Epileptic Encephalopathy Caused by Mutations in the Guanine Nucleotide Exchange Factor DENND5A

Abstract

Epileptic encephalopathies are a catastrophic group of epilepsies characterized by refractory seizures and cognitive arrest, often resulting from abnormal brain development. Here, we have identified an epileptic encephalopathy additionally featuring cerebral calcifications and coarse facial features caused by recessive loss-of-function mutations in DENND5A. DENND5A contains a DENN domain, an evolutionarily ancient enzymatic module conferring guanine nucleotide exchange factor (GEF) activity to multiple proteins serving as GEFs for Rabs, which are key regulators of membrane trafficking. DENND5A is detected predominantly in neuronal tissues, and its highest levels occur during development. Knockdown of DENND5A leads to striking alterations in neuronal development. Mechanistically, these changes appear to result from upregulation of neurotrophin receptors, leading to enhanced downstream signaling. Thus, we have identified a link between a DENN domain protein and neuronal development, dysfunction of which is responsible for a form of epileptic encephalopathy.

Figure 1

Two Families Affected by Epileptic Encephalopathy Featuring Cerebral Calcification and Coarse Facial Features (A and B) Pedigrees of family 1 (A) and family 2 (B). (C–E) Photographs of affected individuals II-6 (C) and II-7 (D) of family 1 and II-4 (E) of family 2. Note the common coarse facial features, including nasal prominence and frontal bossing. (F–H) CT scans of the same individuals as above. Note the generalized atrophy and calcification (white spots) of the basal ganglia (F, II-6; G, II-7) and periventricular cortex (H). (I) Schematic overview of the structure of DENND5A, which contains DENN, RUN, and PLAT domains. The positions of the identified truncating variants are indicated.

Figure 2

EEG and MRI Analysis of Individuals from Family 1 (A) Wakeful EEG of individual II-7 from family 1. Interictal activity (green background) is characterized by near-continuous independent or synchronous bilateral or generalized bifrontally dominant spike-wave discharges. Ictus (seizure; yellow background) is associated with continuous generalized bifrontally dominant rhythmic activity. (B and C) MRI demonstrating dysgenesis of the corpus callosum (arrows) and ventriculomegaly in affected individuals II-6 (B) and II-7 (C), as well as Dandy-Walker malformation, a posterior fossa cyst, hypoplastic vermis, and diffuse hydrocephalus in individual II-6.

Figure 3

DENND5A Is Detected Predominantly in Developing Neurons (A) Equal protein aliquots of lysates from various tissues were blotted with antibodies recognizing DENND5A (polyclonal antibody, Abcam) and actin (monoclonal antibody C4, Millipore) as indicated. Lysates from HEK293T cells expressing FLAG-tagged DENND5A (FLAG-DENND5A) were included as a control. (B) Crude lysates were prepared from brains dissected from embryonic day 18 (E18) or postnatal day 5, 30, or 60 (P5, P30, or P60) rats. Equal protein aliquots were processed for western blot with antibodies recognizing DENND5A and GAPDH (monoclonal antibody, Santa Cruz) as indicated. (C) Primary rat cortical neurons were seeded in culture dishes, and lysates were prepared from the neurons at the indicated days in vitro. Equal protein aliquots were processed for western blots with antibodies recognizing DENND5A, GAPDH, and synapsin. (D) Quantification of the relative amounts of DENND5A and synapsin from blots as in (C). The highest-intensity values were set as 1.0. Data represent the mean ± SEM from eight independent experiments.

Figure 4

DENND5A Knockdown Enhances Process Outgrowth in Neurons and PC12 Cells (A) Primary cultured hippocampal neurons at 1 day in vitro (DIV) were transduced with lentivirus expressing shRNAmiRs targeting rat DENND5A mRNA (GenBank: NM_001107546.2). The 21 nt inhibitory sequences match DENND5A starting at nucleotide 1,758 (shRNAmir DENND5A nt1758: 5′-ACTCAGGATTTACCAGCTAAA-3′) and nucleotide 2,652 (shRNAmiR DENND5A nt2652: 5′-GAGCCACGGGCTACAAGTAAA-3′). Non-targeting shRNAmiR virus was used as a control (shRNAmiR ctrl). At 6 DIV, transduced neurons were fixed and processed for GFP fluorescence and for indirect immunofluorescence with antibody recognizing MAP2 (chicken polyclonal antibody, EnCor Biotechnology) to reveal the somatodendritic region of the neurons. GFP was expressed as part of the viral expression cassette to verify transduction. Images were captured on a laser-scanning confocal microscope (LSM 710, Carl Zeiss) equipped with a Plan Apochromat 40× oil objective (numerical aperture 1.3; Carl Zeiss). Acquisition was performed with ZEN 11.0 software. The scale bar represents 50 μm. (B) Sholl analysis for dendritic complexity of transduced hippocampal neurons from representative images as in (A) was performed with ImageJ with the ShollAnalysis plugin. Data represent the mean ± SEM from two independent experiments measuring a minimum of 18 neurons per condition per experiment; repeated-measure one-way ANOVA followed by a Dunnett’s post-test revealed a significant difference between control and knockdown neurons. ^∗p < 0.05, ^∗∗p < 0.01. (C) PC12 cells were transduced with control lentivirus (shRNAmiR ctrl) or lentivirus to knockdown rat DENND5A (shRNAmiR DENND5A nt1758 and shRNAmiR DENND5A nt2652). Transduced PC12 cells were serum starved for 24 hr and then treated with 50 ng/mL NGF (2.5S NGF, Cederlane) in serum-free medium. After 24 hr treatment, cells were fixed and processed for GFP fluorescence and for indirect immunofluorescence with antibody against α-tubulin (polyclonal antibody, ICN Biomedicals). GFP was expressed as part of the viral expression cassette to verify transduction. The scale bar represents 50 μm. (D) Length of the longest neurite (length) and total area of neurites (area) for each cell from representative images as in (C) were analyzed with ImageJ. Data show the mean ± SEM from two independent experiments measuring a minimum of 30 cells per condition per experiment; repeated-measure one-way ANOVA followed by a Dunnett’s post-test revealed a significant difference between control and knockdown cells. ^∗∗p < 0.01, ^∗∗∗p < 0.001.

Figure 5

Knockdown of DENND5A Leads to Enhanced Erk Activation and Increased Amounts of Neurotrophin Receptors (A) PC12 cells were transduced with control lentivirus (shRNAmiR ctrl) or lentivirus to knockdown DENND5A (DENND5A nt1758 and DENND5A nt2652). After 24 hr serum starvation, transduced PC12 cells were harvested (−NGF) or treated for an additional 24 hr with NGF (50 ng/mL) (+NGF). Equal protein aliquots of lysates prepared from these cells were processed by western blot with antibodies recognizing phospho-Erk (monoclonal antibody, Cell Signaling Technology) and total Erk (monoclonal antibody, Cell Signaling Technology). HSP90 (monoclonal antibody, Assay Designs Stressgen) was used as a loading control. (B) Quantification of five experiments performed as in (A). The graphs show the average relative change in phospho Erk1/2 levels normalized to total Erk1/2 levels between cells with knockdown DENND5A and control cells. Error bars represent the SEM, and statistical analysis employed a one-way ANOVA followed by a Bonferroni post-test. ^∗p < 0.05, ^∗∗p < 0.01. (C) After 24 hr serum starvation, transduced PC12 cells were harvested (−NGF) or treated for 24 hr with NGF (50 ng/mL) (+NGF). Equal protein aliquots of lysates prepared from these cells were processed by western blot with antibodies recognizing TrkA (polyclonal antibody for pan Trk [TrkA and TrkB] [C17F1], Cell Signaling Technology) and HSP90. (D) Quantification of TrkA amounts from experiments as in (C) normalized to HSP90 and expressed in relation to those in control shRNAmiR cells. Data show the mean ± SEM from eight independent experiments, and statistical analysis employed a one-way ANOVA followed by a Bonferroni post-test. ^∗p < 0.05, ^∗∗p < 0.01. (E) Rat cortical neurons were transduced with control lentivirus (shRNAmiR ctrl) or lentivirus to knockdown DENND5A (DENND5A nt1758 and DENND5A nt2652) at 1 DIV. Cell lysates were prepared at 8 DIV, and equal protein aliquots were processed by western blot with antibodies recognizing the indicated proteins. (F) Quantification of TrkB levels from experiments as in (E) normalized to GAPDH and expressed in relation to those of control shRNAmiR cells. Data show the mean ± SEM from six independent experiments, and statistical analysis employed a repeated-measure one-way ANOVA followed by a Dunett’s post-test. ^∗p < 0.05.