Neurologic, Gastric, and Opthalmologic Pathologies in a Murine Model of Mucolipidosis Type IV
2007; Elsevier BV; Volume: 81; Issue: 5 Linguagem: Inglês
10.1086/521954
ISSN1537-6605
AutoresB. Venugopal, Marsha F. Browning, Cyntia Curcio‐Morelli, Andrea Varró, Norman Michaud, N. Nanda Nanthakumar, Steven U. Walkley, James Pickel, Susan A. Slaugenhaupt,
Tópico(s)Adenosine and Purinergic Signaling
ResumoMucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene, which encodes the 65-kDa protein mucolipin-1. The most common clinical features of patients with MLIV include severe mental retardation, delayed motor milestones, ophthalmologic abnormalities, constitutive achlorhydria, and elevated plasma gastrin levels. Here, we describe the first murine model for MLIV, which accurately replicates the phenotype of patients with MLIV. The Mcoln1−/− mice present with numerous dense inclusion bodies in all cell types in brain and particularly in neurons, elevated plasma gastrin, vacuolization in parietal cells, and retinal degeneration. Neurobehavioral assessments, including analysis of gait and clasping, confirm the presence of a neurological defect. Gait deficits progress to complete hind-limb paralysis and death at age ∼8 mo. The Mcoln1−/− mice are born in Mendelian ratios, and both male and female Mcoln1−/− mice are fertile and can breed to produce progeny. The creation of the first murine model for human MLIV provides an excellent system for elucidating disease pathogenesis. In addition, this model provides an invaluable resource for testing treatment strategies and potential therapies aimed at preventing or ameliorating the abnormal lysosomal storage in this devastating neurological disorder. Mucolipidosis type IV (MLIV) is an autosomal recessive lysosomal storage disorder caused by mutations in the MCOLN1 gene, which encodes the 65-kDa protein mucolipin-1. The most common clinical features of patients with MLIV include severe mental retardation, delayed motor milestones, ophthalmologic abnormalities, constitutive achlorhydria, and elevated plasma gastrin levels. Here, we describe the first murine model for MLIV, which accurately replicates the phenotype of patients with MLIV. The Mcoln1−/− mice present with numerous dense inclusion bodies in all cell types in brain and particularly in neurons, elevated plasma gastrin, vacuolization in parietal cells, and retinal degeneration. Neurobehavioral assessments, including analysis of gait and clasping, confirm the presence of a neurological defect. Gait deficits progress to complete hind-limb paralysis and death at age ∼8 mo. The Mcoln1−/− mice are born in Mendelian ratios, and both male and female Mcoln1−/− mice are fertile and can breed to produce progeny. The creation of the first murine model for human MLIV provides an excellent system for elucidating disease pathogenesis. In addition, this model provides an invaluable resource for testing treatment strategies and potential therapies aimed at preventing or ameliorating the abnormal lysosomal storage in this devastating neurological disorder. Mucolipidosis type IV (MLIV [MIM 252650]) belongs to a group of inherited metabolic diseases known as the lysosomal storage disorders (LSDs). It was first described in 1974 as a new variant of the mucolipidoses that displayed corneal clouding and abnormal systemic storage bodies.1Berman ER Livni N Shapira E Merin S Levij IS Congenital corneal clouding with abnormal systemic storage bodies: a new variant of mucolipidosis.J Pediatr. 1974; 84: 519-526Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 2Zeevi DA Frumkin A Bach G TRPML and lysosomal function.Biochim Biophys Acta. 2007; 1772: 851-858Crossref PubMed Scopus (65) Google Scholar MLIV is a progressive neurological disease that presents during the 1st year of life with mental retardation, corneal opacities, elevated blood gastrin levels with achlorhydria, and delayed motor milestones.1Berman ER Livni N Shapira E Merin S Levij IS Congenital corneal clouding with abnormal systemic storage bodies: a new variant of mucolipidosis.J Pediatr. 1974; 84: 519-526Abstract Full Text PDF PubMed Scopus (127) Google Scholar, 3Altarescu G Sun M Moore DF Smith JA Wiggs EA Solomon BI Patronas NJ Frei KP Gupta S Kaneski CR et al.The neurogenetics of mucolipidosis type IV.Neurology. 2002; 59: 306-313Crossref PubMed Scopus (129) Google Scholar Biochemical studies demonstrated abnormal storage of sphingolipids, phospholipids, and acid mucopolysaccharides in the lysosomes of patients with MLIV.4Bargal R Bach G Phospholipids accumulation in mucolipidosis IV cultured fibroblasts.J Inherit Metab Dis. 1988; 11: 144-150Crossref PubMed Scopus (33) Google Scholar MLIV is a rare disorder, and the majority of patients reported to date are of Ashkenazi Jewish (AJ) descent.5Bargal R Avidan N Ben-Asher E Olender Z Zeigler M Frumkin A Raas-Rothschild A Glusman G Lancet D Bach G Identification of the gene causing mucolipidosis type IV.Nat Genet. 2000; 26: 118-123Crossref PubMed Scopus (276) Google Scholar The MLIV disease gene, MCOLN1 (GenBank accession number NM_020533), maps to chromosome 19p13.2-13.3 and encodes a 580-aa protein with a predicted molecular weight of 65 kDa, named "mucolipin-1" (TRPML1). Structural analysis of the amino acid sequence predicts that the protein has six transmembrane domains, and a comparison of the protein sequence against known protein motifs identified a transient receptor potential (TRP) cation-channel domain and an internal channel pore. This TRP domain spans transmembrane domains 3–6, with the pore-forming loop between the fifth and sixth domains.5Bargal R Avidan N Ben-Asher E Olender Z Zeigler M Frumkin A Raas-Rothschild A Glusman G Lancet D Bach G Identification of the gene causing mucolipidosis type IV.Nat Genet. 2000; 26: 118-123Crossref PubMed Scopus (276) Google Scholar, 6Slaugenhaupt SA Acierno Jr, JS Helbling LA Bove C Goldin E Bach G Schiffmann R Gusella JF Mapping of the mucolipidosis type IV gene to chromosome 19p and definition of founder haplotypes.Am J Hum Genet. 1999; 65: 773-778Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 7Sun M Goldin E Stahl S Falardeau JL Kennedy JC Acierno Jr, JS Bove C Kaneski CR Nagle J Bromley MC et al.Mucolipidosis type IV is caused by mutations in a gene encoding a novel transient receptor potential channel.Hum Mol Genet. 2000; 9: 2471-2478Crossref PubMed Scopus (307) Google Scholar, 8Bassi MT Manzoni M Monti E Pizzo MT Ballabio A Borsani G Cloning of the gene encoding a novel integral membrane protein, mucolipidin—and identification of the two major founder mutations causing mucolipidosis type IV.Am J Hum Genet. 2000; 67: 1110-1120Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar The major AJ mutation, present on 72% of the AJ MLIV alleles, is an A→G transition at the 3′ acceptor site of intron 3.9Vergarajauregui S Puertollano R Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes.Traffic. 2006; 7: 337-353Crossref PubMed Scopus (126) Google Scholar The minor AJ mutation, found on 23% of the AJ MLIV alleles, is a 6,434-bp genomic deletion that spans exons 1–6 and the first 12 bp of exon 7.10Wang ZH Zeng B Pastores GM Raksadawan N Ong E Kolodny EH Rapid detection of the two common mutations in Ashkenazi Jewish patients with mucolipidosis type IV.Genet Test. 2001; 5: 87-92Crossref PubMed Scopus (13) Google Scholar, 11Goldin E Stahl S Cooney AM Kaneski CR Gupta S Brady RO Ellis JR Schiffmann R Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.Hum Mutat. 2004; 24: 460-465Crossref PubMed Scopus (56) Google Scholar Discovery of the disease gene has led to the successful implementation of genetic screening for MLIV in the AJ population.10Wang ZH Zeng B Pastores GM Raksadawan N Ong E Kolodny EH Rapid detection of the two common mutations in Ashkenazi Jewish patients with mucolipidosis type IV.Genet Test. 2001; 5: 87-92Crossref PubMed Scopus (13) Google Scholar, 12Edelmann L Dong J Desnick RJ Kornreich R Carrier screening for mucolipidosis type IV in the American Ashkenazi Jewish population.Am J Hum Genet. 2002; 70: 1023-1027Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar To date, >20 independent mutations have been described in MCOLN1.11Goldin E Stahl S Cooney AM Kaneski CR Gupta S Brady RO Ellis JR Schiffmann R Transfer of a mitochondrial DNA fragment to MCOLN1 causes an inherited case of mucolipidosis IV.Hum Mutat. 2004; 24: 460-465Crossref PubMed Scopus (56) Google Scholar, 13Slaugenhaupt SA The molecular basis of mucolipidosis type IV.Curr Mol Med. 2002; 2: 445-450Crossref PubMed Scopus (61) Google Scholar, 14Bach G Webb MB Bargal R Zeigler M Ekstein J The frequency of mucolipidosis type IV in the Ashkenazi Jewish population and the identification of 3 novel MCOLN1 mutations.Hum Mutat. 2005; 26: 591Crossref PubMed Scopus (25) Google Scholar, 15Dobrovolny R Liskova P Ledvinova J Poupetova H Asfaw B Filipec M Jirsova K Kraus J Elleder M Mucolipidosis IV: report of a case with ocular restricted phenotype caused by leaky splice mutation.Am J Ophthalmol. 2007; 143: 663-671Abstract Full Text Full Text PDF PubMed Scopus (17) Google Scholar Lipid storage disorders are most often caused by defective lysosomal hydrolases or activator proteins. However, studies performed before the cloning of the MCOLN1 gene suggested that MLIV was more likely the result of a defect in sorting and transport pathways, rather than a degradation defect, since it had been shown that the lysosomal hydrolases involved in the catabolism of the stored material in MLIV were normal.16Bargal R Bach G Mucolipidosis type IV: abnormal transport of lipids to lysosomes.J Inherit Metab Dis. 1997; 20: 625-632Crossref PubMed Scopus (68) Google Scholar, 17Chen CS Bach G Pagano RE Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease.Proc Natl Acad Sci USA. 1998; 95: 6373-6378Crossref PubMed Scopus (159) Google Scholar Lysosomal storage occurs in cells from every tissue and organ of patients with MLIV.4Bargal R Bach G Phospholipids accumulation in mucolipidosis IV cultured fibroblasts.J Inherit Metab Dis. 1988; 11: 144-150Crossref PubMed Scopus (33) Google Scholar, 16Bargal R Bach G Mucolipidosis type IV: abnormal transport of lipids to lysosomes.J Inherit Metab Dis. 1997; 20: 625-632Crossref PubMed Scopus (68) Google Scholar, 17Chen CS Bach G Pagano RE Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease.Proc Natl Acad Sci USA. 1998; 95: 6373-6378Crossref PubMed Scopus (159) Google Scholar, 18Crandall BF Philippart M Brown WJ Bluestone DA Mucolipidosis IV.Am J Med Genet. 1982; 12: 301-308Crossref PubMed Scopus (46) Google Scholar, 19Bach G Cohen MM Kohn G Abnormal ganglioside accumulation in cultured fibroblasts from patients with mucolipidosis IV.Biochem Biophys Res Commun. 1975; 66: 1483-1490Crossref PubMed Scopus (51) Google Scholar, 20Caimi L Tettamanti G Berra B Sale FO Borrone C Gatti R Durand P Martin JJ (1982) Mucolipidosis IV, a sialolipidosis due to ganglioside sialidase deficiency.J Inherit Metab Dis. 1982; 5: 218-224Crossref PubMed Scopus (31) Google Scholar, 21Folkerth RD Alroy J Lomakina I Skutelsky E Raghavan SS Kolodny EH Mucolipidosis IV: morphology and histochemistry of an autopsy case.J Neuropathol Exp Neurol. 1995; 54: 154-164Crossref PubMed Scopus (60) Google Scholar The composition of the stored material varies from tissue to tissue,21Folkerth RD Alroy J Lomakina I Skutelsky E Raghavan SS Kolodny EH Mucolipidosis IV: morphology and histochemistry of an autopsy case.J Neuropathol Exp Neurol. 1995; 54: 154-164Crossref PubMed Scopus (60) Google Scholar is heterogeneous, and includes sphingolipids (mostly gangliosides), phospholipids, and acid mucopolysaccharides. Experiments in cultured fibroblasts suggested that phospholipids and gangliosides accumulate in MLIV because of a defect in the process of endocytosis of membranous components. The defect appears to lead to excessive transport of these macromolecules into the lysosomes, which is consistent with the heterogeneity of the stored materials in MLIV.16Bargal R Bach G Mucolipidosis type IV: abnormal transport of lipids to lysosomes.J Inherit Metab Dis. 1997; 20: 625-632Crossref PubMed Scopus (68) Google Scholar Further studies examined movement of a lipid analogue along the lysosomal pathway and implicated a defect in the late steps of the endocytic pathway.17Chen CS Bach G Pagano RE Abnormal transport along the lysosomal pathway in mucolipidosis, type IV disease.Proc Natl Acad Sci USA. 1998; 95: 6373-6378Crossref PubMed Scopus (159) Google Scholar Numerous studies of TRPML1 function have been performed since it was implicated in MLIV; however, the precise pathogenic mechanism that leads to disease remains to be elucidated. TRPML1 belongs to the TRPML family, which also includes the closely related proteins mucolipin-2 (TRPML2) and mucolipin-3 (TRPML3).5Bargal R Avidan N Ben-Asher E Olender Z Zeigler M Frumkin A Raas-Rothschild A Glusman G Lancet D Bach G Identification of the gene causing mucolipidosis type IV.Nat Genet. 2000; 26: 118-123Crossref PubMed Scopus (276) Google Scholar The TRP superfamily consists of a diverse group of Ca2+-permeable nonselective cation channels that bear structural similarities to the Drosophila melanogaster TRP gene. To date, >20 TRP-related channels have been identified, and they play roles in extremely diverse cellular processes.22Pedersen SF Owsianik G Nilius B TRP channels: an overview.Cell Calcium. 2005; 38: 233-252Crossref PubMed Scopus (568) Google Scholar, 23Nilius B Owsianik G Voets T Peters JA Transient receptor potential cation channels in disease.Physiol Rev. 2007; 87: 165-217Crossref PubMed Scopus (1051) Google Scholar TRP proteins are widely expressed in the nervous system, and, in nonexcitable cells, these channels may be the primary mode of calcium entry. TRPML1 was initially reported as a novel nonselective cation channel that was permeable to Ca2+ and was regulated by changes in Ca2+ concentration.24LaPlante JM Falardeau J Sun M Kanazirska M Brown EM Slaugenhaupt SA Vassilev PM Identification and characterization of the single channel function of human mucolipin-1 implicated in mucolipidosis type IV, a disorder affecting the lysosomal pathway.FEBS Lett. 2002; 532: 183-187Abstract Full Text Full Text PDF PubMed Scopus (124) Google Scholar Subsequent studies performed in endosomal vesicles from MLIV cells, as well as in liposomes containing the in vitro translated protein, suggested that the channel can be inhibited by pH reduction and Ca2+ transport.25Raychowdhury MK Gonzalez-Perrett S Montalbetti N Timpanaro GA Chasan B Goldmann WH Stahl S Cooney A Goldin E Cantiello HF Molecular pathophysiology of mucolipidosis type IV: pH dysregulation of the mucolipin-1 cation channel.Hum Mol Genet. 2004; 13: 617-627Crossref PubMed Scopus (111) Google Scholar, 26Cantiello HF Montalbetti N Goldmann WH Raychowdhury MK Gonzalez-Perrett S Timpanaro GA Chasan B Cation channel activity of mucolipin-1: the effect of calcium.Pflugers Arch. 2005; 451: 304-312Crossref PubMed Scopus (47) Google Scholar Recently, demonstration of a strong outwardly rectifying current in whole-cell recordings has led to the suggestion that TRPML1 might function to regulate lysosomal pH and to maintain the required acidity for normal lysosomal hydrolase function.27Kiselyov K Chen J Rbaibi Y Oberdick D Tjon-Kon-Sang S Shcheynikov N Muallem S Soyombo A TRP-ML1 is a lysosomal monovalent cation channel that undergoes proteolytic cleavage.J Biol Chem. 2005; 280: 43218-43223Crossref PubMed Scopus (120) Google Scholar, 28Soyombo AA Tjon-Kon-Sang S Rbaibi Y Bashllari E Bisceglia J Muallem S Kiselyov K TRP-ML1 regulates lysosomal pH and acidic lysosomal lipid hydrolytic activity.J Biol Chem. 2006; 281: 7294-7301Crossref PubMed Scopus (174) Google Scholar Several studies have shown that TRPML1 is localized to late endosomes and lysosomes.29Manzoni M Monti E Bresciani R Bozzato A Barlati S Bassi MT Borsani G Overexpression of wild-type and mutant mucolipin proteins in mammalian cells: effects on the late endocytic compartment organization.FEBS Lett. 2004; 567: 219-224Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 30Pryor PR Reimann F Gribble FM Luzio JP Mucolipin-1 is a lysosomal membrane protein required for intracellular lactosylceramide traffic.Traffic. 2006; 7: 1388-1398Crossref PubMed Scopus (126) Google Scholar, 31Miedel MT Weixel KM Bruns JR Traub LM Weisz OA Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1.J Biol Chem. 2006; 281: 12751-12759Crossref PubMed Scopus (50) Google Scholar An N-terminal dileucine motif is sufficient for lysosomal targeting of full-length TRPML1, and this targeting is AP1 dependent.9Vergarajauregui S Puertollano R Two di-leucine motifs regulate trafficking of mucolipin-1 to lysosomes.Traffic. 2006; 7: 337-353Crossref PubMed Scopus (126) Google Scholar, 31Miedel MT Weixel KM Bruns JR Traub LM Weisz OA Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1.J Biol Chem. 2006; 281: 12751-12759Crossref PubMed Scopus (50) Google Scholar, 32Venkatachalam K Hofmann T Montell C Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1.J Biol Chem. 2006; 281: 17517-17527Crossref PubMed Scopus (113) Google Scholar Endogenous TRPML1 is cleaved in its long luminal loop between the first and second transmembrane domains; however, the role of this cleavage in regulating protein function has yet to be determined.31Miedel MT Weixel KM Bruns JR Traub LM Weisz OA Posttranslational cleavage and adaptor protein complex-dependent trafficking of mucolipin-1.J Biol Chem. 2006; 281: 12751-12759Crossref PubMed Scopus (50) Google Scholar, 33Kiselyov K Soyombo A Muallem S TRPpathies.J Physiol. 2007; 578: 641-653Crossref PubMed Scopus (63) Google Scholar Homo- and heteromultimerization of TRPML1 with TRPML2 and TRPML3 was recently described in cells overexpressing all three proteins.32Venkatachalam K Hofmann T Montell C Lysosomal localization of TRPML3 depends on TRPML2 and the mucolipidosis-associated protein TRPML1.J Biol Chem. 2006; 281: 17517-17527Crossref PubMed Scopus (113) Google Scholar Significant mitochondrial fragmentation and decreased mitochondrial Ca2+ buffering efficiency have also been described in MLIV cells. Jennings et al. speculate that a defect in mitochondrial recycling leads to increased sensitivity to apoptosis induced by Ca2+, possibly explaining the degenerative cell death found in MLIV and other LSDs.34Jennings Jr, JJ Zhu JH Rbaibi Y Luo X Chu CT Kiselyov K Mitochondrial aberrations in mucolipidosis type IV.J Biol Chem. 2006; 281: 39041-39050Crossref PubMed Scopus (119) Google Scholar Defective lysosomal exocytosis has also been reported in fibroblasts isolated from patients with MLIV.35LaPlante JM Sun M Falardeau J Dai D Brown EM Slaugenhaupt SA Vassilev PM Lysosomal exocytosis is impaired in mucolipidosis type IV.Mol Genet Metab. 2006; 89: 339-348Crossref PubMed Scopus (102) Google Scholar The Caenorhabditis elegans mucolipin-1 gene ortholog, cup-5, was isolated during a screen for endocytosis-defective mutants.36Fares H Greenwald I Regulation of endocytosis by CUP-5, the Caenorhabditis elegans mucolipin-1 homolog.Nat Genet. 2001; 28: 64-68Crossref PubMed Google Scholar Mutations in cup-5 result in an enhanced rate of uptake of fluid-phase markers, decreased degradation of endocytosed protein, and accumulation of large vacuoles. Interestingly, human TRPML1 was able to rescue the abnormal lysosomal accumulation in these mutants. Recent studies in C. elegans revealed that a mutation in the ATP-binding cassette transporter MRP-4 compensates the degradation defect caused by the absence of cup-5.37Schaheen L Dang H Fares H Basis of lethality in C. elegans lacking CUP-5, the mucolipidosis type IV orthologue.Dev Biol. 2006; 293: 382-391Crossref PubMed Scopus (34) Google Scholar The phenotype of MLIV is variable, with psychomotor delay and corneal opacities by age 1 year as the most common presentation.21Folkerth RD Alroy J Lomakina I Skutelsky E Raghavan SS Kolodny EH Mucolipidosis IV: morphology and histochemistry of an autopsy case.J Neuropathol Exp Neurol. 1995; 54: 154-164Crossref PubMed Scopus (60) Google Scholar, 38Tellez-Nagel I Rapin I Iwamoto T Johnson AB Norton WT Nitowsky H Mucolipidosis IV: clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy.Arch Neurol. 1976; 33: 828-835Crossref PubMed Scopus (78) Google Scholar The brain pathology in patients with MLIV is characterized by pigmented cytoplasmic granules in nondistended neurons and microglial cells.21Folkerth RD Alroy J Lomakina I Skutelsky E Raghavan SS Kolodny EH Mucolipidosis IV: morphology and histochemistry of an autopsy case.J Neuropathol Exp Neurol. 1995; 54: 154-164Crossref PubMed Scopus (60) Google Scholar Electron microscopy (EM) reveals accumulation of lamellated membrane structures and amorphous material in lysosomes in every cell type examined.21Folkerth RD Alroy J Lomakina I Skutelsky E Raghavan SS Kolodny EH Mucolipidosis IV: morphology and histochemistry of an autopsy case.J Neuropathol Exp Neurol. 1995; 54: 154-164Crossref PubMed Scopus (60) Google Scholar, 38Tellez-Nagel I Rapin I Iwamoto T Johnson AB Norton WT Nitowsky H Mucolipidosis IV: clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy.Arch Neurol. 1976; 33: 828-835Crossref PubMed Scopus (78) Google Scholar Cultured skin fibroblasts derived from patients with MLIV contain autofluorescent lysosomes.39Goldin E Blanchette-Mackie EJ Dwyer NK Pentchev PG Brady RO Cultured skin fibroblasts derived from patients with mucolipidosis 4 are auto-fluorescent.Pediatr Res. 1995; 37: 687-692Crossref PubMed Scopus (40) Google Scholar Storage vacuoles are present in the corneal epithelium, as well as in many other cell types, including pancreatic acinar cells, macrophages, and chondrocytes, and in the renal collecting ducts and hepatic bile duct.38Tellez-Nagel I Rapin I Iwamoto T Johnson AB Norton WT Nitowsky H Mucolipidosis IV: clinical, ultrastructural, histochemical, and chemical studies of a case, including a brain biopsy.Arch Neurol. 1976; 33: 828-835Crossref PubMed Scopus (78) Google Scholar, 40Livni N Merin S Mucolipidosis IV: ultrastructural diagnosis of a recently defined genetic disorder.Arch Pathol Lab Med. 1978; 102: 600-604PubMed Google Scholar Diagnosis of MLIV is suggested for patients with characteristic neurodevelopmental delay, elevated blood gastrin levels, and corneal clouding. Currently, confirmatory molecular diagnosis can be made via targeted mutation analysis of the two most common mutations, IVS3-2 A→G and 511→6994 del, which together account for 95% of mutations in the AJ population.10Wang ZH Zeng B Pastores GM Raksadawan N Ong E Kolodny EH Rapid detection of the two common mutations in Ashkenazi Jewish patients with mucolipidosis type IV.Genet Test. 2001; 5: 87-92Crossref PubMed Scopus (13) Google Scholar To understand the pathogenic mechanism by which complete loss of TRPML1 causes MLIV, we generated a mouse with a targeted disruption of the Mcoln1 gene (GenBank accession number NM_053177). This Mcoln1 knockout, designated "Mcoln1−/−," exhibits many clinical and cellular features of the human disease. Murine Mcoln1 is highly similar to human MCOLN1, showing 86% nucleotide identity and 91% amino acid identity. In addition, all six of the transmembrane domains, the putative cation channel, and the dileucine (L-L-X-X) motif at the C-terminus are all highly conserved.41Falardeau JL Kennedy JC Acierno Jr, JS Sun M Stahl S Goldin E Slaugenhaupt SA Cloning and characterization of the mouse Mcoln1 gene reveals an alternatively spliced transcript not seen in humans.BMC Genomics. 2002; 3: 3Crossref PubMed Scopus (16) Google Scholar Our mouse model will be an invaluable resource for understanding the role of TRPML1 in lysosomal function and vesicular trafficking, mitochondrial recycling, and other cellular functions that play a role in the pathophysiology of MLIV. Furthermore, Mcoln1−/− mice will be crucial for testing therapeutic agents aimed at ameliorating the abnormal lysosomal storage in MLIV. The Mcoln1−/− knockout mice were generated and propagated following National Institutes of Health (NIH) guidelines for animal care under an approved protocol of the Animal Care and Use Committee of the National Institutes of Mental Health. All breeding and other procedures performed were reviewed and approved by the Massachusetts General Hospital Subcommittee on Research Animal Care. Mcoln1 genomic DNA clones were isolated by plaque hybridization of a mouse 129/Sv genomic DNA library with full-length mouse Mcoln1 cDNA probes. The targeting vector was constructed as follows (fig. 1A): a neomycin resistance gene (Neor cassette) for positive selection was inserted between exons 2 and 5 of Mcoln1. The herpes simplex virus–thymidine kinase gene (HSV-TK) for negative selection was inserted 3′ of Mcoln1 within exon 13. Methods for generating targeted embryonic stem cells and producing mice from these cells have been described elsewhere.42Nagy A Gertsenstein M Vintersten K Behringer R Manipulating the mouse embryo. Cold Spring Laboratory Press, Cold Spring Harbor, NY2003Google Scholar Hybrid LC3 (C57BL6×129S6) embryonic stem cells (GlobalStem) were transfected with the Mcoln1 targeting vector by electroporation. After 24 h, cells were fed with medium containing 200 βg/ml of G418. G418-resistant clones were picked after 7 d and were screened for homologous recombination by Southern blotting. Three clones carrying a Mcoln-1 targeted allele were injected into C57BL6 blastocysts, which were then transferred into oviducts or uterus of pseudopregnant outbreed recipient females. Pups were evaluated for chimerism. Chimeras from all three lines were mated to C57BL/6 females. Germline transmission was evidenced by coat color and was confirmed by PCR. For Southern-blot analysis, 5′ and 3′ probes were generated using the following sets of primers: forward 5′-TAAACTTGGTGGAGGCTTGC-3′ and reverse 5′-GTGCTCAGCCACTGGGTAAC-3′ and forward 5′-TAGGCTGATCAACGTCACCA-3′ and reverse 5′-GGGTCTGAGGATGGGAGAGT-3′. Mouse genomic DNA was used as a template for the reactions. DNA extracted from embryonic stem cells was digested with AseI (for 5′-probe hybridization) and with PinA and PspI (for 3′-probe hybridization), was run on a 0.8% agarose gel, was blotted onto GeneScreen Plus membrane (Perkin Elmer), and was hybridized with the respective radiolabeled probes. The 5′ probe detected a 7.7-kb band in the targeted allele and an 11.2-kb band in the normal allele, and the 3′ probe detected a 9.6-kb band in the targeted allele and an 11.2-kb band in the normal allele. For expression analysis, 1–2 βg of total RNA extracted from brain was used to synthesize cDNA with SuperScript III (Invitrogen Life Technologies) and was used as template for PCR and quantitative real-time PCR (Q-PCR), as follows. For PCR, exons 2–6 of Mcoln1 were amplified with the primer set forward 5′-CCCCACAGAAGAGGAAGA-3′ and reverse 5′-GTGGATGGTGACATTGACC-3′. PCR products were sequenced to confirm Mcoln1 identity. For Q-PCR, analysis of Mcoln1 was performed using SYBR Green PCR kit in I-Cycler (Bio-Rad). Standard curves representing five-point 1:3 serial dilution of cDNA of one of the samples in the control group were analyzed in each assay. The r2 values were ⩾0.99 for all standard curves, and the amplification efficiency varied between 90% and 100%. Sample relative quantification was analyzed by the ΔCT method, using CT values from Mcoln1+/+ mice as calibrators, and was corrected by the internal control—hypoxanthine guanine phosphoribosyl transferase (HPRT)—in all experiments. Primers were designed to detect Mcoln1 message within exon 4 forward primer 5′-ATGTGGACCCAGCCAATGATACCT-3′ and exon 6 reverse primer 5′-TGTCTTCAGCTGGAAGTGGATGGT-3′. Mouse HPRT primers were forward 5′-TCGAAGTGTTGGATACAGGCC-3′ and reverse 5′-CAACAGGACTCCTCGTATTTG-3′. All primers were designed using PrimerQuest software (Integrated DNA Technologies). Genotyping was done by PCR with use of genomic DNA from mouse tail snips, prepared using the method with hot sodium hydroxide and Tris-HCl.43Truett GE Heeger P Mynatt RL Truett AA Walker JA Warman ML Preparation of PCR-quality mouse genomic DNA with hot sodium hydroxide and Tris (HotSHOT).Biotechniques. 2000; 29: 52-54PubMed Google Scholar PCR was performed with HotStart-Red Taq polymerase (Sigma). Three primers were used in the reaction to easily identify Mcoln1+/+ (wild-type), Mcoln1+/− (heterozygote), and Mcoln1−/− (knockout) mice: forward 5′-GACCCAGGAATGACACCTTC-3′, located in intron 3 position 9060 of the Mcoln1 sequence; reverse 5′-CCCCTTGCTGCCATGTAATA-3′, located in the Neor cassette present in the targeted allele; and reverse 5′-GCGCAAACCACATGTGCTTT-3′, located in intron 3 position 9441 of Mcoln1. Mcoln1+/+ and Mcoln1−/− mice were sacrificed, and gastric tissues were harvested and washed. The tissues were dissected from mice and were fixed in 2.0% glutaraldehyde in 0.1 M sodium cacodylate buffer (pH 7.4) overnight at 4°C. Tissues were stained with hematoxylin and eosin for light microscopy or were postfixed in 1.0% osmium tetroxide (Electron Microscopy Sciences) in cacodylate buffer for EM and infiltrated with Epon resin (Ted Pella). The following day, samples were placed in fresh 100% EPON resin and were embedded in an oven at 60°C overnight. Thin sections were stained with uranyl acetate and lead citrate and were examined at 80 kV in a JEOL 1011 transmission electron microscope (Advanced Microscopy Techniques). For studies of brain, mice were deeply anesthetized with sodium pentobarbital and were perfused systemically with 0.9% saline followed by 4.0% paraformaldehyde in 0.1 M phosphate buffer. Tissue blocks for EM were postfixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer. Before the embedding in EPON, tissues were fixed in 2% osmium tetroxide, we
Referência(s)