Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2013 Nov;16(11):1608-17.
doi: 10.1038/nn.3526. Epub 2013 Sep 22.

Neuroprotection and lifespan extension in Ppt1(-/-) mice by NtBuHA: therapeutic implications for INCL

Chinmoy Sarkar  1 , Goutam Chandra, Shiyong Peng, Zhongjian Zhang, Aiyi Liu, Anil B Mukherjee
Affiliations

Neuroprotection and lifespan extension in Ppt1(-/-) mice by NtBuHA: therapeutic implications for INCL

Chinmoy Sarkar et al. Nat Neurosci. 2013 Nov.

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.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Hydroxylamine derivatives cleave thioester linkage in [14C]palmitoyl~CoA. (Panel a) Method of screening of hydroxylamine-derivatives for the cleavage of thioester linkage in [14C]palmitoyl~CoA. Palmitoyl~CoA is a model substrate of PPT1 in which [14C]palmitic acid (asterisk) is attached to CoA via thioester linkage (arrow). PPT1 or nucleophilic compound cleaving the thioester linkage would release radioactive palmitate from [14C]-palmitoyl~CoA. (Panel b) Water-soluble and (Panel c) DMSO-soluble hydroxylamine-derivatives were screened by incubating [14C]palmitoyl~CoA with each of the 12 hydroxylamine-derivatives (Supplementary Table 1) for 1 h at room temperature. Varying degrees of [14C]palmitate were released by both water-soluble (Panel b, lanes 3–9) and DMSO-soluble (Panel c, lanes 3–7) hydroxylamine-derivatives resolved by TLC. (Panel b: lanes: (1): [14C] palmitic acid standard; (2) [14C]-palmitoyl~CoA standard; (3) N-(tert-Butyl)hydroxylamine; (4): N-Benzylhydroxylamine; (5): N-Methylhydroxylamine; (6): N-Cyclohexylhydroxylamine; (7): N,N-Dimethylhydroxylamine; (8): N,N-Diethylhydroxylamine (9): N,O-Bis(trimethylsilyl) hydroxylamine; (10): hydroxylamine (control); (11): recombinant human PPT1 (control); (12): [14C]palmitate standard. (Panel c), lanes: (1): [14C] palmitic acid standard; (2) [14C]-palmitoyl~CoA standard; (3) N-Benzyloxycarbonyl hydroxylamine; (4), N,N-Dibenzylhydroxylamine; (5): N-Benzoyl-N-phenyl hydroxylamine; (6): N-tert-Butyl-O-[1-[4-(chloromethyl) phenyl]ethyl]- N-(2-methyl-1-phenylpropyl) hydroxylamine; (7): N,O-Di-Boc-hydroxylamine; (8): hydroxylamine (control); (9): recombinant human PPT1 (control); (10): [14C]palmitate standard. The arrow indicates the released free [14C] palmitate band. The densitometric quantitation of released free [14C]palmitate bands is represented graphically. (Panel d) Effect of varying concentrations of NtBuHA treatment for 48 h on the viability of cultured lymphoblasts from an INCL patient was evaluated by MTT assay. (Panel e). Plating efficiency of the NtBuHA-treated INCL fibroblasts compared with that of the normal control cells. Note that compared with the cells treated with up to 1 mM NtBuHA, those treated with 2.5mM NtBuHA manifested slightly lower level of plating efficiency. (Panel f). Time-course of NtBuHA-mediated depletion of [35S]labeled lipid thioester compounds from INCL lymphoblasts. Cultured lymphoblasts from an INCL patient were treated with 500 µM NtBuHA for varying lengths of time (12–48h) after being labeled with [35S]-cysteine. Fresh NtBuHA was added every 12 h. The [35S]-labeled lipid thioesters were extracted and resolved by TLC. Note a marked decrease in intensity of the radioactive bands at 48h. The arrows indicate the positions of each of the 8 identifiable lipid thioester bands (1–8: counting from the top) and the quantitations of these bands are presented graphically (Supplementary Fig. 5a). (Panel g) Depletion of [35S]Cysteine-labeled lipid thioesters in lymphoblasts from INCL patients, which were either untreated or treated with NtBuHA for 48 h. The culture medium was replaced every 12h with fresh NtBuHA containing medium. Because all the normal controls and 9 INCL patients’ cells could not be accommodated in one TLC plate the samples are resolved using two TLC plates. The smaller TLC (left): INCL, INCL patient’s lymphoblast; N1–N3, 3 normal control lymphoblasts; Larger TLC (right) N4, normal lymphoblasts (control), which in addition to N1–N3 (smaller TLC) show virtually no dense thioester bands. However, lymphoblasts from 9 INCL patients (Supplementary Table 2) show clearly identifiable lipid thioester bands. The [35S]labeled lipid thioesters were resolved by TLC as previously reported. Lanes marked ‘–’ or ‘+’ indicate untreated and NtBuHA-treated samples, respectively. Note the reduction in density of the radioactive lipid thioester bands in NtBuHA-treated samples. Quantitative analyses of each of the 8 identifiable lipid thioester bands marked in panel f were performed and the results are shown in Supplementary Fig.5b.
Figure 2
Figure 2
NtBuHA mediates the depletion of ceroid and GRODs in cultured INCL cells. (Panel a) Electron micrographs of cultured lymphoblasts from INCL patients showing the intracellular accumulation of GRODs (inset) and effect of NtBuHA treatment on the accumulation of GRODs. The left panel (with magnified inset below) represents untreated control. The middle and right panels and corresponding magnified insets represent two representative cells treated with NtBuHA (250 µM) for 3 weeks. Note that in most NtBuHA-treated cells GRODs are completely depleted [Panel a (1)] while an infrequent cell contained a few GRODs [Panel a (2); Scale bar: 500nm]. Box plot analysis of GRODs (Panel b) counted from the TEM in untreated (n = 23) and NtBuHA-treated (n = 56) cells. Untreated- versus NtBuHA-treated group (t (77) =7.61, P<0.001, permutation t-test). Morphometric analysis of the GRODs in untreated- (n=47) and NtBuHA-treated (n=21) cells (Panel c) (t (66) =4.68, P<0.001 permutation t-test) when the uuntreated-INCL cells were compared with. NtBuHA-treated cells. Electron micrographs of cerebral cortices of 6-month old WT (Panel d, left panel) and those of untreated- (Panel d, middle panel) and NtBuHA-treated Ppt1−/− mice (Panel d, right panel). Note that compared with WT mouse cortices, which show no GRODs (left panel), those of the untreated Ppt1−/− mice showed a very high level of GRODs (middle panel), whereas those treated with NtBuHA showed significantly reduced number (t (16) =2.89, P=0.012, permutation t-test) of GRODs (right panel; Scale bar: 2µm). (Panel e) Quantitation of GRODs/cell in the brain sections from WT, untreated- and NtBuHA-treated Ppt1−/− mice is presented graphically. Representative electron micrographs from 3–6 animals from each group were analyzed. (Panel f) Autofluorescence visualized by dark field microscopic analysis of cortical tissue sections of 6-month old WT mice (left panel) and untreated- (middle panel) as well as NtBuHA-treated (right panel) Ppt1−/− mice. Densitometric analyses of autofluorescence in the brain tissues of untreated- and NtBuHA-treated Ppt1−/− mice (Panel g) show significant decline in autofluorescence in treated mice; (t(28)=11.78, P<0.001, permutation t-test). Scale bar=50µm.
Figure 3
Figure 3
NtBuHA prevents H2O2-induced apoptosis in INCL lymphoblasts. (Panel a) Flow cytometric analysis of apoptotic cells using apoptosis-marker, annexin V and necrotic cells using necrotic cell marker PI: Untreated control INCL lymphoblasts (Upper left panel); NtBuHA-pretreated (Upper middle panel); and NtBuHA pre- as well as co-treated (Upper right panel). INCL lymphoblasts were either untreated and exposed to oxidative-stress by H2O2 (untreated control, Lower left panel) or the cells were first pretreated with NtBuHA for 12h and then incubated with H2O2 in the absence (Lower middle panel) or presence of NtBuHA (Lower right panel) for 3h. (Panel b) Graphical presentation of level of apoptotic cells in untreated and NtBuHA-treated INCL cells. Apoptotic cells represent annexinV positive cells detected by FACS (Summation of cell number in Q2 + Q3).
Figure 4
Figure 4
NtBuHA suppresses apoptosis in Ppt1−/− mouse brain. Apoptotic cells were detected by TUNEL assay in brain sections (Panel a) from 6-month old WT (n=9); (Panel b) untreated- (n=9) and (Panel c) NtBuHA-treated Ppt1−/− mice (n=12). TUNEL-positive apoptotic cells appear reddish brown. (Panel d) Graphic representation of the levels of apoptotic cells in the brain of WT, untreated- as well as NtBuHA-treated Ppt1−/− mice. (t(16)=27.63, P<0.001, permutation t-test) for WT vs. untreated-Ppt1−/−; (t(19) = 15.74, P<0.001, permutation t-test) for untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/−. (Panel e) Western blot analysis of cortical tissue homogenates prepared from 6-month old WT, untreated- as well as NtBuHA-treated Ppt1−/− mice for cleaved caspase-9 and cleaved PARP-1. Full-length blots of the cropped gel images used in 4e are provided in Sypplementary Fig. 10a. Densitometric quantitation of cleaved caspase-9 (Panel f) and cleaved PARP-1 (Panel g) from three independent experiments are presented. For panel f, WT vs. untreated-Ppt1−/− (t(10)=11.65, P=0.002, permutation t-test); Untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/− (t(10) = 8.23, P=0.002, permutation t-test); For panel g, WT vs. untreated Ppt1−/− (t(9) = 5.08, P=0.004, permutation t-test); Untreated Ppt1−/− vs. NtBuHA-treated Ppt1−/−(t(10) =4.73, P=0.015, permutation t-test).
Figure 5
Figure 5
The effects of NtBuHA on brain size, weight, neuron density, GFAP and NeuN expression. (Panel a) The representative brain size of a WT (left panel), untreated-Ppt1−/−(middle panel) and NtBuHA-treated Ppt1−/− mouse (right panel). (Panel b) The brain weights of WT (n=12), untreated Ppt1−/− (n=5) and NtBuHA-treated Ppt1−/− (n=12) mice were determined. WT vs. untreated-Ppt1−/− (t(15) = 13.3, P<0.001, permutation t-test); Untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/− group (t(15) = 5.1, P<0.001, permutation t-test). (Panel c) The neuron densities in the cerebral cortex of representative WT (left panel), untreated- (middle panel) and NtBuHA-treated Ppt1−/− mouse (right panel) were determined after staining the cortical tissue sections using antibodies against a neuron specific marker protein, NeuN. Sections from at least 4 mice in each category (WT, untreated Ppt1−/− and NtBuHA-treated Ppt1−/−) were analyzed. (Panel d) Graphic representation of the levels of neurons in WT, untreated Ppt1−/− and NtBuHA-treated Ppt1−/− mice. WT vs. untreated-Ppt1−/−(t(8) = 21.8, P=0.008, permutation t-test); Untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/− group (t(9) = 8.17, P=0.002, permutation t-test). (Panel e) Western blot analysis of brain lysates from WT, untreated-Ppt1−/− and NtBuHA-treated Ppt1−/− mice for GFAP as well as NeuN and their densitometric quantitations (Panel f and Panel g). For Panels f, WT vs. untreated-Ppt1−/− (t(10) = 49.7, P=0.002, permutation t-test); Untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/− group (t(10) = 15.1, P=0.002, permutation t-test). For Panels g, WT vs. untreated-Ppt1−/− (t(10) = 8.7, P=0.002, permutation t-test); Untreated-Ppt1−/− vs. NtBuHA-treated Ppt1−/− group (t(10) = 8.76, P=0.002, permutation t-test). Full-length blots of the cropped gel images used in 5e are presented in Supplementary Fig. 10b. (Panel h) Immunohistochemistry of brain sections showing GFAP-positive cells and (Panel i) quantitation of GFAP-positive cells per mm2. All mice used in these experiments were 6 months old. WT vs. untreated-Ppt1−/− (t(6) = 36.35, P=0.03, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(6) = 5.19, P=0.03, permutation t-test).
Figure 6
Figure 6
NtBuHA ameliorates neuropathology and extends lifespan in Ppt1−/− mice. Motor coordination in WT, untreated- and NtBuHA-treated Ppt1−/− mice were evaluated using the rotarod test. Mice at 6 months of age were tested on rotarod at three different speeds: (Panel a) 4-rpm, (Panel b) 8-rpm and (Panel c) 12-rpm. Mice at 8 months of age were tested using only one speed of 4-rpm (Panel d) as the untreated mice could not withstand a higher speed at this age. At all three speeds, the direction of rotation of the rotarod was reversed every 15 s. Prior to the final test, the animals were trained for 3 d (1 min for each condition) and allowed to rest for 1 min between two successive trials. Each test was performed for 1 min. Latency (defined as the amount of time the animals stayed on the rotating rod) was recorded. For panel a, WT vs. untreated-Ppt1−/− (t(16) = 4.62, P<0.001, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(14) = 4.0, P=0.006, permutation t-test). For panel b, WT vs untreated Ppt1−/− (t(15) = 6.4, P<0.001, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(15) = 4.85, P<0.001, permutation t-test). For panel c, WT vs untreated-Ppt1−/− (t(14) = 13.4, P<0.001, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(16) = 4.3, P=0.002, permutation t-test). For panel d, WT vs. untreated-Ppt1−/− (t(8) = 14.96, P=0.008, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(7) = 9.74, P=0.008, permutation t-test). Exploratory behavior (open field test) of WT, untreated Ppt1−/− and NtBuHA-treated Ppt1−/− mice at 6 months of age (Panel e) and at 8 months of age (Panel f). The number of squares travelled by the animals over 3 minutes of time were counted. For 6-month old groups, WT vs untreated-Ppt1−/−(t(8) = 8.6, P=0.005, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(13) = 3.2, P=0.01, permutation t-test). For 8-month old groups, WT vs. untreated-Ppt1−/− (t(10) = 37.0, P=0.002, permutation t-test); Untreated-Ppt1−/− vs NtBuHA-treated Ppt1−/− group (t(11) = 10.9, P=0.001, permutation t-test). (Panel g) Survival plots (Kaplan-Meyer analysis) of untreated and NtBuHA-treated Ppt1−/− mice. Untreated- (n=110) or NtBuHA treated- Ppt1−/− (n=80) animals were euthanized when they could not reach for food or water. Median Survival: 242d for untreated; 277d for treated, chi-square (161, df=1), P<0.001.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Cox TM, Cachón-González MB. The cellular pathology of lysosomal diseases. J. Pathol. 2012;226:241–254. - PubMed
    1. Jeyakumar M, Dwek RA, Butters TD, Platt FM. Storage solutions: treating lysosomal disorders of the brain. Nat. Rev. Neurosci. 2005;6:713–725. - PubMed
    1. Anderson GW, Goebel HH, Simonati A. Human pathology in NCL. Biochim. Biophys. Acta. 2013;1832:1807–1826. - PubMed
    1. Kousi M, Lehesjoki AE, Mole SE. Update of the mutation spectrum and clinical correlations of over 360 mutations in eight genes that underlie the neuronal ceroid lipofuscinoses. Hum.Mutat. 2012;33:42–63. - PubMed
    1. Haltia M, Goebel HH. The neuronal ceroid lipofuscinoses: A historical introduction. Biochim Biophys Acta. 2013;1832:1795–1800. - PubMed

Publication types

MeSH terms

Supplementary concepts