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
Comparative Study
. 2009 Nov 18;29(46):14646-51.
doi: 10.1523/JNEUROSCI.2536-09.2009.

Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1

Florian S Eichler  1 , Thorsten Hornemann, Alex McCampbell, Dika Kuljis, Anke Penno, Daniel Vardeh, Eric Tamrazian, Kevin Garofalo, Ho-Joon Lee, Lohit Kini, Martin Selig, Matthew Frosch, Ken Gable, Arnold von Eckardstein, Clifford J Woolf, Guiman Guan, Jeffrey M Harmon, Teresa M Dunn, Robert H Brown Jr
Affiliations
Comparative Study

Overexpression of the wild-type SPT1 subunit lowers desoxysphingolipid levels and rescues the phenotype of HSAN1

Florian S Eichler et al. J Neurosci. .

Abstract

Mutations in the SPTLC1 subunit of serine palmitoyltransferase (SPT) cause an adult-onset, hereditary sensory, and autonomic neuropathy type I (HSAN1). We previously reported that mice bearing a transgene-expressing mutant SPTLC1 (tgSPTLC1(C133W)) show a reduction in SPT activity and hyperpathia at 10 months of age. Now analyzed at a later age, we find these mice develop sensory loss with a distal small fiber neuropathy and peripheral myelinopathy. This phenotype is largely reversed when these mice are crossed with transgenic mice overexpressing wild-type SPTLC1 showing that the mutant SPTLC1 protein is not inherently toxic. Simple loss of SPT activity also cannot account for the HSAN1 phenotype, since heterozygous SPTLC1 knock-out mice have reduced SPT activity but are otherwise normal. Rather, the presence of two newly identified, potentially deleterious deoxysphingoid bases in the tgSPTLC1(C133W), but not in the wild-type, double-transgenic tgSPTLC1(WT + C133W) or SPTLC1(+/-) mice, suggests that the HSAN1 mutations alter amino acid selectivity of the SPT enzyme such that palmitate is condensed with alanine and glycine, in addition to serine. This observation is consistent with the hypothesis that HSAN1 is the result of a gain-of-function mutation in SPTLC1 that leads to accumulation of a toxic metabolite.

PubMed Disclaimer

Figures

Figure 1.
Figure 1.
Overexpression of the wild-type SPT1 subunit rescues the phenotype of HSAN1 mice. Western blots of transgene (A) as well as endogenous SPTLC1 (B) expression in liver (L), brain (B), and pancreatic tissue (P) in WT, wild-type SPTLC1 overexpressing (TGWT), mutant SPTLC1C133W (TGC133W) and dTG mice (arrows point to anticipated molecular weight of SPTLC1). An HA tag was introduced to facilitate identification of the transgene. C, Western blots of liver and brain SPTLC1 in heterozygote knock-out mice (+/−) and wild-type (+/+) animals. D, In the liver, brain, and spinal cord of the double-transgenic mice, the level of WT SPTLC1 transcript is twice as high as those for the mutant SPTLC1 transgene. E, SPT activity in brain tissue of transgenic and knock-out mice: SPT of SPTLC1C133W mice (TGC133W) is inhibited but restored in dTG. In brain, SPT is decreased by 25% in heterozygous knock-out mice (+/−). SPTLC1C133W mice (14-month-old) (TGC133W) were significantly slower to react in the hot plate test at 55°C (p < 0.05; F) and less sensitive to mechanical stimuli (von Frey hair thresholds) (p = 0.3; G). In the dTG animals, statistically significant improvements are seen. H, Rotorod performance is impaired in SPTLC1C133W mice (TGC133W) with a trend toward improvement in the double transgenics. Sperm count in mice at 2 months (I) and 15 months (J). The mutant (TGC133W) animals have significantly reduced sperm count compared with WT. This is restored in the dTG mice.
Figure 2.
Figure 2.
Mutant SPTLC1C133W mice (TGC133W) have smaller axons and myelin abnormalities in the distal sciatic nerve. A, Low- and high-power electron microscopic images show cross-sections of the distal sciatic nerves. Frequent indentures are seen in the myelin sheaths of the mutant SPLTC1 mice (arrowhead) but absent in all other groups; Scale bars: 10 and 5 μm. B, Histogram of the distribution of axon diameter of the distal sciatic nerve in 15-month-old WT, wild-type overexpressing (TGWT), SPTLC1C133W (TGC133W), and dTG mice. Myelinated axons in the SPTLC1C133W mice appear smaller than in wild-type animals (TGC133W mean axon diameter of 2.64 μm vs WT mean of 3.16 μm; p < 0.05; see open arrow). There is a dropout of small unmyelinated axons in the mutant (TGC133W) animals <0.2 μm (see closed arrow in B and EM images in C; scale bar, 50 nm) and a significant reduction in total unmyelinated axons by ∼35% [p < 0.05; D; number of unmyelinated axons in 25 field-of-view (25,000×)]. These findings are reversed in the dTG animals. The g-ratios of mutant (TGC133W) versus WT versus wild-type SPTLC1 (TGWT) overexpressing animals (E) and dTG versus mutant (TGC133W) (F) are plotted relative to the axon diameter with the best fit, linear regression indicated for each of the mice. Myelin thinning occurs in distal sciatic axons of mutant SPTLC1C133W mice and is restored in double-transgenic mice.
Figure 3.
Figure 3.
Distal sciatic nerves of mutant SPTLC1C133W mice (TGC133W) reveal a mononuclear phagocytic cell reaction. A, Sciatic nerves of TGC133W show more TNF-α staining compared with the wild-type mice. This is associated with the presence of CD68-positive macrophages; scale bar, 50 μm. B, The boxed area of mutant sciatic nerve is magnified; scale bar, 20 μm. C, Ultrastructural analysis reveals lipid droplets within macrophages in the TGC133W mutant; scale bar, 0.25 μm. D, Dorsal root ganglion neurons at L3 are surrounded by abundant TNF-α in the mutant TGC133W mice.
Figure 4.
Figure 4.
Accumulation of deoxysphingoid bases in C133W mutant transgenic mice. Total lipids from WT, wild-type overexpressing (TGWT), heterozygote knock-out (+/−), SPTLC1C133W mice (TGC133W), and dTG were extracted and subjected to an acid and base hydrolyzes. A, All C133W mutants show plasma levels for m18:0 bases in excess of 200 pmol/ml (p < 0.05, by t test). Some dTG mice have mild accumulation of m18:0 but not beyond this threshold. Levels of m18:0 are <100 pmol/ml in the other conditions. In all mice, levels of 18:0 are dramatically higher than m18:1. B, Similar to humans with the C133W mutation (Hornemann et al., 2008) m17:0 is present in plasma of SPTLC1C133W mutant mice but not other animals. However, levels of m17:1 are not detectable (n.d.). C, D, In SPTLC1C133W mutant mice, high levels of m17:0 are present in the sciatic nerve and lower levels in brain, testes, spinal cord, and liver. Similar to plasma, double-transgenic mice have mild accumulation of m18:0 in sciatic nerve but much reduced compared with the C133W mutant mice.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Auer-Grumbach M, De Jonghe P, Verhoeven K, Timmerman V, Wagner K, Hartung HP, Nicholson GA. Autosomal dominant inherited neuropathies with prominent sensory loss and mutilations: a review. Arch Neurol. 2003;60:329–334. - PubMed
    1. Bejaoui K, Wu C, Scheffler MD, Haan G, Ashby P, Wu L, de Jong P, Brown RH., Jr SPTLC1 is mutated in hereditary sensory neuropathy, type 1. Nat Genet. 2001;27:261–262. - PubMed
    1. Broom DC, Samad TA, Kohno T, Tegeder I, Geisslinger G, Woolf CJ. Cyclooxygenase 2 expression in the spared nerve injury model of neuropathic pain. Neuroscience. 2004;124:891–900. - PubMed
    1. Dedov VN, Dedova IV, Merrill AH, Jr, Nicholson GA. Activity of partially inhibited serine palmitoyltransferase is sufficient for normal sphingolipid metabolism and viability of HSN1 patient cells. Biochim Biophys Acta. 2004;1688:168–175. - PubMed
    1. Hanada K. Serine palmitoyltransferase, a key enzyme of sphingolipid metabolism. Biochim Biophys Acta. 2003;1632:16–30. - PubMed

Publication types

MeSH terms

LinkOut - more resources