Reversal of cell, circuit and seizure phenotypes in a mouse model of DNM1 epileptic encephalopathy

Pathogenic heterozygous missense mutations in the DNM1 gene result in a novel form of epileptic encephalopathy. DNM1 encodes for the large GTPase dynamin-1, an enzyme with an obligatory role in the endocytosis of synaptic vesicles (SVs) at mammalian nerve terminals. Pathogenic DNM1 mutations cluster within regions required for its essential GTPase activity, implicating disruption of this enzyme activity as being central to epileptic encephalopathy. We reveal that the most prevalent pathogenic mutation in the GTPase domain of DNM1, R237W, disrupts dynamin-1 enzyme activity and SV endocytosis when overexpressed in central neurons. To determine how this dominant-negative heterozygous mutant impacted cell, circuit and behaviour when expressed from its endogenous locus, we generated a mouse carrying the R237W mutation. Neurons isolated from heterozygous mice displayed dysfunctional SV endocytosis, which translated into altered excitatory neurotransmission and seizure-like phenotypes. Importantly, these phenotypes were corrected at the cell, circuit and in vivo level by the drug, BMS-204352, which accelerates SV endocytosis in wild-type neurons. This study therefore provides the first direct link between dysfunctional SV endocytosis and epilepsy, and importantly reveals that SV endocytosis is a viable therapeutic route for monogenic intractable epilepsies.

Synaptosomes were prepared from two-month-old Dnm1+/+ or Dnm1+/R237W male mice as described (Ivanova, D. et al. Control of synaptic vesicle release probability via VAMP4 targeting to endolysosomes. Sci Adv 7 (2021)). Synaptosome pellets were dissolved in Urea lysis buffer (8M Urea in 50mM Tris-Cl and 1% sodium deoxycholate) and were quantified using the BCA method. 20 μg of total protein was used for proteomic sample preparation by suspension trapping (S-Trap), as recommended by the supplier (ProtiFi, Huntington NY, USA). Samples were reduced with 5 mM Tris (2-carboxyethyl)phosphine (Pierce) for 30 min at 37°C, and subsequently alkylated with 5 mM IAM (Iodoacetamide) for 30 min at 37°C in the dark. After acidification with phosphoric acid, sample was cleaned and digested using Trypsin (1:20) as mentioned by in the manufacturer’s protocol using S-trap filter for 2 hours at 47°C and the digested peptides are eluted using 0.2% Formic acid and 50% Acetonitrile:0.2% formic acid. The eluted digested peptides were dried in speed vac and stored at -80°C.  The peptides were reconstituted in 30 µL of 0.1% formic acid and vortexed and 5 µL of each sample was injected on the mass spectrometer. Peptides were analysed by nanoflow-LC-MS/MS using a Orbitrap Q-Exactive-HF™ Mass Spectrometer (Thermo Scientific TM) coupled to a Dionex™ Ultimate™ 3000. Samples were injected on a 100 μm ID × 5mm trap (Thermo Trap Cartridge 5mm) and separated on a 75 μm × 50 cm nano LC column (EASY-Spray™ LC Columns #ES803). All solvents used were HPLC or LC-MS Grade (Millipore™). Peptides were loaded for 5 minutes at 10 μL/min using 0.1% FA, 2% Acetonitrile in Water. The column was conditioned using 100% Buffer A (0.1% FA, 3% DMSO in Water) and the separation was performed on a linear gradient from 0 to 35% Buffer B (0.1% FA, 3% DMSO, 20% Water in Acetonitrile), over 140 minutes at 250 nL/min. The column was then washed with 90% Buffer B for 5 minutes and equilibrated 10 minutes with 100% Buffer A in preparation for the next analysis. Full MS scans were acquired from 350 to 1500 m/z at resolution 60,000 at m/z 200, with a target AGC of 3x106 and a maximum injection time of 50 ms. MS/MS scans were acquired in HCD mode with a normalized collision energy of 25 and resolution 15000 using a Top 20 method, with a target AGC of 2x105 and a maximum injection time of 50 ms. The MS/MS triggering threshold was set at 5E3 and the dynamic exclusion of previously acquired precursor was enabled for 45 s for DDA (Data-Dependent Acquisition) mode. For DIA (Data Independent Acquisition) mode the scan range was 385 to 1015 m/z, where MS/MS data was acquired in 24 m/z isolation windows at a resolution of 30,000.Pooled peptides from all samples were fractionated on a Basic Reverse Phase column (Gemini C18, 3um particle size, 110A pore, 3 mm internal diameter, 250 mm length, Phenomenex #00G-4439-Y0) on a Dionex Ultimate 3000 Off-line LC system. All solvent used were HPLC grade (Fluka). Peptides were loaded on column for 1 minute at 250 μL/min using 99% Buffer A (20mM Ammonium Formate, pH=8) and eluted for 48 minutes on a linear gradient from 2 to 50% Buffer B (100% ACN). The column is then washed with 90% Buffer B for 5 minutes and equilibrated for 5 minutes for the next injection. Peptide elution was monitored by UV detection using at 214 nm. Fractions were collected every 45 s from 2 min to 60 min for a total of 12 fractions. Non-consecutive concatenation of every 13th fraction was used to obtain 12 pooled fractions (Pooled Fraction 1: Fraction 1 + 13 + 25 + 37, Pooled Fraction 2 : Fraction 2 + 14 + 26 + 38 ...). MTD data_processing_protocol Data Analysis  Label-free quantitative analysis was performed using the data set acquired in DIA mode. Peptide identification was carried out using a library generated using both DDA and DIA datasets using SpectronautTM version 15.0. The library was generated using the Pulsar algorithm integrated in Spectronaut using Mus musculus FASTA using 1% FDR. The maximum of missed cleavage was set to 2 using Trypsin/P enzyme. Carbamidomethylation (C) was set as fixed modification and acetylation (Protein N term), oxidation (M), deamination (NQ), were set as variable modifications. The library consisted spectra information of 5906 proteins in total. DIA data set for both WT and HET was searched using this library quantified 4237 proteins in total. Statistical analysis was done using R script and limma package was used for making contrasts.

Data Analysis  Label-free quantitative analysis was performed using the data set acquired in DIA mode. Peptide identification was carried out using a library generated using both DDA and DIA datasets using SpectronautTM version 15.0. The library was generated using the Pulsar algorithm integrated in Spectronaut using Mus musculus FASTA using 1% FDR. The maximum of missed cleavage was set to 2 using Trypsin/P enzyme. Carbamidomethylation (C) was set as fixed modification and acetylation (Protein N term), oxidation (M), deamination (NQ), were set as variable modifications. The library consisted spectra information of 5906 proteins in total. DIA data set for both WT and HET was searched using this library quantified 4237 proteins in total. Statistical analysis was done using R script and limma package was used for making contrasts.