Skip to main content

Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

  • Article
  • Published:

Neuroprotection and lifespan extension in Ppt1−/− mice by NtBuHA: therapeutic implications for INCL

Nature Neuroscience volume 16, pages 1608–1617 (2013)Cite this article

Subjects

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.

This is a preview of subscription content, access via your institution

Access options

Buy this article

  • Purchase on Springer Link
  • Instant access to full article PDF
Buy now

Prices may be subject to local taxes which are calculated during checkout

Figure 1: Hydroxylamine derivatives cleave thioester linkage in [14C]palmitoyl∼CoA.
Figure 2: NtBuHA mediates the depletion of ceroid and GRODs in cultured INCL cells.
Figure 3: NtBuHA prevents H2O2-induced apoptosis in INCL lymphoblasts.
Figure 4: NtBuHA suppresses apoptosis in Ppt1−/− mouse brain.
Figure 5: The effects of NtBuHA on brain size, weight, neuron density, GFAP and NeuN expression.
Figure 6: NtBuHA ameliorates neuropathology and extends lifespan in Ppt1−/− mice.

Similar content being viewed by others

References

  1. Cox, T.M. & Cachón-González, M.B. The cellular pathology of lysosomal diseases. J. Pathol. 226, 241–254 (2012).

    CAS  Google Scholar 

  2. 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).

    Article  Google Scholar 

  3. Anderson, G.W., Goebel, H.H. & Simonati, A. Human pathology in NCL. Biochim. Biophys. Acta 1832, 1807–1826 (2013).

    Article  CAS  Google Scholar 

  4. 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).

    Article  CAS  Google Scholar 

  5. Haltia, M. & Goebel, H.H. The neuronal ceroid lipofuscinoses: a historical introduction. Biochim. Biophys. Acta 1832, 1795–1800 (2013).

    Article  CAS  Google Scholar 

  6. 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).

    Article  CAS  Google Scholar 

  7. 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).

    CAS  PubMed  Google Scholar 

  8. 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).

    CAS  PubMed  Google Scholar 

  9. Verkruyse, L.A. & Hofmann, S.L. Lysosomal targeting of palmitoyl protein thioesterase. J. Biol. Chem. 271, 15831–15836 (1996).

    Article  CAS  Google Scholar 

  10. 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).

    Article  CAS  Google Scholar 

  11. Järvela, I. Infantile form of neuronal ceroid lipofuscinosis (CLN1) maps to the short arm of chromosome 1. Genomics 9, 170–173 (1991).

    Article  Google Scholar 

  12. Vesa, J. et al. Mutations in the palmitoyl protein thioesterase gene causing infantile neuronal ceroid lipofuscinosis. Nature 376, 584–587 (1995).

    Article  CAS  Google Scholar 

  13. 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).

  14. Smotrys, J.E. & Linder, M.E. Palmitoylation of intracellular signaling proteins: regulation and function. Annu. Rev. Biochem. 73, 559–587 (2004).

    Article  CAS  Google Scholar 

  15. Resh, M.D. Palmitoylation of ligands receptors and intracellular signaling molecules. Sci. STKE 2006, re14 (2006).

    Article  Google Scholar 

  16. Salaun, C., Greaves, J. & Chamberlain, L.H. The intracellular dynamic of protein palmitoylation. J. Cell Biol. 191, 1229–1238 (2010).

    Article  CAS  Google Scholar 

  17. Iwanaga, T., Tsutsumi, R., Noritake, J., Fukata, Y. & Fukata, M. Dynamic protein palmitoylation in cellular signaling. Prog. Lipid Res. 48, 117–127 (2009).

    Article  CAS  Google Scholar 

  18. 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).

    Article  CAS  Google Scholar 

  19. Jocelyn, P.C. Biochemistry of the SH Groups (Academic Press, New York, 1972).

  20. Gross, P. Biologic activity of hydroxylamine: a review. Crit. Rev. Toxicol. 14, 87–99 (1985).

    Article  CAS  Google Scholar 

  21. Kirby, A.J. et al. Hydroxylamine as an oxygen nucleophile. Structure and reactivity of ammonia oxide. J. Am. Chem. Soc. 128, 12374–12375 (2006).

    Article  CAS  Google Scholar 

  22. Drisdel, R.C., Alexander, J.K., Sayeed, A. & Green, W.N. Assays of protein palmitoylation. Methods 40, 127–134 (2006).

    CAS  Google Scholar 

  23. Mosmann, T. Rapid colorimetric assay for cellular growth and survival. J. Immunol. Methods 65, 55–63 (1983).

    Article  CAS  Google Scholar 

  24. 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).

    Article  CAS  Google Scholar 

  25. 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).

    Article  CAS  Google Scholar 

  26. 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).

    Article  CAS  Google Scholar 

  27. 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).

    Article  CAS  Google Scholar 

  28. 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).

    Article  CAS  Google Scholar 

  29. 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).

    Article  CAS  Google Scholar 

  30. Kaur, P. et al. Metabolomic profiling for biomarker discovery in pancreatic cancer. Int. J. Mass Spectrom. 310, 44–51 (2012).

    Article  CAS  Google Scholar 

  31. 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).

    Article  Google Scholar 

  32. 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).

    Article  CAS  Google Scholar 

  33. Isosomppi, J. et al. Developmental expression of palmitoyl protein thioesterase in normal mice. Brain Res. Dev. Brain Res. 118, 1–11 (1999).

    Article  CAS  Google Scholar 

  34. 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).

    Article  CAS  Google Scholar 

  35. 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).

    Article  CAS  Google Scholar 

  36. Crawley, J.N. Exploratory behavior model of anxiety in mice. Neurosci. Biobehav. Rev. 9, 37–44 (1985).

    Article  CAS  Google Scholar 

  37. El-Husseini, A.E.-D. & Bredt, D.S. Protein palmitoylation: a regulator of neuronal development and function. Nat. Rev. Neurosci. 3, 791–802 (2002).

    Article  CAS  Google Scholar 

  38. 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).

    Article  CAS  Google Scholar 

  39. 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).

    Article  CAS  Google Scholar 

  40. Greaves, J. et al. Palmitoylation-induced aggregation of cysteine-string protein mutants that cause neuronal ceroid lipofuscinosis. J. Biol. Chem. 287, 37330–37339 (2012).

    Article  CAS  Google Scholar 

  41. Platt, F.M. & Lachmann, R.H. Treating lysosomal storage disorders: current practice and future prospects. Biochim. Biophys. Acta 1793, 737–745 (2009).

    Article  CAS  Google Scholar 

  42. Kohan, R. et al. Therapeutic approaches to the challenge of neuronal ceroid lipofuscinoses. Curr. Pharm. Biotechnol. 12, 867–883 (2011).

    Article  CAS  Google Scholar 

  43. 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).

    Article  CAS  Google Scholar 

  44. 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).

    Article  CAS  Google Scholar 

  45. 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).

    Article  CAS  Google Scholar 

  46. 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).

    Article  CAS  Google Scholar 

  47. Lönnqvist, T. et al. Hematopoietic stem cell transplantation in infantile neuronal ceroid lipofuscinosis. Neurology 57, 1411–1416 (2001).

    Article  Google Scholar 

  48. 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).

    Article  CAS  Google Scholar 

  49. Srikanth, M. & Kessler, J.A. Nanotechnology-novel therapeutics for CNS disorders. Nat. Rev. Neurol. 8, 307–318 (2012).

    Article  CAS  Google Scholar 

  50. 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).

    Article  CAS  Google Scholar 

Download references

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.

Author information

Author notes

  1. Chinmoy Sarkar and Goutam Chandra: These authors contributed equally to this work.

Authors and Affiliations

  1. Section on Developmental Genetics, Program on Developmental Endocrinology and Genetics, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Chinmoy Sarkar, Goutam Chandra, Shiyong Peng, Zhongjian Zhang & Anil B Mukherjee

  2. Biostatistics and Bioinformatics Branch, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland, USA

    Aiyi Liu

Authors
  1. Chinmoy Sarkar
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Goutam Chandra
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Shiyong Peng
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Zhongjian Zhang
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Aiyi Liu
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Anil B Mukherjee
    View author publications

    You can also search for this author in PubMed Google Scholar

Contributions

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.

Corresponding author

Correspondence to Anil B Mukherjee.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

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.

Supplementary information

Supplementary Text and Figures

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)

Rights and permissions

Reprints and permissions

About this article

Cite this article

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

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1038/nn.3526

This article is cited by

Search

Advanced search

Quick links

Nature Briefing: Translational Research

Sign up for the Nature Briefing: Translational Research newsletter — top stories in biotechnology, drug discovery and pharma.

Get what matters in translational research, free to your inbox weekly. Sign up for Nature Briefing: Translational Research