L444P ve N370S mutasyonları taşıyan gaucher hastalarında makrootofaji-lizozomal sistem mals

Gaucher hastalığı, lizozomal bir enzim olan glukoserebrosidazın EC 3.2.1.45 aktivitesinin bozukluğu sonucu ortaya çıkar. Lizozomlara hücre dışından endositoz ve fagositoz ve hücre içinden makrootofaji-lizozomal sistem makromoleküller taşınmaktadır. Bu çalışmada, Gaucher hastalığında biriken glukozilseramid ve glukozilsfingozinin normal otofajik füzyon süreci ve/veya otofajik akışı bozabilme ve klinik fenotipi şiddetlendirme olasılığı araştırıldı. Otofaji indüksiyonu ve otofagozomların otolizozomlara bozuk maturasyonu/füzyonu durumunun, otofajik akış otolizozomlara kargonun taşınması bozukluğundan ayırt edilmesi için, en sık gözlenen L444P ve N370S mutasyonlarını taşıyan Gaucher fibroblastlarında LC3B-II düzeyleri ve lizozomlarda floresan DQ-BSA’nın proteolizi Cellomics KSR kullanılarak izlendi. LC3B-I ve GAPDH’a göre artmış düzeydeki LC3B-II, hastaların fibroblastlarında otofagozomların sayısında artışı ve/veya azalmış lizozoma akış hızını gösterdi. Bu durum hasta fenotipleri ile uyumlu bulundu. Hasta fibroblastlarında DQ-BSA’nın düşük proteolizi, Gaucher hastalığında otofajik akış hızının azaldığını göstermektedir. Azalmış lizozoma akış hızı, L444P mutasyonu taşıyan Tip II, akut nöropatik formda daha belirgindi. Sonuç olarak, L444P ve N370S mutasyonlarında otofajik ve LC3B-II’yi de içeren diğer substratların lizozomlara akış hızı azalmıştır ve bu azalma klinik şiddet ile kabaca uyumludur.

Anahtar Kelimeler:

Makrootofaji-lizozomal sistem, Gaucher, LC3B-II, DQ-BSA

Macroautophagy-Lysosomal System Mals in Gaucher Patients Carrying L444P and N370S Mutations

Gaucher disease is caused by defects in the activity of the lysosomal enzyme, glucocerebrosidase EC 3.2.1.45 . Since lysosomes ultimately are responsible for turning over macromolecules transported to them from both outside endocytosis and phagocytosis and inside the cell macroautophagy-lysosomal system , we explored the possibility that the glucosylceramide and glucosylsphingosine accumulation resulting from Gaucher disease may disrupt the normal autophagic fusion process and/or autophagic flux exacerbating the clinical phenotype. To discriminate between an induction of autophagy and defective maturation/fusion of autophagosomes to autolysosomes, versus autophagic flux turnover of cargo in the autolysosome , LC3B-II levels and proteolysis of internalized fluorescent DQ-BSA in lysosomes with the use of high throughput instrument Cellomics KSR were monitored in Gaucher fibroblasts carrying the most prevelant mutations L444P and N370S. Increased levels of LC3B-II relative to LC3B-I and GAPDH demonstrated an increase in the number of autophagosomes and/or a decrease rate of lysosomal turn-over flux in fibroblasts of patients which were correlated with the phenotypes. Lower turnover of the DQ-BSA in patient fibroblasts indicated that the flux rate was decreased in Gaucher disease. The decrease of lysosomal flux was more significant in the Type II, acute neurophathic form of L444P mutation. We can conclude that L444P and N370S mutations decreased the the rate of turn-over of autophagy and other substrates, including LC3B-II, in the lysosome and that the decreases roughly correlated with the clinical severity.

Keywords:

Macroautophagy-lysosomal system, Gaucher, LC3B-II, DQ-BSA,

PDF

___

Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baeh- recke EH, Bahr BA, Ballabio A, et al. Guidelines for the use and interpretation of as- says for monitoring autophagy in higher eukaryotes. Autophagy 2008; 4:151-175
Tatti M, Motta M, Salvioli R.: Autophagy in Gaucher disease due to saposin C defi- ciency. Autophagy 2011; 7:94-5.
Pacheco CD, Kunkel R, Lieberman AP.: Autophagy in Niemann-Pick C disease is depen- dent upon Beclin-1 and responsive to lipid trafficking defects. Hum Mol Genet 2007; 16:1495-503.
Ballabio A. Disease pathogenesis explained by basic science: lysosomal storage dis- eases as autophagocytic disorders. Int J Clin Pharmacol Ther 2009;47Suppl 1:S34-8.
Kim I, Rodriguez-Enriquez S, Lemasters JJ. Selective degradation of mitochondria by mitophagy. Arch Biochem Biophys 2007; 462:245-253.
Xie Z, Klionsky DJ.: Autophagosome formation: core machinery and adaptations. Nat Cell Biol. 2007; 9:1102-1109.
Ding WX, Yin XM. Sorting, recognition and activation of the misfolded protein degrada- tion pathways through macroautophagy and the proteasome. Autophagy. 2008; 4:141- 150
Sun Y, Grabowski GA. Impaired autophagosomes and lysosomes in neuronopathic Gaucher disease. Autophagy 2010; 6(5):648-9
Ballabio A, Gieselmann V. Lysosomal disorders: from storage to cellular damage. Bio- chim Biophys Acta 2009; 1793:684-696.
Settembre C, Fraldi A, Jahreiss L, Spampanato C, Venturi C, Medina D, de Pablo R, Tacchetti C, Rubinsztein DC, Ballabio A. A block of autophagy in lysosomal storage disorders. Hum Mol Genet 2008; 17:119-129.
Deganuto M, Pittis MG, Pines A, Dominissini S, Kelley MR, Garcia R, Quadrifoglio F, Bembi B, Tell G. Altered intracellular redox status in Gaucher disease fibroblasts and impairment of adaptive response against oxidative stress. J Cell Physiol. 2007 Jul;212(1):223-35.
Tanaka Y, Guhde G, Suter A, Eskelinen EL, Hartmann D, Lüllmann-Rauch R, Janssen PM, Blanz J, von Figura K, Saftig P. Accumulation of autophagic vacuoles and cardio- myopathy in LAMP-2-deficient mice. Nature 2000; 406:902-66.
Koike M, Shibata M, Waguri S, Yoshimura K, Tanida I, Kominami E, Gotow T, Peters C, von Figura K, Mizushima N et al. Participation of autophagy in storage of lysosomes in neurons from mouse models of neuronal ceroid-lipofuscinoses (Batten disease). Am. J. Pathol 2005; 167:1713–1728.
Cao Y, Espinola JA, Fossale E, Massey AC, Cuervo AM, MacDonald ME, Cotman SL. Autophagy is disrupted in a knock-in mouse model of juvenile neuronal ceroid lipofus- cinosis. J. Biol Chem 2006; 281:20483–20493.
Fukuda T, Ewan L, Bauer M, Mattaliano RJ, Zaal K, Ralston E, Plotz PH, Raben N. Dysfunction of endocytic and autophagic pathways in a lysosomal storage disease. Ann Neurol 2006; 59:700–708.
Jennings JJ, Jr Zhu JH, Rbaibi Y, Luo X, Chu CT, Kiselyov K. Mitochondrial aberra- tions in mucolipidosis Type IV. J Biol Chem 2006; 281:39041–39050.
Tatti M, Motta M, Salvioli R. Autophagy in Gaucher disease due to saposin C deficiency. Autophagy 2011; 7:94-5.
Sun Y, Liou B, Ran H, Skelton MR, Williams MT, Vorhees CV, Kitatani K, Hannun YA, Witte DP, Xu YH, Grabowski GA. Neuronopathic Gaucher disease in the mouse: viable combined selective saposin C deficiency and mutant glucocerebrosidase (V394L) mice with glucosylsphingosine and glucosylceramide accumulation and progressive neuro- logical deficits. Hum Mol Genet 2010; 19:1088-97.
Pacheco CD, Kunkel R, Lieberman AP. Autophagy in Niemann-Pick C disease is depen- dent upon Beclin-1 and responsive to lipid trafficking defects. Hum Mol Genet 2007; 16:1495-503.
Wei H, Kim SJ, Zhang Z, Tsai PC, Wisniewski KE, Mukherjee AB. ER and oxidative stresses are common mediators of apoptosis in both neurodegenerative and non-neu- rodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum Mol Genet. 2008 Feb 15;17(4):469-77.