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. 2019 Feb 15;28(4):584-597.
doi: 10.1093/hmg/ddy370.

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons

Mattéa J Finelli  1 , Davide Aprile  2 , Enrico Castroflorio  3 , Alexander Jeans  4 , Matteo Moschetta  2   3 , Lauren Chessum  5 , Matteo T Degiacomi  6 , Julia Grasegger  1 , Alexis Lupien-Meilleur  7 , Andrew Bassett  8 , Elsa Rossignol  7 , Philippe M Campeau  7 , Michael R Bowl  5 , Fabio Benfenati  3   9 , Anna Fassio  2   9 , Peter L Oliver  1   5
Affiliations

The epilepsy-associated protein TBC1D24 is required for normal development, survival and vesicle trafficking in mammalian neurons

Mattéa J Finelli et al. Hum Mol Genet. .

Abstract

Mutations in the Tre2/Bub2/Cdc16 (TBC)1 domain family member 24 (TBC1D24) gene are associated with a range of inherited neurological disorders, from drug-refractory lethal epileptic encephalopathy and DOORS syndrome (deafness, onychodystrophy, osteodystrophy, mental retardation, seizures) to non-syndromic hearing loss. TBC1D24 has been implicated in neuronal transmission and maturation, although the molecular function of the gene and the cause of the apparently complex disease spectrum remain unclear. Importantly, heterozygous TBC1D24 mutation carriers have also been reported with seizures, suggesting that haploinsufficiency for TBC1D24 is significant clinically. Here we have systematically investigated an allelic series of disease-associated mutations in neurons alongside a new mouse model to investigate the consequences of TBC1D24 haploinsufficiency to mammalian neurodevelopment and synaptic physiology. The cellular studies reveal that disease-causing mutations that disrupt either of the conserved protein domains in TBC1D24 are implicated in neuronal development and survival and are likely acting as loss-of-function alleles. We then further investigated TBC1D24 haploinsufficiency in vivo and demonstrate that TBC1D24 is also crucial for normal presynaptic function: genetic disruption of Tbc1d24 expression in the mouse leads to an impairment of endocytosis and an enlarged endosomal compartment in neurons with a decrease in spontaneous neurotransmission. These data reveal the essential role for TBC1D24 at the mammalian synapse and help to define common synaptic mechanisms that could underlie the varied effects of TBC1D24 mutations in neurological disease.

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Figures

Figure 1
Figure 1
Disease-causing mutations in TBC1D24 influence neuronal cell differentiation and sensitivity to oxidative stress. (A) Three-dimensional structural model of TBC1D24 indicating the positions of published pathogenic mutations classified into three general disease classes (yellow, red, blue) as shown (12,13). E549, the most conserved residue of the TLDc domain is also shown (white). (B) The TBC1D24 mutations investigated in this study include those situated just before (R40L) and within the TBC (R242C) domain and in the TLDc domain (R360L). S324X represents a frame-shift mutation (S324Tfs*3) that results in a premature stop codon (12,13). The E549A mutation has been described previously as disrupting the function of TBC1D24 (20). (C) Western blot demonstrated a complete loss of TBC1D24 protein expression in CRISPR-deleted N2a cells (Tbc1d24Δ/Δ). GAPDH was used as a loading control. (D,E) Untransfected Tbc1d24Δ/Δ cells were more sensitive to arsenite treatment than control N2a cells (D) and grew shorter neurites (N > 100 neurites from 3 independent experiments) (E). (F,G)Tbc1d24Δ/Δ cells transfected with TBC1D24 mutants presented an increased sensitivity to arsenite treatment compared to Tbc1d24Δ/Δ cells transfected with WT TBC1D24 (F) and grew shorter neurites (G), similar to cells transfected with an empty control vector (N > 70 neurites). All data are expressed as mean ± SEM. (F,G) *P < 0.05; **P < 0.01; ***P < 0.001; ANOVA followed by Dunnett’s multiple comparison test compared to TBC1D24 WT vector. #P < 0.05; ##P < 0.01; ###P < 0.001; ANOVA followed by Dunnett’s multiple comparison test compared to empty vector. (D,E) Unpaired t-test.
Figure 2
Figure 2
Genetically reduced levels of Tbc1d24 expression alters primary neuronal maturation and survival. (A)In situ hybridization demonstrating a reduction in Tbc1d24 expression in the postnatal (P15) mouse brain from Tbc1d24tm1b mice versus littermate Tbc1d24WT controls. Scale bar: 0.5 mm. A significant reduction in expression in the hippocampus at P15 is confirmed by qRT-PCR (N = 3 animals of each genotype) (B) and western blot (N = 5 animals of each genotype) (C); GAPDH was used as a loading control. (D) Representative immunostaining of primary hippocampal cultures from Tbc1d24WT or Tbc1d24tm1b with dendritic (Map2) and axonal (Tau) markers. Scale bar: 100 µM. (E,F) Quantification of dendrite (E) and axonal (F) length in hippocampal neurons (N > 100 axons/dendrites from 3 independent preparations of each genotype). (G) In the same cultures, the number of apoptotic cells was quantified by cleaved caspase-3 immunostaining after arsenite treatment to induce oxidative stress. (H,I) Quantification of dendrite (H) and axonal (I) length in primary cortical (N > 100 axons/dendrites from 4 independent preparations of each genotype). (J) In the same cultures the number of apoptotic cells was quantified as in (G). Data are expressed as the mean ± SEM. (B, C, E–J) *P < 0.05; **P < 0.05; ***P < 0.001 unpaired t-test compared to Tbc1d24WT.
Figure 3
Figure 3
Genetic disruption of Tbc1d24 expression results in decreased mEPSC frequency with no effect on excitatory synapse number in hippocampal primary neurons. (A) Representative current-clamp recordings of spike trains evoked by somatic current injection of 300 pA for 500 ms from hippocampal WT Tbc1d24WT (black) and mutant Tbc1d24tm1b (red) mice. (B) AP in 500 ms (or mean firing frequency in Hz) versus injected current (pA; left) and averaged instantaneous frequency at 300 pA (right) (N = 31 neurons from 4 independent preparations). (C) Representative traces of mEPSCs recorded at −70 mV in 14 DIV hippocampal neurons from Tbc1d24WT (black) and Tbc1d24tm1b (red) mice. (D) Histograms showing average peak frequency and amplitude of mEPSCs. (E) Average amplitude of mEPSCs calculated as peak area. (F) Cumulative plot of peak frequency (left) and amplitude (right) showing a selective defect in the peak frequency (N = 25 and 27 neurons from Tbc1d24WT and Tbc1d24tm1b mice respectively from 4 independent preparations). (G) Representative images of Tbc1d24WT and Tbc1d24tm1b hippocampal culture immunolabelled with the glutamatergic presynaptic and postsynaptic markers v-GLUT1 and Homer1. Nuclei are stained with DAPI. Number of v-GLUT1 and Homer1 positive puncta are automatically counted and normalized to the number of cells in the field (N = 28–24 fields per experimental condition from 3 independent preparations). Scale bar: 20 µM. All data are expressed as the mean ± SEM. (B, D, E, G) *P < 0.05; ***P < 0.001; unpaired t-test.
Figure 4
Figure 4
Genetic disruption of Tbc1d24 expression results in defective endocytosis in primary neurons. (A) Average fluorescence traces of hippocampal neurons at 15 DIV expressing the pHluorin SypH 2× in response to 100 stimuli at 10 Hz [N = 188 boutons from 8 coverslips (Tbc1d24WT) or 87 boutons from 5 coverslips (Tbc1d24tm1b)] indicating no difference in the post-stimulus endocytic time constant. (B) Normalized average traces of neurons expressing SypH 2x in response to 300 stimuli at 10 Hz. (C) Comparison of average post-stimulus endocytic time constants following the 300 AP stimulus. The decay phases of ΔF traces were fitted with single exponential functions before normalization and the time constants were calculated from the fits [N = 113 boutons from 5 coverslips (Tbc1d24WT) or 190 boutons from 6 coverslips (Tbc1d24tm1b)]. (D) Time course and extent of endocytosis during neuronal activity were measured using folimycin (Fol). Neurons expressing SypH 2× were stimulated at 10 Hz for 30 s in the absence of Fol. After a 10 min rest, neurons were stimulated at 10 Hz for 120 s in the presence of Fol. Images were acquired during each phase of stimulation. Graph shows average SypH 2× traces from Tbc1d24WT neurons in response to each stimulus. All values were normalized to the maximum fluorescence change at the end of 1200 stimuli in presence of Fol. The short and long vertical arrows indicate the extent of endocytosis and exocytosis at the end of the 300 stimuli (vertical dashed line) at 10 Hz, respectively (N = 162 boutons from 5 coverslips). The same experiment was repeated in Tbc1d24tm1b neurons (N = 200 boutons from 6 coverslips). (E) Time courses of during-stimulus endocytosis for Tbc1d24WT and Tbc1d24tm1b neurons during stimulation (30 s, 10 Hz). Each endocytosis time-course was calculated by subtracting the SypH 2× trace that was acquired in the absence of Fol, from the Fol trace in (D). These traces were fitted with linear functions, the slope of which corresponds to the endocytic rate in arbitrary units per second. (F) Average magnitude of endocytosis calculated during delivery of 300 pulses. Data are expressed as the mean ± SEM. (C,F) **P < 0.01; ***P < 0.001; unpaired t-test.
Figure 5
Figure 5
Genetic disruption of Tbc1d24 expression results in increased endosomal volume in primary neurons. (A) Representative TEM images of synapses of cultured cortical neurons from WT Tbc1d24WT and mutant Tbc1d24tm1b mice, fixed at 17 DIV. Scale bar: 200 nm. (B–D) Conventional TEM analysis of nerve terminals from Tbc1d24tm1b (red bars) revealed a preserved density of both total (H) and docked (I) SVs with respect to control neurons (black bars), with a significant increase in the density of endosomal-like structures. (E) Three-dimensional reconstructions of synaptic terminals from serial ultrathin sections; shown are representative three-dimensional reconstructions from 60 nm-thick serial sections obtained WT Tbc1d24WT and mutant Tbc1d24tm1b synapses. Total SVs and SVs physically docked at the AZ are depicted as blue and yellow spheres, respectively. The AZ and endosomes are shown in red and black, respectively. Scale bar, 200 nm. (F–H) Ultrastructural morphometric analysis of three-dimensionally reconstructed synapses displayed similar amount of both total (F) and docked (G) SVs and endosomal number (H). (I) The endosomal-like volume was calculated from each 2D analysis of synaptic profile and is expressed as mean ± SEM area of endosomes per μm3. Nerve terminal areas and AZ lengths were similar in the two experimental groups (N = 105 and 126 synapses for Tbc1d24WT and Tbc1d24tm1b, respectively, from three independent preparations). Data are expressed as the mean ± SEM. (D, I) *P < 0.05; Mann–Whitney test.
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