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. 2011 Oct 18:10:182.
doi: 10.1186/1476-511X-10-182.

In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor

Paul L Wood  1 , M Amin Khan, Tara Smith, Greg Ehrmantraut, Wei Jin, Wei Cui, Nancy E Braverman, Dayan B Goodenowe
Affiliations

In vitro and in vivo plasmalogen replacement evaluations in rhizomelic chrondrodysplasia punctata and Pelizaeus-Merzbacher disease using PPI-1011, an ether lipid plasmalogen precursor

Paul L Wood et al. Lipids Health Dis. .

Abstract

Background: Childhood peroxisomal disorders and leukodystrophies are devastating diseases characterized by dysfunctional lipid metabolism. Plasmalogens (ether glycerophosphoethanolamine lipids) are decreased in these genetic disorders. The biosynthesis of plasmalogens is initiated in peroxisomes but completed in the endoplasmic reticulum. We therefore undertook a study to evaluate the ability of a 3-substituted, 1-alkyl, 2-acyl glyceryl ether lipid (PPI-1011) to replace plasmalogens in rhizomelic chrondrodysplasia punctata type 1 (RCDP1) and rhizomelic chrondrodysplasia punctata type 2 (RCDP2) lymphocytes which possess peroxisomal mutations culminating in deficient plasmalogen synthesis. We also examined plasmalogen synthesis in Pelizaeus-Merzbacher disease (PMD) lymphocytes which possess a proteolipid protein-1 (PLP1) missense mutation that results in abnormal PLP1 folding and it's accumulation in the endoplasmic reticulum (ER), the cellular site of the last steps in plasmalogen synthesis. In vivo incorporation of plasmalogen precursor into tissue plasmalogens was also evaluated in the Pex7 mouse model of plasmalogen deficiency.

Results: In both RCDP1 and RCDP2 lymphocytes, PPI-1011 repleted the target ethanolamine plasmalogen (PlsEtn16:0/22:6) in a concentration dependent manner. In addition, deacylation/reacylation reactions resulted in repletion of PlsEtn 16:0/20:4 in both RCDP1 and RCDP2 lymphocytes, repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP2 lymphocytes, and partial repletion of PlsEtn 16:0/18:1 and PlsEtn 16:0/18:2 in RCDP1 lymphocytes. In the Pex7 mouse, oral dosing of labeled PPI-1011 demonstrated repletion of tissue levels of the target plasmalogen PlsEtn 16:0/22:6 with phospholipid remodeling also resulting in significant repletion of PlsEtn 16:0/20:4 and PlsEtn 16:0/18:1. Metabolic conversion of PPI-1011 to the target plasmalogen was most active in the liver.

Conclusions: Our data demonstrate that PPI-1011 is activated (removal of 3-substitution) and converted to PlsEtn in vitro in both RCDP1 and RCDP2 lymphocytes and in vivo in the Pex7 mouse model of RCPD1 effectively bypassing the peroxisomal dysfunction present in these disorders. While PPI-1011 was shown to replete PlsEtns 16:0/x, ether lipid precursors of PlsEtn 18:0/x and PlsEtn 18:1/x may also be needed to achieve optimal clinical benefits of plasmalogen replacement in these complex patient populations. In contrast, only limited plasmalogen replacement was observed in PMD lymphocytes suggesting that the effects of protein misfolding and accumulation in the ER negatively affect processing of plasmalogen precursors in this cellular compartment.

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Figures

Figure 1
Figure 1
Ethanolamine plasmalogen levels in RCDP1 and RCDP2 lymphocytes with 0, 20 or 100 μM PPI-1011 for 72 hr. N = 8. Mean ± SEM. All basal decrements and PPI-1011-dependent increases were significantly (p < 0.01) from control lymphocytes and from 0 μM PPI-1011, respectively. 16:0 (palmitic acid), 18:1 (oleic acid), 18:2 (linoleic acid), 20:4 (arachidonic acid), 22:6 (docosahexaenoic acid; DHA).
Figure 2
Figure 2
Incorporation of PPI-1038 (100 μM; 72 hr) into the 16:0/22:6, 18:0/22:6 and 18:1/22:6 plasmalogens and DHA of RCDP1 and RCDP2 lymphocytes. N = 6. Mean ± SEM. P =[13C16]palmitic acid; G =[13C3]glycerol; D = [13C3]DHA. *, p < 0.01 vs. control.
Figure 3
Figure 3
Incorporation of PPI-1038 (100 μM; 72 hr) into the 16:0/22:6, 18:0/22:6 and 18:1/22:6 plasmalogens and DHA of PMD lymphocytes. N = 6. Mean ± SEM. P =[13C6]palmitic acid; G =[13C3]glycerol; D = [13C3]DHA. *, p < 0.01 vs. control.
Figure 4
Figure 4
Ethanolamine plasmalogen levels in Pex7 mouse tissues (liver, kidney, heart, lung, neocortex and eye). N = 5 Pex 7; N = 10 controls (2 heterozygotes + 8 wild-type). Mean ± SEM. All plasmalogen decreases were statistically significant (p < 0.05).
Figure 5
Figure 5
Incorporation of [13C16]palmitic acid and [13C3]glycerol from PPI-1038 (100 mg/kg/day for 3 days) into Pex7 tissue PlsEtn 16:0/22:6. N = 4. Mean ± SD. Increases in labeling in the Pex7 mice were significant in all cases (p < 0.01). HT, heterozygote controls; HO, homozygotes.
Figure 6
Figure 6
Incorporation of [13C6]palmitic acid and [13C3]glycerol (*) from PPI-1038 (100 mg/kg/day for 3 days) into PlsEtn 16:0/22:6 in the neocortex and eyes of Pex7 mice. Similarly the incorporation of [13C3]DHA (**) into PlsEtn 16:0/22:6, PlsEtn 18:0/22:6 and PlsEtn 18:1/22:6 is presented. N = 4. Mean ± SD. Increases in labeling in the Pex7 mice were significant in all cases (p < 0.01). HT, heterozygote controls; HO, homozygotes. * = [13C16]palmitic acid + [13C3]glycerol labeled PlsEtn x/22:6; ** = [13C3]DHA labeled PlsEn x/22:6.
Figure 7
Figure 7
Conversion of PPI-1011 to the target plasmalogen PlsEtn 16:0/22:6. Removal of the sn-3 lipoic acid by lipases is followed in the endoplasmic reticulum by addition of phosphoethanolamine to the glycerol backbone at sn-3 (EC 3.1.3.4) and desaturation of the ether linked fatty acid at sn-1 (EC 1.14.99.19). Subsequent lipid remodeling at sn-2 is mediated by tightly coupled deacylation/reacylation enzyme reactions [10].
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