AAV Vector-Mediated Overexpression of CB1 Cannabinoid Receptor in Pyramidal Neurons of the Hippocampus Protects against Seizure-Induced Excitoxicity

The CB1 cannabinoid receptor is the most abundant G-protein coupled receptor in the brain and a key regulator of neuronal excitability. There is strong evidence that CB1 receptor on glutamatergic hippocampal neurons is beneficial to alleviate epileptiform seizures in mouse and man. Therefore, we hypothesized that experimentally increased CB1 gene dosage in principal neurons would have therapeutic effects in kainic acid (KA)-induced hippocampal pathogenesis. Here, we show that virus-mediated conditional overexpression of CB1 receptor in pyramidal and mossy cells of the hippocampus is neuroprotective and moderates convulsions in the acute KA seizure model in mice. We introduce a recombinant adeno-associated virus (AAV) genome with a short stop element flanked by loxP sites, for highly efficient attenuation of transgene expression on the transcriptional level. The presence of Cre-recombinase is strictly necessary for expression of reporter proteins or CB1 receptor in vitro and in vivo. Transgenic CB1 receptor immunoreactivity is targeted to glutamatergic neurons after stereotaxic delivery of AAV to the dorsal hippocampus of the driver mice NEX-cre. Increased CB1 receptor protein levels in hippocampal lysates of AAV-treated Cre-mice is paralleled by enhanced cannabinoid-induced G-protein activation. KA-induced seizure severity and mortality is reduced in CB1 receptor overexpressors compared with AAV-treated control animals. Neuronal damage in the hippocampal CA3 field is specifically absent from AAV-treated Cre-transgenics, but evident throughout cortical areas of both treatment groups. Our data provide further evidence for a role of increased CB1 signaling in pyramidal hippocampal neurons as a safeguard against the adverse effects of excessive excitatory network activity

Targeting neuronal populations by AAV-mediated gene transfer for studying the endocannabinoid system

The cannabinoid type 1 (CB1) receptor is involved in a plethora of physiological functions and heterogeneously expressed on different neuronal populations. Several conditional loss-of-function studies revealed distinct effects of CB1 receptor signaling on glutamatergic and GABAergic neurons, respectively. To gain a comprehensive picture of CB1 receptor-mediated effects, the present study aimed at developing a gain-of-function approach, which complements conditional loss-of-function studies. Therefore, adeno-associated virus (AAV)-mediated gene delivery and Cre-mediated recombination were combined to recreate an innovative method, which ensures region- and cell type-specific transgene expression in the brain. This method was used to overexpress the CB1 receptor in glutamatergic pyramidal neurons of the mouse hippocampus. Enhanced CB1 receptor activity at glutamatergic terminals caused impairment in hippocampus-dependent memory performance. On the other hand, elevated CB1 receptor levels provoked an increased protection against kainic acid-induced seizures and against excitotoxic neuronal cell death. This finding indicates the protective role of CB1 receptor on hippocampal glutamatergic terminals as a molecular stout guard in controlling excessive neuronal network activity. Hence, CB1 receptor on glutamatergic hippocampal neurons may represent a target for novel agents to restrain excitotoxic events and to treat neurodegenerative diseases. Endocannabinoid synthesizing and degrading enzymes tightly regulate endocannabinoid signaling, and thus, represent a promising therapeutic target. To further elucidate the precise function of the 2-AG degrading enzyme monoacylglycerol lipase (MAGL), MAGL was overexpressed specifically in hippocampal pyramidal neurons. This genetic modification resulted in highly increased MAGL activity accompanied by a 50 % decrease in 2-AG levels without affecting the content of arachidonic acid and anandamide. Elevated MAGL protein levels at glutamatergic terminals eliminated depolarization-induced suppression of excitation (DSE), while depolarization-induced suppression of inhibition (DSI) was unchanged. This result indicates that the on-demand availability of the endocannabinoid 2-AG is crucial for short-term plasticity at glutamatergic synapses in the hippocampus. Mice overexpressing MAGL exhibited elevated corticosterone levels under basal conditions and an increase in anxiety-like behavior, but surprisingly, showed no changes in aversive memory formation and in seizure susceptibility. This finding suggests that 2 AG-mediated hippocampal DSE is essential for adapting to aversive situations, but is not required to form aversive memory and to protect against kainic acid-induced seizures. Thus, specific inhibition of MAGL expressed in hippocampal pyramidal neurons may represent a potential treatment strategy for anxiety and stress disorders. Finally, the method of AAV-mediated cell type-specific transgene expression was advanced to allow drug-inducible and reversible transgene expression. Therefore, elements of the tetracycline-controlled gene expression system were incorporated in our “conditional” AAV vector. This approach showed that transgene expression is switched on after drug application and that background activity in the uninduced state was only detectable in scattered cells of the hippocampus. Thus, this AAV vector will proof useful for future research applications and gene therapy approaches.Der Cannabinoid Typ 1 (CB1) Rezeptor ist an einer Vielzahl physiologischer Funktionen beteiligt und heterogen in verschiedenen neuronalen Populationen exprimiert. Die unterschiedlichen Auswirkungen der CB1 Rezeptoraktivierung in glutamatergen bzw. GABAergen Neuronen wurden in verschiedenen Studien an Zelltyp-spezifischen CB1 Rezeptor-Knockout-Mäusen gezeigt. Um ein umfassendes Bild der CB1 Rezeptor-vermittelten Effekte zu gewinnen, wurde in dieser Studie ein Mausmodell entwickelt, das eine Zelltyp-spezifische Erhöhung der CB1 Rezeptorexpression aufweist und Zelltyp-spezifische CB1 Rezeptor-Knockout-Modelle komplementieren soll. Hierfür wurde eine innovative Methode durch Kombination von Adeno-assoziierten Virus (AAV)-vermitteltem Gentransfer und Cre-vermittelter Rekombination entwickelt, die Hirnregion- und Zelltyp-spezifische Transgenexpression gewährleistet. Mithilfe dieser Methode konnte eine Überexpression des CB1 Rezeptors in glutamatergen Pyramidenneuronen des Hippocampus der Maus erzeugt werden. Es wurde gezeigt, dass eine Steigerung der CB1 Rezeptoraktivität in glutamatergen Neuronen zu einer Beeinträchtigung der Hippocampus-abhängigen Gedächtnisleistung führte. Andererseits schützten erhöhte CB1 Rezeptormengen vor Kainat-induzierten epileptischen Krampfanfällen und vor excitotoxischem Zelltod. Diese Erkenntnis deutet an, dass der CB1 Rezeptor in glutamatergen Neuronen eine protektive Funktion vor übermäßiger Netzwerkaktivität einnimmt und somit einen Ansatzpunkt für die Therapie von neurodegenerativen Erkrankungen darstellt. Endocannabinoid-synthetisierende und -degradierende Enzyme regulieren das Ausmaß der Endocannabinoid-vermittelten Rezeptoraktivierung und stellen dadurch einen vielversprechenden therapeutischen Ansatzpunkt dar. Um die exakte Funktion des 2-AG-degradierenden Enzyms Monoacylglycerol-Lipase (MAGL) weiter aufzuklären, wurde MAGL spezifisch in hippocampalen Pyramidenneuronen überexprimiert. Diese genetische Modifikation führte zu einer gesteigerten MAGL-Aktivität und zu einer Reduzierung von 2 AG um 50 % ohne den Gehalt an Arachidonsäure und Anandamid zu beeinflussen. Erhöhte MAGL Mengen in glutamatergen Neuronen unterbanden die synaptische Depression der Glutamatausschüttung, genannt „depolarization-induced suppression of excitation” (DSE), während die synaptische Depression der GABA-Ausschüttung, genannt „depolarization-induced suppression of inhibition“ (DSI), unverändert war. Dieses Ergebnis deutet an, dass die Verfügbarkeit des Endocannabinoids 2-AG entscheidend für die Kurzzeitdepression an glutamatergen Synapsen im Hippocampus ist. MAGL-überexprimierende Mäuse wiesen erhöhte Werte des Stresshormons Corticosteron unter Basalbedingungen auf und zeigten erhöhtes Angstverhalten. Überraschenderweise hatte die MAGL-Überexpression keinen Einfluss auf die Bildung eines aversiven Gedächtnisses und auf die Anfälligkeit für Krampfanfälle. Die gewonnen Erkenntnisse weisen darauf hin, dass 2-AG-vermitteltes hippocampales DSE für eine Adaptierung auf eine aversive Situation erforderlich ist, aber nicht für die Bildung eines aversiven Gedächtnisses und die Protektion vor Kainat-induzierten Krampfanfällen benötigt wird. Folglich könnte eine spezifische Inhibition von MAGL in hippocampalen Pyramidenneuronen eine potentielle Behandlungsstrategie für Angst- und Belastungsstörungen darstellen. Schließlich wurde die Methode der AAV-vermittelten Zelltyp-spezifischen Transgenexpression weiterentwickelt, um eine Pharmakon-induzierbare und reversible Transgenexpression zu ermöglichen. Hierfür wurden Elemente des Tetracyclin-induzierbaren Genexpressionssystems in unseren „konditionalen“ AAV Vektor integriert. Dieser Ansatz zeigte, dass die Transgenexpression nach Gabe des Tetracyclin-Analogons Doxycyclin angeschaltet werden kann, und dass unspezifische Hintergrundexpression in Abwesenheit von Doxycyclin nur in vereinzelten Zellen des Hippocampus nachgewiesen werden konnte. Dieser AAV Vektor wird sich für künftige Forschungsansätze und Gentherapiestudien als nützlich erweisen

Recombination of AAV genomes is restricted to Cre-expressing neurons in vivo.

<p>AAV-Stop-GFP was injected to the hippocampus of NEX-Cre transgenic mice and GFP expression visualized in brain sections two months later. Transduced CA1 pyramidal neurons express GFP (A) and Cre recombinase (B). As expected, the merge picture (C) shows segregated subcellular expression domains of the cytosolic GFP and the nuclear Cre. Bar: 25 µm.</p

Transgenic HA-tagged CB1 receptor is expressed in hippocampal pyramidal neurons.

<p>AAV-Stop-CB1 was injected bilaterally to the hippocampus of NEX-Cre transgenic (AAV-Glu-CB1) mice and their respective wild-type (AAV-WT) littermates. <b>A</b>, Cre-activated CB1 expression occurred in pyramidal neurons and appeared in a similar pattern compared to AAV-Stop-GFP confirming the integrity of the system. GC, granule cell layer; Hil, hilar region of dentate gyrus; LMol, stratum lacunosum-molecularis; Mol, stratum molecularis; Or, stratum oriens; Pyr, CA1/CA3 pyramidal cell layer; Rad, stratum radiatum. Bar: 250 µm. <b>B-D</b>, Higher magnification micrographs of dentate granule cells shown in A. Immunohistochemistry for the HA-tag reveals co-expression of CB1 and VGluT1 in the inner molecular layer of the dentate gyrus, validating the presynaptic localization of transgenic CB1 receptor. Bar in D: 25 µm. <b>E</b>, Western blot analysis of hippocampal homogenates of AAV-Glu-CB1 (n = 3) and AAV-WT (n = 3) mice. AAV-Glu-CB1 mice express significantly increased levels of CB1 receptor protein. Data are normalized for α-tubulin (p<0.001; unpaired t test analysis, two-tailed). <b>F</b>, Stimulation of [³⁵S]GTPγS binding in hippocampal homogenates of AAV-Glu-CB1 mice (n = 6) and AAV-WT littermates (n = 3) was determined by various concentrations of the CB1 agonist HU-210. Basal binding was measured in absence of HU-210 and defined as 0% in each experiment. Data are presented as percentage stimulation above basal [³⁵S]GTPγS binding and are the means ± SEM, all performed in duplicates. The non-linear regression curve illustrates that overexpression of the CB1 receptor in hippocampal pyramidal neurons resulted in significantly enhanced cannabinoids-induced G-protein activation and thus increased cannabinoid signaling. EC₅₀ = 3.26±0.08 nM (AAV-Glu-CB1), 2.97±0.13 nM (AAV-WT); E_max  = 224.4±7.39 (AAV-Glu-CB1), 93.77±4.99 (AAV-WT). Unpaired t test analysis, two-tailed: p<0.0001.</p

Behavioural effects of elevated CB1 receptor levels in hippocampal pyramidal neurons and mossy cells.

<p>Seizures were induced in AAV-Glu-CB1 mice (n = 15) and AAV-WT controls (n = 11) by i.p. injection of kainic acid (30 mg/kg). <b>A</b>, Seizure severity was reduced in AAV-Glu-CB1 mice at every time point of scoring compared to AAV-WT controls without reaching significance (p = 0.065, Mann Whitney test, two-tailed). <b>B</b>, The average behavioral score over a period of 120 min is significantly decreased in AAV-Glu-CB1 mice (p = 0.0007, Mann Whitney test, two-tailed) indicating improved protection against KA-induced seizures. <b>C</b>, Kaplan–Meier survival curves of AAV-Glu-CB1 and AAV-WT mice during KA treatment are significantly different between both genotypes (p = 0.0494, log rank test). The survival rate at 180 min after KA injection was 53% of AAV-Glu-CB1 vs. 18% of AAV-WT mice.</p

Cre recombinase-activated transgene expression.

<p><b>A</b>, AAV expression cassette. Top, Silencing of transgene expression by transcriptional termination cassette containing three polyadenylation sites flanked by loxP sites (triangles). Bottom, Cre recombinase-mediated excision of the Stop cassette leading to transcription of the transgene. ITR, inverted terminal repeat; CBA, chicken-β-actin promoter; 3xpA, three polyadenylation (pA) signals; GOI, gene of interest; WPRE, woodchuck hepatitis virus post-transcriptional regulatory element. <b>B–G</b>, HEK cells were co-transfected with pAAV-Stop-GFP and pAAV-Cre (B–D) or pAAV-empty (E–G) and GFP immunofluorescence is strictly limited to Cre transfectants. Transgene expression is tightly inhibited when pAAV-Stop-GFP is co-transfected with pAAV-empty. Blue: cell nucleus staining with DAPI, Green: GFP immunostaining, Red: Cre recombinase immunostaining. Bar in G: 50 µm. <b>H–P</b>, Two months after stereotaxic vector delivery (AAV-GFP or AAV-Stop-GFP) to the dorsal hippocampus of adult wild-type or NEX-Cre mice, GFP epifluorescence was assessed in brain sections. <b>H–J</b>, AAV-GFP efficiently transduces all types of neurons of the hippocampal formation, in particular in CA1, CA2, CA3, the hilar region and the dentate gyrus. Note that transduced interneurons (arrowheads in H) can be visualized in areas of low GFP abundance. GC, granule cell layer; Hil, hilar region; LMol, stratum lacunosum-molecularis; Mol, stratum molecularis; Or, stratum oriens; Pyr, CA1/CA3 pyramidal cell layer; Rad, stratum radiatum. <b>K–M</b>, After AAV-Stop-GFP injection, GFP expression was not detectable in wild-type mice. <b>N–P</b>, In NEX-Cre mice, neurons of the pyramidal cell layer express the reporter gene, while granule cells of the dentate gyrus are spared. Note that in this mouse line, Cre recombinase is not expressed in the adult dentate gyrus (see Goebbels et al., 2006). Bar in M: 250 µm.</p

Impaired anandamide/palmitoylethanolamide signaling in hippocampal glutamatergic neurons alters synaptic plasticity, learning, and emotional responses

Endocannabinoid signaling via anandamide (AEA) is implicated in a variety of neuronal functions and considered a promising therapeutic target for numerous emotion-related disorders. The major AEA degrading enzyme is fatty acid amide hydrolase (FAAH). Genetic deletion and pharmacological inhibition of FAAH reduce anxiety and improve emotional responses and memory in rodents and humans. Complementarily, the mechanisms and impact of decreased AEA signaling remain to be delineated in detail. In the present study, using the Cre/loxP system combined with an adeno-associated virus (AAV)-mediated delivery system, FAAH was selectively overexpressed in hippocampal CA1-CA3 glutamatergic neurons of adult mice. This approach led to specific FAAH overexpression at the postsynaptic site of CA1-CA3 neurons, to increased FAAH enzymatic activity, and, in consequence, to decreased hippocampal levels of AEA and palmitoylethanolamide (PEA), but the levels of the second major endocannabinoid 2-arachidonoyl glycerol (2-AG) and of oleoylethanolamide (OEA) were unchanged. Electrophysiological recordings revealed an enhancement of both excitatory and inhibitory synaptic activity and of long-term potentiation (LTP). In contrast, excitatory and inhibitory long-term depression (LTD) and short-term synaptic plasticity, apparent as depolarization-induced suppression of excitation (DSE) and inhibition (DSI), remained unaltered. These changes in hippocampal synaptic activity were associated with an increase in anxiety-like behavior, and a deficit in object recognition memory and in extinction of aversive memory. This study indicates that AEA is not involved in hippocampal short-term plasticity, or eLTD and iLTD, but modulates glutamatergic transmission most likely via presynaptic sites, and that disturbances in this process impair learning and emotional responses

Impaired 2-AG signaling in hippocampal glutamatergic neurons : aggravation of anxiety-like behavior and unaltered seizure susceptibility

Background: Postsynaptically generated 2-arachidonoylglycerol activates the presynaptic cannabinoid type-1 receptor, which is involved in synaptic plasticity at both glutamatergic and GABAergic synapses. However, the differential function of 2-arachidonoylglycerol signaling at glutamatergic vs GABAergic synapses in the context of animal behavior has not been investigated yet. Methods: Here, we analyzed the role of 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons. Monoacylglycerol lipase, the primary degrading enzyme of 2-arachidonoylglycerol, is expressed at presynaptic sites of excitatory and inhibitory neurons. By adeno-associated virus-mediated overexpression of monoacylglycerol lipase in glutamatergic neurons of the mouse hippocampus, we selectively interfered with 2-arachidonoylglycerol signaling at glutamatergic synapses of these neurons. Results: Genetic modification of monoacylglycerol lipase resulted in a 50% decrease in 2-arachidonoylglycerol tissue levels without affecting the content of the second major endocannabinoid anandamide. A typical electrophysiological read-out for 2-arachidonoylglycerol signaling is the depolarization-induced suppression of excitation and of inhibition. Elevated monoacylglycerol lipase levels at glutamatergic terminals selectively impaired depolarization-induced suppression of excitation, while depolarization-induced suppression of inhibition was not significantly changed. At the behavioral level, mice with impaired hippocampal glutamatergic 2-arachidonoylglycerol signaling exhibited increased anxiety-like behavior but showed no alterations in aversive memory formation and seizure susceptibility. Conclusion: Our data indicate that 2-arachidonoylglycerol signaling selectively in hippocampal glutamatergic neurons is essential for the animal’s adaptation to aversive situations