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. 2024 Jun 26;22(1):142.
doi: 10.1186/s12915-024-01940-y.

Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior

Constanza Ahumada-Marchant  1 , Carlos Ancatén-Gonzalez  2   3 , Henny Haensgen  1 , Bastian Brauer  1 , Nicolas Merino-Veliz  1 , Rita Droste  4 , Felipe Arancibia  1 , H Robert Horvitz  4 , Martha Constantine-Paton  5 , Gloria Arriagada  1 , Andrés E Chávez  3 , Fernando J Bustos  6   7
Affiliations

Deletion of VPS50 protein in mouse brain impairs synaptic function and behavior

Constanza Ahumada-Marchant et al. BMC Biol. .

Abstract

Background: The VPS50 protein functions in synaptic and dense core vesicle acidification, and perturbations of VPS50 function produce behavioral changes in Caenorhabditis elegans. Patients with mutations in VPS50 show severe developmental delay and intellectual disability, characteristics that have been associated with autism spectrum disorders (ASDs). The mechanisms that link VPS50 mutations to ASD are unknown.

Results: To examine the role of VPS50 in mammalian brain function and behavior, we used the CRISPR/Cas9 system to generate knockouts of VPS50 in both cultured murine cortical neurons and living mice. In cultured neurons, KO of VPS50 did not affect the number of synaptic vesicles but did cause mislocalization of the V-ATPase V1 domain pump and impaired synaptic activity, likely as a consequence of defects in vesicle acidification and vesicle content. In mice, mosaic KO of VPS50 in the hippocampus altered synaptic transmission and plasticity and generated robust cognitive impairments.

Conclusions: We propose that VPS50 functions as an accessory protein to aid the recruitment of the V-ATPase V1 domain to synaptic vesicles and in that way plays a crucial role in controlling synaptic vesicle acidification. Understanding the mechanisms controlling behaviors and synaptic function in ASD-associated mutations is pivotal for the development of targeted interventions, which may open new avenues for therapeutic strategies aimed at ASD and related conditions.

Keywords: ASD; Gene editing; Synaptic vesicles; VPS50; Vesicle acidification.

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Conflict of interest statement

The authors declare that they have no competing interests.

Figures

Fig. 1
Fig. 1
VPS50 gene editing causes a decrease in synaptic vesicle acidification but no change in synaptic vesicle number. Cortical neurons from Cas9KI mice were transduced with sgRNA targeting VPS50 at 3 DIV to produce VPS50 KO. A T7 endonuclease I assay of control or VPS50 KO cortical neurons. B RT-qPCR to quantify relative mRNA expression of VPS50. C VPS50 protein expression in VPS50 KO and control neurons. N-cadherin was used as loading control. D Quantification of VPS50 expression in western blots (n = 6 biological replicates). E, F Representative images of synaptic terminals as visualized by electron microscopy and quantification of the number of synaptic vesicles in control and VPS50 KO neurons (n = 50 cells per condition from 3 independent experiments). G Representative images of the Ratio-SyPhy signal used to determine synaptic vesicle acidification in control and VPS50 mKO neurons. Red signal shows all synaptic vesicles and green signal shows vesicles with basic pH. H Quantification of Ratio-SyPhy signal showing Mander’s coefficient. Thirteen samples from 3 biological replicates were used for Ratio-SyPhy quantification. Scale bar: 400 nm (E); 25 μm (G). Unpaired t-test was used for statistical analysis; ***p < 0.001. Error bars represent ± SEM. ***p < 0.001
Fig. 2
Fig. 2
VPS50 KO causes mislocalization of v-ATPaseV1 domain in cortical neurons. Proximity-ligation assays (PLAs) was used to determine proximity between studied proteins as indicated in each case. A positive PLA signal, thus proximity between the proteins, is observed as red puncta in images (red channel, 546 nm). In all experiments, an antibody against the microtubule-associated protein 2 (MAP2) was used to stain processes of all neurons (green channel, 488 nm). Merge shows superimposed images of MAP2 (green), PLA signals (red), and the signal for a protein of interest as shown in the figure (blue, 633 nm). A PLA using PSD95 or Synapsin1 (Syn1) antibodies to define pre- or post-synaptic localization of VPS50 in control neurons. B Quantification of PLA puncta per field of VPS50/PSD95 and VPS50/Syn1. C PLA to assess proximity of VPS50 and v-ATPAseV1 domain in control and VPS50 KO neurons. D Quantification of PLA puncta per field of VPS50/v-ATPaseV1 in control and VPS50 KO neurons. E PLA to determine proximity of v-ATPaseV1 domain and Synaptophysin (Syn) in control and VPS50 KO neurons. F Quantification of PLA puncta per field of v-ATPaseV1 domain/Syn in Control and VPS50 KO neurons. Six low magnification (20 ×) fields from 3 independent biological samples were used for quantification in each condition. Scale bars, 25 μm. Unpaired t-test was used for statistical analysis; ***p < 0.001. Error bars represent ± SEM
Fig. 3
Fig. 3
VPS50 KO neurons show a reduction in synaptic activity that can be recovered by inducing synaptic vesicle acidification. Cortical neuronal cultures from Cas9KI animals were transduced at 3DIV and at 12–13 DIV used for electrophysiology or calcium imaging. A Representative traces and B, C quantification of amplitude (B) and frequency (C) of spontaneous AMPA-mediated EPSCs. D Representative traces and E, F quantification of the frequency (E) and amplitude (F) of miniature AMPA-mediated currents. G Representative traces and quantification of the frequency (H) of current clamp recordings of control and VPS50 KO neurons. I–K Representative traces of current clamp recordings of control and VPS50 KO neurons expressing pHoenix. Orange bars under the traces show the periods pHoenix was activated. K Quantification of the frequency of spiking of control and VPS50 KO neurons before, during, and after stimulus with pHoenix to artificially acidify synaptic vesicles. For all experiments, at least 18 neurons from 3 independent experiments were analyzed. Unpaired t-test was used for statistical analysis; ***p < 0.001. Error bars represent mean ± SEM
Fig. 4
Fig. 4
Systemic injection of AAV at P1 to produce VPS50 mKO. A, B Representative image of coronal sections of a mouse brain (VPS50 mKO) injected at P1 with AAV after 15 weeks to show infection efficiency by tdTomato fluorescent signal. Two different sections are shown to show broad infection. A, B (right) Anatomical annotations from the Allen Reference Atlas – Mouse Brain (atlas.brain-map.org), at the same slice position as A and B (left). Scheme of the relative positions are shown to identify brain areas. C Higher magnification image of a hippocampus section showing transduction efficiency. D Representative western blot analyses for the detection of VPS50 expression in control and VPS50 mKO animals in both cortex and hippocampus. B-tubulin was used as loading control. E, F Quantification of VPS50 expression in western blots from cortex (E) or hippocampus (F) n = 6 animals per condition. G, H RT-qPCR to quantify expression of VPS50 in cortex (G) or hippocampus (H), n = 13 animals per condition. Scale bar, 500 μm. Unpaired t-test was used for statistical analysis; ***p < 0.001. Error bars represent ± SEM
Fig. 5
Fig. 5
Hippocampal spontaneous excitatory synaptic activity is impaired in VPS50 mKO mouse. A Representative traces of mEPSCs recorded from CA1 pyramidal neurons in acute hippocampal slices of control and VPS50 mKO animals. B, C Quantification of the frequency (B) and amplitude (C) of mEPSCs. D Representative traces of sEPSCs in hippocampal slices of control and VPS50 mKO animals. E, F Quantification of the frequency (E) and amplitude (F) of mEPSCs. In each representative trace (A–D), the numbers of cells (c) and animals (a) are indicated in parentheses. Two-samples Student t-test was used for statistical analysis; *p < 0.05, ***p < 0.001. Summary data consist of mean ± SEM
Fig. 6
Fig. 6
Hippocampal synaptic function and plasticity are impaired in VPS50 mKO mice. Electrophysiological recordings characterizing Schaffer collateral-to-CA1 synapses in acute hippocampal slices of control and VPS50 mKO animals. A Representative traces fEPSP of input–output curve elicited at different stimulus intensities. B Input–output curves reveal a strong reduction in the amplitude of fEPSC at all stimulus intensities tested. C Representative traces and D quantification of paired-pulse facilitation at different inter-stimulus intervals. E Representative synaptic responses and F quantification showing a strong synaptic depression in response to a single high-frequency stimulus train (100 pulses at 100 Hz) that likely reflect a reduce vesicular content in VPS50 mKO synapses compared to control synapses. G Representative traces before and after LTP induction elicited by four trains of high-frequency stimulation. Sample traces were taken at times indicated by numbers in summary plot. H Summary plot showing that the magnitude of LTP is reduced in VPS50 mKO animals. In each representative trace, the number of slices (s) or cells (c) and animals (a) are indicated in parenthesis. Two-samples Student t-test was used for statistical analysis; *p < 0.05. Summary data consist of mean ± SEM
Fig. 7
Fig. 7
VPS50 mKO animals show impaired hippocampal memory formation. Brain-wide control and VPS50 mKO animals were subjected to behavioral testing. A Quantification of the time spent on the ramp on the accelerated rotarod apparatus. B Scheme of the Barnes maze memory paradigm in which clues attached to the wall, escape hole, and escape zone are shown. C Quantification of the numbers of primary errors made by control and VPS50 mKO animals before finding the escape hole. D Quantification of the primary latency of control and VPS50 mKO animals to find the escape hole. E Quantification of the time control and VPS50 mKO animals spent in the escape zone. F Quantification of freezing percentage time shown by control and VPS50 mKO animals in the fear conditioning paradigm. For behavioral experiments, a total of N = 13 animals per condition were assayed. Unpaired t-test was used for statistical analysis; *p < 0.05. Error bars represent ± SEM
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