Andreas T. Sørensen

Optogenetic control of epileptiform activity

The optogenetic approach to gain control over neuronal excitability both in vitro and in vivo has emerged as a fascinating scientific tool to explore neuronal networks, but it also opens possibilities for developing novel treatment strategies for neurologic conditions. We have explored whether such an optogenetic approach using the light-driven halorhodopsin chloride pump from Natronomonas pharaonis (NpHR), modified for mammalian CNS expression to hyperpolarize central neurons, may inhibit excessive hyperexcitability and epileptiform activity. We show that a lentiviral vector containing the NpHR gene under the calcium/calmodulin-dependent protein kinase IIα promoter transduces principal cells of the hippocampus and cortex and hyperpolarizes these cells, preventing generation of action potentials and epileptiform activity during optical stimulation. This study proves a principle, that selective hyperpolarization of principal cortical neurons by NpHR is sufficient to curtail paroxysmal activity in transduced neurons and can inhibit stimulation train-induced bursting in hippocampal organotypic slice cultures, which represents a model tissue of pharmacoresistant epilepsy. This study demonstrates that the optogenetic approach may prove useful for controlling epileptiform activity and opens a future perspective to develop it into a strategy to treat epilepsy.

Wnt5a-treated midbrain neural stem cells improve dopamine cell replacement therapy in parkinsonian mice.

Dopamine (DA) cell replacement therapy in Parkinson disease (PD) can be achieved using human fetal mesencephalic tissue; however, limited tissue availability has hindered further developments. Embryonic stem cells provide a promising alternative, but poor survival and risk of teratoma formation have prevented their clinical application. We present here a method for generating large numbers of DA neurons based on expanding and differentiating ventral midbrain (VM) neural stem cells/progenitors in the presence of key signals necessary for VM DA neuron development. Mouse VM neurospheres (VMNs) expanded with FGF2, differentiated with sonic hedgehog and FGF8, and transfected with Wnt5a (VMN-Wnt5a) generated 10-fold more DA neurons than did conventional FGF2-treated VMNs. VMN-Wnt5a cells exhibited the transcriptional and biochemical profiles and intrinsic electrophysiological properties of midbrain DA cells. Transplantation of these cells into parkinsonian mice resulted in significant cellular and functional recovery. Importantly, no tumors were detected and only a few transplanted grafts contained sporadic nestin-expressing progenitors. Our findings show that Wnt5a improves the differentiation and functional integration of stem cell-derived DA neurons in vivo and define Wnt5a-treated neural stem cells as an efficient and safe source of DA neurons for cell replacement therapy in PD.

Functional Integration of Grafted Neural Stem Cell-Derived Dopaminergic Neurons Monitored by Optogenetics in an In Vitro Parkinson Model

Intrastriatal grafts of stem cell-derived dopamine (DA) neurons induce behavioral recovery in animal models of Parkinsons disease (PD), but how they functionally integrate in host neural circuitries is poorly understood. Here, Wnt5a-overexpressing neural stem cells derived from embryonic ventral mesencephalon of tyrosine hydroxylase-GFP transgenic mice were expanded as neurospheres and transplanted into organotypic cultures of wild type mouse striatum. Differentiated GFP-labeled DA neurons in the grafts exhibited mature neuronal properties, including spontaneous firing of action potentials, presence of post-synaptic currents, and functional expression of DA D2 autoreceptors. These properties resembled those recorded from identical cells in acute slices of intrastriatal grafts in the 6-hydroxy-DA-induced mouse PD model and from DA neurons in intact substantia nigra. Optogenetic activation or inhibition of grafted cells and host neurons using channelrhodopsin-2 (ChR2) and halorhodopsin (NpHR), respectively, revealed complex, bi-directional synaptic interactions between grafted cells and host neurons and extensive synaptic connectivity within the graft. Our data demonstrate for the first time using optogenetics that ectopically grafted stem cell-derived DA neurons become functionally integrated in the DA-denervated striatum. Further optogenetic dissection of the synaptic wiring between grafted and host neurons will be crucial to clarify the cellular and synaptic mechanisms underlying behavioral recovery as well as adverse effects following stem cell-based DA cell replacement strategies in PD.

Adeno-associated viral vector-induced overexpression of neuropeptide Y Y2 receptors in the hippocampus suppresses seizures.

Gene therapy using recombinant adeno-associated viral vectors overexpressing neuropeptide Y in the hippocampus exerts seizure-suppressant effects in rodent epilepsy models and is currently considered for clinical application in patients with intractable mesial temporal lobe epilepsy. Seizure suppression by neuropeptide Y in the hippocampus is predominantly mediated by Y2 receptors, which, together with neuropeptide Y, are upregulated after seizures as a compensatory mechanism. To explore whether such upregulation could prevent seizures, we overexpressed Y2 receptors in the hippocampus using recombinant adeno-associated viral vectors. In two temporal lobe epilepsy models, electrical kindling and kainate-induced seizures, vector-based transduction of Y2 receptor complementary DNA in the hippocampus of adult rats exerted seizure-suppressant effects. Simultaneous overexpression of Y2 and neuropeptide Y had a more pronounced seizure-suppressant effect. These results demonstrate that overexpression of Y2 receptors (alone or in combination with neuropeptide Y) could be an alternative strategy for epilepsy treatment.

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

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.

Differential suppression of seizures via Y2 and Y5 neuropeptide Y receptors

Neuropeptide Y (NPY) prominently inhibits epileptic seizures in different animal models. The NPY receptors mediating this effect remain controversial partially due to lack of highly selective agonists and antagonists. To circumvent this problem, we used various NPY receptor knockout mice with the same genetic background and explored anti-epileptic action of NPY in vitro and in vivo. In Y2 (Y2-/-) and Y5 (Y5-/-) receptor knockouts, NPY partially inhibited 0 Mg2+-induced epileptiform activity in hippocampal slices. In contrast, in double knockouts (Y2Y5-/-), NPY had no effect, suggesting that in the hippocampus in vitro both receptors mediate anti-epileptiform action of NPY in an additive manner. Systemic kainate induced more severe seizures in Y5-/- and Y2Y5-/-, but not in Y2-/- mice, as compared to wild-type mice. Moreover, kainate seizures were aggravated by administration of the Y5 antagonist L-152,804 in wild-type mice. In Y5-/- mice, hippocampal kindling progressed faster, and afterdischarge durations were longer in amygdala, but not in hippocampus, as compared to wild-type controls. Taken together, these data suggest that, in mice, both Y2 and Y5 receptors regulate hippocampal seizures in vitro, while activation of Y5 receptors in extra-hippocampal regions reduces generalized seizures in vivo.

NPY gene transfer in the hippocampus attenuates synaptic plasticity and learning

Recombinant adeno‐associated viral (rAAV) vector‐induced neuropeptide Y (NPY) overexpression in the hippocampus exerts powerful antiepileptic and antiepileptogenic effects in rats. Such gene therapy approach could be a valuable alternative for developing new antiepileptic treatment strategies. Future clinical progress, however, requires more detailed evaluation of possible side effects of this treatment. Until now it has been unknown whether rAAV vector‐based NPY overexpression in the hippocampus alters normal synaptic transmission and plasticity, which could disturb learning and memory processing. Here we show, by electrophysiological recordings in CA1 of the hippocampal formation of rats, that hippocampal NPY gene transfer into the intact brain does not affect basal synaptic transmission, but slightly alters short‐term synaptic plasticity, most likely via NPY Y2 receptor‐mediated mechanisms. In addition, transgene NPY seems to be released during high frequency neuronal activity, leading to decreased glutamate release in excitatory synapses. Importantly, memory consolidation appears to be affected by the treatment. We found that long‐term potentiation (LTP) in the CA1 area is partially impaired and animals have a slower rate of hippocampal‐based spatial discrimination learning. These data provide the first evidence that rAAV‐based gene therapy using NPY exerts relative limited effect on synaptic plasticity and learning in the hippocampus, and therefore this approach could be considered as a viable alternative for epilepsy treatment.

Novel approaches to epilepsy treatment

The aim of epilepsy treatment is to achieve complete seizure freedom. Nonetheless, numerous side effects and seizure resistance to antiepileptic drugs (AEDs) affecting about 30–40% of all patients are main unmet needs in today’s epileptology. For this reason, novel approaches to treat epilepsy are highly needed. Herein, we highlight recent progress in stem‐cell–based and gene transfer–based therapies in epilepsy according to findings in animal models and address their potential clinical application. Multiple therapeutic targets are described, including neuropeptides, neurotrophic factors, and inhibitory neurotransmitters. We also address new molecular‐genetic approaches utilizing optogenetic technology. The therapeutic strategies presented herein are predominately aimed toward treatment of partial/focal epilepsies, but could also be envisaged for targeting key seizure propagation areas in the brain. These novel strategies provide proof‐of‐principle for developing effective treatments for refractory epilepsy in the foreseeable future.

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

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. Stem Cells 2014;32:3088–3098

Functional properties and synaptic integration of genetically labelled dopaminergic neurons in intrastriatal grafts.

Intrastriatal grafts of fetal ventral mesencephalic tissue, rich in dopaminergic neurons, can reverse symptoms in Parkinsons disease. For development of effective cell replacement therapy, other sources of dopaminergic neurons, e.g. derived from stem cells, are needed. However, the electrophysiological properties grafted cells need to have in order to induce substantial functional recovery are poorly defined. It has not been possible to prospectively identify and record from dopaminergic neurons in fetal transplants. Here we used transgenic mice expressing green fluorescent protein under control of the rat tyrosine hydroxylase promoter for whole‐cell patch‐clamp recordings of endogenous and grafted dopaminergic neurons. We transplanted ventral mesencephalic tissue from E12.5 transgenic mice into striatum of neonatal rats with or without lesions of the nigrostriatal dopamine system. The transplanted cells exhibited intrinsic electrophysiological properties typical of substantia nigra dopaminergic neurons, i.e. broad action potentials, inward rectifying currents with characteristic ‘sag’, and spontaneous action potentials. The grafted dopaminergic neurons also received functional excitatory and inhibitory synaptic inputs from the host brain, as shown by the presence of both spontaneous and stimulation‐evoked excitatory and inhibitory postsynaptic currents. Occurrence of spontaneous excitatory and inhibitory currents was lower, and of spontaneous action potentials was higher, in neurons placed in the dopamine‐depleted striatum than of those in the intact striatum. Our findings define specific electrophysiological characteristics of transplanted fetal dopaminergic neurons, and we provide the first direct evidence of functional synaptic integration of these neurons into host neural circuitries.