not mimic genetic heterogeneity of patients. In contrast, direct conversion constitutes an attractive approach to generate human astrocytes that capture age and genetic diversity. We describe efficient direct conversion of human fibroblasts to functional induced astrocytes (iAs). Expression of the minimal combination Sox9 and Nfib generates iAs with molecular, phenotypic, and functional properties resembling primary human astrocytes. iAs could be obtained by conversion of fibroblasts covering the entire human lifespan. Importantly, iAs supported function of induced neurons obtained through direct conversion from the same fibroblast population. Fibroblast-derived iAs will become a useful tool to elucidate the biology of astrocytes and complement current in vitro models for studies of late-onset neurological disorders.

Lipid mediator n-3 docosapentaenoic acid-derived protectin D1 enhances synaptic inhibition of hippocampal principal neurons by interaction with a G-protein-coupled receptor.

Epilepsy is a severe neurological disease manifested by spontaneous recurrent seizures due to abnormal hyper-synchronization of neuronal activity. Epilepsy affects about 1% of the population and up to 40% of patients experience seizures that are resistant to currently available drugs, thus highlighting an urgent need for novel treatments. In this regard, anti-inflammatory drugs emerged as potential therapeutic candidates. In particular, specific molecules apt to resolve the neuroinflammatory response occurring in acquired epilepsies have been proven to counteract seizures in experimental models, and humans. One candidate investigational molecule has been recently identified as the lipid mediator n-3 docosapentaenoic acid-derived protectin D1 (PD1n-3DPA ) which significantly reduced seizures, cell loss, and cognitive deficit in a mouse model of acquired epilepsy. However, the mechanisms that mediate the PD1n-3DPA effect remain elusive. We here addressed whether PD1n-3DPA has direct effects on neuronal activity independent of its anti-inflammatory action. We incubated, therefore, hippocampal slices with PD1n-3DPA and investigated its effect on excitatory and inhibitory synaptic inputs to the CA1 pyramidal neurons. We demonstrate that inhibitory drive onto the perisomatic region of the pyramidal neurons is increased by PD1n-3DPA , and this effect is mediated by pertussis toxin-sensitive G-protein coupled receptors. Our data indicate that PD1n-3DPA acts directly on inhibitory transmission, most likely at the presynaptic site of inhibitory synapses as also supported by Xenopus oocytes and immunohistochemical experiments. Thus, in addition to its anti-inflammatory effects, PD1n-3DPA anti-seizure and neuroprotective effects may be mediated by its direct action on neuronal excitability by modulating their synaptic inputs.

Myo-Inositol Limits Kainic Acid-Induced Epileptogenesis in Rats.

Epilepsy is a severe neurological disease characterized by spontaneous recurrent seizures (SRS). A complex pathophysiological process referred to as epileptogenesis transforms a normal brain into an epileptic one. Prevention of epileptogenesis is a subject of intensive research. Currently, there are no clinically approved drugs that can act as preventive medication. Our previous studies have revealed highly promising antiepileptogenic properties of a compound-myo-inositol (MI) and the present research broadens previous results and demonstrates the long-term disease-modifying effect of this drug, as well as the amelioration of cognitive comorbidities. For the first time, we show that long-term treatment with MI: (i) decreases the frequency and duration of electrographic SRS in the hippocampus; (ii) has an ameliorating effect on spatial learning and memory deficit associated with epileptogenesis, and (iii) attenuates cell loss in the hippocampus. MI treatment also alters the expression of the glial fibrillary acidic protein, LRRC8A subunit of volume-regulated anion channels, and protein tyrosine phosphatase receptor type R, all expected to counteract the epileptogenesis. All these effects are still present even 4 weeks after MI treatment ceased. This suggests that MI may exert multiple actions on various epileptogenesis-associated changes in the brain and, therefore, could be considered as a candidate target for prevention of epileptogenesis.

Human Stem Cell-Derived GABAergic Interneurons Establish Efferent Synapses onto Host Neurons in Rat Epileptic Hippocampus and Inhibit Spontaneous Recurrent Seizures.

Epilepsy is a complex disorder affecting the central nervous system and is characterised by spontaneously recurring seizures (SRSs). Epileptic patients undergo symptomatic pharmacological treatments, however, in 30% of cases, they are ineffective, mostly in patients with temporal lobe epilepsy. Therefore, there is a need for developing novel treatment strategies. Transplantation of cells releasing γ-aminobutyric acid (GABA) could be used to counteract the imbalance between excitation and inhibition within epileptic neuronal networks. We generated GABAergic interneuron precursors from human embryonic stem cells (hESCs) and grafted them in the hippocampi of rats developing chronic SRSs after kainic acid-induced status epilepticus. Using whole-cell patch-clamp recordings, we characterised the maturation of the grafted cells into functional GABAergic interneurons in the host brain, and we confirmed the presence of functional inhibitory synaptic connections from grafted cells onto the host neurons. Moreover, optogenetic stimulation of grafted hESC-derived interneurons reduced the rate of epileptiform discharges in vitro. We also observed decreased SRS frequency and total time spent in SRSs in these animals in vivo as compared to non-grafted controls. These data represent a proof-of-concept that hESC-derived GABAergic neurons can exert a therapeutic effect on epileptic animals presumably through establishing inhibitory synapses with host neurons.

Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks.

Gamma-aminobutyric acid (GABA)-releasing interneurons modulate neuronal network activity in the brain by inhibiting other neurons. The alteration or absence of these cells disrupts the balance between excitatory and inhibitory processes, leading to neurological disorders such as epilepsy. In this regard, cell-based therapy may be an alternative therapeutic approach. We generated light-sensitive human embryonic stem cell (hESC)-derived GABAergic interneurons (hdIN) and tested their functionality. After 35 days in vitro (DIV), hdINs showed electrophysiological properties and spontaneous synaptic currents comparable to mature neurons. In co-culture with human cortical neurons and after transplantation (AT) into human brain tissue resected from patients with drug-resistant epilepsy, light-activated channelrhodopsin-2 (ChR2) expressing hdINs induced postsynaptic currents in human neurons, strongly suggesting functional efferent synapse formation. These results provide a proof-of-concept that hESC-derived neurons can integrate and modulate the activity of a human host neuronal network. Therefore, this study supports the possibility of precise temporal control of network excitability by transplantation of light-sensitive interneurons.

Gene Therapy Vector Encoding Neuropeptide Y and Its Receptor Y2 for Future Treatment of Epilepsy: Preclinical Data in Rats.

Gene therapy to treat pharmacoresistant temporal lobe epilepsy in humans is now being developed using an AAV vector (CG01) that encodes the combination of neuropeptide Y and its antiepileptic receptor Y2. With this in mind, the present study aimed to provide important preclinical data on the effects of CG01 on the duration of transgene expression, cellular tropism, and potential side effects on body weight and cognitive function. The CG01 vector was administered unilaterally into the dorsal and ventral hippocampus of adult male rats and expression of both transgenes was found to remain elevated without a sign of decline at 6 months post-injection. CG01 appeared to mediate expression selectively in hippocampal neurons, without expression in astrocytes or oligodendrocytes. No effects were seen on body weight as well as on short- or long-term memory as revealed by testing in the Y-maze or Morris water maze tests. Thus these data show that unilateral CG01 vector treatment as future gene therapy in pharmacoresistant temporal lobe epilepsy patients should result in stable and long-term expression predominantly in neurons and be well tolerated without side effects on body weight and cognitive function.

Leaky Optoelectrical Fiber for Optogenetic Stimulation and Electrochemical Detection of Dopamine Exocytosis from Human Dopaminergic Neurons.

In Parkinson's disease, the degeneration of dopaminergic neurons in substantia nigra leads to a decrease in the physiological levels of dopamine in striatum. The existing dopaminergic therapies effectively alleviate the symptoms, albeit they do not revert the disease progression and result in significant adverse effects. Transplanting dopaminergic neurons derived from stem cells could restore dopamine levels without additional motor complications. However, the transplanted cells disperse in vivo and it is not possible to stimulate them on demand to modulate dopamine release to prevent dyskinesia. In order to address these issues, this paper presents a multifunctional leaky optoelectrical fiber for potential neuromodulation and as a cell substrate for application in combined optogenetic stem cell therapy. Pyrolytic carbon coated optical fibers are laser ablated to pattern micro-optical windows to permit light leakage over a large area. The pyrolytic carbon acts as an excellent electrode for the electrochemical detection of dopamine. Human neural stem cells are genetically modified to express the light sensitive opsin channelrhodopsin-2 and are differentiated into dopaminergic neurons on the leaky optoelectrical fiber. Finally, light leaking from the micro-optical windows is used to stimulate the dopaminergic neurons resulting in the release of dopamine that is detected in real-time using chronoamperometry.

Inhibition of epileptiform activity by neuropeptide Y in brain tissue from drug-resistant temporal lobe epilepsy patients.

In epilepsy patients, drug-resistant seizures often originate in one of the temporal lobes. In selected cases, when certain requirements are met, this area is surgically resected for therapeutic reasons. We kept the resected tissue slices alive in vitro for 48 h to create a platform for testing a novel treatment strategy based on neuropeptide Y (NPY) against drug-resistant epilepsy. We demonstrate that NPY exerts a significant inhibitory effect on epileptiform activity, recorded with whole-cell patch-clamp, in human hippocampal dentate gyrus. Application of NPY reduced overall number of paroxysmal depolarising shifts and action potentials. This effect was mediated by Y2 receptors, since application of selective Y2-receptor antagonist blocked the effect of NPY. This proof-of-concept finding is an important translational milestone for validating NPY-based gene therapy for targeting focal drug-resistant epilepsies, and increasing the prospects for positive outcome in potential clinical trials.

Concentration- and time-dependent effects of myo-inositol on evoked epileptic afterdischarge in the hippocampus in vivo.

Epilepsy is one of the most widespread neurological diseases characterized by spontaneous recurrent seizures. There is no cure for epilepsy, and available pharmacological treatments with anti-seizure drugs are only symptomatic. Moreover, about third of epilepsy patients are resistant to the anti-seizure drugs. Thus, it is essential to discover new anti-epilepsy drugs. Recently, myo-inositol has been identified as a promising antiepileptic compound. In the present study, using electrophysiological method, we examined for the first time, the effect of myo-inositol on the generation of epileptic afterdischarges in the hippocampus evoked by a local electrical stimulation. This was achieved by implanting two electrodes with a cannula into the same dorsal hippocampus, which allowed for simultaneous local injection of myo-inositol or saline and afterdischarges induction and recording from the same hippocampus. We found that myo-inositol has time- and concentration-dependent effects on the evoked afterdischarges. Specifically, 5 minutes after 1 M myo-inositol infusion, the afterdischarges duration was significantly decreased as compared to preinjection durations in the same animals and also as compared to preinjection level durations in saline injected or contralateral hippocampus myo-inositol infused animals. Further, 0.055 M myo-inositol significantly decreased afterdischarges duration at 5 minutes as compared to 40 minutes post-injection. At both concentrations, the afterdischarges duration recovered to the pre-injection value at 40 minutes after the myo-inositol injection. The present data, taken together with our previous results, strongly suggest that myo-inositol has significant local seizure-suppressant effect.

Disease Modification by Combinatorial Single Vector Gene Therapy: A Preclinical Translational Study in Epilepsy.

Gene therapy has been suggested as a plausible novel approach to achieve seizure control in patients with focal epilepsy that do not adequately respond to pharmacological treatment. We investigated the seizure-suppressant potential of combinatorial neuropeptide Y and Y2 receptor single vector gene therapy based on adeno-associated virus serotype 1 (AAV1) in rats. First, a dose-response study in the systemic kainate-induced acute seizure model was performed, whereby the 1012 genomic particles (gp)/mL titer of the vector was selected as an optimal concentration. Second, an efficacy study was performed in the intrahippocampal kainate chronic model of spontaneous recurrent seizures (SRSs), designed to reflect a likely clinical scenario, with magnetic resonance image (MRI)-guided focal unilateral administration of the vector in the hippocampus during the chronic stage of the disease. The efficacy study demonstrated a favorable outcome of the gene therapy, with a 31% responder rate (more than 50% reduction in SRS frequency) and 13% seizure-freedom rate, whereas no such effects were observed in the control animals. The inter-SRS and SRS cluster intervals were also significantly prolonged in the treated group compared to controls. In addition, the SRS duration was significantly reduced in the treated group but not in the controls. This study establishes the SRS-suppressant ability of the single vector combinatorial neuropeptide Y/Y2 receptor gene therapy in a clinically relevant chronic model of epilepsy.

Advancing research toward faster diagnosis, better treatment, and end of stigma in epilepsy.

Seven large European Union (EU)-funded epilepsy-related research projects joined forces in May 2018 in Brussels, Belgium, in a unique community building event-the epiXchange conference. During this conference, 170 investigators from the projects DESIRE, EpimiRNA, EPISTOP, EpiTarget, EpiXchange, and EpiPGX as well as the European Reference Network EpiCARE, met up with key stakeholders including representatives of the European Commission, patient organizations, commercial partners, and other European and International groups. The epiXchange conference focused on sharing and reviewing the advances made by each project in the previous 5 years; describing the infrastructures generated; and discussing the innovations and commercial applications across five thematic areas: biomarkers, genetics, therapeutics, comorbidities, and biobanks and resources. These projects have, in fact, generated major breakthroughs including the discovery of biofluid-based molecules for diagnosis, elucidating new genetic causes of epilepsy, creating advanced new models of epilepsy, and the pre-clinical development of novel compounds. Workshop-style discussions focused on how to overcome scientific and clinical challenges for accelerating translation of research outcomes and how to increase synergies between the projects and stakeholders at a European level. The resulting advances would lead toward a measurable impact of epilepsy research through better diagnostics, treatments, and quality-of-life for persons with epilepsy. In addition, epiXchange provided a unique forum for examining how the different projects could build momentum for future novel groundbreaking epilepsy research in Europe and beyond. This report includes the main recommendations that resulted from these discussions.

Long-Term Effects of Myoinositol on Behavioural Seizures and Biochemical Changes Evoked by Kainic Acid Induced Epileptogenesis.

Epilepsy is one of the most devastating neurological diseases and despite significant efforts there is no cure available. Occurrence of spontaneous seizures in epilepsy is preceded by numerous functional and structural pathophysiological reorganizations in the brain-a process called epileptogenesis. Treatment strategies targeting this process may be efficient for preventing spontaneous recurrent seizures (SRS) in epilepsy, or for modification of disease progression. We have previously shown that (i) myoinositol (MI) pretreatment significantly decreases severity of acute seizures (status epilepticus: SE) induced by kainic acid (KA) in experimental animals and (ii) that daily post-SE administration of MI for 4 weeks prevents certain biochemical changes triggered by SE. However it was not established whether such MI treatment also exerts long-term effects on the frequency of SRS. In the present study we have shown that, in KA-induced post-SE epilepsy model in rats, MI treatment for 28 days reduces frequency and duration of behavioural SRS not only during the treatment, but also after its termination for the following 4 weeks. Moreover, MI has significant effects on molecular changes in the hippocampus, including mi-RNA expression spectrum, as well as mRNA levels of sodium-MI transporter and LRRC8A subunit of the volume regulated anionic channel. Taken together, these data suggest that molecular changes induced by MI treatment may counteract epileptogenesis. Thus, here we provide data indicating antiepileptogenic properties of MI, which further supports the idea of developing new antiepileptogenic and disease modifying drug that targets MI system.

Long-Term, Targeted Delivery of GDNF from Encapsulated Cells Is Neuroprotective and Reduces Seizures in the Pilocarpine Model of Epilepsy.

Neurotrophic factors are candidates for treating epilepsy, but their development has been hampered by difficulties in achieving stable and targeted delivery of efficacious concentrations within the desired brain region. We have developed an encapsulated cell technology that overcomes these obstacles by providing a targeted, continuous, de novo synthesized source of high levels of neurotrophic molecules from human clonal ARPE-19 cells encapsulated into hollow fiber membranes. Here we illustrate the potential of this approach for delivering glial cell line-derived neurotrophic factor (GDNF) directly to the hippocampus of epileptic rats. In vivo studies demonstrated that bilateral intrahippocampal implants continued to secrete GDNF that produced high hippocampal GDNF tissue levels in a long-term manner. Identical implants robustly reduced seizure frequency in the pilocarpine model. Seizures were reduced rapidly, and this effect increased in magnitude over 3 months, ultimately leading to a reduction of seizures by 93%. This effect persisted even after device removal, suggesting potential disease-modifying benefits. Importantly, seizure reduction was associated with normalized changes in anxiety and improved cognitive performance. Immunohistochemical analyses revealed that the neurological benefits of GDNF were associated with the normalization of anatomical alterations accompanying chronic epilepsy, including hippocampal atrophy, cell degeneration, loss of parvalbumin-positive interneurons, and abnormal neurogenesis. These effects were associated with the activation of GDNF receptors. All in all, these results support the concept that the implantation of encapsulated GDNF-secreting cells can deliver GDNF in a sustained, targeted, and efficacious manner, paving the way for continuing preclinical evaluation and eventual clinical translation of this approach for epilepsy.SIGNIFICANCE STATEMENT Epilepsy is one of the most common neurological conditions, affecting millions of individuals of all ages. These patients experience debilitating seizures that frequently increase over time and can associate with significant cognitive decline and psychiatric disorders that are generally poorly controlled by pharmacotherapy. We have developed a clinically validated, implantable cell encapsulation system that delivers high and consistent levels of GDNF directly to the brain. In epileptic animals, this system produced a progressive and permanent reduction (>90%) in seizure frequency. These benefits were accompanied by improvements in cognitive and anxiolytic behavior and the normalization of changes in CNS anatomy that underlie chronic epilepsy. Together, these data suggest a novel means of tackling the frequently intractable neurological consequences of this devastating disorder.