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

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
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     ©   2   0   1         5    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d . NATURE GENETICS   ADVANCE ONLINE PUBLICATION 1 LETTERS 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. The hereditary cerebellar ataxias are a group of clinical conditions presenting with imbalance, poor coordination and atrophy and/or hypoplasia of the cerebellum, most often with deterioration of neu-rological function. A common hallmark of the cerebellar ataxias is a progressive cerebellar neurodegeneration due to Purkinje cell loss. A combination of dominant, recessive and X-linked forms of disease, including the spinocerebellar ataxias, Friedreich ataxia and ataxia telangiectasia, contribute to the estimated prevalence of 8.9 per 100,000 (ref. 1). In addition to the dominant trinucleotide-repeat disorders that lead to toxic accumulation of unfolded protein 2,3 , the recessive forms of disease are associated with inactivating mutations and early-onset presentations. The genes implicated thus far suggest defects in neuronal survival pathways 4,5 , but knowledge of many mechanisms is still lacking, and most patients elude genetic diagnosis.Recessive ataxias often show clinical overlap with lysosomal disor-ders; in fact, many lysosomal diseases such as Niemann-Pick disease, Tay-Sachs disease and I-cell disease show evidence of Purkinje cell loss and clinical features of ataxia, in addition to the well-established Biallelic mutations in SNX14  cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction Naiara Akizu 1–3 , Vincent Cantagrel 4 , Maha S Zaki 5 , Lihadh Al-Gazali 6 , Xin Wang 1,2 , Rasim Ozgur Rosti 1,2 , Esra Dikoglu 1,2 , Antoinette Bernabe Gelot 7,8 , Basak Rosti 1,2 , Keith K Vaux  1,2 , Eric M Scott 1,2 , Jennifer L Silhavy  1,2 , Jana Schroth 1,2 , Brett Copeland 1,2 , Ashleigh E Schaffer 1,2 , Philip L S M Gordts 9 , Jeffrey D Esko 9 , Matthew D Buschman 10 , Seth J Field 10 , Gennaro Napolitano 11 , Ghada M Abdel-Salam 5 , R Koksal Ozgul 12 , Mahmut Samil Sagıroglu 13 , Matloob Azam 14 , Samira Ismail 5 , Mona Aglan 5 , Laila Selim 15 , Iman G Mahmoud 15 , Sawsan Abdel-Hadi 15 , Amera El Badawy  15 , Abdelrahim A Sadek  16 , Faezeh Mojahedi 17 , Hulya Kayserili 18 , Amira Masri 19 , Laila Bastaki 20 , Samia Temtamy  5 , Ulrich Müller 3 , Isabelle Desguerre 21 , Jean-Laurent Casanova 2,22,23 , Ali Dursun 24 , Murat Gunel 25–27 , Stacey B Gabriel 28 , Pascale de Lonlay  29  & Joseph G Gleeson 1,2,30 1 Laboratory for Pediatric Brain Disease, The Rockefeller University, New York, New York, USA. 2 Howard Hughes Medical Institute, Chevy Chase, Maryland, USA. 3 Dorris Neuroscience Center, Scripps Research Institute, La Jolla, California, USA. 4 Institut Imagine, INSERM U1163, Hôpital Necker Enfants Malades, Paris, France. 5 Human Genetics and Genome Research Division, Clinical Genetics Department, National Research Centre, Cairo, Egypt. 6 Department of Pediatrics, College of Medicine and Health Sciences, United Arab Emirates University, Al Ain, Abu Dhabi, United Arab Emirates. 7 Assistance Publique–Hôpitaux de Paris, Hôpital Armand Trousseau, Département de Génétique, UF Génétique du Développement, Neuropathologie, Paris, France. 8 Institut de Neurobiologie de la Méditerranée (INMED) INSERM U901, Marseille, France. 9 Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, California, USA. 10 Division of Endocrinology and Metabolism, Department of Medicine, University of California, San Diego, La Jolla, California, USA. 11 Department of Molecular and Experimental Medicine, The Scripps Research Institute, La Jolla, California, USA. 12 Pediatric Metabolism, Institute of Child Health, Hacettepe University, Ankara, Turkey. 13 Tübitak Bilgem Uekae, Gebze/Kocaeli, Turkey. 14 Wah Medical College, Wah, Pakistan. 15 Department of Pediatric Neurology, Children’s Hospital, Cairo University, Cairo, Egypt. 16 Pediatric Neurology Department, Faculty of Medicine, Sohag University, Sohag, Egypt. 17 Mashhad Medical Genetic Counseling Center, Mashhad, Iran. 18 Medical Genetics Department, Istanbul University, Istanbul Medical Faculty, Istanbul, Turkey. 19 Division of Child Neurology, Department of Pediatrics, University of Jordan, Amman, Jordan. 20 Kuwait Medical Genetics Centre, Maternity Hospital, Safat, Kuwait. 21 Department of Pediatric Neurology, Necker Enfants Malades Hospital, Paris Descartes University, Paris, France. 22 Génétique Humaine des Maladies Infectieuses, INSERM U1163, Université Paris Descartes, Institut Imagine, Paris, France. 23 St. Giles Laboratory of Human Genetics of Infectious Diseases, The Rockefeller University, New York, New York, USA. 24 Pediatric Metabolism, Hacettepe University Faculty of Medicine, Ankara, Turkey. 25 Department of Neurosurgery, Yale University, School of Medicine, New Haven, Connecticut, USA. 26 Department of Neurobiology, Yale University, School of Medicine, New Haven, Connecticut, USA. 27 Department of Genetics, Yale University, School of Medicine, New Haven, Connecticut, USA. 28 Broad Institute of MIT and Harvard, Cambridge, Massachusetts, USA. 29 Reference Center of Inherited Metabolic Diseases, Paris Descartes University, Necker Enfants Malades Hospital, Assistance Publique–Hôpitaux de Paris, Paris, France. 30 New York Genome Center, New York, New York, USA. Correspondence should be addressed to J.G.G. ( 24 October 2014; accepted 2 March 2015; published online 6 April 2015; doi:10.1038/ng.3256     ©   2   0   1         5    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d . 2 ADVANCE ONLINE PUBLICATION NATURE GENETICS LETTERS features of enlarged organs and coarsening of facial features 6–8 . These overlaps suggest that cerebellar cells are exquisitely sensitive to otherwise generalized perturbations of lysosomal function.Autophagy is the major pathway for intracellular catabolic deg-radation of most long-lived proteins and organelles, thus provid-ing nutrients during starvation 9 . When core components of this pathway are impaired, the result is multisystem organ involvement that includes neurodegeneration 9–13 . In the major pathway, termed macroautophagy, the autophagosome fuses with the multivesicular body (MVB) or lysosome, and the contents are degraded by acidic hydrolases. Fusion events are at least partially regulated by the phos-phatidylinositol lipid components of the respective membranes, with phosphatidylinositol 3-phosphate (PI(3)P) associated with autophago-somes and phosphatidylinositol (3,5)-bisphosphate (PI(3,5)P 2 ) associated with MVBs and lysosomes 14 . However, the proteins regu-lating these relatively late-stage fusion events are mostly unknown.We studied a cohort of 96 families presenting with likely auto-somal recessive childhood-onset cerebellar atrophy with ataxia, 81 of which had a history of parental consanguinity and 76 of which had 2 or more affected members without congenital malformations or environmental risk factors. We performed whole-exome sequencing on at least one member of each of the families, according to pub-lished protocols 15 . For families with documented consanguinity, we prioritized homozygous, rare (allele frequency <0.2% in our in-house exome database of 3,000 individuals) and potentially damaging  variants (Genomic Evolutionary Rate Profile (GERP) score >4 or phastCons (genome conservation) score >0.9). Many of the families displayed damaging mutations in genes already implicated in cer-ebellar atrophy, including NPC1  and GRID2 . Overall, 15% of cases 2935-ll-3 3423-ll-2 525-ll-3 2892-ll-1 1382-ll-1c.645dupAp.Glu216Argfs*25c.645dupAp.Glu216Argfs*25c.1132C>Tp.Arg378*c.1132C>Tp.Arg378*c.1182delGp.Lys395Argfs*22Control(wild type)ABD-ll-2(p.Arg378*)1 24444969 130 304 336 468 570 690 807 912 946    T   M   T   M  PXAp.Leu143* p.Arg378* p.Cys890*p.IIe270Argfs*17p.Glu216Argfs*25 p.Lys395Argfs*22 p.Asp922*RGS PX PXCchr.6: 86,215,215–86,303,629NM_153816NM_02046850 kb hg19c.428T>A c.645dupA c.1132C>Tc.912+5G>A c.1182delG c.2764_2770delGACATTGc.2670delTc.809_813delTAAGATotal = 8161.73% Unidentified a bcde 2.47% GRID2 1.23% SPTBN2 1.23% SEPSECS 1.23% WDR62 1.23% NPC1 1.23%  ACOX1 1.23% CLN6 12.35% New1.23% HUWE1 1.23% ZFYVE26 1.23% SETX  1.23% EXOSC3 1.23% PIGN 9.88% SNX14 Figure 1   SNX14   mutations cause a syndromic form of severe cerebellar atrophy and coarsened facial features. ( a ) Summary of the exome sequencing results from 81 families with cerebellar atrophy. SNX14   mutations accounted for 9.9% of the families, with other genes making individual contributions. ( b ) Midline sagittal (top) or axial (middle) magnetic resonance imaging (MRI) and facies of affected individuals from representative families. Prominent atrophy of the cerebellum was 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 subjects was obtained. ( c ) SNX14   exons are shown as boxes, with the location of mutations indicated. ( d ) The location of 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*) cerebellum stained with hematoxylin and eosin compared with a control showing a reduction in the internal granule cell layer (arrows; top), near-complete depletion of Purkinje cells (arrows; middle) and dystrophic, degenerating remnant Purkinje cells (arrows; bottom). Scale bars, 100  m. Table 1 Clinical findings in individuals with biallelic SNX14   mutations Clinical featureFraction of patients displaying featureDevelopment Delayed gross motor22/22 Delayed fine motor22/22 Delayed or absent language22/22 Delayed or absent social22/22 Autistic-like behavior12/22Neurological findings Epileptic seizures8/22 Hypotonia22/22 Nystagmus11/22 Gait wide-based or absent22/22 Cerebellar atrophy on brain MRI22/22Storage disorder phenotype Coarse facies22/22 Hearing loss (SNHL)5/22 Kyphoscoliosis, clinodactyly10/22 Hepatosplenomegaly5/22 Hypertrichosis12/22 Macroglossia12/22 Atrial septal defect or patent ductus2/22 Altered urine oligosaccharides or glycosylaminoglycans5/22 See Supplementary Table 4  for detailed clinical information. SNHL, sensorineuronal hearing loss.     ©   2   0   1         5    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d . NATURE GENETICS   ADVANCE ONLINE PUBLICATION 3 LETTERS showed mutations in genes that fully explained their presentation ( Supplementary Table 1 ), 60% of families showed no obvious can-didates and 16% of families displayed putative mutations in a gene or genes not previously implicated in human disease ( Fig. 1a ).To identify causative mutations, we focused on family 468, with three similarly affected children and one healthy child, which allowed for parametric linkage analysis, defining a single major locus at chr. 6: 55,153,677–91,988,281 (hg19) (logarithm of odds (LOD) = 2.528) ( Supplementary Fig. 1 ). Alignment of all loci having LOD >–2 with whole-exome sequencing data from two affected individu-als highlighted a single c.1132C>T variant in the SNX14  gene pre-dicting a p.Arg378* alteration. Turning our attention to this gene for the remaining patients analyzed by whole-exome sequencing, we identified a total of 16 patients from 8 families with truncating  variants throughout the coding region, nearly all in constitutively spliced exons and predicted to cause loss of function ( Fig. 1b – d , Supplementary Fig. 2  and Supplementary Table 2 ). All patients displayed a block of homozygosity on chromosome 6 containing the SNX14  gene ( Supplementary Fig. 1 ), and mutations segregated according to a recessive mode of inheritance. Variants in other genes in these patients were either previously described SNPs in other populations or were of unknown effect ( Supplementary Table 3 ). Four families shared the same p.Arg378* alteration, and analysis confirmed a common 1.5-Mb haplotype, supportive of this altera-tion representing a founder mutation ( Supplementary Fig. 1 ). Overall, patients with SNX14  variants accounted for 10% of the fami-lies, making it the single most commonly mutated gene in our cohort. Furthermore, while preparing this manuscript, whole-exome sequenc-ing from an additional consanguineous family with four children SNX14 a GAPDH    F  e   t  a   l    b  r  a   i  n   B  r  a   i  n  C  e  r  e   b  e   l   l  u  m   L  u  n  g    K   i  d  n  e  y   T  e  s   t   i  s  S   k  e   l  e   t  a   l   m  u  s  c   l  e   L   i  v  e  r   F   i   b  r  o   b   l  a  s   t   H   E   K  2  9  3   T  – b RibophorinGM130IIILC3LAMP1Cathepsin DEEA1SNX14AutophagosomesEndosomes  Cis  GolgiLysosomes ERInput c Merge Merge MergeSNX14LAMP2 EEA1 GM130SNX14 SNX14 d PI(3)PPI(4)PPI(5)PPI(3,4)P 2 PI(4,5)P 2 PI(3,4,5)P 2 PI(3,5)P 2   p  4  0    P   X  S   N   X  1  4    P   X 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 and the HEK293T cell line. GAPDH   was used as a loading control. No template (−) served as a negative control. ( b ) Cell fractionation of human NPCs. SNX14 was enriched in lysosomal-endosomal compartments (red). ER, endoplasmic reticulum. ( c ) Immunostaining for LAMP2, EEA1 and GM130 (green) in NPCs expressing DsRed-tagged SNX14. SNX14 overlapped in its localization with the LAMP2 lysosomal marker (arrows). Scale bars, 10  m. ( d ) A lipid-binding assay with recombinant SNX14 PX domain on a phosphoinositide-spotted membrane showed preferential binding to PI(3,5)P 2  (red) compared with a p40 phox  PX domain control. b  468-l-2 Lysosomes Lysosomes 1382-l-2468-ll-2 1382-ll-4 SNX14 a   4  6  8  -   l  -  2  4  6  8  -   l   l  -  2  1  3  8  2  -   l  -  2   c   l  o  n  e   1  1  3  8  2  -   l  -  2   c   l  o  n  e   2  1  3  8  2  -   l   l  -  4   c   l  o  n  e   2  1  3  8  2  -   l   l  -  4   c   l  o  n  e   1 GAPDH 155040302010010    R  e   l  a   t   i  v  e  a  r  e  a   L  y  s  o  s  o  m  e  n  u  m   b  e  r 50U *** NSA U A Figure 3  Patient-derived SNX14  -mutant NPCs display enlarged lysosomes. ( a ) Immunoblot of iPSC-derived NPCs for families 468 (p.Arg378*) and 1382 (p.Lys395Argfs*22), with affected (red) and unaffected (black) samples labeled. Affected individuals had undetectable levels of SNX14 protein. GAPDH was used as a loading control. ( b ) Top, LysoTracker Green DND-26 staining with engorged lysosomes in NPCs derived from affected individuals (arrows). Scale bars, 5  m. Bottom left, the dot plot shows the relative area for individual LysoTracker-positive lysosomes ( n   = 223 and 194 lysosomes from the unaffected (U) and affected (A) NPCs of 2 families, respectively). Bottom right, graph bars represent the average number of LysoTracker-positive lysosomes per cell ( n   = 17 and 18 cells from the unaffected and affected NPCs of 2 families, respectively). Error bars, s.d. *** P   < 0.0005; NS, not significant (two-tailed t   test).     ©   2   0   1         5    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d . 4 ADVANCE ONLINE PUBLICATION NATURE GENETICS LETTERS with cerebellar atrophy independently identified a homozygous truncating mutation in SNX14  ( Supplementary Fig. 2 ). SNX14  encodes a 946-residue protein containing 2 transmembrane domains, a regulator of G protein signaling (RGS) domain predicted to act as a GTPase-activating protein (GAP) and a phox homology (PX) domain predicted to bind phosphatidylinositol lipids and function in intracellular trafficking. Alternative splicing results in transcript  variants encoding distinct isoforms. The SNX14  variants identified in patients predicted both early and late truncating events, suggesting loss of function as the disease mechanism ( Fig. 1c , d ).Patients showed several common features in addition to age-dependent atrophy of the cerebellum, with evidence of cerebral cortical atrophy in about half of the affected individuals ( Table 1  and Supplementary Table 4 ). One deceased patient studied neuropatho-logically showed almost complete absence of Purkinje cells. The few Purkinje cells remaining were ectopically located and atrophic, with enlarged apical neurites. Bergmann gliosis was prominent in the depopulated Purkinje cell layer, and neurofilament immunostaining showed radially oriented bundles of distended axons located on the superficial part of the internal granule layer. Forebrain also presented with neuronal loss, although this loss was less severe than in the cerebellum ( Fig. 1e  and Supplementary Fig. 3 ).Most patients with SNX14  mutations presented between birth and 1 year of age with global developmental delay and hypotonia. Seizures developed in half by 2 years and were well controlled with anticonvulsant medication. Nystagmus, difficulty ambulating and reduced deep tendon reflexes were present in most children, and sen-sorineuronal hearing loss was present in about one-third. Coarsened facial features with prominent forehead, epicanthal folds, upturned nares, long philtrum and full lips were seen in all, features approxi-mating those of mucopolysaccharidosis or other lysosomal storage disorders (LSDs) ( Fig. 1b  and Supplementary Fig. 2b ). Likewise, ultrastructural analysis of spinal cord tissue found axonal spheroids filled with membranous structures reminiscent of the cytoplasmic membranous bodies observed in LSDs 16  ( Supplementary Fig. 3c ). Palpable liver or spleen edge was detected in 5 of 18 patients, but no evidence of abnormal liver, urine or hematological chemistries was apparent. Urine oligosaccharides showed an abnormal pattern in one affected individual, and two patients showed elevated urinary glycosoaminoglycan levels. However, detailed lysosomal enzyme analysis in plasma and leukocytes from two affected individuals proved unremarkable ( Supplementary Note ). Although whole-exome sequenc-ing was initially required to identify patients with SNX14  mutations, as the clinical presentation was clarified, we were able to predict muta-tions with 100% accuracy, identifying an additional four patients from three families with homozygous SNX14  mutations ( Fig. 1 , Supplementary Fig. 2  and Supplementary Table 2 ). These findings suggest that this disorder represents a heretofore unknown, clinically recognizable condition. SNX14  mRNA showed nearly uniform expression in human fetal and adult tissues ( Fig. 2a ). Cellular fractionation aimed at distin-guishing the major membrane-bound pools of SNX14 protein in wild-type human neural precursor cells identified SNX14 predominantly associated with a lysosomal-rich fraction ( Fig. 2b ). Overexpression of tagged SNX14 confirmed overlapping localization with lysosomes ( Fig. 2c  and Supplementary Fig. 4 ) but not with other endosomal or Golgi markers that were present in the SNX14 fraction, suggesting a role for SNX14 in lysosomal function. Furthermore, a lipid-binding assay with recombinant PX domain from SNX14 showed specific (albeit relatively weak) direct binding with PI(3,5)P 2 , the predomi-nant phosphoinositide associated with lysosomes ( Fig. 2d ).To identify lysosomal defects associated with SNX14  mutations, we generated induced pluripotent stem cells (iPSCs) and differentiated them into neural precursor cells (NPCs) through the reprogramming 1382-l-2 468-l-2 2 µ m2 µ m500 nm 500 nm c  468-ll-21382-ll-1 2 µ m 2 µ m500 nm 500 nm LC3-II a  468-l-21382-l-2468-ll-2rescue1382-ll-1rescue468-ll-21382-ll-1 α -tubulinSNX14    F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d   F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d   F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d LC3-II α -tubulinSNX14 b  468-l-2 468-ll-21382-l-2 1382-ll-1    F  e  d   F  e  d  S   t  a  r  v  e  d  S   t  a  r  v  e  d  S   t  a  r  v  e  d  +    P   I   3  0   m   i  n  S   t  a  r  v  e  d  +    P   I   3  0   m   i  n  S   t  a  r  v  e  d  +    P   I   1    h  S   t  a  r  v  e  d  +    P   I   1    h LC3-II α -tubulinLC3-II α -tubulin *    L   C   3   f   l  u  x 1050U A    A  u   t  o  p   h  a  g  o  s  o  m  e   f  o  r  m  a   t   i  o  n 3210NSU A300 ***** 250200150    L   C   3  -   l   l  r  e   l  a   t   i  v  e   l  e  v  e   l  s 10050    F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d   F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d   F  e  d   R  a  p  a  m  y  c   i  n  S   t  a  r  v  e  d 0Unaffected Affected Rescue Figure 4  Patient-derived SNX14  -mutant NPCs display an abnormal starvation-induced autophagic response. ( a ) Immunoblot analysis of LC3-II in affected and unaffected NPCs and in affected NPCs transduced to express Flag-SNX14 (rescue) upon induction of autophagy by starvation for 1.5 h with EBSS (Eagle’s balanced salt solution) or treatment for 2 h with rapamycin (1  M). Right, graph bars represent the average LC3-II/   -tubulin levels relative to the fed condition. Error bars, s.d. ( n   = 3 clones). * P   < 0.05, ** P   < 0.005 (two-tailed t   test). Affected cells displayed an accumulation of LC3-II levels upon autophagic induction, which is partially rescued by forced SNX14 expression. ( b ) LC3 immunoblot (left) for quantification of autophagic flux measured by the LC3-II ratio in lane 3 versus lane 2 for unaffected cells (black) and lane 7 versus lane 6 for affected cells (red; middle) and quantification of autophagosome formation assessed as the increase in LC3-II levels at two time points (lane 4 versus lane 3 for controls and lane 8 versus lane 7 for affected cells) after inhibition of lysosomal proteolysis with 200  M leupeptin and 20 mM NH 4 Cl (PI, protease inhibitor) (right). The graphs present means ±  s.d. ( n   = 3 clones). * P   < 0.05; NS, not significant (two-tailed t   test). ( c ) Transmission electron microscopy analysis of unaffected (black) and affected (red) NPCs treated for 2 h with EBSS showing autophagic structures in affected NPCs (arrowheads). Data represent the results from one NPC clone for each affected or unaffected sample.     ©   2   0   1         5    N  a   t  u  r  e   A  m  e  r   i  c  a ,   I  n  c .   A   l   l  r   i  g   h   t  s  r  e  s  e  r  v  e   d . NATURE GENETICS   ADVANCE ONLINE PUBLICATION 5 LETTERS of fibroblasts from controls and SNX14 -mutated patients (from families 468 and 1382) 17,18 . As in the fibroblasts from patients, SNX14 protein was absent from patient-derived NPCs ( Fig. 3a  and Supplementary Fig. 5 ). Although we noted no difference in reprogramming, differentiation or cel-lular survival in culture for the control and patient-derived cells ( Supplementary Fig. 5 ), lysosomes appeared increased in size in patient-derived NPCs ( Fig. 3b  and Supplementary Fig. 6 ). To quantify this effect, we performed flow cytometry analysis to gate for fluorescent signal upon LysoTracker labeling, which stains intracellular acidic compartments (lysosomes and late endo-somes), and we found that about twice as many patient-derived cells as controls fell outside the normalized intensity distribution ( Supplementary Fig. 6a ).To assess whether this lysosomal enlargement affected lysosomal activity, we tested NPCs for active cathepsin D (whose levels depend upon both the lysosomal localization of the enzyme and acidification) by its specific binding to pepstatin A conjugated to BODIPY FL 19  and found no obvious differences in the intensity of stained lysosomes for control and patient-derived cells ( Supplementary Fig. 6d ). However, immunoblot analysis detected a slight but significant ( P   < 0.05) reduction in cathepsin D levels in affected NPCs in comparison to unaffected cells ( Supplementary Fig. 7c ), suggesting that a fraction of lysosomes may be defective for cathepsin D. Although we did not test for defects in other lysosomal enzyme activities in NPCs, our findings are reminiscent of those for LSDs.Autophagy requires the fusion of lysosomes with autophagosomes, so lysosomal abnormalities could result in autophagic defects such as those observed in LSDs 6–8 . To test for potential autophagic defects, we cultured patient-derived NPCs under starvation conditions and then assessed them for the levels of lipidated LC3 (LC3-II), which marks autophagosomes. Although all lines showed increased LC3-II levels upon serum starvation, patient-derived cells showed a more dra-matic response, which was reproduced under alternative induction of autophagy through inhibition of the mTOR pathway with rapamycin. Notably, the increased LC3-II levels were restored to basal rates by forced expression of tagged SNX14 in patient-derived cells ( Fig. 4a ). By LC3 flux analysis under nutrient-deprived conditions, where LC3-II ratios in the presence and absence of lysosomal inhibitors (leupep-tin and NH 4 Cl) were calculated 20 , we identified slower LC3 flux in patient-derived cells in comparison to controls. This result, together ************ Scrambled MO 6 ng  snx14  MO 6 ng1 µ m 500 µ m 1 µ m 500 µ mScrambled MO 6 ng    R  e   l  a   t   i  v  e  o  p   t   i  c   t  e  c   t  u  m   w   i   d   t   h   R  e   l  a   t   i  v  e  r   i  g   h   t  e  y  e  w   i   d   t   h 100806040200    N   I  S  c  r  a  m   b   l  e  d   s  n  x  1  4    M  O   3   n  g    s  n  x  1  4    M  O   6   n  g    N   I  S  c  r  a  m   b   l  e  d   s  n  x  1  4    M  O   3   n  g    s  n  x  1  4    M  O   6   n  g  100806040200 snx14  MO 3 ng  snx14  MO 6 ng    R  e  s  c  u  e      s     n     x       1       4    M   O    6  n  g      s     n     x       1       4    M   O    3  n  g   S  c  r  a  m   b   l  e   d  Zebrin II ********    R  e   l  a   t   i  v  e  z  e   b  r   i  n   I   I  a  r  e  a 100806040200   S  c  r  a  m   b   l  e  d   R  e  s  c  u  e   s  n  x  1  4    M  O   3   n  g    s  n  x  1  4    M  O   6   n  g       s     n     x       1       4     M   O    6  n  g   S  c  r  a  m   b   l  e   d Ptf1a / DNA      s     n     x       1       4     M   O    6  n  g   S  c  r  a  m   b   l  e   d Casp3 / DNA abec d Figure 5  Morphant snx14   zebrafish show apoptosis, excessive numbers of autophagic vesicles and loss of neural tissue, including cerebellar primordium. ( a ) Comparison of zebrafish injected with scrambled (6 ng) or snx14   (3 ng and 6 ng) morpholino (MO) 48 hours post-fertilization (h.p.f.). Calipers were used to measure the indicated distance. Scale bars, 250  m. Right, graphs present the reduced optic tectum and right eye widths in morphants. Mean ±  s.e.m. ( n   = 15 embryos with no injection (NI), 16 embryos with the scrambled morpholino, and 31 (3 ng) and 18 (6 ng) embryos with the snx14   morpholino). *** P   < 0.0005 (two-tailed t   test). ( b ) Immuno-staining of zebrafish injected with scrambled or snx14   morpholino for zebrin II (a Purkinje cell marker), with snx14   morphants rescued by coinjection with human SNX14   mRNA (50 pg). Scale bars, 50  m. Bottom, the graph presents the zebrin II compartment area relative to embryos injected with the scrambled morpholino. Mean ±  s.e.m. ( n   = 10 embryos for the scrambled morpholino, 6 embryos (3 ng) and 9 (6 ng) embryos for the snx14   morpholino, and 9 embryos for the rescue). ** P   < 0.005 (two-tailed t   test). ( c ) Maximum confocal projection from Tg( ptf1a  : EGFP  ) zebrafish at 36 h.p.f. injected with scrambled or snx14   morpholino showing reduced numbers of Purkinje cell progenitors. Scale bars, 50  m. ( d ) Maximum confocal projection with increased numbers of cells positive for caspase-3 in snx14   morphants at 36 h.p.f. DNA was stained with DAPI (4 ′ ,6-diamidino-2-phenylindole). Scale bars, 50  m. ( e ) Transmission electron microscopy showing autophagic structures in neurons residing between the optic lobes in snx14   morphants and embryos injected with scrambled morpholino at 48 h.p.f. Arrowheads, autophagic structures.
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