Both proteins co-localized and showed a similar distribution (mainly present in SLIs) in both genotypes == Normal Length of Nodes of Ranvier and Paranodes in Fa2h/Mice == Because the paranodal structure is disturbed in mice lacking sulfatide [34], we wondered whether absence of 2-hydroxylated sulfatide may also affect the paranodes in older mice. incisures (SLIs). Accordingly, the number of SLIs was significantly increased in 17-month-old but not 4-month-oldFa2h/mice compared to age-matched wild-type mice. On the other hand, the relative increase in the SLI frequency was less pronounced than expected from Cadm4, Lin7, Mpp6 (Pals2), and band 4.1G (Epb41l2) protein levels. This suggests that the latter not only reflect the higher SLI frequency but that the concentration of the Cadm4 containing complex itself is increased in the SLIs or compact myelin ofFa2h/mice and may potentially play a role in the pathogenesis of the disease. The Eicosapentaenoic Acid proteome data are available via ProteomeXchange with identifier PXD030244. == Supplementary Information == The online version contains supplementary material available at 10.1007/s12035-022-02832-4. Keywords:Myelin, Schmidt-Lanterman incisure, Spastic paraplegia, Sphingolipid == Introduction == Schmidt-Lanterman incisures (SLIs), also known as myelin incisures or Schmidt-Lanterman clefts, are cytoplasmic channels of the Schwann cells in the myelin internodes. It is generally assumed that SLIs facilitate transport of metabolites, ions, and signaling molecules between peri-nuclear and adaxonal cytoplasmic regions by reducing diffusion distances because of radial diffusion through gap junctions [1]. Galactosylceramide and its sulfated derivative sulfatide are abundant sphingolipids in the nervous system [2]. A large percentage of galactosylceramide and sulfatide in CNS and PNS myelin of mammals contains 2-hydroxylated fatty acyl residues [3,4]. In myelinating cells, the 2-hydroxylation reaction is exclusively catalyzed by the enzyme fatty acid 2-hydroxylase (FA2H), a cytochrome b5 domain-containing enzyme of the endoplasmic reticulum [5,6]. Although free fatty acids are substrates for the enzyme in an in vitro activity assay [7], X-ray structural analyses suggest that ceramides may be additional in vivo substrates [8]. The functional role of the 2-hydroxylation modification of sphingolipids is not fully understood. Hydroxylated sphingolipids appear to have unique roles in signal transduction [9] and may affect the turnover of membrane proteins by their influence on the mobility of lipids in membrane subdomains (or lipid rafts) [1012]. Mutations in theFA2Hgene that reduce or KLHL22 antibody abolish activity of the enzyme cause a complicated form of hereditary spastic paraplegia type 35 (SPG35) associated with leukodystrophy, which is also known as fatty acid hydroxylase-associated neurodegeneration (FAHN) and as a subtype of Eicosapentaenoic Acid neurodegeneration with brain iron accumulation (NBIA) [13]. More than 40 disease-associated humanFA2Hmutations have been reported [14].Fa2h-deficient (Fa2h/) mice serve as animal model of SPG35/FAHN and develop a phenotype that is reminiscent of symptoms of the human disease [15,16]. In a previous study, we found evidence for alterations in the CNS myelin proteome ofFa2h/mice [17]. Although SPG35, like hereditary spastic paraplegias in general, is characterized by degeneration of upper motor neurons, peripheral neuropathy has been described in about 30% of the patients [14]. Karle et al. [18] estimated a prevalence of peripheral neuropathy of about 60% in all cases of hereditary spastic paraplegia together. In the present report, we performed a myelin proteome study of sciatic nerves, in order to examine possible molecular changes in the PNS myelin ofFa2h/mice. == Experimental Procedures Eicosapentaenoic Acid == == Animals == Generation ofFa2h/mice (Fa2htm1Meck; MGI:3829000) and genotyping Eicosapentaenoic Acid has been described previously (Zller et al., 2008). Animal experiments have been approved by the national authorities (reference number: 8402.04.2014-A039). == Antibodies == Antibodies used in this study are listed in Table1and were kind gifts from Peter Prophy and Arthur M. Butt or were purchased from the following companies: Abcam (Cambridge, UK), Antibodies Incorporated (Davis, California, USA), Biorbyt (Cambridge, UK), GeneTex (Irvine, California, USA), Jackson ImmunoResearch (Philadelphia, Pennsylvania, USA), Merck (Darmstadt, Germany), and Thermo Fisher (Waltham, Massachusetts, USA). == Table 1. == Antibodies used in this study Abbreviations:WBWestern blot,IFimmunofluorescence == Purification of Myelin from Sciatic Nerves == Myelin from sciatic nerves was isolated according to Eicosapentaenoic Acid Caroni and Schwab [19] with the following modifications. Sciatic nerves were removed from mice that had been killed.