Abstract
Infantile neuronal ceroid lipofuscinosis (INCL) is a devastating childhood neurodegenerative lysosomal storage disease (LSD) that has no effective treatment. It is caused by inactivating mutations in the palmitoyl-protein thioesterase-1 (PPT1) gene. PPT1 deficiency impairs the cleavage of thioester linkage in palmitoylated proteins (constituents of ceroid), preventing degradation by lysosomal hydrolases. Consequently, accumulation of lysosomal ceroid leads to INCL. Thioester linkage is cleaved by nucleophilic attack. Hydroxylamine, a potent nucleophilic cellular metabolite, may have therapeutic potential for INCL, but its toxicity precludes clinical application. We found that a hydroxylamine derivative, N-(tert-Butyl) hydroxylamine (NtBuHA), was non-toxic, cleaved thioester linkage in palmitoylated proteins and mediated lysosomal ceroid depletion in cultured cells from INCL patients. In Ppt1−/− mice, which mimic INCL, NtBuHA crossed the blood-brain barrier, depleted lysosomal ceroid, suppressed neuronal apoptosis, slowed neurological deterioration and extended lifespan. Our findings provide a proof of concept that thioesterase-mimetic and antioxidant small molecules such as NtBuHA are potential drug targets for thioesterase deficiency diseases such as INCL.
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References
Cox, T.M. & Cachón-González, M.B. The cellular pathology of lysosomal diseases. J. Pathol. 226, 241–254 (2012).
Jeyakumar, M., Dwek, R.A., Butters, T.D. & Platt, F.M. Storage solutions: treating lysosomal disorders of the brain. Nat. Rev. Neurosci. 6, 713–725 (2005).
Anderson, G.W., Goebel, H.H. & Simonati, A. Human pathology in NCL. Biochim. Biophys. Acta 1832, 1807–1826 (2013).
Kousi, M., Lehesjoki, A.E. & Mole, S.E. Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses. Hum. Mutat. 33, 42–63 (2012).
Haltia, M. & Goebel, H.H. The neuronal ceroid lipofuscinoses: a historical introduction. Biochim. Biophys. Acta 1832, 1795–1800 (2013).
Wong, A.M., Rahim, A.A., Waddington, S.N. & Cooper, J.D. Current therapies for the soluble lysosomal forms of neuronal ceroid lipofuscinosis. Biochem. Soc. Trans. 38, 1484–1488 (2010).
Camp, L.A. & Hofmann, S.L. Purification and properties of a palmitoyl-protein thioesterase that cleaves palmitate from H-Ras. J. Biol. Chem. 268, 22566–22574 (1993).
Camp, L.A., Verkruyse, L.A., Afendis, S.J., Slaughter, C.A. & Hofmann, S.L. Molecular cloning and expression of palmitoyl-protein thioesterase. J. Biol. Chem. 269, 23212–23219 (1994).
Verkruyse, L.A. & Hofmann, S.L. Lysosomal targeting of palmitoyl protein thioesterase. J. Biol. Chem. 271, 15831–15836 (1996).
Hellsten, E., Vesa, J., Olkkonen, V.M., Jalanko, A. & Peltonen, L. Human palmitoyl protein thioesterase: evidence for lysosomal targeting of the enzyme and disturbed cellular routing in infantile neuronal ceroid lipofuscinosis. EMBO J. 15, 5240–5245 (1996).
Järvela, I. Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1. Genomics 9, 170–173 (1991).
Vesa, J. et al. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature 376, 584–587 (1995).
Hofmann, S.L. & Peltonen, L. The neuronal ceroid lipofuscinosis. in The Metabolic and Molecular Basis of Inherited Disease 8th edn (eds. Scriver, C.R., Beudet, A.L., Sly, W.S. & Valle, D.) 3877–3894 (McGraw-Hill, New York, 2001).
Smotrys, J.E. & Linder, M.E. Palmitoylation of intracellular signaling proteins: regulation and function. Annu. Rev. Biochem. 73, 559–587 (2004).
Resh, M.D. Palmitoylation of ligands receptors and intracellular signaling molecules. Sci. STKE 2006, re14 (2006).
Salaun, C., Greaves, J. & Chamberlain, L.H. The intracellular dynamic of protein palmitoylation. J. Cell Biol. 191, 1229–1238 (2010).
Iwanaga, T., Tsutsumi, R., Noritake, J., Fukata, Y. & Fukata, M. Dynamic protein palmitoylation in cellular signaling. Prog. Lipid Res. 48, 117–127 (2009).
Lu, J.Y., Verkruyse, L.A. & Hofmann, S.L. Lipid thioesters derived from acylated proteins accumulate in infantile neuronal ceroid lipofuscinosis: correction of the defect in lymphoblasts by recombinant palmitoyl-protein thioesterase. Proc. Natl. Acad. Sci. USA 93, 10046–10050 (1996).
Jocelyn, P.C. Biochemistry of the SH Groups (Academic Press, New York, 1972).
Gross, P. Biologic activity of hydroxylamine: a review. Crit. Rev. Toxicol. 14, 87–99 (1985).
Kirby, A.J. et al. Hydroxylamine as an oxygen nucleophile. Structure and reactivity of ammonia oxide. J. Am. Chem. Soc. 128, 12374–12375 (2006).
Drisdel, R.C., Alexander, J.K., Sayeed, A. & Green, W.N. Assays of protein palmitoylation. Methods 40, 127–134 (2006).
Mosmann, T. Rapid colorimetric assay for cellular growth and survival. J. Immunol. Methods 65, 55–63 (1983).
Atamna, H., Robinson, C., Ingersoli, R., Elliott, H. & Aimes, B.N. N-t-butyl hydroxylamine is an antioxidant that reverses age-related changes in mitochondria in vivo and in vitro. FASEB J. 15, 2196–2204 (2001).
Gupta, P. et al. Disruption of PPT1 or PPT2 causes neuronal ceroid lipofuscinosis in knockout mice. Proc. Natl. Acad. Sci. USA 98, 13566–13571 (2001).
Zhang, Z. et al. Palmitoyl-protein thioesterase-1 deficiency mediates the activation of the unfolded protein response and neuronal apoptosis in INCL. Hum. Mol. Genet. 15, 337–346 (2006).
Kim, S.J., Zhang, Z., Lee, Y.C. & Mukherjee, A.B. Palmitoyl-protein thioesterase-1 deficiency leads to the activation of caspase-9 and contributes to rapid neurodegeneration in INCL. Hum. Mol. Genet. 15, 1580–1586 (2006).
Wei, H. et al. ER- and oxidative-stresses are common mediators of apoptosis in both neuro- degenerative and non-neurodegenerative lysosomal storage disorders and are alleviated by chemical chaperones. Hum. Mol. Genet. 17, 469–477 (2008).
Riikonen, R., Vanhanen, S.L., Tyynela, J., Santavuori, P. & Turpeinen, U. CSF insulin-like growth factor-1 in infantile neuronal ceroid lipofuscinosis. Neurology 54, 1828–1832 (2000).
Kaur, P. et al. Metabolomic profiling for biomarker discovery in pancreatic cancer. Int. J. Mass Spectrom. 310, 44–51 (2012).
Munasinghe, J., Zhang, Z., Kong, E., Heffer, A. & Mukherjee, A.B. Evaluation of neurodegeneration in a mouse model of infantile Batten disease by magnetic resonance imaging and magnetic resonance spectroscopy. Neurodegener. Dis. 9, 159–169 (2012).
Zhang, Z. et al. Palmitoyl-protein thioesterase gene expression in the developing mouse brain and retina: implications for early loss of vision in infantile neuronal ceroid lipofuscinosis. Gene 231, 203–211 (1999).
Isosomppi, J. et al. Developmental expression of palmitoyl protein thioesterase in normal mice. Brain Res. Dev. Brain Res. 118, 1–11 (1999).
Macauley, S.L., Pekny, M. & Sands, M.S. The role of attenuated astrocyte activation in infantile neuronal ceroid lipofuscinosis. J. Neurosci. 31, 15575–15585 (2011).
Hamm, R.J., Pike, B.R., O'Dell, D.M., Lyeth, V. & Jenkins, L.W. The rotarod test: an evaluation of its effectiveness in assessing motor deficits following traumatic brain injury. J. Neurotrauma 11, 187–196 (1994).
Crawley, J.N. Exploratory behavior model of anxiety in mice. Neurosci. Biobehav. Rev. 9, 37–44 (1985).
El-Husseini, A.E.-D. & Bredt, D.S. Protein palmitoylation: a regulator of neuronal development and function. Nat. Rev. Neurosci. 3, 791–802 (2002).
Duncan, J.A. & Gilman, A.G. A cytoplasmic acyl-protein thioesterase that removes palmitate from G protein alpha subunits and p21(RAS). J. Biol. Chem. 273, 15830–15837 (1998).
Tomatis, V.M., Trenchi, A., Gomez, G.A. & Daniotti, J.L. Acyl-protein thioesterase 2 catalyzes the decimation of peripheral membrane-associated GAP-43. PLoS ONE 5, e15045 (2010).
Greaves, J. et al. Palmitoylation-induced aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis. J. Biol. Chem. 287, 37330–37339 (2012).
Platt, F.M. & Lachmann, R.H. Treating lysosomal storage disorders: current practice and future prospects. Biochim. Biophys. Acta 1793, 737–745 (2009).
Kohan, R. et al. Therapeutic approaches to the challenge of neuronal ceroid lipofuscinoses. Curr. Pharm. Biotechnol. 12, 867–883 (2011).
Lu, J.Y., Hu, J. & Hofmann, S.L. Human recombinant palmitoyl-protein thioesterase-1 (PPT1) for preclinical evaluation of enzyme replacement therapy for infantile neuronal ceroid lipofuscinosis. Mol. Genet. Metab. 99, 374–378 (2010).
Hu, J. et al. Intravenous high-dose enzyme replacement therapy with recombinant palmitoyl-protein thioesterase reduces visceral lysosomal storage and modestly prolongs survival in a preclinical mouse model of infantile neuronal ceroid lipofuscinosis. Mol. Genet. Metab. 107, 213–221 (2012).
Griffey, M.A. et al. CNS-directed AAV2-mediated gene therapy ameliorates functional deficits in a murine model of infantile neuronal ceroid lipofuscinosis. Mol. Ther. 13, 538–547 (2006).
Chen, Y.H., Chang, M. & Davidson, B. L Molecular signatures of disease brain endothelia provide new sites for CNS-directed enzyme therapy. Nat. Med. 15, 1215–1218 (2009).
Lönnqvist, T. et al. Hematopoietic stem cell transplantation in infantile neuronal ceroid lipofuscinosis. Neurology 57, 1411–1416 (2001).
Tamaki, S.J. et al. Neuroprotection of host cells by human central nervous system stem cells in a mouse model of infantile neuronal ceroid lipofuscinosis. Cell Stem Cell 5, 310–319 (2009).
Srikanth, M. & Kessler, J.A. Nanotechnology-novel therapeutics for CNS disorders. Nat. Rev. Neurol. 8, 307–318 (2012).
Franken, N.A., Rodermond, H.M., Stap, J., Haverman, J. & van Bree, C. Clonogenic assay of cells in vitro. Nat. Protoc. 1, 2315–2319 (2006).
Acknowledgements
We thank S.L. Hofmann (University of Texas Southwestern Medical Center) for the generous gift of Ppt1−/− mice and for a sample of recombinant human PPT1 enzyme that we used as a positive control. M. Sands (Washington University School of Medicine) provided a pair of C57 congenic Ppt1−/− mice that we used to establish our colony at the US National Institutes of Health. We thank S.W. Levin, J.Y. Chou and I. Owens for critical review of the manuscript and helpful suggestions. We also thank A. Bouchelion and A. Chen for editorial assistance. We thank L. Dye at the Microscopy and Imaging Core facility for his expert assistance in performing TEM of cultured lymphoblasts and mouse brain tissues. We are grateful to H.-S. Jun for helping us with the FACS analysis. The mass spectrometric analyses for the detection of NtBuHA in brain tissues were performed in the laboratory of A.K. Cheema (Georgetown University School of Medicine). This research was supported in whole by the Intramural Research Program of the Eunice Kennedy Shriver National Institute of Child Health and Human Development.
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C.S. and G.C. designed and performed the majority of the experiments. S.P. performed genotyping of the mice, densitometric analyses, motor function and behavior testing, and analyzed the data. Z.Z. designed some of the experiments, performed data analysis and prepared the illustrations. A.L. provided statistical analyses of the data. A.B.M. conceived the project, designed some of the experiments and wrote major portions of the manuscript. All of the authors participated in the writing and editing of the manuscript.
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Integrated supplementary information
Supplementary Figure 1 Schematic depiction of the mechanism of ceroid accumulation in INCL.
In normal cells, the membrane-anchored palmitoylated proteins are depalmitoylated by lysosomal PPT1 and degraded by lysosomal hydrolases clearing the lysosomes. However in PPT1-deficient (INCL) cells failure of depalmitoylation makes these proteins refractory to lysosomal hydrolases. Consequently, accumulation of the palmitoylated proteins leads to lysosomal storage of ceroid causing INCL pathogenesis.
Supplementary Figure 2 Effect of hydroxylamine derivatives on viability of INCL fibroblasts.
INCL fibroblasts were incubated with increasing concentrations of (a) N-t-butyl hydroxylamine, (b) N-benzylhydroxylamine, (c) N-Methyl hydroxylamine, (d) N-cyclohexyl hydroxylamine, (e) N-benzyloxycarbonyl hydroxylamine, (f) N,N-dimethyl hydroxylamine, (g) N,N-diethyl hydroxylamine, (h) N-benzoyl-N-phenyl hydroxylamine, (i) N,O-bis(trimethylsilyl) hydroxylamine, (j) N,O-Di-BOC hydroxylamine for 48 hours. Viability of the treated cells was estimated by MTT assay (see method section). Note that most of the derivatives are nontoxic up to 1 mM concentration. * P<0.05.
Supplementary Figure 3 Viability and plating efficiency of INCL lymphoblasts treated with NtBuHA.
(a). Lymphoblasts isolated from INCL patients were cultured for varying length of time in presence of 500 μM NtBuHA. Viability of those cells at the end of incubation period was estimated by MTT assay and expressed as percent of viability of untreated control. The data is presented as the mean of three independent experiments ±SD. (b). Skin fibroblasts from an INCL patient were treated with varying doses (0 to 2.5 mM) of NtBuHA for 48 h. At the end of the treatment period the cells were trypsinized and re-plated at a density of 100 cells per plate. The colonies formed from 100 cells in more than 10 replicates for each treatment were stained with Crystal violet, air dried and photographed.
Supplementary Figure 4 NtBuHA-mediated cleavage of thioester linkage in [14C] Palmitoyl CoA.
(a) Dose-response: The release of free [14C]palmitic acid from [14C] palmitoyl CoA by varying doses of NtBuHA at room temperature for 1 h. Lanes: (1) [14C]palmitic acid standard; (2) [14C]-palmitoyl CoA standard; (3) 10 μM NtBuHA; (4) 50 μM NtBuHA; (5) 100 μM NtBuHA; (6) 250 μM NtBuHA and (7) 500 μM NtBuHA. (b) Time-course of [14C]-palmitic acid release from [14C] palmitoyl CoA by 500 μM of NtBuHA. Lanes: (1) [14C]palmitic acid standard; (2) 0 min; (3) 15 min; (4) 30 min; (5) 60 min; (6) 120 min. Arrows indicate free [14C]Palmitate released. The densitometric quantitation of free [14C]palmitate bands are shown graphically below the autoradiographs of the TLCs (n=3). Note that the release of [14C]palmitate from [14C]-palmitoyl CoA by NtBuHA is dose- and time-dependent. The results were analyzed using student t test and P<0.01 was considered significant (in panel a) and P<0.05 in panel b at 60 and 120 minutes.
Supplementary Figure 5 Densitometric quantitation of lipid thioester bands in untreated- and NtBuHA-treated lymphoblasts from normal subjects and INCL patients.
[35S]Cysteine-labeled lipid thioester bands resolved by TLC as shown in Figure 1f. Eight bands were readily identifiable (arrows) and marked as bands 1 to 8 (from the top). The density of each of these bands from untreated and NtBuHA-treated INCL patients' cells were quantified using QuantityOne software (Biorad). The densitometric data are presented as the mean intensity/mm2 (INT/mm2) from three independent experiments ±SD. (b). [35S]Cysteine-labeled lipid thioester bands in untreated and NtBuHA-treated lymphoblasts from 9 INCL patients are resolved by TLC as shown in Figure 1g (right panel). Each of the 8 readily identifiable bands as indicated in Fig.1f was marked as bands 1 to 8 (from the top). The results of densitometric quantitation are presented as the mean of 3 independent experiments ±SD.
Supplementary Figure 6 Depletion of GRODs in INCL fibroblasts after treatment with NtBuHA.
INCL fibroblast cells were treated without (a) or with (b) 250 μM of NtBuHA for 3 weeks. Cells were then fixed in 2.5% glutaraldehyde and processed for electron microscopy. The number of GRODs/cell from untreated- (n=7) and NtBuHA-treated (n=10) INCL fibroblasts are graphically represented in panel c. Note a clear reduction in GROD level in INCL cells treated with NtBuHA. Scale bar: 1μm.
Supplementary Figure 7 Western blot analyses of ER- and oxidative stress markers in WT and Ppt1-/- mouse brains.
Cortical tissue homogenates prepared from 6-month old WT, untreated-Ppt1-/- and NtBuHA-treated Ppt1-/- mice (n=6 in each group) were analyzed for ER-stress markers: ATF6, Grp94 and Grp78 and oxidative stress-markers: SOD2 and catalase, respectively. The results of densitometric quantitation of the protein bands (mean ±SDs) are represented in the bar graphs.
Supplementary Figure 8 Protection of superficial and deep cortical neurons of Ppt1-/- mice by NtBuHA.
Superficial (II-IV) and deep (IV-VI) cortical layers of WT (a; upper panel), untreated Ppt1-/- (a; middle panel), and NtBuHA-treated Ppt1-/- (a; lower panel) brain sections were immunostained with Cux1 and Ctip2 respectively. For each group, representative images of Cux1 and Ctip2 –positive cells in layers II & III and layer V respectively are shown (b). Quantification of Cux1- (c) and Ctip2- (d) positive cells per 0.01 mm2 in these layers revealed significant protection of both these cells in NtBuHA-treated Ppt1-/- mice over their untreated littermates (n=4).
Supplementary Figure 9 Cortical thickness and rotarod performance of WT, untreated- and NtBuHA-treated Ppt1-/- mice.
The sections of the brain from 6-month old WT (n= 4), untreated Ppt1-/- (n=4) and NtBuHA-treated Ppt1-/- (n=4) mice were stained with cresyl violet and photomicrographs were analyzed for cortical thickness (a). Graphic representation of the brain region (V2ML, secondary visual cortex mediolateral) where the cortical thickness was measured (Arrow); (b) cresyl violet stained cortical sections from WT, untreated- and NtBuHA-treated Ppt1-/- mice; (c) graphic representation of the cortical thickness among WT mice and their untreated- and NtBuHA-treated littermates. The results are presented as the mean ±SD; (d) Three-month old WT (n=6) and Ppt1-/- mice (n=6) were tested on rotarod at 4, 8 and 12 rpm for 60 seconds. No significant difference in motor coordination was observed at this age between WT and Ppt1-/- mice; (e) Six-months old WT (n= 6), untreated Ppt1-/- (n=8) and NtBuHA-treated Ppt1-/- (n=7) mice were allowed to stay on rotarod at 12 rpm for up to 300 seconds. Note a significant, albeit modest, improvement in performance of NtBuHA-treated Ppt1-/- mice in the rotarod endurance test.
Supplementary Figure 10 Full length pictures of the cropped blots presented in the main figures: 4e and 5e.
panel a, full length picture of the cropped gel in main Figure 4e; panel b, full length picture of the cropped gel in main Figure 5e.
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Supplementary Figures 1–10 and Supplementary Tables 1 and 2 (PDF 2757 kb)
Rotarod test in WT, untreated Ppt1-/- and NtBuHA-treated Ppt1-/- mice.
Rotarod test (60 seconds) in WT, untreated Ppt1-/- and NtBuHA-treated Ppt1-/- mice. Mouse #1 & 2 (left), NtBuHA-treated; Mouse #3 & 4 (middle), untreated and Mouse #5 (right), WT. (WMV 12677 kb)
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Sarkar, C., Chandra, G., Peng, S. et al. Neuroprotection and lifespan extension in Ppt1−/− mice by NtBuHA: therapeutic implications for INCL. Nat Neurosci 16, 1608–1617 (2013). https://doi.org/10.1038/nn.3526
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DOI: https://doi.org/10.1038/nn.3526
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