The CHD Protein, Kismet, is Important for the Recycling of Synaptic Vesicles during Endocytosis

Abstract

Chromatin remodeling proteins of the chromodomain DNA-binding protein family, CHD7 and CHD8, mediate early neurodevelopmental events including neural migration and differentiation. As such, mutations in either protein can lead to neurodevelopmental disorders. How chromatin remodeling proteins influence the activity of mature synapses, however, is relatively unexplored. A critical feature of mature neurons is well-regulated endocytosis, which is vital for synaptic function to recycle membrane and synaptic proteins enabling the continued release of synaptic vesicles. Here we show that Kismet, the Drosophila homolog of CHD7 and CHD8, regulates endocytosis. Kismet positively influenced transcript levels and bound to dap160 and endophilin B transcription start sites and promoters in whole nervous systems and influenced the synaptic localization of Dynamin/Shibire. In addition, kismet mutants exhibit reduced VGLUT, a synaptic vesicle marker, at stimulated but not resting synapses and reduced levels of synaptic Rab11. Endocytosis is restored at kismet mutant synapses by pharmacologically inhibiting the function of histone deacetyltransferases (HDACs). These data suggest that HDAC activity may oppose Kismet to promote synaptic vesicle endocytosis. A deeper understanding of how CHD proteins regulate the function of mature neurons will help better understand neurodevelopmental disorders.

Figure 1

Kismet promotes endocytosis of presynaptic vesicles. (A) Confocal micrographs of the 6/7 NMJ as indicated by immunolabeling with HRP (magenta). Arrows denote satellite boutons as quantified in the right histogram. Scale bar = 20 μm. (B) High resolution confocal micrographs showing the presynaptic motor neuron (HRP, magenta) and internalization of the lipophilic dye FM 1–43FX (green) after 90 s stimulation with 90 mM KCl and 2 mM Ca²⁺ in controls (w¹¹¹⁸), the endocytosis mutant dap160^EP2543, and kis mutants. Scale bar = 5 µm. (C) Quantification of FM 1–43FX fluorescence intensity relative to controls indicates that kis mutants exhibit a significant reduction in endocytosis as indicated by internalization of FM1–43FX compared with controls. (D) Representative recordings of eEJCs resulting from a 20 Hz, 1 min HFS train in genotypes as indicated. (E) eEJC amplitudes in controls and kis mutants during HFS followed by a recovery period of 0.2 Hz stimulation. kis mutants exhibit a significant reduction in eEJCs evoked after HFS suggesting that vesicle recycling is impaired in kis mutants.

Figure 2

Kismet affects the readily releasable and recycling pool of vesicles. (A) Control (w¹¹¹⁸) and kis mutant NMJs were immunolabeled for the vesicular glutamate transporter (VGLUT) at rest (Ca²⁺-free HL-3 with 50 mM EDTA) or after stimulation (60 mM KCl and 1.5 mM Ca²⁺ for 10 min). Confocal images of Drosophila 6/7 NMJ terminal boutons labeled with HRP to delineate neurons (magenta) and VGLUT (green). Scale bar = 5 µm. (B) Quantification of VGLUT fluorescence intensity normalized to controls at rest indicates that VGLUT levels are significantly reduced in kis mutants but not controls after stimulation. (C) Quantification of Rab11 fluorescence levels shows that there is a slight but significant reduction in synaptic Rab11 in kis mutants compared with controls. (D) Histogram of rab transcript levels in kis mutants normalized to controls as determined by RT-qPCR. (E) High magnification confocal micrographs of Drosophila 6/7 NMJ terminal boutons labeled with HRP to identify neurons (magenta) and Rab11 (green). Scale bar = 5 µm.

Figure 3

Kismet mutants exhibit altered levels of transcripts and proteins involved in endocytosis. (A) Several transcripts important for clathrin-mediated endocytosis were examined in the CNS of controls and kis mutants via qRT-PCR. Transcript levels are expressed relative to w¹¹¹⁸ controls. (B) High magnification confocal images of A3 or A4 NMJ terminal boutons labeled with HRP to denote neurons (magenta) and Dap160 (B1, green), Dynamin/Shibire (B2, Dyn, green), Endophilin A (B3, EndoA, green), or Endophilin B (B4, EndoB, green). Right histograms show quantification of fluorescence intensities relative to controls (w¹¹¹⁸). Scale bar = 5 µm. (C) Promoter and transcription start site (TSS) occupancy was assessed for genes whose transcript and protein levels were consistently altered in kis mutants. Kismet likely binds to endoB promoters (pro) and the TSS and pro of dap160.

Figure 4

Kismet regulates the localization of Dynamin. (A–D) The localization of Dynamin/Shibire (Dyn) was assessed relative to the active zone protein, Bruchpilot (Brp), at rest (Ca²⁺-free HL-3 with 50 mM EDTA) or after stimulation (60 mM KCl and 1.5 mM Ca²⁺ for 10 min). Confocal images of z-projected A3 or A4 terminal NMJ boutons immunolabeled with Brp (green) and Dyn (magenta) in controls (w¹¹¹⁸, A), dap160 mutants (B), or kis mutants (C,D). Dyn is redistributed in controls from active zones at rest to periactive zones during synaptic activity (see arrow in A). Dyn is mislocalized to periactive zones in kis^LM27/kis^k13416 mutants at rest (see arrow in D) and migrates toward active zones during synaptic activity. Scale bar = 2 µm. (E) Cumulative frequency histograms depicting the distance Dynamin was relative to Brp at rest and stimulated conditions in the indicated genotypes. (F) Quantification of the distance between Brp and Dyn in synaptic boutons indicating Dyn is mislocalized in kis mutants at rest.

Figure 5

The histone deacetylase inhibitors, SAHA and SBHA, suppress the deficit in endocytosis observed in kismet mutants. (A–C) High resolution confocal micrographs showing the presynaptic motor neuron (HRP, magenta) and internalization of the lipophilic dye FM 1–43FX (green) after 1 min stimulation with 90 mM KCl and 2 mM Ca²⁺ in controls (w¹¹¹⁸) and kis mutants. Eggs were laid and larvae were allowed to develop on instant food containing DMSO (A), SBHA (B), or SAHA (C). Scale bar = 5 µm. (D) Quantification of FM 1–43FX fluorescence intensity relative to DMSO controls indicates that endocytosis is restored in kis^LM27/kis^k13416 mutants raised on either SBHA or SAHA.