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Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder

Molecular Autism volume 16, Article number: 26 (2025) Cite this article

Abstract

Background

The key pathological mechanisms underlying autism spectrum disorder (ASD) remain relatively undetermined, potentially due to the heterogenous nature of the condition. Targeted studies of a series of monogenic ASDs have revealed postsynaptic dysfunction as a central conserved mechanism. Presynaptic dysfunction is emerging as an additional disease locus in neurodevelopmental disorders; however, it is unclear whether this dysfunction drives ASD or is an adaptation to the altered brain microenvironment.

Methods

To differentiate between these two competing scenarios, we performed a high content analysis of key stages of the synaptic vesicle lifecycle in primary neuronal cultures derived from a series of preclinical rat models of monogenic ASD. These five independent models (Nrxn1+/−, Nlgn3−/y, Syngap+/−, Syngap+/Δ−GAP, Pten+/−) were specifically selected to have perturbations in a diverse palette of genes that were expressed either at the pre- or post-synapse. Synaptic vesicle exocytosis and cargo trafficking were triggered via two discrete trains of activity and monitored using the genetically-encoded reporter synaptophysin-pHluorin. Activity-dependent bulk endocytosis was assessed during intense neuronal activity using the fluid phase marker tetramethylrhodamine-dextran.

Results

Both synaptic vesicle fusion events and cargo trafficking were unaffected in all models investigated under all stimulation protocols. However, a key convergent phenotype across neurons derived from all five models was revealed, a depression in activity-dependent bulk endocytosis.

Limitations

The study is exclusively conducted in primary cultures of hippocampal neurons; therefore, the impact on neurons from other brain regions or altered brain microcircuitry was not assessed. No molecular mechanism has been identified for this depression.

Conclusion

This suggests that depression of activity-dependent bulk endocytosis is a presynaptic homeostatic mechanism to correct for intrinsic dysfunction in ASD neurons.

Background

Autism spectrum disorder (ASD) impacts up to 1 in 150 children, with intellectual disability (ID) being a frequent co-morbidity [1]. Despite this high prevalence, the mechanisms that result in brain dysfunction at the molecular, cell and circuit level in these neurodevelopmental disorders are largely undetermined. The study of monogenic ASD provides an opportunity to decipher these key mechanisms via the identification of a molecular locus [2]. Interestingly, a number of the most prevalent genetic causes of these disorders concentrate on genes that control synaptic events [3]. In support, multiple preclinical monogenic ASD models display defects at the synaptic and circuit level, consistent with perturbations observed in humans with similar mutations [4, 5].

The synapse is comprised of the presynapse, which releases chemical neurotransmitters during invasion of action potentials (APs), and the postsynapse which integrates this input and adapts output via a series of plastic changes. Postsynaptic alterations are commonly observed in rodent models of ASD, supporting the view that this subcellular structure is a physical convergence point in the genesis and expression of ASD [6]. Key proteins include synaptic Ras GTPase-activating protein 1 (SynGAP1) and fragile X messenger ribonucleoprotein (FMRP), which control synaptic plasticity via excitatory α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor trafficking and protein translation downstream from postsynaptic metabotropic receptors respectively [7,8,9]. Furthermore, dysfunction in cell adhesion molecules such as presynaptic neurexins and postsynaptic neuroligins also commonly result in ASD via incorrect stabilisation and maintenance of synapse function [10,11,12]. Therefore, dysfunction in a series of genes, including those encoding key postsynaptic molecules is a highly prevalent cause of ASD.

In contrast to the postsynapse, presynaptic dysfunction in neurodevelopmental disorders is still relatively under-researched. This is in spite of a cohort of key genes essential for neurotransmitter release having been identified as causal in conditions such as epilepsy, ID and ASD [13, 14]. Neurotransmitter release occurs in response to the activity-dependent influx of calcium, via voltage-gated channels, which triggers the fusion of neurotransmitter-containing synaptic vesicles (SVs) [15]. The functional pool of SVs is relatively small in typical central nerve terminals, making their efficient regeneration essential for the maintenance of neurotransmission. This is mediated by a series of endocytosis modes, which are recruited in both time and space by different patterns of neuronal activity [16, 17]. SV cargo retrieval is dependent on clathrin-mediated endocytosis (CME), which can occur at either the presynaptic plasma membrane or on intracellular endosomes generated by either ultrafast endocytosis (UFE) or activity-dependent bulk endocytosis (ADBE [18,19,20]. Ultrafast endocytosis saturates rapidly during AP trains [21], whereas ADBE is the dominant endocytosis mode during intense neuronal activity [22, 23].

We recently discovered a selective defect in ADBE in neurons lacking FMRP [24], which translated into a decrease in presynaptic performance during periods of high activity. Since this phenotype could be corrected via agonism of big potassium (BK) channels, we hypothesized that the depression of ADBE was an adaptation to the hyperexcitability observed in Fmr1−/y neurons and circuits [25,26,27]. In this study, we tested this hypothesis by examining SV recycling in a cohort of five independent monogenic rat ASD models the majority of which display hyperexcitability at either the cell or circuit level. We discovered that neurons from all models displayed depressed ADBE, with no obvious deficit in SV exocytosis or cargo trafficking. This therefore supports the hypothesis that a reduction in ADBE is a specific homeostatic adaptation to intrinsic synaptic dysfunction in ASD models.

Methods

Materials

Unless otherwise specified, all cell culture reagents were obtained from Invitrogen (Paisley, UK). Foetal bovine serum was from Biosera (Nuaille, France). Papain was obtained from Worthington Biochemical (Lakewood, NJ, USA). All other reagents were obtained from Sigma-Aldrich (Poole, UK) unless specified. Rabbit anti-SV2A was obtained from Abcam (Cambridge, UK; ab32942 RRID: AB_778192). Anti-rabbit Alexa Fluor 488 (A11008 RRID: AB_143165) was obtained from Invitrogen (Paisley, UK). Synaptophysin-pHluorin (sypHy) was a gift from Prof. L. Lagnado (University of Sussex, UK).

Rat models

Procedures were performed in accordance with the UK Animal (Scientific Procedures) Act 1986, under Project and Personal Licence authority and were approved by the Animal Welfare and Ethical Review Body at the University of Edinburgh (Home Office project licence – 7008878). Similarly, procedures were conducted in accordance with protocols approved by the Institutional Animal Ethics Committee of Institute for Stem Cell Science and Regenerative Medicine, Bangalore. All animals were killed by Schedule 1 procedures in accordance with UK Home Office Guidelines; adults were killed by exposure to CO2 followed by decapitation, whereas embryos were killed by decapitation followed by destruction of the brain. In Edinburgh, rats were housed on a 12/12 h light/dark cycle with a 21 ± 2 °C room temperature and food/water ad libitum. In Bangalore, rats were maintained on a 14 h light/10 h dark cycle with ad-libitum access to diet and water.

All transgenic rats in this study were generated by Horizon Discovery (now Envigo).

Sprague–Dawley Nlgn3−/y transgenic rats were created as previously described [28]. Long Evans-SGem1/PWC, referred to in the manuscript as Syngap+/− rats, were created as described [29]. Colony founders of Long Evans-SGem2/PWC, referred to as Syngap+/Δ−GAP, were produced by zinc finger nuclease-mediated deletion of the GAP domain of Syngap as described [30, 31]. Sprague Dawley Nrxn1tm1sage rats, referred to as Nrxn1+/− rats [32, 33] were purchased from Horizon Discovery. Sprague Dawley Pten+/− rats were purchased from Horizon Discovery and were generated as described previously [34].

Hippocampal cultures

Hippocampi from each embryo (e18.5-e19.5) were processed separately to avoid contamination across genotypes. For rats with an X chromosome mutation (Nlgn3), male embryos were taken for hippocampal dissection. For all other rat lines embryos of both sexes were used.

Dissociated primary hippocampal cultures were prepared from embryos as previously described [24]. Briefly, isolated hippocampi were digested in a 10 U/mL papain solution (Worthington Biochemical, LK003178) at 37 °C for 20 min. The papain was then neutralised using DMEM F12 (ThermoFisher Scientific, 21331-020) supplemented with 10% Foetal bovine serum (BioSera, S1810-500) and 1% penicillin/streptomycin (ThermoFisher Scientific, 15140-122). Cells were triturated to form a single cell suspension and plated at 5 × 104 cells (with the exception of single cell tetramethylrhodamine (TMR)-dextran uptake experiments, 2.5 × 104 cells) per coverslip on laminin (10 µg/ mL; Sigma Aldrich, L2020) and poly-D‐lysine (Sigma Aldrich, P7886) coated 25 mm glass coverslips (VWR International Ltd, Lutterworth, UK). Cultures were maintained in Neurobasal media (ThermoFisher Scientific, 21103-049) supplemented with 2% B-27 (ThermoFisher Scientific, 17504-044), 0.5 mM L‐glutamine (ThermoFisher Scientific, 25030-024) and 1% penicillin/streptomycin. After 2–3 days in vitro (DIV), 1 µM of cytosine arabinofuranoside (Sigma Aldrich, C1768) was added to each well to inhibit glial proliferation. Hippocampal neurons were transfected with sypHy at DIV 7 using Lipofectamine 2000 (ThermoFisher Scientific, 11668027) prior to imaging at DIV 13–15.

High content screening of SV recycling using sypHy

SypHy-transfected neurons were visualised at 500 nm band pass excitation with a 515 nm dichroic filter and a long-pass > 520 nm emission filter on a Zeiss Axio Observer D1 inverted epifluorescence microscope (Cambridge, UK). Images were captured using an AxioCam 506 mono camera (Zeiss) with a Zeiss EC Plan Neofluar 40x/1.30 oil immersion objective. Image acquisition was performed using Zen Pro software (Zeiss). Hippocampal cultures were mounted in a Warner Instruments (Hamden, CT, USA) imaging chamber with embedded parallel platinum wires (RC-21BRFS) while undergoing constant perfusion with imaging buffer (119 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM HEPES, 30 mM glucose at pH 7.4, supplemented with 10 µM 6-cyano-7-nitroquinoxaline-2,3-dione (Abcam, Cambridge, UK, ab120271) and 50 µM DL-2-Amino-5-phosphonopentanoic acid (Abcam, Cambridge, UK, ab120044). Images were acquired at 4 s intervals. After acquisition of a 1 min baseline, neurons were challenged with an impermeant acidic buffer (69 mM NaCl, 2.5 mM KCl, 2 mM CaCl2, 2 mM MgCl2, 25 mM MES, 30 mM glucose at pH 5.5) for 1 min. After returning to imaging buffer for 2 min, cultures were challenged with two field stimuli (delivered using a Digitimer LTD MultiStim system-D330 stimulator, current output 100 mA, current width 1 ms) separated by 5 min. Neurons were first stimulated at 10 Hz for 30 s (300 APs) then 40 Hz for 10 s (400 APs). Finally, after a 3 min recovery period, alkaline buffer (50 mM NH4Cl substituted for 50 mM NaCl in imaging buffer) was used to reveal the maximal pHluorin response.

Time traces were analysed using the FIJI distribution of Image J (National Institutes of Health). Images were aligned using the Rigid body model of the StackReg plugin (https://imagej.net/StackReg). Nerve terminal fluorescence was measured using the Time Series Analyser plugin (https://imagej.nih.gov/ij/plugins/time-series.html). Regions of interest (ROIs) 5 pixels in diameter were placed over nerve terminals that responded to the electrical stimulus. A response trace was calculated for each cell by averaging the individual traces from each selected ROI. For sypHy time traces, fluorescence decay time constants (tau, τ, s) were calculated by fitting a monoexponential decay curve to data from the time point after the end of electrical stimulation.

Tetramethylrhodamine (TMR)-dextran uptake

TMR-dextran (ThermoFisher Scientific, D1842) uptake was performed as described previously [24]. Neurons were mounted on a Zeiss Axio Observer D1 microscope as described above before challenging with 400 action potentials (40 Hz) in the presence of 50 µM of TMR-dextran (40,000 MW) in imaging buffer. The TMR-dextran solution was immediately washed away after stimulation terminated, and images were acquired using 556/25 nm excitation and 630/98 nm emission bandpass filters (Zeiss) while undergoing constant perfusion. Per coverslip of cells, 3–6 different fields of view were imaged. The TMR-dextran puncta in each image were quantified using the Analyze Particles plugin of Image J (NIH, https://imagej.nih.gov/ij/developer/api/ij/plugin/filter/ParticleAnalyzer.html) to select and count particles of 0.23–0.91 µm2. For all experiments, for each condition, at least one unstimulated coverslip was imaged to correct for the background level of TMR-dextran uptake.

SV2A immunofluorescence staining

Immunofluorescence staining was performed as previously described [24]. Briefly, neurons were fixed with 4% paraformaldehyde (Sigma Aldrich, 47608) in PBS for 15 min. Excess paraformaldehyde was quenched with 50 mM NH4Cl in PBS. Cells were then permeabilized in 1% bovine serum albumin (BSA; Roche Diagnostics GmbH, Germany, 10735078001) in PBS-Triton 0.1% solution for 5 min and blocked in 1% BSA in PBS at room temperature for 1 h. After blocking, cells were incubated in rabbit anti-SV2A (1:200 dilution) for 1 h, after which the cultures were washed with PBS and incubated with fluorescently conjugated secondary antibodies (anti-rabbit Alexa Fluor 488; 1:1000 dilution) for 1 h. The coverslips were mounted on slides for imaging with FluorSave (Millipore, Darmstadt, Germany, 345789). SV2A puncta were visualised at 500 nm band pass excitation with a 515 nm dichroic filter and a long-pass > 520 nm emission filter on a Zeiss Axio Observer D1 inverted epifluorescence microscope (Cambridge, UK). Images were captured using an AxioCam 506 mono camera (Zeiss) with a Zeiss EC Plan Neofluar 40x/1.30 oil immersion objective. SV2A puncta in each image were quantified using the Analyze Particles plugin of Image J to select and count particles of 0.23–3.18 µm2.

Preparation of synaptosomes

Rat brains were sliced in ice cold ACSF (124 mM NaCl, 2.5 mM KCl, 1.2 mM NaH2PO4, 25 mM NaHCO3, 20 mM Glucose, 2 mM CaCl2, 1 mM MgCl2) using a vibrating blade microtome (VT1200S, Leica). Hippocampi were dissected in ice-cold ACSF from P28 Nrxn1+/−, Nlgn3−/y and P60 Pten+/− rats with wild-type male littermate controls. Tissue from three rats was pooled together to produce one preparation of synaptosomes. A total tissue lysate was prepared in a sucrose/EDTA buffer (0.32 M Sucrose, 1 mM EDTA, 5 mM Tris, 4oC, pH 7.4) using a pre-chilled motorized Teflon glass homogenizer, followed by centrifugation at 1075 g for 10 min at 4oC. Pure synaptosomes were isolated by adding supernatant on top of a discontinuous Percoll-density gradient (3% top, 10% middle, and 23% bottom; Percoll, P1644, Sigma-Aldrich, UK) and centrifuged at 47,807 g for 8 min at 4oC. The fraction between 23% and 10% was collected and re-suspended in HEPES-Buffered-Krebs (118.5 mM NaCl, 4.7 mM KCl, 1.18 mM MgSO4, 10 mM Glucose, 1 mM Na2HPO4, 20 mM HEPES, pH 7.4) and synaptosomes were pelleted by centrifugation at 20,198 g for 15 min at 4oC. Synaptosome pellets were dissolved in RIPA buffer (50 mM, Tris-HCl pH 7.4, 150 mM NaCl, 1% Triton, 0.5% Na-deoxycholate, 0.1% SDS, 1 mM sodium-orthovanadate, 1 mM PMSF, 1 mM EDTA) supplemented with protease inhibitors (Roche complete mini EDTA-free protease inhibitor cocktail 4693159001, Sigma-Aldrich, UK) and phosphatase inhibitors (cocktail II P5726, cocktail III P0044, Sigma-Aldrich, UK). Protein levels were estimated by a MicroBCA Assay (Pierce BCA protein estimation kit, Cat # 23225, Thermofisher, UK).

Western blotting

Approximately 10 µg of each protein extract was separated on a precast gradient gel (NuPAGE 4–12% Bis-Tris Protein Gels, NP0336BOX, Thermofisher) and transferred to PVDF membrane (GE10600022, Thermofisher, UK). After protein transfer, membranes were stained with a reversible protein stain kit (memcode 24585, Thermofisher Scientific) according to the manufacturer’s instructions. The exception was for Pten+/−, where nitrocellulose membrane (1620115, Bio-Rad) and reversible protein stain kit (memcode 24580, theromofisher Scientifc) were used. All membranes were blocked with Odyssey Blocking Buffer (Cat #-927-50003, LI-COR Biotech.) for 1-hour at room temperature followed by incubation at 4oC overnight with primary antibodies (PTEN, 1:1000, Cat # sc393186, RRID AB_2923140, Santa Cruz; NLGN3, 1:1000, Cat # 129113, RRID AB_2619816, Synaptic Systems; NRXN1, 1:1000, Cat # 175103, RRID AB_10697816, Synaptic Systems). Membranes were washed with TBST (0.1% Tween 20) followed by a 1-hour incubation with secondary antibodies (IRDye 800CW Goat anti-Rabbit IgG- 1: 10,000, Cat # 925-32211, RRID AB_2651127; IRDye 680LT Goat anti Mouse IgG- 1: 10,000, Cat # 925-68020, RRID AB_2687826, LI-COR Biotechnology) at room temperature. After washing the membranes with TBST, immunoblots were dried and digitally scanned by using a CLx Odyssey Infrared Imaging System, LI-COR, UK Ltd. The density of individual bands was calculated using Licor Image Studio Lite software. Each value was normalised to total protein and then to their control littermates. Data analysis for immunoblotting was done using Graphpad Prism version 6.0e software.

Experimental design and statistical analysis

Microsoft Excel (Microsoft, Washington, USA) and Prism 6 software (GraphPad software Inc., San Diego USA) were used for data processing and analysis. The experimenter was blinded to genotype during data acquisition and analysis. For all figures, results are presented with error bars as ± SEM, and the n for each condition represents the number of coverslips imaged. For all assays, cells were obtained from at least three independent cultures. In sypHy assays, at least 10 ROIs were collected from each coverslip. The number of ROIs examined was comparable for all experiments. Normality was determined using a D’Agostino & Pearson omnibus normality test. For comparison between genotypes, an unpaired Students t test was performed where data followed a normal distribution, with a Mann-Whitney test performed for those that did not. For comparison between > 2 conditions, a one-way ANOVA was performed. Results were corrected for multiple comparisons. Full statistical reporting is provided in Table 1.

Table 1 Collated table of statistical analysis
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Fig. 1
figure 1

High content monitoring of SV recycling. Hippocampal neurons were transfected with synaptophysin-pHluorin (sypHy) on day in vitro (DIV) 7 and imaged at DIV 13–15. Transfected neurons were challenged with a pulse of impermeant acidic buffer for 1 min (to reveal the surface fraction of sypHy, shaded pink). After returning to imaging buffer for 2 min, neurons were stimulated with a train of 300 action potentials (10 Hz) followed by a 5 min rest period. After this rest period neurons were stimulated with a further train of 400 action potentials (40 Hz). Finally, after a further 3 min neurons were exposed to an alkaline buffer (NH4Cl, to reveal the total SV pool, blue shaded region). Stimulation is indicated by bars. Mean trace displays the average sypHy fluorescent response of wild-type neurons ± SEM. Traces are ΔF/F0 and are presented as a fraction of the total SV pool

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Fig. 2
figure 2

Nrxn1+/− neurons display depressed ADBE. A-C) Hippocampal synaptosome lysates from either wild-type (WT) or Nrxn1+/− rats were probed for the presence of Neurexin-1 (Nrxn1, A) and total protein (B). (C) Quantification of Nrxn1 levels in both are displayed, normalised to total protein ± SEM, n = 3 independent synaptosome preparations for both WT and Nrxn1+/−, * p = 0.0365, unpaired t test. D-J) Hippocampal neurons derived from either WT or Nrxn1+/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. D, H) Mean sypHy fluorescence traces of WT and Nrxn1+/− hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (D) or peak fluorescence during electrical stimulation (H) ± SEM. E) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F, G) Mean peak sypHy response in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. (I, J) Mean sypHy retrieval time constants (t) in response to either 10 Hz (I) or 40 Hz (J) action potential trains ± SEM. For D-Jn = 9 coverslips for both WT and Nrxn1+/− from 3 independent cultures. (K, L) Primary hippocampal cultures derived either WT or Nrxn1+/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. (K) Representative images of TMR-dextran uptake in WT and Nrxn1+/− cultures. Scale bar = 50 μm. L) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (n = 13 coverslips from 3 independent cultures for WT and Nrxn1+/−). (M) Primary hippocampal cultures derived from either WT or Nrxn1+/− rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. (N) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 12 coverslips, Nrxn1+/−n = 15 coverslips from 3 independent cultures). In all cases an unpaired two-sided students t test was performed, ** p = 0.0037, unpaired t test

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Fig. 3
figure 3

Nlgn3−/y neurons display depressed ADBE. A-C) Hippocampal synaptosome lysates from either wild-type (WT) or Nlgn3−/y rats were probed for the presence of Neuroligin-3 (Nlgn3, A) and total protein (B). (C) Quantification of Nlgn3 levels in both are displayed, normalised to total protein ± SEM, n = 4 independent synaptosome preparations for both WT and Nlgn3−/y, *** p < 0.0001, unpaired t test. (D-J) Hippocampal neurons derived from either WT or Nlgn3−/y rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. (D, H) Mean sypHy fluorescence traces of WT and Nlgn3−/y hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (D) or peak fluorescence during electrical stimulation (H) ± SEM. (E) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F, G) Mean peak sypHy response in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. (I, J) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (I) or 40 Hz (J) action potential trains ± SEM. For D-J WT n = 11 coverslips, Nlgn3−/yn = 14 from 3 independent cultures. (K, L) Primary hippocampal cultures derived either WT or Nlgn3−/y rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. (K) Representative images of TMR-dextran uptake in WT and Nlgn3−/y cultures. Scale bar = 50 μm. (L) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 11 coverslips, Nlgn3−/yn = 12 from 3 independent cultures). (M) Primary hippocampal cultures derived from either WT or Nlgn3−/y rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. (N) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 14 coverslips, Nlgn3−/yn = 15 coverslips from 3 independent cultures). In all cases an unpaired two-sided students t test was performed, except E, F and J, * p = 0.0112, unpaired t test

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Fig. 4
figure 4

Syngap−/−neurons display depressed ADBE. Hippocampal neurons derived from either wild-type (WT), Syngap+/− or Syngap−/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. A, E) Mean sypHy fluorescence traces of WT, Syngap+/− or Syngap−/− hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (A) or peak fluorescence during electrical stimulation (E) ± SEM. B) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. C, D) Mean peak sypHy response in response to either 10 Hz (C) or 40 Hz (D) action potential trains ± SEM. F, G) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. For A-G, WT n = 12 coverslips, Syngap+/−n = 11 and Syngap−/−n = 12 from 3 independent cultures. H-I) Primary hippocampal cultures derived either wild-type (WT), Syngap+/− or Syngap−/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. H) Representative images of TMR-dextran uptake in WT, Syngap+/− or Syngap−/− cultures. Scale bar = 50 μm. I) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 15 coverslips, Syngap+/− and Syngap−/−n = 16 from 3 independent cultures). J) Primary hippocampal cultures derived from either WT, Syngap+/− or Syngap−/− rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. K) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT and Syngap+/−n = 15 coverslips, Syngap−/−n = 13 from 3 independent cultures). In all cases a one-way ANOVA was performed, ** p = 0.01

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Fig. 5
figure 5

SyngapΔ − GAP/Δ−GAP neurons display depressed ADBE. Hippocampal neurons derived from either wild-type (WT), Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. A, E) Mean sypHy fluorescence traces of WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (A) or peak fluorescence during electrical stimulation (E) ± SEM. B) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. C, D) Mean peak sypHy response in response to either 10 Hz (C) or 40 Hz (D) action potential trains ± SEM. F, G) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. For A-G, WT n = 14 coverslips, Syngap+/Δ−GAPn = 12 and SyngapΔ − GAP/Δ−GAPn = 10 from 3 independent cultures. H-I) Primary hippocampal cultures derived either wild-type (WT), Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. H) Representative images of TMR-dextran uptake in WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP cultures. Scale bar = 50 μm. I) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 8 coverslips, Syngap+/Δ−GAP and SyngapΔ − GAP/Δ−GAPn = 9 from 3 independent cultures). J) Primary hippocampal cultures derived from either WT, Syngap+/Δ−GAP or SyngapΔ − GAP/Δ−GAP rat embryos were fixed at DIV13-15 and stained for the presence of SV2A. K) Mean number of SV2A puncta per field of view normalised to WT ± SEM (WT n = 10 coverslips, Syngap+/Δ−GAPn = 11 and SyngapΔ − GAP/Δ−GAPn = 13 from 3 independent cultures). In all cases a one-way ANOVA was performed, * p = 0.017

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Results

High content screening of SV recycling

To determine whether the observed depression of ADBE in Fmr1−/y neurons [24] was a wider synaptic signature of ASD, we examined SV recycling using a battery of optical assays in primary cultures of hippocampal neurons derived from monogenic rat ASD models. A high content protocol was designed to capture a series of different SV recycling parameters using the genetically-encoded reporter sypHy (Fig. 1). SypHy consists of the abundant SV cargo protein synaptophysin, that has a pH-sensitive EGFP (pHluorin) inserted into an intralumenal loop [35]. It reports the pH of its immediate environment, with fluorescence quenched in the acidic SV interior and unquenched during SV fusion. Therefore, the extent of SV fusion can be estimated by the evoked increase in fluorescence during stimulation. SypHy is then retrieved from the plasma membrane and packaged to SVs, which are acidified to permit neurotransmitter filling. SV cargo retrieval is rate limiting when compared to acidification [35, 36] but see [37], meaning that the kinetics and extent of the former can be estimated from the fluorescence decrease after stimulation terminates.

Fig. 6
figure 6

Pten+/− neurons display depressed ADBE. A-C) Hippocampal synaptosome lysates from either wild-type (WT) or Pten+/− rats were probed for the presence of PTEN (A) and total protein (B). (C) Quantification of PTEN levels in both are displayed, normalised to total protein ± SEM, n = 5 independent synaptosome preparations for both WT and Pten+/−, * p = 0.0178, unpaired t test. D-J) Hippocampal neurons derived from either WT or Pten+/− rat embryos were transfected with synaptophysin-pHluorin (sypHy) after 7 days in vitro (DIV) and imaged at DIV 13–15. D, H) Mean sypHy fluorescence traces of WT and Pten+/− hippocampal neurons normalised to either the total SV pool as revealed by NH4Cl (D) or peak fluorescence during electrical stimulation (H) ± SEM. E) Mean sypHy surface fraction presented as a percentage of the total SV pool ± SEM. F, G) Mean peak sypHy response in response to either 10 Hz (F) or 40 Hz (G) action potential trains ± SEM. I, J) Mean sypHy retrieval time constants (τ) in response to either 10 Hz (I) or 40 Hz (J) action potential trains ± SEM. For D-J, WT n = 15 coverslips, Pten+/−n = 19 from 3 independent cultures. K, L) Primary hippocampal cultures derived either wild-type (WT) or Pten+/− rat embryos were challenged with a train of action potentials (40 Hz, 10 s) in the presence of tetramethylrhodamine (TMR)-dextran (50 µM). TMR-dextran was immediately washed away and the number of TMR-dextran puncta were counted. K) Representative images of TMR-dextran uptake in WT and Pten+/− cultures. Scale bar = 50 μm. L) Mean number of TMR-dextran puncta per field of view normalised to WT ± SEM (WT n = 24 coverslips, Pten+/−n = 20 from 3 independent cultures)

Full size image

First, sypHy-expressing neurons were exposed to an impermeant acidic buffer to quench sypHy fluorescence on the presynaptic plasma membrane, since the altered surface fraction of SV cargo can be indicative of chronic dysfunction in either their clustering or retrieval [38, 39]. Second, after recovery of the sypHy response to the acid buffer, neurons were challenged with two sequential AP trains (10 Hz, 30 s or 40 Hz 10 s) to reveal whether SV fusion or cargo retrieval was disrupted during periods of low and high activity. Finally, neurons were exposed to buffer containing NH4Cl, to unquench all sypHy within acidic compartments, to estimate the extent of SV fusion as a proportion of the total SV pool. In parallel, activity-dependent uptake of the large fluid phase marker TMR-dextran (40 kDa), was measured, since it exclusively reports ADBE due to size exclusion from SVs [40].

ADBE is depressed in both Nrxn1 +/− and Nlgn3 −/y neurons

We first tested our high content imaging protocol on two preclinical rodent models - Nrxn1+/− and Nlgn3−/y rats. These rat models replicate the genetic alterations in the human NRXN1 or NLGN3 genes that are primarily responsible for ASD [10, 41], due to haploinsufficiency of the Nrxn1 gene, or deletion of the X-linked Nlgn3 gene respectively. An additional reason for investigating these rat models is that an unbiased screen of molecules on bulk endosomes generated via ADBE revealed that synaptic adhesion molecules were disproportionately represented [42]. Furthermore, the gene products of Nrxn1 and Nlgn3, Neurexin-1 and Neuroligin-3, were present on bulk endosomes purified via two independent approaches [42]. This suggests the potential for a direct mechanistic link between adhesion molecules, ASD and ADBE. Therefore, we first determined whether these construct-valid rat models of ASD displayed dysfunctional SV recycling and ADBE.

Neurexins are presynaptic cell adhesion molecules that stabilise synapses via trans-synaptic interactions with postsynaptic partners such as neuroligins [41]. Their conditional knockout has pleotropic effects on evoked neurotransmitter release, with the primary deficit being an inefficient coupling of voltage-gated calcium influx to SV fusion [43]. Interestingly, patient-derived NRXN1+/− induced pluripotent neurons display an intrinsic hyperexcitability when investigated [44]. Since ADBE is also triggered via activity-dependent calcium influx [45, 46], we first determined the impact of Nrxn1 haploinsufficiency on ADBE and SV recycling in primary hippocampal cultures from the Nrxn1+/− rat [32, 33]. Western blotting confirmed a reduction in Neurexin-1 levels to approximately 50% (Fig. 2A-C). When the high content sypHy assay was performed on Nrxn1+/− neurons and wild-type littermate controls, no significant difference in the extent of the evoked sypHy response was observed to either AP train (10–40 Hz, Fig. 2D, F-H). This suggests that SV fusion was not significantly impacted by the absence of a single Nrxn1 allele. Furthermore, the kinetics and extent of SV cargo retrieval in Nrxn1+/− neurons were not significantly impacted when compared to wild-type neurons (Fig. 2I, J). This absence of effect was corroborated by the comparable extent of sypHy surface fraction between wild-type and Nrxn1+/− neurons (Fig. 2E). However, when ADBE was assessed, a significant reduction in the number of nerve terminals displaying activity-dependent uptake of TMR-dextran was observed in Nrxn1+/− neurons when compared to wild-type littermate controls (Fig. 2K, L). This decrease in TMR-dextran puncta number was not due to a reduction in nerve terminals, since staining with the SV marker SV2A revealed no change in this parameter when Nrxn+/− and wild-type neurons were compared (Fig. 2M, N). Therefore Nrxn1+/− neurons display a selective deficit in ADBE, but not SV fusion or cargo retrieval.

Neurexin-1 forms complexes with postsynaptic adhesion molecules to drive synaptogenesis and synaptic stability [12]. Key proteins in this context are the neuroligin family, of which neuroligin-3 is an important member. Neuroligin-3 is located at both excitatory and inhibitory synapses and performs essential roles in synaptic development, function and maintenance [47]. As stated above, NLGN3 gene mutations are associated with ASD, with the majority of pathogenic mutations resulting in a loss of neuroligin-3 function [10]. Therefore, we next determined whether ADBE was depressed in hippocampal neurons derived from a recently generated model of neuroligin-3 dysfunction, the Nlgn3−/y rat [28]. After validating the absence of Neuroligin-3 using semi-quantitative Western blotting (Fig. 3A-C), we first addressed whether Nlgn3−/y neurons displayed altered SV fusion or cargo retrieval using our high content sypHy screen. Nlgn3−/y neurons displayed no defect in SV fusion in response to either stimulation protocol, when compared to wild-type littermate controls (Fig. 3D, F-H). Furthermore, there was no difference between Nlgn3−/y and wild-type neurons in terms of either the extent or kinetics of sypHy retrieval (Fig. 3I, J). Finally, there was no significant change in the surface fraction of sypHy between the two genotypes (Fig. 3E), suggesting both SV cargo retrieval and clustering are unaffected by the absence of neuroligin-3. In contrast, the number of nerve terminals displaying an activity-dependent accumulation of TMR-dextran was reduced in Nlgn3−/y neurons when compared to wild-type controls (Fig. 3K, L). Since the number of nerve terminals was unchanged in Nlgn3−/y cultures (revealed by SV2A immunostaining, Fig. 3M, N), this meant that, similar to Nrxn1+/− neurons, Nlgn3−/y neurons display reduced ADBE.

Loss of syngap results in depressed ADBE

Neurexin-1 is a presynaptic protein, whereas neuroligin-3 forms essential complexes that modify and maintain presynaptic function [12]. Therefore, the observed depression of ADBE may result from a direct mechanistic involvement of these molecules in this endocytosis mode. To test the hypothesis that depression in ADBE was a consequence of intrinsic synaptic dysfunction in ASD, and not to loss of a key regulatory molecule, we next examined ASD models where the affected gene product is expressed exclusively at the postsynapse. The models chosen for these experiments were the Syngap+/− rat and the Syngap+/Δ−GAP rat [29,30,31]. SynGAP performs key roles at the postsynapse, principally in the control of AMPA receptor trafficking and synaptic plasticity [7]. Furthermore, SYNGAP haploinsufficiency is a highly prevalent cause of ASD, resulting from loss of function mutations in the SYNGAP gene [48, 49]. The Syngap+/− rat model was generated by introducing a frameshift mutation in the Syngap gene, resulting in nonsense mediated decay of mRNA encoding the mutant allele [29]. In contrast, the Syngap+/Δ−GAP rat has a deletion in the exons encoding the calcium/lipid binding C2, and GTPase activating protein (GAP) domain [30, 31]. The level of SynGAP protein expression for both models has been validated previously [29, 31].

As stated above, the exclusive postsynaptic localisation of SynGAP provides an excellent opportunity to test the hypothesis that depression of ADBE is a consequence of the ASD brain microenvironment. We first determined the ability of Syngap+/− hippocampal neurons to perform efficient SV fusion and cargo trafficking using our sypHy assay. In this instance, we also examined the performance of Syngap−/− neurons in this assay, to determine if the complete absence of the protein exacerbated any potential phenotype. When SV fusion was assessed, neither Syngap+/− nor Syngap−/− neurons displayed any deficit when compared to wild-type littermate controls during either the low or high frequency stimulus train (Fig. 4A, C-E). Furthermore, neither genotype displayed dysfunctional SV cargo retrieval in response to these trains, or an alteration in the surface distribution of sypHy (Fig. 4B, F,G). In contrast, Syngap+/− neurons displayed a reduction the number of activity-dependent TMR-dextran puncta, a decrease which became significant in Syngap−/− neurons when compared to wild-type controls (Fig. 4H, I). This was not a result of a decrease in nerve terminals in these primary cultures, since the number of SV2A puncta was unchanged across all genotypes (Fig. 4J, K). Therefore, a depression in ADBE is still observed even in neurons where the affected gene product is exclusively postsynaptic.

SynGAP functions as a GAP that negatively regulates Ras and Rap GTPases to control both F-actin dynamics (RasGAP) and p38 MAPK (RapGAP) activity [50,51,52]. However, SynGAP has other functions outside of its GAP activity. Its C-terminus alters synaptic strength via PDZ interactions with PSD-95, which in turn control AMPA receptor recruitment and synaptogenesis [53,54,55]. While the role of the GAP activity in brain function remains poorly understood, recent work has suggested that this catalytic domain may not be obligatory for synaptic plasticity [56]. To delineate these more structural roles from its GAP activity, we next exploited the Syngap+/Δ-GAP rat model [30, 31], which shares many behavioural traits with the Syngap+/- rat. Primary hippocampal cultures were prepared from both Syngap+/Δ-GAP and SyngapΔ-GAP/Δ-GAP rats in addition to wild-type littermate controls. When the three genotypes were assessed for SV fusion phenotypes using the sypHy assay, there was no significant difference for either stimulus train (Fig. 5A, C-E). There was also no significant effect of genotype on SV cargo retrieval after either stimulus and no impact on the surface fraction of sypHy (Fig. 5B, F,G). When the number of activity-dependent TMR-dextran puncta were determined, Syngap+/Δ-GAP neurons displayed a reduction, which again became significant when SyngapΔ-GAP/Δ-GAP neurons were examined (Fig. 5H, I). Furthermore, the number of nerve terminals in culture, identified via SV2A staining, was unchanged across all genotypes (Fig. 5J, K). This result therefore confirms that loss of postsynaptic SynGAP function in two independent model systems results in depression of ADBE.

Pten+/− neurons display depressed ADBE

The demonstration of depression of ADBE in SynGAP models, which have exclusively postsynaptic deficits, provides strong support for the hypothesis that a reduction in ADBE is a convergent consequence of ASD. As a final proof, we exploited a different rat ASD model that has no discernible presynaptic locus of dysfunction, the Pten+/− rat [34]. PTEN (phosphatase and tensin homologue deleted on chromosome 10) is a tumour suppressor, that negatively regulates the AKT / mTOR signalling pathway [57]. Haploinsufficiency resulting from loss of function mutations in the PTEN gene result in PTEN hamartoma tumor syndrome (PHTS [58]), with common links to ASD, macrocephaly, epilepsy and neurodevelopmental impairment [59,60,61].

Semi-quantitative Western blotting confirmed a reduction in PTEN expression in Pten+/− rats (Fig. 6A-C). Primary hippocampal cultures from either Pten+/− rat embryos or wild-type littermate controls were entered into the sypHy high content assay. Consistent with other models of ASD, Pten+/− neurons displayed no defect in SV fusion, cargo retrieval or surface stranding of sypHy (Fig. 6D-J). Intriguingly, Pten+/− neurons did display a reduction in the number of activity-dependent TMR-dextran puncta when compared to littermate controls (Fig. 6K, L). Therefore, in an ASD model system with no overt presynaptic dysfunction, depression of ADBE still occurs.

Discussion

The complex aetiology of ASD complicate the investigation of their causal mechanisms. Because of this, the study of monogenic ASD, where the genetic locus is known, has provided a series of key insights into potential convergent signalling pathways [3]. In this study we have revealed that neurons derived from a range of rat models of monogenic ASD display a depression of ADBE, a presynaptic endocytosis mode that is dominant during periods of high neuronal activity. This depression was observed regardless of whether the mutated gene was presynaptic, postsynaptic or neither, suggesting it is a convergent strategy to ameliorate disrupted synaptic function.

We initially revealed depression in ADBE in Fmr1−/y neurons, a model for fragile X syndrome [24]. Similar to this study, SV fusion and cargo trafficking were unaffected. A BK channel agonist corrected the depression of ADBE in these neurons, suggesting the alteration could be a result of the direct regulation of BK channels by the Fmr1 gene product, FMRP [62,63,64]. However, a BK channel antagonist did not recapitulate the depression in wild-type neurons, suggesting that FMRP had no direct mechanistic role in ADBE [24]. In this work, we determined whether ADBE was perturbed in other ASD models that did not display overt defects in presynaptic endocytosis. The large fluid phase marker TMR-dextran was used for these studies, since it reports ADBE due to size exclusion from single SVs [22]. TMR-dextran is an excellent reporter of ADBE, since interventions that block this endocytosis mode selectively reduce TMR-dextran uptake [45, 46, 65,66,67].

Specific cell adhesion molecules such as CHL1 and N-cadherins have proposed roles at the presynapse in both activity-dependent SV retrieval and ADBE [68,69,70,71]. In fact, cell adhesion molecules including both neurexin-1 and neuroligin-3 are greatly over-represented on ADBE-generated bulk endosomes [42]. This suggests that they might be required for optimal ADBE. In support, neurexin-1 controls localised calcium channel coupling to SV fusion at the active zone [41, 43] and both neurexin-1 and neuroligin-3 are synaptogenic [12]. However, a direct role via these presynaptic functions is unlikely. This is because ADBE is triggered via delocalised calcium increases, rather than localised calcium influx at the active zone [45] and there was no significant change in synapse number in either Nrxn1+/− or Nlgn3−/y cultures, suggesting synaptogenesis was not modulating this effect (this study). No change in synapse number was also observed in cultured human Nrxn1+/− neurons, however these neurons did display a considerable decrease in both spontaneous and evoked neurotransmitter release [72]. Intriguingly, mouse neurons that were engineered for the same Nrxn1+/− genotype displayed no significant neurotransmitter release defect [72], in a similar manner to the rat Nrxn1+/− neurons in this study. Therefore, it is possible that human neurons are exquisitely sensitive to depletion of neurexin-1 and that rodent neurons are more resilient to this insult.

Two independent models of SYNGAP haploinsufficiency disorder displayed depression of ADBE. The exclusive postsynaptic location of SynGAP provides strong support for the depression of ADBE being a compensatory adjustment in ASD. The expression of specific isoforms of SynGAP are driven via neuronal activity and have downstream effects on mEPSC frequency [73], suggesting a potential presynaptic role. However, it is more likely that these effects are mediated via the nano-organisation of AMPA receptors at the postsynapse. The observation that a depression of ADBE was observed in Syngap+/Δ−GAP neurons suggests that this depression resulted from a loss of enzyme activity, rather than interactions with postsynaptic partners such as PSD-95, LRRTMs and neuroligins [53,54,55]. In this context, key enzymatic roles for SynGAP could include either its RasGAP, which regulates F-actin and spine dynamics [74] or Rap-GAP activity which controls the p38 MAPK signalling pathway ultimately regulating postsynaptic AMPA receptor trafficking [51, 75, 76]. The obligatory role of the GAP domain in SYNGAP function has recently been questioned [56], however the GAP rat displays a series of circuit and behavioural phenotypes that suggest its enzymatic role of required for optimal function [31]. Regardless, the depression of ADBE in two independent models where the gene product is expressed exclusively at the postsynapse provides strong support for this depression to be a homeostatic adaptation to disrupted synaptic function.

The depression of ADBE in a novel Pten+/− model could potentially be explained by the fact that PTEN is expressed at growth cones during axonal navigation and synaptogenesis. However, its expression is restricted to the postsynapse in mature neurons [77]. The synaptic locus of dysfunction in neurons lacking PTEN appears to be due to dysregulation of the AKT/mTOR signalling pathway, specifically mTORC1/RAPTOR [78]. Deficiency in PTEN results in neuronal hypertrophy and hyperexcitability [79,80,81,82], which is corrected in multiple models via inhibition of AKT/mTOR signalling via rapamycin [78, 83, 84]. PTEN deficient neurons display enhanced excitatory neurotransmission, which appears to be due to enhanced postsynaptic function. Intriguingly, an increase in the size of the RRP and mEPSC frequency was also observed, however this was most likely due to an increase in the number of available synapses in these neurons [78, 79, 84]. In agreement with this hypothesis, no change in evoked SV fusion events were detected in this study.

The advent of monogenic rat models of ASD has transformed our understanding of social interaction deficits and cognitive behaviour, especially those that are difficult to replicate in mouse models with the same genetic mutations [85]. The study of neurons derived from these models can provide important insight into the fundamental cellular mechanisms that underpin these subtle alterations. This study reveals one such mechanism, the depression of a single presynaptic endocytosis mode, which is observed in all five independent rat models used. We suggest that the convergence upon depression of ADBE is a conserved and scalable lever through which to sculpt synaptic strength and circuit hyperexcitability regardless of genetic insults, differences in cellular localisation, and mechanisms of pathogenicity of the genes mutated in ASD.

The central question arising from this study is, what the molecular mechanism responsible for the depression of ADBE in ASD model neurons? ADBE is the dominant endocytosis mode during high activity and appear to have a similar molecular mechanism to ultrafast endocytosis, which is triggered by sparse stimulation [22, 46, 86,87,88]. Since both pathways occur with a timescale that is an order of magnitude faster than SV cargo clustering and retrieval via CME, an emerging view is that CME initiates on the presynaptic plasma membrane but completes on endosomes formed via either UFE or ADBE [18,19,20]. Since SV fusion and cargo retrieval are not impacted in ASD models, it suggests that the depression is intrinsic to ADBE or mechanisms that control it. One compelling explanation is that the observed depression is a compensatory mechanism to limit the intrinsic circuit hyperexcitability observed in many ASD models including many of the models used in this study [26, 27, 44, 78,79,80,81,82, 89,90,91] but see [92]. It will be key to determine the impact of elevated excitability in the long-term on presynaptic events such as ADBE.

Out of all potential presynaptic endocytosis modes, a depression in ADBE is the most intriguing, since its depression would be manifested disproportionately across specific cell types. This is because it is triggered during periods of high activity [22], meaning that GABAergic neurons and inhibitory circuits (which typically display higher firing rates [93]), may be excessively impacted. In agreement, reduced ADBE results in an inability to sustain neurotransmission during high activity [24, 67, 94]. Therefore, a depression of ADBE may not be a homogenous adaptation across the brain, but rather a circuit-specific correction in neuronal function. It will be essential in future work to determine how, (1) ADBE is impacted in intact circuits in ASD model system and (2) how circuit-specific modulation of this activity-dependent endocytosis mode may sculpt ASD-like behaviours.

Limitations

There are a number of limitations to the current work. The principal limitation is that the work has been performed exclusively in primary neuronal culture. Therefore, the impact of altered brain microcircuitry observed in these models of ASD cannot be assessed in this experimental system. Furthermore, this work has been performed on embryonic hippocampal neurons, therefore, there is a possibility that these findings do not extend to neurons from distinct brain regions which may display different phenotypes. However, the fact that a convergent depression of ADBE is observed across all hippocampal neurons derived from these models strongly suggests that it is an adaptation to the intrinsic dysfunction due to a specific monogenic insult.

One intrinsic defect observed in almost all of these rat models or in patient-derived neurons is hyperexcitability. However, at this current time Nlgn3−/y circuits and isolated neurons have not been reported to display this phenotype. Therefore, the suggestion that the depression of ADBE is a consequence of hyperexcitability in ASD requires further validation, both in a demonstration of hyperexcitability of all ASD neurons in culture, and in artificially modulating neuronal culture excitability and examining the impact on ADBE.

One final limitation of the study is that the mechanism of ADBE depression in ASD neurons was not determined. This is an ongoing line of enquiry however, and requires a detailed interrogation of molecular alterations at the synapse coupled to targeted interventions to modulate expression of known molecules essential for ADBE.

Conclusions

The depression of ADBE observed across five independent monogenic rat models of ASD in this study, in addition to that previously observed in Fmr1−/y neurons, suggests that it is a homeostatic mechanism to correct for some aspect of intrinsic dysfunction. Intrinsic hyperexcitability is a potential instigator of this depression and will be the focus of future investigations both in vitro and in vivo.

Data availability

The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Abbreviations

ADBE:

Activity-dependent bulk endocytosis

AMPA:

α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid

AP:

Action potential

ASD:

Autism spectrum disorder

BK channels:

Big potassium channels

CME:

Clathrin-mediated endocytosis

DIV:

Days in vitro

FMRP:

Fragile X messenger ribonucleoprotein

GAP:

GTPase activating protein

ID:

Intellectual disability

SV:

Synaptic vesicle

sypHy:

Synaptophysin-pHluorin

TMR-dextran:

Tetramethylrhodamine-dextran

UFE:

Ultrafast endocytosis

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Funding

This work was supported by grants from the Simons Foundation (529508), Epilepsy Research UK (P2003) and the RS McDonald Fund.

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Author notes

  1. Katherine Bonnycastle

    Present address: Service de Génétique Médicale, Centre Hospitalier Universitaire (CHU) Sainte-Justine, Université de Montréal, Montreal, QC, Canada

Authors and Affiliations

  1. Centre for Discovery Brain Sciences, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland, EH8 9XD, UK

    Katherine Bonnycastle, Mohammed Sarfaraz Nawaz, Peter C. Kind & Michael A. Cousin

  2. Simons Initiative for the Developing Brain, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland, EH8 9XD, UK

    Katherine Bonnycastle, Mohammed Sarfaraz Nawaz, Peter C. Kind & Michael A. Cousin

  3. Muir Maxwell Epilepsy Centre, University of Edinburgh, Hugh Robson Building, George Square, Edinburgh, Scotland, EH8 9XD, UK

    Katherine Bonnycastle, Peter C. Kind & Michael A. Cousin

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Contributions

KB and MAC were responsible for the conception and design of the experiments. KB and MSN were responsible for the collection and assembly of data. KB was responsible for the analysis and interpretation of data. KB, PCK, MAC wrote the manuscript, and all authors had the opportunity to contribute to its editing. All persons designated as authors qualify for authorship, and all those who qualify for authorship are listed. All authors read and approved the final manuscript.

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Correspondence to Michael A. Cousin.

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Procedures were performed in accordance with the UK Animal (Scientific Procedures) Act 1986, under Project and Personal Licence authority and were approved by the Animal Welfare and Ethical Review Body at the University of Edinburgh (Home Office project licence – 7008878). Similarly, procedures were conducted in accordance with protocols approved by the Institutional Animal Ethics Committee of Institute for Stem Cell Science and Regenerative Medicine, Bangalore.

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Competing interests

Peter Kind is an Associate Editor for Molecular Autism.

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Bonnycastle, K., Nawaz, M.S., Kind, P.C. et al. Convergent depression of activity-dependent bulk endocytosis in rodent models of autism spectrum disorder. Molecular Autism 16, 26 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13229-025-00660-6

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Keywords

Molecular Autism

ISSN: 2040-2392