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

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.

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 (TG^WT), mutant SPTLC1^C133W (TG^C133W) 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 SPTLC1^C133W mice (TG^C133W) is inhibited but restored in dTG. In brain, SPT is decreased by 25% in heterozygous knock-out mice (+/−). SPTLC1^C133W mice (14-month-old) (TG^C133W) 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 SPTLC1^C133W mice (TG^C133W) with a trend toward improvement in the double transgenics. Sperm count in mice at 2 months (I) and 15 months (J). The mutant (TG^C133W) animals have significantly reduced sperm count compared with WT. This is restored in the dTG mice.

Figure 2.

Mutant SPTLC1^C133W mice (TG^C133W) 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 (TG^WT), SPTLC1^C133W (TG^C133W), and dTG mice. Myelinated axons in the SPTLC1^C133W mice appear smaller than in wild-type animals (TG^C133W 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 (TG^C133W) 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 (TG^C133W) versus WT versus wild-type SPTLC1 (TG^WT) overexpressing animals (E) and dTG versus mutant (TG^C133W) (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 SPTLC1^C133W mice and is restored in double-transgenic mice.

Figure 3.

Distal sciatic nerves of mutant SPTLC1^C133W mice (TG^C133W) reveal a mononuclear phagocytic cell reaction. A, Sciatic nerves of TG^C133W 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 TG^C133W mutant; scale bar, 0.25 μm. D, Dorsal root ganglion neurons at L3 are surrounded by abundant TNF-α in the mutant TG^C133W mice.

Figure 4.

Accumulation of deoxysphingoid bases in C133W mutant transgenic mice. Total lipids from WT, wild-type overexpressing (TG^WT), heterozygote knock-out (+/−), SPTLC1^C133W mice (TG^C133W), 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 SPTLC1^C133W mutant mice but not other animals. However, levels of m17:1 are not detectable (n.d.). C, D, In SPTLC1^C133W 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.