Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction - PubMed (original) (raw)

doi: 10.1038/ng.3256. Epub 2015 Apr 6.

Vincent Cantagrel 2, Maha S Zaki 3, Lihadh Al-Gazali 4, Xin Wang 5, Rasim Ozgur Rosti 5, Esra Dikoglu 5, Antoinette Bernabe Gelot 6, Basak Rosti 5, Keith K Vaux 5, Eric M Scott 5, Jennifer L Silhavy 5, Jana Schroth 5, Brett Copeland 5, Ashleigh E Schaffer 5, Philip L S M Gordts 7, Jeffrey D Esko 7, Matthew D Buschman 8, Seth J Field 8, Gennaro Napolitano 9, Ghada M Abdel-Salam 3, R Koksal Ozgul 10, Mahmut Samil Sagıroglu 11, Matloob Azam 12, Samira Ismail 3, Mona Aglan 3, Laila Selim 13, Iman G Mahmoud 13, Sawsan Abdel-Hadi 13, Amera El Badawy 13, Abdelrahim A Sadek 14, Faezeh Mojahedi 15, Hulya Kayserili 16, Amira Masri 17, Laila Bastaki 18, Samia Temtamy 3, Ulrich Müller 19, Isabelle Desguerre 20, Jean-Laurent Casanova 21, Ali Dursun 22, Murat Gunel 23, Stacey B Gabriel 24, Pascale de Lonlay 25, Joseph G Gleeson 26

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Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction

Naiara Akizu et al. Nat Genet. 2015 May.

Abstract

Pediatric-onset ataxias often present clinically as developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a new clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in the sorting nexin gene SNX14, encoding a ubiquitously expressed modular PX domain-containing sorting factor. We found SNX14 localized to lysosomes and associated with phosphatidylinositol (3,5)-bisphosphate, a key component of late endosomes/lysosomes. Patient-derived cells showed engorged lysosomes and a slower autophagosome clearance rate upon autophagy induction by starvation. Zebrafish morphants for snx14 showed dramatic loss of cerebellar parenchyma, accumulation of autophagosomes and activation of apoptosis. Our results characterize a unique ataxia syndrome due to biallelic SNX14 mutations leading to lysosome-autophagosome dysfunction.

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Figures

Figure 1

Figure 1. SNX14 mutations cause a syndromic form of severe cerebellar atrophy and coarsened facial features

(a) Summary of exome results from 81 families with cerebellar atrophy. SNX14 accounted for 9.88% of the total families, with other genes making individual contributions. (b) Midline sagittal (top) or axial (middle) MRI and facies of affected individuals from representative families. Prominent atrophy of cerebellum evidenced by reduced volume and apparent folia (arrows and circles). Facies show prominent forehead, epicanthal folds, long philtrum and full lips. Consent to publish images of the subject was obtained. (c) SNX14 exons as ticks and location of mutations indicated. Scale bar 50 kb. (d) Truncating mutations relative to predicted protein domains. TM: Transmembrane, PXA: Phox homology associated, RGS: Regulator of G protein signaling, PX: Phox homology, PXC: Sorting Nexin, C-terminal. (e) ABD-II-2 (p.Arg378*) hematoxylin-eosin stained cerebellum compared with control showing reduction in internal granule cell layer (arrow, top), near complete depletion of Purkinje cells (arrow, middle), and dystrophic degenerating remnant Purkinje cell (arrow, bottom). Scale bar 100 µm.

Figure 2

Figure 2. SNX14 localizes to late endosome/lysosome compartments

(a) RT-PCR expression pattern of human SNX14 showing ubiquitous expression in representative fetal and adult human tissues. GAPDH: loading control. (b) Cell fractionation from human neural progenitor cells (NPCs). SNX14 was enriched in lysosomal-endosomal compartments (red). (c) LAMP2, EEA1 and GM130 (green) in dsRED tagged SNX14 expressing NPCs. SNX14 overlapped in localization with LAMP2 lysosomal marker (arrows). Scale bar 10 µm. (d) Lipid binding assay with SNX14 PX domain on phosphoinositides-spotted membrane, showed preferential binding to PI(3,5)P2 (red), compared with p40phox PX domain control.

Figure 3

Figure 3. Patient-derived SNX14 mutant neural progenitor cells display enlarged lysosomes

(a) Immunoblot of IPSC-derived neural progenitor cells (NPCs) from families 468 (p.Arg378*) and 1382 (p.Lys395Arg_fs_*22), with affected (A, red) and unaffected (U, black) labeled. Affecteds showed undetectable SNX14 protein. GAPDH: loading control. (b) Lysotracker Green DND-26 staining with engorged lysosomes in affecteds NPCs (arrows). Scale bar 5 um. Dot plot shows relative area for individual LysoTracker positive lysosomes (n = 223 and 194 lysosomes from 2 families unaffected and affected NPCs respectively, N = 2). Graph bars represent average number of LysoTracker-positive lysosomes per cell (n = 17 and 18 from 2 families unaffected and affected NPCs respectively, N = 2). Error bars, S.D. *** p < 0.0005, N.S. not significant (two tiled t-test).

Figure 4

Figure 4. Patient-derived SNX14 mutant neural progenitor cells display abnormal starvation-induced autophagic response

(a) Immunoblot analysis of LC3 II in affected (red), unaffected and affected transduced with SNX14 (grey) NPCs upon induction of autophagy by starvation with 1 hr 30 min EBSS (Eagle’s Balanced Salt Solution) or rapamycin (1 µM for 2 hr). Graph bars represent average LC3II/αTubulin levels relative to feeding condition. Error bars S.D. (N = 3 clones) * p < 0.05, ** p < 0.005, N.S. not significant (two tiled t-test). Affected cells display an accumulation of LC3 II levels upon autophagic induction, partially rescued by exogenous SNX14 expression. (b) LC3 immunoblot (left) for quantification of autophagic flux measured by LC3 II ratio in lane 3 vs. lane 2 for unaffected and lane 7 vs. lane 6 for affected (red) (middle), and quantification of autophagosome formation (left) assessed as the increase in LC3-II levels at two time points (lane 4 vs. lane 3 for control, and lane 8 vs. lane 7 for affected) after inhibition of lysosomal proteolysis with Leupeptin 200 µM and NH4Cl 20 mM (PI 30 min/PI 1 hr). Graph represent mean ± S.D. (N = 3 clones) * p < 0.05, N.S. not significant (two tailed t-test) (c) Transmission electron microscopic analysis of 2 hr EBSS treated unaffected (black) and affected (red) NPCs showing autophagic structures in affecteds (arrowheads). Data represents results from one NPC clones from each affected or unaffected.

Figure 5

Figure 5. Morphant snx14 zebrafish show apoptosis, excessive autophagic vesicles, and loss of neural tissue including cerebellar primordium

(a) Comparison of scrambled (6ng) and snx14 (3ng and 6ng) morphant zebrafish 48 hours postfertilization (hpf). Calipers: measured distance. Scale bar 250 µm. Graphs: Reduced optic tectum and right eye width in morphants. Mean ± SEM (n = 15 embryos for NI, 16 for Scramble, 31 for MO 3ng and 18 for MO 6ng, N = 2). *p < 0.05; **p < 0.005 (two tiled t-test). (b) Scramble or snx14 morphants for Zebrin II (Purkinje cell marker), rescued with human SNX14 (50 pg). Scale bar 50 µm. Graph: Zebrin II compartment area relative to scramble MO injected embryos. Mean ±SEM (n = 10 embryos for Scramble, 6 for MO 3ng, 9 for MO 6ng and 9 for rescue) *p < 0.05; **p < 0.005 (two tiled t-test). (c) Maximum confocal projection from 36 hpf Tg(ptf1a:eGFP) (green) zebrafish with scramble or snx14 MO showing reduced Purkinje cell progenitors. (d) Maximum confocal projection with increased caspase 3 (red) positive cells in 36 hpf snx14 morphants. Blue: DAPI. Scale bar 50 µm. (e) Transmission electron microscopy showing autophagic structures in 48 hpf snx14 and scrambled morphant neurons residing between the optic lobes. Box: Highlighted areas. Arrowheads: autophagic structures.

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