Evaluating Transduction Efficiency of Medial Septal Neurons: A Comparative Study of Adeno-Associated Viruses with Three Distinct Promoters

Evaluating Transduction Efficiency of Medial Septal Neurons: A Comparative Study of Adeno-Associated Viruses with Three Distinct Promoters | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 13 February 2025 V1 Latest version Share on Evaluating Transduction Efficiency of Medial Septal Neurons: A Comparative Study of Adeno-Associated Viruses with Three Distinct Promoters Authors : Alena Koryagina , Yulia Dobryakova1 , Konstantin Gerasimov , Ghofran Alkhalabi , Alexandr Moshchenko , Vsevolod Belousov , and Alexey Bolshakov 0000-0001-5915-8847 [email protected] Authors Info & Affiliations https://doi.org/10.22541/au.173943977.73866826/v1 337 views 123 downloads Contents Abstract Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract We evaluated the transduction efficacy of adeno-associated viruses (AAVs) carrying the green fluorescent protein (GFP) marker gene under three distinct promoters—CAG, synapsin, and the mouse choline acetyltransferase (Chat) promoter—in cholinergic and parvalbumin-positive (PV+) neurons within the medial septal area of mice and rats. We quantified the proportion of Chat-positive and PV+ neurons expressing GFP in both species. In mice, the CAG and synapsin promoters demonstrated extremely low efficacy for GFP expression in Chat-positive neurons but were highly effective in transducing PV+ neurons. In contrast, these promoters were equally effective in transducing both Chat-positive and PV+ neurons in rats. The Chat promoter yielded moderate GFP expression in cholinergic neurons in mice, with negligible expression in PV+ neurons, though it also led to off-target expression in other cell types. In rats, the Chat promoter produced moderate GFP expression in both cholinergic and PV+ neurons; however, the majority of GFP-expressing cells were unrelated to these specific neuronal subtypes. Evaluating Transduction Efficiency of Medial Septal Neurons: A Comparative Study of Adeno-Associated Viruses with Three Distinct Promoters Alena A. Koryagina 1 , Yulia V. Dobryakova 1 , Konstantin A. Gerasimov 1, 2 , Ghofran Alkhalabi 1 , Alexandr A. Moshchenko 3 , Vsevolod V. Belousov 2,3,4,5 , Alexey P. Bolshakov 1 1 Institute of Higher Nervous Activity and Neurophysiology, Russian Academy of Sciences, Moscow, Russia 2 Russian National Medical Research University, Moscow, Russia 3 Federal Center of Brain Research and Neurotechnologies, Federal Medical Biological Agency, Moscow, Russia. 4 Shemyakin-Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, Russia 5 Life Improvement by Future Technologies (LIFT) Center, Moscow, Russia Correspondence should be addressed to A.P.B. ( [email protected] ) Moscow, Russia Corresponding author’s address: Institute of Higher Nervous Activity and Neurophysiology, RAS, 5A Butlerova street, 117485 Moscow, Russia; phone: +7 (495) 334-70-00; fax: +7 (499) 743-00-56; email: [email protected] Short running title: Comparison of three promoters ACKNOWLEDGMENTS This research was funded by the Russian Science Foundation (RSF), grant number 23-75-30023. Abstract We evaluated the transduction efficacy of adeno-associated viruses (AAVs) carrying the green fluorescent protein (GFP) marker gene under three distinct promoters—CAG, synapsin, and the mouse choline acetyltransferase (Chat) promoter—in cholinergic and parvalbumin-positive (PV+) neurons within the medial septal area of mice and rats. We quantified the proportion of Chat-positive and PV+ neurons expressing GFP in both species. In mice, the CAG and synapsin promoters demonstrated extremely low efficacy for GFP expression in Chat-positive neurons but were highly effective in transducing PV+ neurons. In contrast, these promoters were equally effective in transducing both Chat-positive and PV+ neurons in rats. The Chat promoter yielded moderate GFP expression in cholinergic neurons in mice, with negligible expression in PV+ neurons, though it also led to off-target expression in other cell types. In rats, the Chat promoter produced moderate GFP expression in both cholinergic and PV+ neurons; however, the majority of GFP-expressing cells were unrelated to these specific neuronal subtypes. KEYWORDS cholinergic neuron, CAG promoter, hSyn promoter, medial septum, parvalbumin-positive neuron, adeno-associated virus INTRODUCTION Recombinant adeno-associated viruses (AAV) are frequently used to achieve efficient neuronal transduction in the central nervous system (CNS). They have low immunogenicity (Verdera et al. , 2020; Chandler et al. , 2021; Whitehead et al. , 2021) which makes them more suitable for use in clinical trials for therapy of various diseases including neurological diseases. Many studies showed that AAV serotype and promoter in virus cassette are two main factors that determine transgene expression in the desired cell population(Naso et al. , 2017; Challis et al. , 2022). The efficacy of promotor strongly depends on the cell type that was transduced by AAV since every type of cell expresses a unique set of transcription factors that determine its phenotype and ability to initiate transcription from given promotor. Many studies in CNS used AAVs with CAG or synapsin promoters that are considered as universal for infection of neurons and are frequently used as reference promoters for comparison (Santoscoy et al. , 2023). Other studies employ promoters specific for some neuronal subpopulations, like promoters of genes encoding choline acetyltransferase (Chat) (Gamage et al. , 2023; Santoscoy et al. , 2023), tyrosine hydroxylase (Th)(Oh et al. , 2009), and tryptophan hydroxylase (Tph2)(Benzekhroufa et al. , 2009), to provide specific expression in cholinergic, catecholaminergic, and serotonergic neurons, respectively. Basal forebrain cholinergic neurons, including neurons in the nucleus basalis of Meynert, medial septal nucleus, and diagonal band of Broca, play an important role in processes of memory formation, learning, and stress response and their loss occurs in several neurodegenerative diseases such as Alzheimer’s disease and dementia with Lewy bodies (Liu et al. , 2018; Geula et al. , 2021; Kniffin et al. , 2024). It was proposed that salvation of these neurons may help to prevent or slow down cognitive decline in people with the mentioned diseases. Therefore, development of approaches that provide selective expression of genes, whose protein products have neuroprotective characteristics, in these cholinergic neurons is considered as relevant task not only for experimental science but also for possible use in human gene therapy. Targeted expression of desired gene in cholinergic neurons may be achieved using promoter selective for these neurons. Since Chat is a gene selectively expressed in cholinergic neurons, its promoter region was used to provide selective transgene expression in cholinergic neurons in entire CNS of transgenic mice (Gamage et al. , 2023). Mouse Chat promoter was also used to create AAVs for transgene expression in cholinergic neurons in the medial septal area (MSA) (Mu et al. , 2022). AAV-based transduction of cholinergic striatal neurons was performed using human truncated CHAT promoter (Santoscoy et al. , 2023).Other studies used AAVs with undescribed variants of Chat promoter either from commercial source (Mei et al. , 2024) or friendly laboratory (Martel et al. , 2020) with unclear specificity. Specificity of the mentioned promoters was analyzed in mice and never compared with specificity in other model animals. Here, we compared efficacy of expression of green fluorescent protein (eGFP) in the MSA of mice and rats transduced with AAV9 vectors carrying eGFP under synthetic CAG promotor, synapsin promotor, and mouse Chat promotor. We analyzed expression of eGFP in two major MSA subpopulations, GABAergic parvalbumin-positive neurons and cholinergic neurons. MATERIALS AND METHODS Design of plasmids for viral production Vector PT-3825 that carries Chat promoter was received from BrainVTA. It contains mCherry under Chat promoter (the first intron of mouse Chat gene) fused to Cre recombinase with P2A. Fragment mCherry-p2A-CRE was excised with SalI and EcoRI restriction enzymes and replaced with sequence of enhanced green fluorescent protein (EGFP). As a result, we obtained the vector pAAV-Chat_promoter-EGFP. Then two vectors with other promoters were received using backbone of vector pAAV-Chat_promoter-EGFP: Chat promoter was excised with SalI and MluI restriction enzymes and replaced with CAG or hSyn promoters. All three newly synthesized vectors (pAAV-Chat-EGFP, pAAV-CAG-EGFP, pAAV-hSyn-EGFP) with confirmed sequences were used for further virus packaging. AAV production AAV were produced using the HEK293TN adherent cell line. Cells were cultured in DMEM (Thermo Fisher, USA) with 10% FBS (Biosera, France) and 1% Pen/Strep (Paneco, Russia) at 37 °C with 5% CO₂. Prior to transfection, 75,000 cells/cm² were seeded in T-175 flasks (SPL, South Korea) and incubated overnight. Transient transfection was performed using PEI MAX 40K (Polysciences, USA) with GOI, pHelper, and RepCap9 plasmids in a 1:1:1 molar ratio. After 18–22 hours, the medium was replaced with fresh growth medium containing 2% FBS. Four days post-transfection, cells were harvested, centrifuged at 2300 × g for 5 minutes, and lysed by freezing/thawing. The lysate was treated with Denarase endonuclease (c-Lecta, Germany) for 1 hour at 37 °C, then centrifuged at 12,000 × g for 10 minutes. The clarified lysate was purified using an iodixanol gradient (Zolotukhin et al. , 1999) in a 6 ml ultracentrifuge tube. The AAV-containing 40% layer was collected and concentrated with Amicon Ultra-4 (Merck, Germany). Viral particles were diluted in PBS with 0.001% Pluronic F68 (Applichem, Germany) and 150 mM NaCl (NeoFroxx, Germany) and stored at -80 °C. Genome copy numbers was determined by qPCR. AAV samples were treated with DNase I (Thermo Fisher, USA) and analyzed using the Rotor-Gene Q system (Qiagen, USA). PCR reactions used qPCRmix-HS (Evrogen, Russia) with primers and FAM/BHQ probe (Aurnhammer et al. , 2012). Animals The experiments were performed with adult male Wistar rats (250–350 g) and adult mice (25–30 g). Rats and mice were received from “Stolbovaya” Nursery (Russia). All experiments were performed in accordance with the ethical principles stated in the Directive 2010/63/EU of the European Parliament and of the Council of 22 September 2010 and were approved by the Ethical Committee of the Institute of Higher Nervous Activity and Neurophysiology of the Russian Academy of Sciences (protocol no. 3 of May 4, 2023). Before the surgery, the animals were randomly divided into groups. Rats and mice were randomly subdivided into the following three groups: animals with AAV expressing EGFP with the CAG promotor (CAG-rats, n = 7, CAG-mice, n = 5), animals treated with AAV-EGFP with Syn promoter (Syn-rats, n = 5, Syn-mice, n = 4) and animals that received AAV-EGFP with Chat promoter (Chat-rats, n = 6, Chat-mice, n = 4). A total of 18 rats and 13 mice were involved in the study. During the experiments, all animals were maintained with a 12-h light–dark cycle and had ad libitum access to food and water. Stereotaxic surgery and virus administration AAV administration was performed using standard stereotaxic methods (CAG-EGFP titer was 6.5•10 11 gc/ml; hSyn-EGFP titer was 4.5•10 12 gc/ml; Chat-EGFP titer was 1.5•10 11 gc/ml). Animals were anesthetized with isoflurane (Baxter, United States) and mounted in a Kopf stereotaxic frame. AAV suspension (1 μL) was injected into the medial septum area (Rats: 0.4 mm anterior, 1.5 lateral to bregma, angle 14°; mice: 0.8 mm anterior, 1 lateral to bregma, angle 14°). All injections were performed through the Hamilton syringe (Hamilton company, United States) using a microinfusion pump (Stoelting Co., United States) at a rate of 0.2 μL/min. After each injection, the needle was left in situ for 10 min. Animals were allowed to recover for 14 days after the surgery. Immunohistochemistry All animals were anesthetized, the brains were removed, and the frontal part of the brain, which contains septum, was dissected and postfixed in 4% formalin (Panreac, Spain) solution in PBS (Biolot, Russia) for at least 2 days. The full extent of the septum was sectioned at a 50 μm thick coronal brain slices using a vibrating blade microtome (Leica VT1200 S). Two sections from the septum area (from bregma AP +1.2–+0.36 mm) were selected and stained for choline acetyltransferase (CHAT). Sections were incubated in 0.3% Тriton X-100 (SERVA, Germany) in 0.01 M PBS (PBS-T) and then for 1 h in blocking solution (5% normal goat serum in PBS, abcam, United States) in PBS-T and later in blocking solution with primary antibodies (rabbit anti-Choactase 1:500, abcam, United States) at 4°C overnight. The next day, sections were washed in PBS-T and incubated with Alexa 546 conjugated antibodies (diluted as 1:500 in the same blocking solution at room temperature for 2 h). Two additional sections from the MSA were stained for parvalbumin (PV). The staining procedure was the same as for CHAT, except the primary antibodies were for PV (rabbit anti-parvalbumin 1:500, Millipore, United States). In both cases, EGFP fluorescence was enhanced using immunostaining. In brief, after the preincubation step as above, the sections were placed for 1 h in blocking solution (5% normal goat serum in PBS, Sigma-Aldrich) and after that in blocking solution with primary chicken anti-GFP antibodies diluted as 1:1500 (Abcam, United States) at 4°C overnight. The next day, the sections were washed in PBS-T and incubated with secondary goat anti-chiсken Alexa 488 conjugated antibodies (diluted as 1:1000 in the same blocking solution at room temperature for 2 h). Fluorescent microscopy and image analysis and quantification Images of the medial septal area were taken with a Keyence microscope with a 10× dry objective for GFP and RFP intensity measurements. Z-stack images were taken in 1μm intervals and then used for construction of full focus images using Keyence BZ-II Analyzer 2.1 software. For the analysis of CHAT+ or PV+ neuron transduction efficiency, two different MSA sections were taken for each animal. The number of CHAT-positive, PV-positive, CHAT-positive/EGFP-positive, and PV-positive/EGFP-positive cells were counted using Fiji in a blinded fashion. The transduction efficiency of two neuron types was calculated as a proportion between the number of neurons double positive for EGFP and CHAT or PV and total number of CHAT- or PV-positive neurons, respectively. Statistical Analysis Non-parametric Kruskal-Wallis test, followed by Mann–Whitney U test, was used to reveal the differences between the groups. The differences were considered significant at p < 0.05. RESULTS Injection of AAVs into the MSA leads to infection of cells in MSA and expression of marker protein EGFP. Analysis of colocalization of EGFP and marker of cholinergic neurons CHAT and marker of GABAergic neurons parvalbumin (PV) showed that the efficacy of transduction of different neurons depended on the promoter in the AAV cassette and type of experimental animal. EGFP expression in cholinergic neurons In mice, AAVs with CAG and synapsin promoter had similar efficacy for EGFP expression in cholinergic neurons. About 10% of cholinergic neurons expressed EGFP after transduction with AAVs carrying these two promoters (Fig. 1A,B,D; Supplementary Fig. S1). In case of AAVs with CHAT promoter, on the average 20% of cholinergic neurons expressed EGFP (Fig. 1B, D; Supplementary Fig. S1). In rats, the efficacy of studied promoters for expression in cholinergic cells was similar to the efficacy observed in mice. CHAT was the most effective among three promoters for expression of EGFP in cholinergic neurons (Fig. 2; Supplementary Fig. S2). EGFP expression in parvalbumin-positive neurons In mice, AAVs with CAG promoter had the highest efficacy for EGFP expression in PV-positive MSA neurons (Fig. 3A-D; Supplementary Fig. S3). On the average, 35% PV-positive neurons expressed EGFP (Fig. 3D). Efficacy of synapsin promoter was smaller; about 20% PV-positive MSA neurons expressed EGFP (Fig. 3C, D). The lowest efficacy had CHAT promoter which provided EGFP expression in about 5% PV-positive cells (Fig. 3B, D). In rats, in contrast to mice, EGFP expression in PV-positive MSA neurons was similar in all groups of rats independently of the promoter in AAV cassette (Fig. 4A-D; Supplementary Fig. S4). DISCUSSION Current studies in neuroscience require development of technical approaches that would provide manipulations with desired cell subpopulation. Usually, these manipulations include expression of given gene, for example proteins for opto- or chemogenetics, after transduction of cells with AAVs. Viral suspensions carrying desired genetic construct may be stereotactically injected in the target brain area. However, this approach requires analysis of efficacy of promoter for transduction of target cell subpopulation as well as other cells located in the targeted brain area. Some promoters, like CAG and human synapsin promoter, were considered as “universal” since they were reported to be effective in the majority of glutamatergic and GABAergic neurons. Our study shows that these two universal promoters provide very low expression of target gene in cholinergic neurons in MSA but they were effective for expression in PV-positive MSA neurons and other MSA cells which, according to their size and morphology, may be related to neurons. Low efficacy of CAG and synapsin promoters is not related to low tropism of AAVs to cholinergic neurons because AAVs of the same serotype with CHAT promoter had higher efficacy, which in some animals reached up to 35-45%. We expected that the efficacy of expression provided by universal promoters and Chat promoter in cholinergic neurons will be comparable since previous studies with CHAT-Cre mice showed that transduction with AAVs with these promoters provided expression in cholinergic MSA neurons when injected to transgenic Chat-Cre mice (Mineur et al. , 2022; Mu et al. , 2022). It was recently described that high viral load during intracerebral injections of AAVs may decrease specificity of promoter in AAV (Furlanis et al. , 2024). Presumably, in our and other mentioned cases, CAG and synapsin promoters were effective only in cholinergic cells that underwent high viral load. The mouse promoter used in our study includes part of the first intron and second exon of mouse Chat gene. In rats, similar position in Chat gene has sequence with 78% homology to mouse gene which, probably, provides basis for functioning of the mouse sequence as a promoter in both rats and mice. The human first intron of CHAT gene contains sequence that is 65% homologous to the Chat promoter used in our study. However, this sequence does not include previously reported promoter sequence in the first intron of human CHAT gene (Santoscoy et al. , 2023). Importantly, mouse and rat sequences of the first intron do not contain sequences that are homologous to the mentioned human promoter sequence. It appears that this human promoter sequence can still provide expression in mouse striatal cholinergic cells (Santoscoy et al. , 2023). Our data together with previous data suggest that both mouse and human promoter sequences contain regulatory elements that can provide specificity for expression in cholinergic neurons. Our study shows that mouse Chat promoter functions equally well in both rats and mice for transduction of cholinergic cells. When we analyzed transduction of non-cholinergic PV-positive MSA neurons, we found that autologous Chat promoter had small efficacy in mouse PV-positive neurons compared to CAG and synapsin promoters. However, in rats, mouse Chat promoter was as effective as other two mentioned promoters in non-cholinergic PV-positive cells. The latter suggests that although heterologous promoter may provide effective expression in the desired cell subpopulation, it still may lose specificity and lead to transgene expression in undesirable cell subpopulations. CONCLUSION Our study shows that in both mice and rats human synapsin promoter and synthetic CAG promoter are effective for transgene expression in parvalbumin-positive MSA neurons but not in cholinergic MSA neurons. Murine Chat promoter is effective for expression in cholinergic MSA neuron in both rats and mice, however, in rats in provides expression also in parvalbumin-positive MSA neurons. DATA AVAILABILITY STATEMENT The data that support the findings of this study are available from the corresponding author upon reasonable request. ACKNOWLEDGMENTS This research was funded by the Russian Science Foundation (RSF), grant number 23-75-30023. AUTHOR CONTRIBUTIONS Koryagina, cloning, surgical operations, immunohistochemical staining, image analysis, writing first draft; Gerasimov, surgical operations, fluorescent microscopy; Al-Khalabi, surgical operations, preparation of sections; Dobryakova, surgical operations, immunohistochemical staining, writing first draft; Moshchenko, viral vectors production; Belousov, study design, supervision, funding acquisition; Bolshakov, supervision, writing first draft. DECLARATION OF INTERESTS The authors declare no conflicts of interests. REFERENCES Aurnhammer, C., Haase, M., Muether, N., Hausl, M., Rauschhuber, C., Huber, I., Nitschko, H., Busch, U., Sing, A., Ehrhardt, A., & Baiker, A. 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Panels A-C show overlay of EGFP immunostaining (green) and Chat immunostaining in the medial septal area. Panel D shows proportion of Chat-positive cells that expressed EGFP after transduction with AAVs with different promoters. *, p<0.0003, Mann-Whitney test. Fig. 3. Transduction of the mouse medial septal area with AAVs carrying EGFP under CAG (A), Chat (B), and synapsin (C) promoters. Panels A-C show overlay of EGFP immunostaining (green) and parvalbumin (pvalb) immunostaining in the medial septal area. Panel D shows proportion of pvalb-positive cells that expressed EGFP after transduction with AAVs with different promoters. *, p<0.0001, Mann-Whitney test. Fig. 4. Transduction of the rat medial septal area with AAVs carrying EGFP under CAG (A), Chat (B), and synapsin (C) promoters. Panels A-C show overlay of EGFP immunostaining (green) and parvalbumin (pvalb) immunostaining in the medial septal area. Panel D shows proportion of pvalb-positive cells in the medial septal area that expressed EGFP after transduction with AAVs with different promoters. The groups with different promoters do not differ significantly. Information & Authors Information Version history V1 Version 1 13 February 2025 Copyright This work is licensed under a Non Exclusive No Reuse License. Keywords cag promoter cholinergic neurons hsyn promoter parvalbumin Authors Affiliations Alena Koryagina FSBSI Institute of Higher Nervous Activity and Neurophysiology of RAS View all articles by this author Yulia Dobryakova1 FSBSI Institute of Higher Nervous Activity and Neurophysiology of RAS View all articles by this author Konstantin Gerasimov FSBSI Institute of Higher Nervous Activity and Neurophysiology of RAS View all articles by this author Ghofran Alkhalabi FSBSI Institute of Higher Nervous Activity and Neurophysiology of RAS View all articles by this author Alexandr Moshchenko Federal Center of Brain Research and Neurotechnologies View all articles by this author Vsevolod Belousov Pirogov Russian National Research Medical University View all articles by this author Alexey Bolshakov 0000-0001-5915-8847 [email protected] FSBSI Institute of Higher Nervous Activity and Neurophysiology of RAS View all articles by this author Metrics & Citations Metrics Article Usage 337 views 123 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Alena Koryagina, Yulia Dobryakova1, Konstantin Gerasimov, et al. Evaluating Transduction Efficiency of Medial Septal Neurons: A Comparative Study of Adeno-Associated Viruses with Three Distinct Promoters. Authorea . 13 February 2025. DOI: https://doi.org/10.22541/au.173943977.73866826/v1 If you have the appropriate software installed, you can download article citation data to the citation manager of your choice. Simply select your manager software from the list below and click Download. For more information or tips please see 'Downloading to a citation manager' in the Help menu . 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