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.

DNM1基因中的致病性杂合错义突变导致了一种新的癫痫性脑病。DNM1编码大GTP酶动蛋白-1，该酶在哺乳动物神经末梢突触囊泡（SVs）的内吞作用中扮演着不可或缺的角色。致病的DNM1突变聚集在其必需的GTP酶活性相关区域，暗示该酶活性的破坏是癫痫性脑病的关键。本研究揭示了DNM1 GTP酶域中最常见的致病突变R237W，当在中央神经元中过表达时，会破坏动蛋白-1酶活性及SV内吞作用。为了确定这种显性负性杂合突变体在其内源位点表达时对细胞、电路和行为的影响，我们生成了一只携带R237W突变的鼠。从杂合鼠中分离的神经元表现出功能障碍的SV内吞作用，这转化为兴奋性神经传递的改变和类似癫痫的表型。值得注意的是，这些表型在细胞、电路和体内水平上通过药物BMS-204352得到纠正，该药物可加速野生型神经元中的SV内吞作用。因此，本研究首次将功能障碍的SV内吞作用与癫痫联系起来，并重要的是揭示了SV内吞作用是治疗单基因难治性癫痫的有效途径。

通过以下方法制备了来自两个月大的Dnm1+/+或Dnm1+/R237W雄性小鼠的突触体：如Ivanova等人所述（Sci Adv 7 (2021)）。将突触体沉淀溶解于脲裂解缓冲液（8M脲在50mM Tris-Cl和1%十二烷基硫酸钠中）中，并使用BCA方法进行定量。使用供应商（ProtiFi，Huntington NY，美国）推荐的方法，通过悬浮捕获（S-Trap）进行20 μg总蛋白的蛋白质组样本制备。样品在37°C下用5 mM Tris（2-羧基乙基）磷氢二铵（Pierce）还原30分钟，随后在37°C下用5 mM IAM（碘代乙酰胺）烷基化30分钟，在黑暗中进行。酸化后，样品被清洗并使用制造商协议中提到的胰蛋白酶（1:20）进行消化，使用S-trap过滤器在47°C下消化2小时，然后使用0.2%甲酸和50%乙腈：0.2%甲酸洗脱消化肽。洗脱的消化肽在快速真空下干燥，并储存在-80°C。将肽重新溶解在30 µL的0.1%甲酸中，涡旋，并注入5 µL的每个样本到质谱仪中。肽通过纳米流-LC-MS/MS分析，使用Thermo Scientific TM Orbitrap Q-Exactive-HF™质谱仪（Thermo Scientific TM）连接到Dionex™ Ultimate™ 3000。样品注入100 μm ID × 5mm陷阱（Thermo Trap Cartridge 5mm）中，并在75 μm × 50cm纳米LC柱（EASY-Spray™ LC Columns #ES803）上进行分离。所有溶剂均为HPLC或LC-MS级（Millipore™）。肽在0.1% FA，2% 乙腈在水中使用5分钟以10 μL/min的速度加载。使用100%缓冲液A（0.1% FA，3% DMSO在水中）进行柱条件化，并在140分钟内进行从0到35%缓冲液B（0.1% FA，3% DMSO，20% 水在乙腈中）的线性梯度分离，流速为250 nL/min。然后使用90%缓冲液B清洗5分钟，并平衡10分钟以准备下一次分析。全MS扫描在350至1500 m/z范围内以60,000的分辨率进行，m/z 200处的目标AGC为3x106，最大注入时间为50ms。MS/MS扫描在HCD模式下进行，使用正常化的碰撞能量25和15,000的分辨率，使用Top 20方法，目标AGC为2x105，最大注入时间为50ms。MS/MS触发阈值设置为5E3，并启用先前获取的前体的动态排除，持续时间为45秒，用于DDA（数据依赖性采集）模式。对于DIA（数据独立采集）模式，扫描范围为385至1015 m/z，在30,000的分辨率下在24 m/z隔离窗口中采集MS/MS数据。

来自所有样本的混合肽在Dionex Ultimate 3000离线LC系统上的Basic Reverse Phase柱（Gemini C18，3um颗粒大小，110A孔径，3mm内径，250mm长度，Phenomenex #00G-4439-Y0）上分级。所有溶剂均为HPLC级（Fluka）。肽在1分钟内以250 μL/min的速度加载到柱上，使用99%缓冲液A（20mM甲酸铵，pH=8），并在48分钟内以线性梯度从2%到50%缓冲液B（100% ACN）洗脱。然后使用90%缓冲液B清洗5分钟，并平衡5分钟以准备下一次注入。通过214nm的UV检测监测肽洗脱。从2分钟到60分钟，每45秒收集一次分数，总共收集12个分数。将每个13个分数的非连续连接用于获得12个混合分数（混合分数1：分数1 + 13 + 25 + 37，混合分数2：分数2 + 14 + 26 + 38 ...）。

使用在DIA模式下获得的数据集进行无标记定量分析。使用SpectronautTM版本15.0生成的库进行肽鉴定，该库使用DDA和DIA数据集生成。该库使用Spectronaut中的Pulsar算法，使用Mus musculus FASTA，以1% FDR生成。使用胰蛋白酶/P酶将最大错切次数设置为2。将羧甲基化（C）设置为固定修饰，乙酰化（蛋白质N端）、氧化（M）、脱氨（NQ）设置为可变修饰。该库包含5906个蛋白质的谱信息。使用此库对WT和HET的DIA数据集进行搜索，总共量化了4237个蛋白质。使用R脚本来进行统计分析，并使用limma包来制作对比。