Dynorphin‐based “release on demand” gene therapy for drug‐resistant temporal lobe epilepsy
2019; Springer Nature; Volume: 11; Issue: 10 Linguagem: Inglês
10.15252/emmm.201809963
ISSN1757-4684
AutoresAlexandra S. Agostinho, Mario Mietzsch, Luca Zangrandi, Iwona Kmieć, Anna Mutti, Larissa Kraus, Pawel Fidzinski, Ulf C. Schneider, Martin Holtkamp, Regine Heilbronn, Christoph Schwarzer,
Tópico(s)Genetics and Neurodevelopmental Disorders
ResumoArticle5 September 2019Open Access Source DataTransparent process Dynorphin-based "release on demand" gene therapy for drug-resistant temporal lobe epilepsy Alexandra S Agostinho Alexandra S Agostinho Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Mario Mietzsch Mario Mietzsch Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Luca Zangrandi Luca Zangrandi Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Iwona Kmiec Iwona Kmiec Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Anna Mutti Anna Mutti Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Larissa Kraus Larissa Kraus Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Pawel Fidzinski Pawel Fidzinski Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Search for more papers by this author Ulf C Schneider Ulf C Schneider Department of Neurosurgery, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Martin Holtkamp Martin Holtkamp Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Regine Heilbronn Corresponding Author Regine Heilbronn [email protected] orcid.org/0000-0001-6412-3278 Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Christoph Schwarzer Corresponding Author Christoph Schwarzer [email protected] orcid.org/0000-0002-6373-3717 Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Alexandra S Agostinho Alexandra S Agostinho Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Mario Mietzsch Mario Mietzsch Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Luca Zangrandi Luca Zangrandi Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Iwona Kmiec Iwona Kmiec Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Anna Mutti Anna Mutti Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Larissa Kraus Larissa Kraus Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Pawel Fidzinski Pawel Fidzinski Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Search for more papers by this author Ulf C Schneider Ulf C Schneider Department of Neurosurgery, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Search for more papers by this author Martin Holtkamp Martin Holtkamp Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Regine Heilbronn Corresponding Author Regine Heilbronn [email protected] orcid.org/0000-0001-6412-3278 Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany Berlin Institute of Health (BIH), Berlin, Germany Search for more papers by this author Christoph Schwarzer Corresponding Author Christoph Schwarzer [email protected] orcid.org/0000-0002-6373-3717 Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria Search for more papers by this author Author Information Alexandra S Agostinho1,‡, Mario Mietzsch2,‡, Luca Zangrandi1,‡, Iwona Kmiec1, Anna Mutti1, Larissa Kraus3,4, Pawel Fidzinski3, Ulf C Schneider5, Martin Holtkamp3,4, Regine Heilbronn *,2,4 and Christoph Schwarzer *,1 1Department of Pharmacology, Medical University of Innsbruck, Innsbruck, Austria 2Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany 3Department of Neurology, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Epilepsy-Center Berlin-Brandenburg, Berlin, Germany 4Berlin Institute of Health (BIH), Berlin, Germany 5Department of Neurosurgery, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Berlin, Germany ‡These authors contributed equally to this work ‡Institute of Virology, Campus Benjamin Franklin, Charité - Universitätsmedizin Berlin, corporate member of Freie Universität Berlin, Humboldt-Universität zu Berlin, and Berlin Institute of Health, Germany *Corresponding author. Tel: +49 30 84453696; E-mail: [email protected] *Corresponding author. Tel: +43 512 9003 71205; E-mail: [email protected] EMBO Mol Med (2019)11:e9963https://doi.org/10.15252/emmm.201809963 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Focal epilepsy represents one of the most common chronic CNS diseases. The high incidence of drug resistance, devastating comorbidities, and insufficient responsiveness to surgery pose unmet medical challenges. In the quest of novel, disease-modifying treatment strategies of neuropeptides represent promising candidates. Here, we provide the "proof of concept" that gene therapy by adeno-associated virus (AAV) vector transduction of preprodynorphin into the epileptogenic focus of well-accepted mouse and rat models for temporal lobe epilepsy leads to suppression of seizures over months. The debilitating long-term decline of spatial learning and memory is prevented. In human hippocampal slices obtained from epilepsy surgery, dynorphins suppressed seizure-like activity, suggestive of a high potential for clinical translation. AAV-delivered preprodynorphin expression is focally and neuronally restricted and release is dependent on high-frequency stimulation, as it occurs at the onset of seizures. The novel format of "release on demand" dynorphin delivery is viewed as a key to prevent habituation and to minimize the risk of adverse effects, leading to long-term suppression of seizures and of their devastating sequel. Synopsis Temporal lobe epilepsy is a prevalent CNS disease with high medical need. In this proof-of-principle study, AAV vector-based expression of dynorphin in the epileptogenic focus leads to long-lasting suppression of seizures and rescues cognitive functions in clinically relevant disease models. AAV-mediated expression of preprodynorphin in the epileptogenic hippocampus suppresses seizures in mouse and rat models of temporal lobe epilepsy (TLE). Dynorphin-based gene therapy rescues cognitive impairment in epileptic mice. Dynorphins suppress induced seizure-like events in human hippocampal slices obtained from TLE surgery. Focally restricted gene therapy combined with excitation-induced release of dynorphins reduces risks of tolerance and side effects. Introduction With a worldwide prevalence of 1–2%, epilepsy represents one of the most frequent chronic neurological diseases affecting patients of all ages (Thurman et al, 2011). Recurrent seizures disrupt normal brain functions, lead to neuronal loss, and result in cognitive and emotional deficits. Patients suffer from stigmatization, social isolation, combined with disability, educational underachievement, and poor employment outcomes (WHO, 2015). About 70% of epilepsy patients experience focal seizures that arise from an epileptogenic focus in the temporal lobe, most frequently in the hippocampus, called mesial temporal lobe epilepsy (mTLE) (Blumcke et al, 2012). Unfortunately, mTLE with hippocampal sclerosis is the hardest to treat with up to 80% of patients not becoming seizure-free with antiepileptic drugs. Moreover, patients suffer from severe adverse side effects (Eadie, 2012; Perucca & Gilliam, 2012). Often, cognitive deficits and emotional blunting develop, potentially facilitated by antiepileptic medication (Ertem et al, 2013). In 2008, the FDA issued a black-box warning that several antiepileptic drugs increase the risk of suicidal tendencies (Mula & Sander, 2013). For patients with drug-refractory epilepsy whose seizures originate from a well-defined and accessible focus, neurosurgery for resection of the epileptogenic focus may remain the ultimate solution (Duncan, 2007; Bergey, 2013). But even then, the outcome is highly variable (Spencer & Huh, 2008). To date, none of the prevailing treatments offers a satisfactory long-term solution. Therefore, medical need for innovative treatment options is high. Accumulating evidence suggests that neuropeptides, including dynorphins, act as endogenous modulators of neuronal excitability (Henriksen et al, 1982; Siggins et al, 1986). The dynorphins represent a family of vesicle-stored endogenous opioids, perceived as natural anticonvulsants (Tortella & Long, 1988; Mazarati & Wasterlain, 2002). During burst stimulation, typical for the onset of seizures, dynorphins are released from neurons and bind to kappa opioid receptors (KOR), thereby preventing seizure development (Schwarzer, 2009). The seizure threshold is lowered in preprodynorphin (pDyn) knockout mice leading to increased susceptibility for the development of epilepsy (Loacker et al, 2007). Similarly, low dynorphin levels in humans correlate with increased vulnerability for the disease (Stogmann et al, 2002). In mouse models of mTLE as well as in affected patients, dynorphin levels are reduced in the epileptogenic focus, but KOR are mostly maintained (de Lanerolle et al, 1997). The aim of the present study was to replenish the exhausted reservoirs of dynorphins in neurons of the epileptogenic focus. This restores the source of seizure-suppressing endogenous KOR agonists. As vector-derived propeptides are identical to the endogenous protein, they should be similarly stored in large dense-core vesicles, processed, and matured peptides release upon high-frequency stimulation. Thus, transduced dynorphins will be "released on demand" similar to endogenous neuropeptides. In well-recognized animal models of unilateral mTLE, a single focal application of AAV vectors transducing human pDyn into an established epileptogenic focus led to long-term suppression of seizures and stopped disease progression. Results Suppression of seizures by AAV-pDyn delivery to the epileptogenic focus in the kainic acid mouse model of mTLE Focal and secondary, generalizing seizures are typical hallmarks of mTLE, as reflected in the widely accepted kainic acid (KA)-induced mouse model of mTLE. Importantly, hippocampal paroxysmal discharges (HPD: unilateral spike trains with a duration of more than 20 s during which animals may display some stereotypies) observed in this model do not respond well to antiepileptic drugs (Riban et al, 2002; Klein et al, 2015; Zangrandi et al, 2016). Therefore, HPDs are considered to model drug-resistant seizures. To study the influence of pDyn overexpression on the frequency and severity of recurrent seizures, an adeno-associated virus (AAV) vector was constructed to express the human preprodynorphin cDNA (Fig 1A). Figure 1. Effect of AAV-pDyn on seizures in mouse and rat models of TLE A. Displayed (sc)AAV2-based vector backbones were packaged in AAV serotype 1 capsids. AAV-ITRs are displayed in gray, and ΔITR refers to the mutated ITR version of scAAVs. The transgene of AAV-pDyn is a codon-optimized version of the full-length human preprodynorphin cDNA enhanced by a WPRE element. Control vectors carry either a truncated, non-functional version of the enhanced GFP gene (AAV-ΔGFP), or its functional counterpart (AAV-eGFP; not displayed). B. Kainic acid mouse model of TLE: Daily EEG recordings obtained from the epileptogenic focus starting from 1 month after KA injection and spanning the period from 2 days before to 7 days after AAV-pDyn delivery (2 × 109 gp). C. Higher time resolution of the indicated section of the Day +1 in (B), representing a hippocampal paroxysmal discharge (HPD). A generalized seizure is depicted in Fig EV1. D, E. The characteristic EEG features of this model, secondary generalized seizures (bars) and HPDs (lines), were reduced in number and in duration by AAV-pDyn (red; n = 3 (from day 60); 7 (till day 30) per time interval), but not by AAV-ΔGFP or after sham treatment (blue; n = 4 (day 90); 5 (day 30 and 60); and 6 (before day 30), per time interval). The relatively high variability of seizure frequencies and duration is typical for this model and reflects the findings in human mTLE. Some animals could not be recorded for the entire period due to loss of implant. **P < 0.01; effect of treatment on HPDs; ##P < 0.01; effect of treatment on generalized seizures; analyzed by two-way ANOVA with Bonferroni correction for both number and time. F. Injection of norBNI (20 mg/kg; i.p.) results in a transient reappearance of HPDs immediately and 24 h after application. One week after norBNI application (washout), suppression of HPDs was re-established. Data obtained from 4 epileptic animals before (black) and after (red) AAV-pDyn delivery (2 × 109 gp) are depicted. *P < 0.05; **P < 0.01; one-way ANOVA for repeated measures with Friedman post hoc test. G. EEG recordings obtained from the ipsilateral dorsal hippocampus of rats after electrical self-sustained status epilepticus (SSSE) before (black) and after (red) AAV-pDyn delivery (4x109 gp) are depicted. Spike trains with a frequency of at least 2.5 Hz induced by SSSE were markedly reduced by AAV-pDyn (n = 4). *P < 0.05 one-way ANOVA for repeated measures with Friedman post hoc test. Data information: Data represent mean ± standard error of the mean. Download figure Download PowerPoint To achieve high per particle gene expression rates, a self-complementary (sc)AAV2 vector backbone equipped with a potent truncated CBA promoter and translation-enhancing WPRE element was chosen to achieve high-level expression of a codon-optimized human pDyn cDNA, as described in the methods. AAV serotype 1 capsids were chosen for packaging due to proven neuronal transduction combined with restricted spread of the vector beyond the site of injection (Murlidharan et al, 2014; Hocquemiller et al, 2016). Highly purified and concentrated AAV vectors, 2 × 109 genomic particles (gp) coding for human pDyn (AAV-pDyn), or non-functional control vectors (AAV-ΔGFP) were injected into the epileptogenic focus about 1 month after KA injection, when focal epilepsy had developed. At this stage, animals displayed numerous HPDs (Fig 1B, top EEG trace, and Fig 1C) and up to 3 generalized seizures (displaying spike trains on the traces of all 4 recording electrodes and tonic–clonic motor seizures; Fig EV1) a day. AAV-pDyn delivery induced a gradual reduction in generalized seizures (Fig 1D) and of HPDs (Fig 1E). Generalized seizures completely disappeared within 1 week, and no further events were observed for the entire observation period (3 months). HPDs were gradually reduced over the entire observation period. By contrast, animals injected with AAV-ΔGFP continued to experience seizures for the entire observation period (Fig 1D and E). To demonstrate that Dyn action was mediated by kappa opioid receptors, mice were treated with the KOR antagonist norBNI (20 mg/kg) 30 days post-AAV-pDyn delivery when seizures had disappeared. Drug treatment led to a transient reinstatement of seizures and their disappearance upon washout of the antagonist (Fig 1F). Click here to expand this figure. Figure EV1. EEG trace of a seizure A. EEG trace of a generalized seizure in a KA-treated mouse; from top to bottom contralateral hippocampus, contralateral motor cortex and ipsilateral hippocampus. B. Blow-up of the EEG trace from the ipsilateral hippocampus in panel (A). Download figure Download PowerPoint Suppression of seizures by AAV-pDyn delivery in an electroconvulsive rat model of mTLE To confirm that AAV-pDyn-induced seizure suppression in kainic acid-induced mTLE in mice is reproduced in another species and differently induced TLE model, we set up the rat model of electrically induced, focal, and self-sustained status epilepticus (Nissinen et al, 1999, 2000). After unilateral electrical stimulation of the lateral amygdala, rats develop spontaneous seizure-like EEG abnormalities (spike trains) mostly originating from either the amygdala or the hippocampus within a few weeks. To avoid overlap with the stimulation site, AAV-pDyn (2 × 109gp) was infused into the dorsal hippocampus. This treatment induced a significant reduction in spike trains already after 1 week that consolidated up to 4 months (Fig 1G). Conservation and restoration of cognitive functions upon AAV-pDyn delivery to the epileptogenic focus Many mTLE patients suffer from progressive deficits in spatial and declarative memory. Similar comorbidities also develop 1–2 months after unilateral KA injection in mice (Groeticke et al, 2008). To test whether the silencing of seizures in the ipsilateral hippocampus starting during epileptogenesis prevents the memory decline, AAV-pDyn was injected into the epileptogenic focus using mice 2 weeks after KA injection. At this stage, EEG abnormalities such as high-voltage sharp waves, spike trains, and HPD characteristic of early-stage epilepsy have established (Riban et al, 2002), yet animals are still able to learn. Subsequently, mice were tested for spatial memory applying the Barnes maze in a repetitive manner, learning a different target at months 1, 2, and 6 after KA injection. Animals treated with AAV-pDyn performed comparably to naïve, age-matched controls at each time interval (Fig 2, upper panel). Learning curves are depicted in Fig EV2. The decline in performance of both groups of animals at the late time interval (Fig 2G–I) is most probably due to impaired vision known to develop in aged C57BL/6N mice. The time interval chosen is the latest where C57Bl/6N mice are still able to learn the task, and AAV-pDyn-treated epileptic animals kept up with the performance of naïve mice. Figure 2. Effects of AAV-pDyn on spatial learning and memory A–I. Spatial learning and memory were tested on the Barnes maze. Quadrant 1 (Q1) contains the target hole (red; A). Unilateral injection of AAV-pDyn (B) or AAV-eGFP (C) into naïve young adult mice (12 weeks age) did not influence the performance as compared to naïve controls (D) when tested 4 weeks after AAV injection. Mice treated 2 weeks after KA with AAV-pDyn (E, H) performed equally to age-matched naïve controls (D, G) 2 weeks (E) and 5.5 months (H) after treatment. By contrast, animals treated 2 weeks after KA with AAV-ΔGFP (F, I) gradually lost this ability. Two-way ANOVA revealed significance between AAV-ΔGFP- and AAV-pDyn-treated groups for interaction 2 weeks (P = 0.0349) and 5.5 months (P = 0.0311) after AAV, respectively, at each time interval and quadrant (P < 0.0001) 2 weeks after AAV. Comparison of AAV-pDyn injected with naïve animals revealed no differences. J–O. Epileptic mice, which were not able to learn the Barnes maze task 1 month after KA (J, M), AAV-pDyn application restored spatial memory 1 (K) and 2 months (L) after treatment. Treatment with AAV-ΔGFP did not result in improved memory (N, O). Two-way ANOVA revealed significance for interaction (P = 0.0049) and quadrant (< 0.0001) comparing AAV-ΔGFP with AAV-pDyn-treated animals at the later time interval. Data information: Data represent mean ± standard deviation. Animal numbers: (B) and (C) n = 9; (D) through (H) n = 8; (I) n = 5, note: Three mice had to be killed due to accelerating seizure activity and resulting weight loss; (J) through (O) n = 7. ***P < 0.001; **P < 0.01; *P < 0.05 by one-way ANOVA and Dunnett post hoc test. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. Learning during Barnes maze testingSpatial learning and memory were tested on the Barnes maze. Mice were trained on 4 consecutive days before testing memory. The acquisition of learning was monitored by assessment of the time needed to find the target hole (A–C; G–I) and the number of wrong holes visited before finding the correct one (primary errors; D–F: J–L) on each training day. A–F. Unilateral injection of AAV-pDyn (red) or AAV-ΔGFP (blue) into naïve young adult mice compared with naive controls (black) is depicted in (A) and (D). Interestingly, AAV-ΔGFP-injected mice took longer to find the hole. Noteworthy, this had no impact on memory retrieval (Fig 2A–C). Mice treated 2 weeks after KA with AAV-pDyn (red) or AAV-ΔGFP (blue) (B, C, E, F) performed differently 2 weeks after vector application (B, E) in respect to time needed to target and 5.5 months (C, F) after treatment in respect to primary errors. The overall reduction in time needed to target is most probably due to the repeated testing of animals on the Barnes maze. The target hole was repositioned in each round, but the mice were familiar with the test per se. G–L. A person not familiar with the mice assigned epileptic animals into two groups before testing on Barnes maze. No differences were observed between the two groups before vector treatment (G, J). By contrast, AAV-pDyn (red)-treated animals reached the target significantly faster than AAV-ΔGFP (blue) 1 and 2 months after treatment (H, I). Primary errors did not differ (K, L). Data information: Animal numbers: (A) and (D) n = 9; (B, C, E, F) n = 8; for AAV-ΔGFP in (C) and (F) n = 5, because 3 mice had to be excluded from evaluation due to accelerating seizure activity and resulting weight loss; (G–L) n = 7; data represent mean ± standard error of 3 training sessions per day. **P < 0.01; *P < 0.05 by two-way ANOVA for repeated measurements depicting only difference between treatments. Besides the differences between treatments, significance of the factor time (i.e., the training days) was observed for data depicted in (A) (P = 0.0002); (B) (P = 0.0009); (D) (P < 0.0001); (E) (P = 0.0010); and (F) (P = 0.0212) suggestive of learning. Download figure Download PowerPoint By contrast, animals treated with AAV-ΔGFP lost this ability (Fig 2, upper panel). Noteworthy, the animals retained an intact working memory. All animal groups performed equally well in a spontaneous alternation test. 60 to 75% of correct answers were observed in AAV-pDyn- or AAV-ΔGFP-treated epileptic and in naïve animals (Fig EV4K). This finding recapitulates data from mTLE patients, in whom the short-term memory regularly remains intact (Silva et al, 2010). In a second experiment, mice in the state of chronic epilepsy, already displaying established deficits in spatial learning and memory, were tested for functional reconstitution. Although the contralateral hippocampus displays only minor neuropathological alterations, its function appears compromised. Due to strong collateral connections between both hippocampi, disturbances of the non-affected, functionally intact, contralateral hippocampus are highly likely. This led to our hypothesis that silencing of the ipsilateral hippocampus by pDyn delivery should prevent this development. Mice were treated by AAV delivery into the epileptogenic focus 5 weeks after KA injection. Animals injected with AAV-pDyn gradually regained lost spatial memory within 1 month after AAV delivery. Performance levels similar to naïve controls were achieved within 2 months (Fig 2J–L). By contrast, no improvement of memory functions was observed in epileptic mice treated with AAV-ΔGFP (Fig 2M–O). The time course of reconstitution fits well to the observed time course of seizure suppression in mice observed before (Fig 1). Neuron-specific long-term expression of pDyn and stimulation-dependent "release on demand" Long-term expression of AAV-delivered pDyn in neurons of the epileptogenic focus was demonstrated at 6 months after KA injection (5.5 months after vector delivery) by double immunofluorescence. Mostly dentate granule cells and pyramidal cells showed cytoplasmatic pDyn immunoreactivity together with nuclear immunoreactivity for the neuronal marker NeuN, but not for the glial marker GFAP (Fig 3A–F). Expression of Dyn peptides was also observed in principle and non-principal neurons. Upon injection of AAV-pDyn into the hilus of naive mice, strong immunoreactivity was observed in the mossy fiber tract, indicative of transduction of granule cells. Moreover, somata of non-principle cells in the polymorph cell layer and corresponding labeling in the outer molecular layer suggests transduction of GABAergic interneurons. Strong labeling of the inner molecular layer also in the contralateral hippocampus suggests transduction of mossy cells (Fig EV3). Figure 3. Distribution and release "on demand" of vector-derived dynorphins A–F. Double-immunofluorescence labeling is depicted for pDyn and NeuN (A–C) or GFAP (D–F) in the ipsilateral dentate gyrus of KA-treated and AAV-pDyn-injected mice. Enlarged view in (F) represents 15 × 30 μm. G. Mature Dyn B content (measured by a Dyn B-specific EIA) in the dorsal hippocampus of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 6 (filled symbols; n = 3) months after vector treatment. Naïve animals were age-matched to the 1.5 months after AAV group. iH stands for ipsilateral hippocampus and cH for contralateral hippocampus. *P < 0.05; paired t-test was used for comparison of ipsi- and contralateral hippocampi. Two-way ANOVA was used to compare Dyn levels between the early and late time interval. H. Mature Dyn B content in the CSF of mice treated with KA (blue symbols) or KA and AAV-pDyn (red symbols) 1.5 (open symbols; n = 6) and 7 (filled symbols; n = 4) months after vector treatment. **P < 0.01; one-way ANOVA with Dunnett post hoc test I. The release of fully processed, mature Dyn B under different stimulation conditions was analyzed in microdialysates collected from the hippocampus of pDyn-deficient (KO) animals 2 weeks after injection of AAV-pDyn. Three baseline samples (BL; 25 min each) were collected. This was followed by 25 min low-frequency stimulation (LF), 25 min baseline, and 25 min high-frequency stimulation (HF; I). J. The microdialysis probe (red cross) was placed in the dentate gyrus, and the stimulation electrode (black cross) in the entorhinal cortex. K. Dyn B was quantified by EIA in the dialysate collected during different stimulation intensities. The red line represents the detection limit of the EIA. *P < 0.05; n = 4; one-way ANOVA with Tukey post hoc test. Data information: Data represent mean ± standard error of the mean. Source data are available online for this figure. Source Data for Figure 3 [emmm201809963-sup-0004-SDataFig3.zip] Download figure Download PowerPoint Click here to expand this figure. Figure EV3. Distribution of vector-derived dynorphins A–D. Immunohistochemistry was performed on PFA-fixed 40-μm sections obtained from a pDyn knockout mouse unilaterally injected with 2 × 109 gp of AAV-pDyn 3 weeks before. The ipsilateral hippocampus (A) displays strong immunoreactivity in the terminal field of mossy fiber
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