These GD1a-containing vesicles that colocalized with LAMP1 were a frequent finding in mutant internodes, but rarely observed in controls (Figure 3a, left)

These GD1a-containing vesicles that colocalized with LAMP1 were a frequent finding in mutant internodes, but rarely observed in controls (Figure 3a, left). Open in a separate window Figure 3. Hallmarks of lysosomal storage disorders in mutant nerves.(A) Double-staining of sciatic teased fibers displayed giant GD1a+ (green) vesicles that colocalized with LAMP1 (red) in internodes only of mutant animals (rectangular inset magnified in right panels). of paranodal loops. We revealed the same pathological features in a mouse model of human Adrenomyeloneuropathy, preceding disease-onset Cambinol by one year. Thus, peroxisomal dysfunction causes secondary failure of local lysosomes, thereby impairing the turnover of gangliosides in myelin. This reveals a new aspect of axon-glia interactions, with Schwann cell Rabbit Polyclonal to SIRT2 lipid metabolism regulating the anchorage of juxtaparanodal Kv1-channels. DOI: http://dx.doi.org/10.7554/eLife.23332.001 mutations of ABCD1 are responsible for the disease X-linked Adrenoleukodystrophy (X-ALD). This peroxisomal ATP-binding cassette (ABC-) transporter mediates the import of very long-chain fatty acids (VLCFA) into the organelle. In consequence, ABCD1-deficient peroxisomes are not capable of importing and degrading VLCFA that are specific substrates of peroxisomal -oxidation (Kemp et al., 2012). A more severe impairment of peroxisomes is caused by lack of the gene (also called multifunctional protein 2; gene) that encodes?a central enzyme of peroxisomal -oxidation. In MFP2-deficient cells, the -oxidation of virtually all peroxisome-specific substrates, including VLCFA, is inhibited (Verheijden et al., 2013). A complete disruption of the organelle is observed in the absence of peroxisome biogenesis factor peroxin 5 (PEX5). This cycling receptor recognizes proteins with a peroxisomal targeting sequence type 1 (PTS1) and is involved in their transfer into peroxisomes. PEX5-dependent protein import applies to the majority of peroxisomal enzymes. Thus, PEX5-deletion disrupts peroxisomal function substantially (Waterham et al., 2016). Schwann cell lipid metabolism is rate-limiting for myelination and is important for maintenance of axonal integrity (Saher et al., 2011; Viader et al., 2013), which requires in addition to membrane wrapping the assembly of nodal, paranodal, and juxtaparanodal membrane proteins (Rasband and Peles, 2015). The juxtaparanodal domain of myelinated axons harbors voltage-gated potassium channels, Kv1.1 (KCNA1) and Kv1.2 (KCNA2; Chiu and Ritchie, 1980; Robbins and Tempel, 2012), which also align the inner mesaxon as a thin band (Arroyo et al., 1999). Associated with connexin-29 hemichannels (Rash et al., 2016), their clustering and anchoring at juxtaparanodes requires the neuronal membrane proteins CASPR2 and TAG-1, the latter expressed by glia and neurons (Poliak et al., 1999b; Traka et al., 2003). Kv1 channels have been proposed to play a role in regulating fiber excitability (Baker et al., 2011; Glasscock et al., 2012), but the exact in vivo function of these fast-opening/slowly?inactivating channels remains unknown (Arancibia-Carcamo and Attwell, 2014). Results mice, termed cKO or Cambinol ‘mutants’ in the following, lack peroxisomal protein import in Schwann cells (Figure 1a; Figure 1figure supplement 1a). The PNS of these mice is well myelinated and unlike the CNS (Kassmann et al., 2007) without immune-mediated injury, in agreement with pilot observations (Kassmann et al., 2011). Upon closer inspection, we determined about 50% genomic recombination, corresponding to the fraction of Schwann?cell?(SC) nuclei in sciatic nerves (Figure 1figure supplement 1b). Teased fiber preparations, stained for PMP70, revealed peroxisomes as puncta. In mutant nerves, these were import-deficient ‘ghosts’, as evidenced by cytoplasmic catalase, normally a luminal peroxisomal marker (Figure 1b). Open in a separate window Figure 1. Schwann cell-specific PEX5-deficiency causes peroxisome dysfunction and peripheral Cambinol neuropathy in the absence of axonal loss or dysmyelination.(A) Scheme of normal (left) and impaired (right) PEX5-dependent peroxisomal protein import. PTS1, peroxisomal targeting signal type 1; PEX5, peroxisomal biogenesis factor peroxin 5. (B) Catalase (red) is present in peroxisomes (PMP70; green) of controls, but is localized in the cytoplasm of mutant fibers.?PMP70, peroxisomal membrane protein 70; DAPI-stained SC nuclei are depicted in blue; Scale bar, 10 m. Cambinol (C, D) Lipid mass spectrometry of nerve lysates from controls and mutants aged 9 months indicates peroxisomal dysfunction. Peroxisomal products (PEO-) and its corresponding plasmalogens (PEP-) are reduced. Specific substrates of peroxisomal -oxidation, VLCFA, are accumulated in mutant nerves. Statistics: means s.d.; Cambinol n?=?6; Students T-test; ***p 0.001. (ECG) Functional impairment of mutant compared to control sciatic nerves is?assessed by electrophysiology at the?age of?2 months (n?=?4). To evoke significant responses of all measured nerves, larger stimulus intensity is required for mutant (0.155mA) as compared to control (0.135mA) nerves. Peak amplitudes plotted against increasing stimulus intensity indicates earlier response saturation of mutant nerves. CAP, compound action potentials; au, arbitrary units; NCV, nerve conduction velocities (each curve representing single-nerve mean.