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Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.

Cullup T, Kho AL, Dionisi-Vici C, Brandmeier B, Smith F, Urry Z, Simpson MA, Yau S, Bertini E, McClelland V, Al-Owain M, Koelker S, Koerner C, Hoffmann GF, Wijburg FA, ten Hoedt AE, Rogers RC, Manchester D, Miyata R, Hayashi M, Said E, Soler D, Kroisel PM, Windpassinger C, Filloux FM, Al-Kaabi S, Hertecant J, Del Campo M, Buk S, Bodi I, Goebel HH, Sewry CA, Abbs S, Mohammed S, Josifova D, Gautel M, Jungbluth H - Nat. Genet. (2012)

Bottom Line: EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes.Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes.These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

View Article: PubMed Central - PubMed

Affiliation: DNA Laboratory, Guy's and St Thomas' Serco Pathology, Guy's Hospital, London, UK.

ABSTRACT
Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 affected individuals. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes. These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

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Fusion of LC3-positive puncta with lysosomes in Vici syndromeIn control fibroblasts subjected to 6h bafilomycin treatment, lysosomal structures were detected by staining with monoclonal anti-LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, Vici patients fibroblasts consistently show smaller LC3-positive puncta that only sporadically colocalise with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar: 5 μm
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Figure 4: Fusion of LC3-positive puncta with lysosomes in Vici syndromeIn control fibroblasts subjected to 6h bafilomycin treatment, lysosomal structures were detected by staining with monoclonal anti-LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, Vici patients fibroblasts consistently show smaller LC3-positive puncta that only sporadically colocalise with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar: 5 μm

Mentions: We hypothesized that the increase in p62/SQSTM1 and Nbr1 in atrophic muscle fibres reflects a block in the autophagy pathway, in keeping with the accumulation of autolysosomes observed after epg-5 knockdown in multicellular organisms10, and the observation of severe muscle atrophy and cardiac failure following autophagy inhibition22,32,33. To further investigate if the increased p62/SQSTM1 and Nbr1 levels are reflective of increased upstream induction or downstream blockade of the autophagy pathway, we exposed patient-derived and healthy control fibroblasts for 12 hours to the autophagy inducer rapamycin (an inhibitor of the mTORC1 complex) and the autophagy inhibitor bafilomycin (an inhibitor of the autolysosomal H+ ATPase required for acidification and hence degradation of lysosomal contents34). Untreated patient-derived cells showed increased levels of p62/SQSTM1, Nbr1 and LC3, particularly of processed, lipidated LC3-II. Induction of autophagy by rapamycin, or block of autophagosomal clearance by bafilomycin, led to strong accumulation of Nbr1 and p62/SQSTM1 in control cells at 12 hours, while the combined upstream activation by rapamycin and the block of clearance by bafilomycin led to further accumulation of these proteins, as well as of LC3-II at 12 hours (Figure 3), as expected. In contrast, the elevated levels of Nbr1 and p62/SQSTM1 increased no further in Vici syndrome patient cells following rapamycin treatment, or combined treatment. This suggests that the induction of early steps in autophagy, including the processing of LC3-I to LC3-II, is unimpaired in Vici syndrome, while the clearance of autophagosomal cargo is nearly saturated. These observations are supported by immunofluorescence microscopy in Vici patient fibroblasts: we observed a high level of baseline Nbr1 and p62/SQSTM1 positive puncta (Supplementary Figure 2) as well as LC3- and p62/SQSTM1 positive puncta (Supplementary Figure 3) compared to control cells, indicative of the accumulation of autophagosomes in the epg-5 deficient cells. In contrast, we found that the fusion of LC3-positive puncta with lysosomes, as indicated by the colocalisation of LC3 with lysosome-associated membrane proteins (LAMP1), is reduced in Vici patient fibroblasts (Figure 4). Similarly, the fusion of Nbr1-positive puncta with LAMP1 positive lysosomal vesicles was strongly reduced (Supplementary Figure 4). Lastly, baseline levels of lysine-63 polyubiquitylated proteins (a measure of ubiquitylation products destined for autolysosomal degradation) were elevated in Vici cells (Supplementary Figure 5). Together, these results indicate a severe deficit in autophagosomal clearance associated with mutations in EPG5, resulting in the accumulation of autophagic cargo in Nbr1 and SQSTM1-positive autophagosomes and impaired fusion with lysosomes.


Recessive mutations in EPG5 cause Vici syndrome, a multisystem disorder with defective autophagy.

Cullup T, Kho AL, Dionisi-Vici C, Brandmeier B, Smith F, Urry Z, Simpson MA, Yau S, Bertini E, McClelland V, Al-Owain M, Koelker S, Koerner C, Hoffmann GF, Wijburg FA, ten Hoedt AE, Rogers RC, Manchester D, Miyata R, Hayashi M, Said E, Soler D, Kroisel PM, Windpassinger C, Filloux FM, Al-Kaabi S, Hertecant J, Del Campo M, Buk S, Bodi I, Goebel HH, Sewry CA, Abbs S, Mohammed S, Josifova D, Gautel M, Jungbluth H - Nat. Genet. (2012)

Fusion of LC3-positive puncta with lysosomes in Vici syndromeIn control fibroblasts subjected to 6h bafilomycin treatment, lysosomal structures were detected by staining with monoclonal anti-LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, Vici patients fibroblasts consistently show smaller LC3-positive puncta that only sporadically colocalise with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar: 5 μm
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Related In: Results  -  Collection

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Figure 4: Fusion of LC3-positive puncta with lysosomes in Vici syndromeIn control fibroblasts subjected to 6h bafilomycin treatment, lysosomal structures were detected by staining with monoclonal anti-LAMP1. Numerous LC3-positive autophagosomes are found engulfed by the LAMP1-positive vesicular structures (arrowheads). In contrast, Vici patients fibroblasts consistently show smaller LC3-positive puncta that only sporadically colocalise with LAMP1, with many isolated LC3-positive puncta (arrows). Note that LC3 signal in Vici cells occurs mostly at the rim of LAMP1-positive structures, not centrally. Scale bar: 5 μm
Mentions: We hypothesized that the increase in p62/SQSTM1 and Nbr1 in atrophic muscle fibres reflects a block in the autophagy pathway, in keeping with the accumulation of autolysosomes observed after epg-5 knockdown in multicellular organisms10, and the observation of severe muscle atrophy and cardiac failure following autophagy inhibition22,32,33. To further investigate if the increased p62/SQSTM1 and Nbr1 levels are reflective of increased upstream induction or downstream blockade of the autophagy pathway, we exposed patient-derived and healthy control fibroblasts for 12 hours to the autophagy inducer rapamycin (an inhibitor of the mTORC1 complex) and the autophagy inhibitor bafilomycin (an inhibitor of the autolysosomal H+ ATPase required for acidification and hence degradation of lysosomal contents34). Untreated patient-derived cells showed increased levels of p62/SQSTM1, Nbr1 and LC3, particularly of processed, lipidated LC3-II. Induction of autophagy by rapamycin, or block of autophagosomal clearance by bafilomycin, led to strong accumulation of Nbr1 and p62/SQSTM1 in control cells at 12 hours, while the combined upstream activation by rapamycin and the block of clearance by bafilomycin led to further accumulation of these proteins, as well as of LC3-II at 12 hours (Figure 3), as expected. In contrast, the elevated levels of Nbr1 and p62/SQSTM1 increased no further in Vici syndrome patient cells following rapamycin treatment, or combined treatment. This suggests that the induction of early steps in autophagy, including the processing of LC3-I to LC3-II, is unimpaired in Vici syndrome, while the clearance of autophagosomal cargo is nearly saturated. These observations are supported by immunofluorescence microscopy in Vici patient fibroblasts: we observed a high level of baseline Nbr1 and p62/SQSTM1 positive puncta (Supplementary Figure 2) as well as LC3- and p62/SQSTM1 positive puncta (Supplementary Figure 3) compared to control cells, indicative of the accumulation of autophagosomes in the epg-5 deficient cells. In contrast, we found that the fusion of LC3-positive puncta with lysosomes, as indicated by the colocalisation of LC3 with lysosome-associated membrane proteins (LAMP1), is reduced in Vici patient fibroblasts (Figure 4). Similarly, the fusion of Nbr1-positive puncta with LAMP1 positive lysosomal vesicles was strongly reduced (Supplementary Figure 4). Lastly, baseline levels of lysine-63 polyubiquitylated proteins (a measure of ubiquitylation products destined for autolysosomal degradation) were elevated in Vici cells (Supplementary Figure 5). Together, these results indicate a severe deficit in autophagosomal clearance associated with mutations in EPG5, resulting in the accumulation of autophagic cargo in Nbr1 and SQSTM1-positive autophagosomes and impaired fusion with lysosomes.

Bottom Line: EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes.Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes.These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

View Article: PubMed Central - PubMed

Affiliation: DNA Laboratory, Guy's and St Thomas' Serco Pathology, Guy's Hospital, London, UK.

ABSTRACT
Vici syndrome is a recessively inherited multisystem disorder characterized by callosal agenesis, cataracts, cardiomyopathy, combined immunodeficiency and hypopigmentation. To investigate the molecular basis of Vici syndrome, we carried out exome and Sanger sequence analysis in a cohort of 18 affected individuals. We identified recessive mutations in EPG5 (previously KIAA1632), indicating a causative role in Vici syndrome. EPG5 is the human homolog of the metazoan-specific autophagy gene epg-5, encoding a key autophagy regulator (ectopic P-granules autophagy protein 5) implicated in the formation of autolysosomes. Further studies showed a severe block in autophagosomal clearance in muscle and fibroblasts from individuals with mutant EPG5, resulting in the accumulation of autophagic cargo in autophagosomes. These findings position Vici syndrome as a paradigm of human multisystem disorders associated with defective autophagy and suggest a fundamental role of the autophagy pathway in the immune system and the anatomical and functional formation of organs such as the brain and heart.

Show MeSH
Related in: MedlinePlus