transplantation-human-stem-cell-derived-gabaergic-neurons-into-early

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 ...

Lund University Hospital

Recently, hippocampal neuropeptide Y (NPY) gene therapy has been shown to effectively suppress both acute and chronic seizures in animal model of epilepsy, thus representing a promising novel antiepileptic treatment strategy, particularly for patients with intractable mesial temporal lobe epilepsy (TLE). However, our previous studies show that recombinant adeno-associated viral (rAAV)-NPY treatment in naive rats attenuates long-term potentiation (LTP) and transiently impairs hippocampal learning process, indicating that negative effect on memory function could be a potential side effect of NPY gene therapy. Here we report how rAAV vector-mediated overexpression of NPY in the hippocampus affects rapid kindling, and subsequently explore how synaptic plasticity and transmission is affected by kindling and NPY overexpression by field recordings in CA1 stratum radiatum of brain slices. In animals injected with rAAV-NPY, we show that rapid kindling-induced hippocampal seizures in vivo are effectively suppressed as compared to rAAV-empty injected (control) rats. Six to nine weeks later, basal synaptic transmission and short-term synaptic plasticity are unchanged after rapid kindling, while LTP is significantly attenuated in vitro. Importantly, transgene NPY overexpression has no effect on short-term synaptic plasticity, and does not further compromise LTP in kindled animals. These data suggest that epileptic seizure-induced impairment of memory function in the hippocampus may not be further affected by rAAV-NPY treatment, and may be considered less critical for clinical application in epilepsy patients already experiencing memory disturbances.

Hippocampal NPY gene transfer attenuates seizures without affecting epilepsy-induced impairment of LTP.

Cholecystokinin (CCK)-expressing basket cells encompass a subclass of inhibitory GABAergic interneurons that regulate memory-forming oscillatory network activity of the hippocampal formation in accordance to the emotional and motivational state of the animal, conveyed onto these cells by respective extrahippocampal afferents. Various excitatory and inhibitory afferent and efferent synapses of the hippocampal CCK basket cells express serotoninergic, cholinergic, cannabinoid, and benzodiazepine sensitive receptors, all contributing to their functional plasticity. We explored whether CCK basket cells are modulated by neuropeptide Y (NPY), one of the major local neuropeptides that strongly inhibits hippocampal excitability and has significant effect on its memory function. Here, using GAD65-GFP transgenic mice for prospective identification of CCK basket cells and whole-cell patch-clamp recordings, we show for the first time that excitatory and inhibitory inputs onto CCK basket cells in the dentate gyrus of the hippocampus are modulated by NPY through activation of NPY Y2 receptors. The frequency of spontaneous and miniature EPSCs, as well as the amplitudes of stimulation-evoked EPSCs were decreased. Similarly, the frequency of both spontaneous and miniature IPSCs, and the amplitudes of stimulation-evoked IPSCs were decreased after NPY application. Most of the effects of NPY could be attributed to a presynaptic site of action. Our data provide the first evidence that the excitatory and inhibitory inputs onto the CCK basket cells could be modulated by local levels of NPY, and may change the way these cells process extrahippocampal afferent information, influencing hippocampal function and its network excitability during normal and pathological oscillatory activities.

Tuning afferent synapses of hippocampal interneurons by neuropeptide Y.

Cholecystokinin (CCK-) positive basket cells form a distinct class of inhibitory GABAergic interneurons, proposed to act as fine-tuning devices of hippocampal gamma-frequency (30-90 Hz) oscillations, which can convert into higher frequency seizure activity. Therefore, CCK-basket cells may play an important role in regulation of hyper-excitability and seizures in the hippocampus. In normal conditions, the endogenous excitability regulator neuropeptide Y (NPY) has been shown to modulate afferent inputs onto dentate gyrus CCK-basket cells, providing a possible novel mechanism for excitability control in the hippocampus. Using GAD65-GFP mice for CCK-basket cell identification, and whole-cell patch-clamp recordings, we explored whether the effect of NPY on afferent synapses to CCK-basket cells is modified in the hyper-excitable dentate gyrus. To induce a hyper-excitable state, recurrent seizures were evoked by electrical stimulation of the hippocampus using the well-characterized rapid kindling protocol. The frequency of spontaneous and miniature excitatory and inhibitory post-synaptic currents recorded in CCK-basket cells was decreased by NPY. The excitatory post-synaptic currents evoked in CCK-basket cells by optogenetic activation of principal neurons were also decreased in amplitude. Interestingly, we observed an increased proportion of spontaneous inhibitory post-synaptic currents with slower rise times, indicating that NPY may inhibit gamma aminobutyric acid release preferentially in peri-somatic synapses. These findings indicate that increased levels and release of NPY observed after seizures can modulate afferent inputs to CCK-basket cells, and therefore alter their impact on the oscillatory network activity and excitability in the hippocampus.

Altered profile of basket cell afferent synapses in hyper-excitable dentate gyrus revealed by optogenetic and two-pathway stimulations.

Optogenetic tools comprise a variety of different light-sensitive proteins from single-cell organisms that can be expressed in mammalian neurons and effectively control their excitability. Two main classes of optogenetic tools allow to either depolarize or hyperpolarize, and respectively generate or inhibit action potentials in selective populations of neurons. This opens unprecedented possibilities for delineating the role of certain neuronal populations in brain processing and diseases. Moreover, optogenetics may be considered for developing potential treatment strategies for brain diseases, particularly for excitability disorders such as epilepsy. Expression of the inhibitory halorhodopsin NpHR in hippocampal principal cells has been recently used as a tool to effectively control chemically and electrically induced epileptiform activity in slice preparations, and to reduce in vivo spiking induced by tetanus toxin injection in the motor cortex. In this review we give a comprehensive summary of what has been achieved so far in the field of epilepsy using optogenetics, and discuss some of the possible strategies that could be envisaged in the future. We also point out some of the challenges and pitfalls in relation to possible outcomes of using optogenetics for controlling network excitability, and associated brain diseases. This article is part of the Special Issue entitled 'New Targets and Approaches to the Treatment of Epilepsy'.

An optogenetic approach in epilepsy.

Optogenetic techniques provide powerful tools for bidirectional control of neuronal activity and investigating alterations occurring in excitability disorders, such as epilepsy. In particular, the possibility to specifically activate by light-determined interneuron populations expressing channelrhodopsin-2 provides an unprecedented opportunity of exploring their contribution to physiological and pathological network activity. There are several subclasses of interneurons in cortical areas with different functional connectivity to the principal neurons (e.g., targeting their perisomatic or dendritic compartments). Therefore, one could optogenetically activate specific or a mixed population of interneurons and dissect their selective or concerted inhibitory action on principal cells. We chose to explore a conceptually novel strategy involving simultaneous activation of mixed populations of interneurons by optogenetics and study their impact on ongoing epileptiform activity in mouse acute hippocampal slices. Here we demonstrate that such approach results in a brief initial action potential discharge in CA3 pyramidal neurons, followed by prolonged suppression of ongoing epileptiform activity during light exposure. Such sequence of events was caused by massive light-induced release of GABA from ChR2-expressing interneurons. The inhibition of epileptiform activity was less pronounced if only parvalbumin- or somatostatin-expressing interneurons were activated by light. Our data suggest that global optogenetic activation of mixed interneuron populations is a more effective approach for development of novel therapeutic strategies for epilepsy, but the initial action potential generation in principal neurons needs to be taken in consideration. 

Global optogenetic activation of inhibitory interneurons during epileptiform activity.

Recent technological advances open exciting avenues for improving the understanding of mechanisms in a broad range of epilepsies. This chapter focuses on the development of optogenetics and on-demand technologies for the study of epilepsy and the control of seizures. Optogenetics is a technique which, through cell-type selective expression of light-sensitive proteins called opsins, allows temporally precise control via light delivery of specific populations of neurons. Therefore, it is now possible not only to record interictal and ictal neuronal activity, but also to test causality and identify potential new therapeutic approaches. We first discuss the benefits and caveats to using optogenetic approaches and recent advances in optogenetics related tools. We then turn to the use of optogenetics, including on-demand optogenetics in the study of epilepsies, which highlights the powerful potential of optogenetics for epilepsy research. 

How might novel technologies such as optogenetics lead to better treatments in epilepsy?

Reprogramming of somatic cells into pluripotency stem cell state has opened new opportunities in cell replacement therapy and disease modeling in a number of neurological disorders. It still remains unknown, however, to what degree the grafted human-induced pluripotent stem cells (hiPSCs) differentiate into a functional neuronal phenotype and if they integrate into the host circuitry. Here, we present a detailed characterization of the functional properties and synaptic integration of hiPSC-derived neurons grafted in an in vitro model of hyperexcitable epileptic tissue, namely organotypic hippocampal slice cultures (OHSCs), and in adult rats in vivo. The hiPSCs were first differentiated into long-term self-renewing neuroepithelial stem (lt-NES) cells, which are known to form primarily GABAergic neurons. When differentiated in OHSCs for 6 weeks, lt-NES cell-derived neurons displayed neuronal properties such as tetrodotoxin-sensitive sodium currents and action potentials (APs), as well as both spontaneous and evoked postsynaptic currents, indicating functional afferent synaptic inputs. The grafted cells had a distinct electrophysiological profile compared to host cells in the OHSCs with higher input resistance, lower resting membrane potential, and APs with lower amplitude and longer duration. To investigate the origin of synaptic afferents to the grafted lt-NES cell-derived neurons, the host neurons were transduced with Channelrhodopsin-2 (ChR2) and optogenetically activated by blue light. Simultaneous recordings of synaptic currents in grafted lt-NES cell-derived neurons using whole-cell patch-clamp technique at 6 weeks after grafting revealed limited synaptic connections from host neurons. Longer differentiation times, up to 24 weeks after grafting in vivo, revealed more mature intrinsic properties and extensive synaptic afferents from host neurons to the lt-NES cell-derived neurons, suggesting that these cells require extended time for differentiation/maturation and synaptogenesis. However, even at this later time point, the grafted cells maintained a higher input resistance. These data indicate that grafted lt-NES cell-derived neurons receive ample afferent input from the host brain. Since the lt-NES cells used in this study show a strong propensity for GABAergic differentiation, the host-to-graft synaptic afferents may facilitate inhibitory neurotransmitter release, and normalize hyperexcitable neuronal networks in brain diseases, for example, such as epilepsy.

Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors.

Development of novel disease-modifying treatment strategies for neurological disorders, which at present have no cure, represents a major challenge for today's neurology. Translation of findings from animal models to humans represents an unresolved gap in most of the preclinical studies. Gene therapy is an evolving innovative approach that may prove useful for clinical applications. In animal models of temporal lobe epilepsy (TLE), gene therapy treatments based on viral vectors encoding NPY or galanin have been shown to effectively suppress seizures. However, how this translates to human TLE remains unknown. A unique possibility to validate these animal studies is provided by a surgical therapeutic approach, whereby resected epileptic tissue from temporal lobes of pharmacoresistant patients are available for neurophysiological studies in vitro. To test whether NPY and galanin have antiepileptic actions in human epileptic tissue as well, we applied these neuropeptides directly to human hippocampal slices in vitro. NPY strongly decreased stimulation-induced EPSPs in dentate gyrus and CA1 (up to 30 and 55%, respectively) via Y2 receptors, while galanin had no significant effect. Receptor autoradiographic binding revealed the presence of both NPY and galanin receptors, while functional receptor binding was only detected for NPY, suggesting that galanin receptor signaling may be impaired. These results underline the importance of validating findings from animal studies in human brain tissue, and advocate for NPY as a more appropriate candidate than galanin for future gene therapy trials in pharmacoresistant TLE patients. 

Differential Effect of Neuropeptides on Excitatory Synaptic Transmission in Human Epileptic Hippocampus.

Chloride ions play an important role in controlling excitability of principal neurons in the central nervous system. When neurotransmitter GABA is released from inhibitory interneurons, activated GABA type A (GABA) receptors on principal neurons become permeable to chloride. Typically, chloride flows through activated GABA receptors into the neurons causing hyperpolarization or shunting inhibition, and in turn inhibits action potential (AP) generation. However, in situations when intracellular chloride concentration is increased, chloride ions can flow in opposite direction, depolarize neurons, and promote AP generation. It is generally recognized that altered chloride homeostasis per se has no effect on the AP threshold. Here, we demonstrate that chloride overload of mouse principal CA3 pyramidal neurons not only makes these cells more excitable through GABA receptor activation but also lowers the AP threshold, further aggravating excitability. This phenomenon has not been described in principal neurons and adds to our understanding of mechanisms regulating neuronal and network excitability, particularly in developing brain and during pathological situations with altered chloride homeostasis. This finding further broadens the spectrum of neuronal plasticity regulated by ionic compositions across the cellular membrane.

Altered Chloride Homeostasis Decreases the Action Potential Threshold and Increases Hyperexcitability in Hippocampal Neurons.

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.

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

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.

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

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 Interneurons Establish Efferent Synapses onto Host Neurons in Rat Epileptic Hippocampus and Inhibit Spontaneous Recurrent Seizures.

Article	Year
Hippocampal NPY gene transfer attenuates seizures without affecting epilepsy-induced impairment of LTP. Experimental neurology\| PubMed ID: 19038255	2009
Tuning afferent synapses of hippocampal interneurons by neuropeptide Y. Hippocampus\| PubMed ID: 20043282	2011
Altered profile of basket cell afferent synapses in hyper-excitable dentate gyrus revealed by optogenetic and two-pathway stimulations. The European journal of neuroscience\| PubMed ID: 22512307	2012
An optogenetic approach in epilepsy. Neuropharmacology\| PubMed ID: 22698957	2013
Global optogenetic activation of inhibitory interneurons during epileptiform activity. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 24573293	2014
How might novel technologies such as optogenetics lead to better treatments in epilepsy? Advances in experimental medicine and biology\| PubMed ID: 25012388	2014
Optogenetics reveal delayed afferent synaptogenesis on grafted human-induced pluripotent stem cell-derived neural progenitors. Stem cells (Dayton, Ohio)\| PubMed ID: 25183299	2014
Differential Effect of Neuropeptides on Excitatory Synaptic Transmission in Human Epileptic Hippocampus. The Journal of neuroscience : the official journal of the Society for Neuroscience\| PubMed ID: 26134645	2015
Altered Chloride Homeostasis Decreases the Action Potential Threshold and Increases Hyperexcitability in Hippocampal Neurons. eNeuro\| PubMed ID: 29379872	2017
Inhibition of epileptiform activity by neuropeptide Y in brain tissue from drug-resistant temporal lobe epilepsy patients. Scientific reports\| PubMed ID: 31852985	2019
Human stem cell-derived GABAergic neurons functionally integrate into human neuronal networks. Scientific reports\| PubMed ID: 34764308	2021
Human Stem Cell-Derived GABAergic Interneurons Establish Efferent Synapses onto Host Neurons in Rat Epileptic Hippocampus and Inhibit Spontaneous Recurrent Seizures. International journal of molecular sciences\| PubMed ID: 34948040	2021

Marco Ledri

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