Loss of Fig4 in both Schwann cells and motor neurons contributes to CMT4J neuropathy

Mutations of FIG4 are responsible for Yunis-Varón syndrome, familial epilepsy with polymicrogyria, and Charcot-Marie-Tooth type 4J neuropathy (CMT4J). Although loss of the FIG4 phospholipid phosphatase consistently causes decreased PtdIns(3,5)P2 levels, cell-specific sensitivity to partial loss of FIG4 function may differentiate FIG4-associated disorders. CMT4J is an autosomal recessive neuropathy characterized by severe demyelination and axonal loss in human, with both motor and sensory involvement. However, it is unclear whether FIG4 has cell autonomous roles in both motor neurons and Schwann cells, and how loss of FIG4/PtdIns(3,5)P2-mediated functions contribute to the pathogenesis of CMT4J. Here, we report that mice with conditional inactivation of Fig4 in motor neurons display neuronal and axonal degeneration. In contrast, conditional inactivation of Fig4 in Schwann cells causes demyelination and defects in autophagy-mediated degradation. Moreover, Fig4-regulated endolysosomal trafficking in Schwann cells is essential for myelin biogenesis during development and for proper regeneration/remyelination after injury. Our data suggest that impaired endolysosomal trafficking in both motor neurons and Schwann cells contributes to CMT4J neuropathy.


INTRODUCTION
In yeast and mammalian cells, the Fig4/FIG4 phospholipid phosphatase controls the generation and turnover of the PtdIns(3,5)P 2 phosphoinositide, a regulator of membrane and protein trafficking at the level of the endosome -lysosome axis. Loss of Fig4/ FIG4 causes a decrease of PtdIns(3,5)P 2 levels and defects in multiple pathways in the endomembrane system. Typical cellular features associated with Fig4/FIG4 loss are the enlargement of late endosomes -lysosomes (LE/LY) and cytosolic vacuolization (1 -3).
Yunis-Varón syndrome is a severe disorder with autosomal recessive inheritance characterised by skeletal and structural brain abnormalities and facial dysmorphism (5). FIG4 mutations identified in Yunis-Varón patients are nonsense or missense mutations that abolish FIG4 enzymatic activity, thus resulting in complete loss of FIG4 function (5,9). Recently, a homozygous missense mutation causing partial loss of FIG4 function was demonstrated to co-segregate with polymicrogyria, psychiatric manifestations and epilepsy in a consanguineous Moroccan family, thus suggesting a role for FIG4 in the regulation of cortical brain development (10). ALS is a severe neurological disorder characterized by selective neurodegeneration of lower † These authors equally contributed to this work. * To whom correspondence should be addressed at: San Raffaele Scientific Institute, 20132 Milano, Italy. Tel: +39 02 26364743; Fax: +39 02 26435093; Email: bolino.alessandra@hsr.it and upper motor neurons. ALS patients carrying mutations in FIG4 are heterozygous for a null allele (deletions or splice site mutations leading to frameshift) or for missense mutations which alter FIG4 enzymatic activity (4). Patients with CMT4J neuropathy display a variable degree of severity. Early onset CMT4J shows asymmetrical motor and sensory neuropathy, which is usually rapid in progression. Late onset CMT4J displays a prevalent motor and asymmetric neuropathy, which is a typical feature of lower motor neuron disease rather than of CMT neuropathy (6). However, in both early and late onset CMT4J, the reduction of nerve conduction velocity (NCV) and the presence of onion bulbs in nerve biopsy suggest a demyelinating type of CMT, thus being classified in the CMT4 subclass (6 -8). CMT4J patients are compound heterozygous for one missense mutation and one loss-of-function mutation. The I41T allele is the most frequent CMT4J missense mutation, and partially affects FIG4 enzymatic activity by destabilizing the protein (3,11).
Overall, these disorders indicate that, despite the ubiquitous expression, loss of FIG4 affects specific cell types with distinct pathogenetic mechanisms. This cell-specific effect might be due to the impact of the different mutations on the FIG4 enzymatic activity/stability and/or to the impairment of cell-specific functions within the endolysosome axis. These aspects have been only partially elucidated using the Fig4-null mouse models generated so far. For example, while available mouse models clearly indicate a predominant role for Fig4 in neurons, the onion bulbs and active demyelination/remyelination observed in CMT4J patient biopsies would be consistent with a cell autonomous role for FIG4 in Schwann cells as well (6 -8).
Here, we report the generation and characterization of mouse mutants with conditional inactivation of Fig4 in either motor neurons or Schwann cells, two cell types affected in the CMT4J neuropathy. We found that Fig4 loss in motor neurons causes neuronal and axonal degeneration, whereas the Fig4-Schwann cell conditional mutant displays a general trafficking impairment, leading to a defect in autophagy-mediated degradation and demyelination. We also exploited the Fig4-Schwann cell conditional mutant to investigate whether trafficking through the endolysosome axis contributes to myelin biogenesis during development and to regeneration/remyelination. Our in vitro and in vivo data suggest that altered LE/LY homeostasis in Schwann cells impairs both active myelination and nerve regeneration.

RESULTS
Loss of Fig4 in motor neurons in vivo leads to neuronal and axonal degeneration CMT4J patients initially display a prevalent motor and asymmetric neuropathy, which is a typical feature of a lower motor neuron disease rather than of demyelinating CMT neuropathies (6,7 (1,3,12). However, in the Fig4 plt/plt mouse, spinal motor neurons were among the last neurons to exhibit vacuolization, being largely preserved at P21 but filled with vacuoles at 6 weeks of age (3,13). The lethality of the Fig4 plt/plt mice 6 weeks of age did not permit further evaluation of the Fig4 loss-of-function phenotype in motor neurons. Thus, for a more specific assessment of Fig4 in motor neurons and their peripheral projections, we generated Fig4 Floxed/plt , HB9-Cre mice, in which the HB9-Cre transgene produces somatic recombination at embryonic day 9.5 (E9.5) in motor neurons and in the pancreas (14 -17). To achieve maximal efficiency of HB9-Cre-mediated recombination, we generated compound heterozygous mice carrying one null allele (Fig4 Fig. S1), where the Olig2 promoter drives Cre expression starting at E10.5 in Shh (Sonic Hedgehog) responsive domains of the neural tube that give rise to motor neurons as well as oligodendrocyte precursor cells (OPCs) (19,20).
These results demonstrate that Fig4 has a cell autonomous role in motor neurons since Fig4 Floxed/plt , HB9-Cre mutants show neuronal and axonal degeneration, leading to mild hypomyelination likely as a secondary consequence of altered axoglial communication. Moreover, the fact that demyelination is not observed in the nerves of the motor neuron conditional   endocytic routes (24,25). Consistent with this, Fig4 Floxed/Floxed , P0-Cre mutant sciatic nerves also displayed reduced myelin thickness and increased g-ratio values at P60 ( Fig. 2C and D), suggesting that the regulation of endocytic trafficking through the endolysosomal axis is essential for myelination (g-ratio:  We attribute the somewhat milder phenotype of Fig4 Floxed/plt , P0-Cre mice, compared with Fig4 Floxed/Floxed , P0-Cre mice, to the greater contribution of genetic background from strain C57BL/6J, which is known to exacerbate Fig4-null associated features (1,3,26). The Fig4 plt allele was maintained in a mixed strain background whereas the Fig4 Floxed/Floxed , P0-Cre genotype was enriched in the C57BL/6J strain (3,12).
Overall, these findings demonstrate that Schwann cells are vulnerable to loss of Fig4 and that loss of Fig4 in Schwann cells reproduces demyelinating features of human CMT4J neuropathy.

Loss of Fig4 in Schwann cells in vivo impairs nerve regeneration
Loss of Fig4 in Schwann cells was shown above to be associated with demyelination and progressive axonal loss. Since impaired regeneration may also contribute to axonal loss in CMT neuropathies, we investigated whether impaired trafficking through the endolysosome axis in Fig4-null Schwann cells could affect remyelination and nerve regeneration. To this aim, we exploited the milder Fig4 Floxed/plt P0-Cre mouse model to prevent developmental degeneration that might interfere with nerve regeneration after injury. Sciatic nerves from Fig4 Floxed/plt , P0-Cre mice and controls were crushed at 2 months of age, a time point at which myelination and axonal integrity are largely normal, and morphological analysis was performed 60 days after injury (Fig. 5A). As expected, regeneration of axons and myelin was nearly complete in control nerves. In contrast, in Fig4 Floxed/plt , P0-Cre crushed nerves, we observed a reduced number of medium and large calibre fibres and total fibres, and the presence of myelin debris ( Fig. 5A  Since specific loss of Fig4 in Schwann cells appears to cause an endolysosomal trafficking impairment, we asked whether the altered regeneration of myelinated fibres could be caused by delayed clearance of myelin debris by Schwann cells. Immunohistochemistry of nerves at 7 days after injury demonstrated that MBP protein level was similar between non-regenerating Fig4 Floxed/plt , P0-Cre nerves and controls, indicating normal myelin clearance (Fig. 5C). This finding was also supported by morphological analysis of semithin sections (data not shown).
In conclusion, loss of Fig4 in Schwann cells impairs regeneration of myelinated fibres suggesting that Fig4 and PtdIns(3,5)P 2 homeostasis are necessary for efficient nerve regeneration.

Loss of Fig4 in Schwann cells impairs endolysosomal homeostasis resulting in hypomyelination in vitro
We showed above that loss of Fig4 specifically in Schwann cells causes a general trafficking defect, an impairment of active myelination during development, and demyelination. To determine how loss of Fig4 in Schwann cells and the consequent impairment of PtdIns(3,5)P 2 -mediated trafficking affect myelination during development, we established myelin-forming Schwann cell/DRG neuron co-culture explants from Fig4 Floxed/plt , P0-Cre mice, in which P0-Cre-mediated recombination should be more efficient in vitro due to the presence of a single Floxed allele. Under myelinating conditions, following ascorbic acid treatment, most Schwann cells displayed enlarged LAMP1positive LE/LY (Fig. 6A -D). Although Fig4 Floxed/plt , P0-Cre explants were able to myelinate, the number of MBP-positive segments was significantly reduced when compared with control cultures (Fig. 6E -G). The Schwann cell number was similar between mutant and control (Fig. 6H). This finding is consistent with the observed hypomyelination in mutant mice and confirms that reduced PtdIns(3,5)P 2 in Schwann cells results in impaired myelination. Note that myelin production was equivalent in To explore the molecular basis of hypomyelination, we determined whether loss of Fig4 in Schwann cells and the enlargement of LE/LY results in impaired exocytosis of myelin proteins, impaired myelin protein degradation or more general defects in the endomembrane system during active myelination. It has been recently suggested that P0 co-localizes with LAMP1positive compartments at early stages of postnatal nerve development (27). Myelin protein zero (P0) may be stored in LE/LY compartments prior to exocytosis during myelin assembly, as proposed for PLP in oligodendrocytes in the CNS (28), or during remyelination after damage. To explore this possibility, we evaluated retention of P0 in the enlarged LE/LY compartment of Fig4-null Schwann cells. Explants were stained with anti-P0 and anti-LAMP1 antibodies after ascorbic acid treatment, at 1 -6 days of ascorbic acid treatment. We did not observe co-localization of P0 and LAMP1 in mutant or control cultures (Fig. 6I -K).
We then investigated whether altered protein degradation or turnover contributes hypomyelination in mutant cells. To this aim, we determined levels of autophagic markers and ubiquitinated substrates by western blot analysis. We did not observe an elevation of LC3II/I, p62 or ubiquitinated substrates in Fig4 Floxed/plt , P0-Cre explants when compared with controls after 7 days of ascorbic acid treatment (Supplementary Material, Fig. S2A -C), suggesting that hypomyelination is not a consequence of impaired protein turnover.
Cholesterol is known to facilitate the exit of P0 from the Golgi compartment and delivery to the plasma membrane, and addition of cholesterol to myelin-forming cultures accelerates myelination (29). We reasoned that if hypomyelination in Fig4 Floxed/plt , P0-Cre mutant cultures was the consequence of impaired membrane trafficking in the endomembrane system, cholesterol delivery to the plasma membrane would itself be impaired and therefore addition of cholesterol to the culture medium would not rescue hypomyelination. Control cultures treated with cholesterol displayed enhanced myelination after 7 days of treatment, but not at 13 days, in agreement with previous reports (29) (Fig. 7A -D, and quantification in G). Consistent with our hypothesis, treatment of mutant explants with ascorbic acid in the presence of 20 mg/ml cholesterol did not rescue hypomyelination after 7 or 14 days of treatment ( Fig. 7E and F, and quantification in H). This finding suggests that in mutant cells cholesterol trafficking and/or P0 delivery and assembly to the plasma membrane is not as efficient as in controls due to defects in the endolysosomal trafficking.

Altered endolysosomal trafficking results in a block of autophagic progression and demyelination
We then asked how loss of Fig4 in Schwann cells and the consequent impairment of PtdIns(3,5)P 2 -mediated trafficking can lead to the observed demyelination in the nerve. Fig4 loss and/or reduced activity of the PIKfyve kinase complex that generates PtdIns(3,5)P 2 from PtdIns3P, leads to decreased PtdIns(3,5)P 2 levels and enlargement of the LE/LY compartment (2,(30)(31)(32). A block in autophagic progression has been reported in astrocytes and, to a lesser extent, in neurons of Fig4 plt/plt mice (1,3,33). This defect may result from reduced fusion between enlarged LE/LY and autophagosomes to form autophagolysosomes (33). We thus asked whether there is a block of autophagy in Fig4-null Schwann cells. Western blot analysis of sciatic nerve lysates from Fig4 Floxed/plt , P0-Cre mice at P30 revealed that the levels of LAMP1, LC3II and p62 proteins were significantly increased, consistent with a block in autophagic flux (Fig. 8A-D). Accumulation of ubiquitinated proteins is characteristic of impaired autophagy (34). We analysed levels of ubiquitinated proteins in P90 Fig4 Floxed/plt , P0-Cre nerve lysates and found an increase of polyubiquitinated proteins in mutant nerves when compared with control (Fig. 8E).
The data demonstrate that Fig4 loss in Schwann cells causes both enlarged LE/LY and a block of autophagic flux, which may lead to demyelination.

The CMT4J neuropathy: cell autonomy of Fig4 in motor neurons and Schwann cells
Loss of human FIG4 causes a spectrum of inherited conditions suggesting that more than one cell type is vulnerable to impairment of FIG4/PtdIns(3,5)P 2 -mediated functions (3 -7,9,10). This cell-specific sensitivity could be related to the impact of the different mutations on FIG4 enzymatic activity and protein stability, as well as to the impairment of different cellular functions in different cell types.
The CMT4J neuropathy is classified as CMT type 4 because of the autosomal recessive inheritance and of demyelinating features such as the reduction of NCV and the presence of onion bulbs in the nerve biopsies (6 -8). However, late onset CMT4J patients initially display a predominantly motor and asymmetric neuropathy, suggesting a lower motor neuron involvement in the pathogenesis of this neuropathy (6).
In the mouse, the phenotypes of the global null  the Fig4 plt/plt mouse beginning by P1-P7, whereas motor neurons do not display typical pathological features related to PtdIns(3,5)P 2 deficiency until 6 weeks of age (3,13). Whether FIG4 has a cell autonomous role in motor neurons and Schwann cells and how loss of FIG4/PtdIns(3,5)P 2 -mediated functions in these cells contribute to CMT4J pathogenesis have not been clearly defined. To address this gap in our knowledge we generated conditional mutants that selectively lack Fig4 in motor neurons or Schwann cells. Spinal cords of Fig4 Floxed/plt , HB9-Cre and Fig4 Floxed/plt , Olig2-Cre mice display motor neuron vacuolization, which is associated with axonal degeneration, hypomyelination and a decrease in the number of myelinated fibres in the quadriceps nerve. These findings suggest that Fig4 has a cell autonomous role in motor neurons and that loss of Fig4 in motor neurons contributes to the CMT4J neuropathy. In the motor neuron-specific knockout demyelinating features were not observed, supporting the hypothesis that demyelination is a consequence of specific loss of Fig4 in Schwann cells. Consistent with this, we also report that Demyelination in peripheral neuropathies can arise as a consequence of impaired endosomal trafficking and/or of a gain of toxic function. In addition to FIG4, several other proteins involved in the regulation of membrane trafficking are mutated in demyelinating CMT neuropathies and dys-regulation of endolysosomal trafficking may be a common mechanism at the basis of these disorders. These proteins regulate or are connected with PI metabolism, and include FRABIN/FGD4, SIMPLE, SH3TC2, MTMR2, MTMR5 and MTMR13 (1,35). For instance, mutated SIMPLE re-localizes from endosomal membranes to the cytosol where it exerts a dominant negative effect on the ESCRT protein complex (36,37). This in turn is thought to lead to defective ESCRT-mediated endosomal sorting and degradation of ErbB2/B3 receptors, loss of ERK signalling attenuation and demyelination.
Demyelination in peripheral neuropathies can also arise from activation of an integrated stress response (ISR) in Schwann cells. Several mutations in the myelin protein P0 responsible for demyelinating CMT1B cause misfolding of the protein, retention in the ER and UPR-mediated activation of ISR, which is maladaptive and leads to demyelination (38,39). Moreover, in the Tfam-KO Schwann cell mutant, mitochondria dysfunction causes ISR and demyelination (40). The  demyelinating features such as myelin debris and degeneration, onion bulbs and decrease of NCV, thus recapitulating demyelination of the CMT4J neuropathy. Demyelination in these models can be the consequence of a general endolysosomal trafficking impairment and loss of LE/LY homeostasis. We indeed demonstrated that Fig4-deficient Schwann cells display a block in autophagy, which progressively leads to an accumulation of ubiquitinated substrates. As in lysosomal storage disorders, accumulation of macromolecules, cholesterol and defective calcium release from the LE/LY compartment may affect the functioning of several organelles such as Golgi, ER and mitochondria thus ultimately leading to demyelination (41).

Endolysosomal trafficking and myelination
Myelin biogenesis relies on polarized trafficking and assembly of bulk of lipids and proteins (25). We exploited the mouse model with conditional ablation of Fig4 in Schwann cells to investigate whether the resulting impairment of endolysosomal trafficking would influence myelin biogenesis during development. We indeed observed decreased myelin thickness and hypomyelination both in vivo and in vitro as a result of the Fig4 deficiency in Schwann cells. During postnatal nerve development, the P0 myelin protein has been found to co-localize with the lysosomal cathepsin D in Schwann cells in vivo. Calcium-dependent lysosomal exocytosis has been suggested as a mechanism of P0 delivery to the plasma membrane during myelin biogenesis (27). We thought that hypomyelination in the conditionally deleted Schwann cells could result from the reduced delivery of P0 to the plasma membrane from the LE/LY compartment. However, we did not observe co-localization of P0 with LAMP1-positive LE/LY in either control or mutant Schwann cells at various stages of differentiation. We then hypothesized that the hypomyelination could result from a general impairment of trafficking occurring in Fig4-null Schwann cells. The observation that cholesterol supplementation does not rescue the hypomyelination in vitro supports this hypothesis. In normal conditions, cholesterol promotes the insertion of myelin P0 and other proteins into vesicles specifically destined for the developing myelin sheath. In Fig4-null Schwann cells, myelin proteins and/or cholesterol may be mis-trafficked and not properly delivered to the nascent myelin sheath causing hypomyelination.
Thus, we conclude that FIG4-mediated regulation of endolysosome trafficking in Schwann cells is important for generation of myelin during development, although the molecular mechanism remains to be clarified.
Therapeutical perspectives for CMT4J and pathologies associated to FIG4 loss FIG4 loss leads to decreased PtdIns(3,5)P 2 levels and to the enlargement of LE/LY. Loss of LE/LY homeostasis and vacuolization in models of PtdIns(3,5)P 2 deficiency may arise as a consequence of Ca 2+ retention in the lysosome, which is the other Ca 2+ buffering organelle in addition to the ER (41,42). Indeed, PtdIns(3,5)P 2 activates the cation channels TPC1 (two pore channel), TPC2 and MCONL1/TRPML1 (Mucolipin 1, transient receptor potential cation channel subfamily M, member 1) localized at LE/LY compartments (43). An exciting and intriguing therapeutic strategy for CMT4J and other human disorders associated with FIG4 deficiency would be to increase the activity of these channels to restore LE/LY homeostasis and dynamics. Indeed, synthetic compounds have been recently identified as activators of the MCONL1/TRPML1 channels, which promote the efflux of Ca 2+ from the lysosomal lumen into the cytosol and trigger lysosomal exocytosis (43). However, different cell types may respond differently to these treatments depending on the level of expression of these channels and the elicited electrophysiological response. Mouse models of cell-specific Fig4 deficiency will therefore be useful for preclinical validation of these strategies.

Ethics statement
All experiments involving animals were performed in accordance with Italian national regulations and covered by experimental protocols reviewed by local Institutional Animal Care and Use Committees (IACUC #525). Animals were generated and maintained in a SPF (specific pathogen free) Institutional facility. Genotyping was performed as already described (3,12,17). For all the experiments involving animals at least n ¼ 4 animals per genotype of either sex were analysed. Fig4 Floxed/ Floxed mice have normal physiology and are indistinguishable from controls since they display normal nerve morphology including myelin thickness, and the absence of cell vacuolization in all cells analysed in both CNS and PNS also in aged mice (data not shown and as previously reported) (12). Fig4 plt/+ heterozygous mice are normal as no vacuolization has been observed in any tissue analysed and myelin thickness is normal in peripheral nerves, as previously reported (3,18).

Morphological analysis
Semithin section and ultrastructural analysis of sciatic nerves was performed as previously reported (44). To perform morphometric analysis, digitalized images of fibre cross sections were obtained from corresponding levels of the sciatic nerves with a ×100objective and digital camera Leica DFC300F. Five images per animal were acquired and analysed with the Leica QWin software (Leica Microsystem). The g-ratio was determined by dividing the mean diameter of an axon (without myelin) by the mean diameter of the same axon including myelin. Statistical analysis was performed on the mean g-ratio values of the different nerves (animals) analysed per genotype. The quadriceps nerve, the motor branch of the femoral nerve, was dissected at the level of the inguinal ligament (45).

Immunohistochemistry on sections
Spinal cord Juvenile (P21) and adult (P120 -210) mice were overdosed with ketamine/xylosine and transcardially perfused with ice-cold PBS and 4% PFA. Spinal cords were post-fixed in 4% PFA overnight, cryoprotected in 30% sucrose and frozen in OCT compound on dry ice. Lumbar spinal cords were sectioned at 30-40 mm at levels L3 -L6 and stored in PBS. Free-floating immunohistochemistry was performed on 5 -10 spinal cord sections per mouse. Sections were incubated overnight in primary antibody solution (Goat anti-ChAT, Millipore 1:100, 1.5% NDS in PBST-x) at 48C on a rocker. After three washes in PBST-x sections were incubated in biotinylated donkey antigoat secondary antibody in PBST-x (1:500, Jackson Immunoresearch) for 1 h. Followed by three washes in PBST-x, sections were incubated in ABC Vectashield kit solution (Vector, as per manufacturer's instructions) for 1 h. Antibody binding was visualized using diaminobenzidine kit (Vector Labs, as per manufacturer's instructions). Sections were mounted on glass slides, dehydrated in series of ethanol solutions and imaged using a Leica inverted bright field microscope.

Sciatic nerve
Immunofluorescence on cryosections was performed as described (47) and examined with confocal TCS SP5 laser-scanning confocal (Leica) or Olympus BX (Olympus Optical) fluorescent microscope, and Zeiss Axiovert S100 TV2 with Hamamatsu OrcaII-ER. For immunohistochemistry, sciatic nerves were removed and rapidly snap-frozen in liquid nitrogen, either unfixed or previously fixed in buffered 4% PFA.
Myelin-forming Schwann cell/DRG neuron co-cultures were established from embryonic Day 13.5 mouse embryos as previously described (48,49). Following NB (Neurobasal, B27 supplement, glucose, NGF) medium treatment for 7 days to allow neuritogenesis and Schwann cell migration, myelination was induced by treatment for 7 -15 days with ascorbic acid (final concentration, 50 mg/ml) (Sigma). Cholesterol (dissolved in ethanol) was added every other day to C-media (MEM, 10% serum, NGF, glucose) plus ascorbic acid at 20 mg/ml final concentration. Analysis of autophagy on co-cultures was performed by starving Schwann cell/DRG explants in HBSS for 6 h and then by adding 100 nM bafilomycin A1 (LC laboratories) for 2 h to block LC3/p62 lysosomal degradation.
Analysis of proteasome-mediated degradation was performed by treating co-cultures using MG132 proteasome inhibitor at 25 mM final concentration for 6 h.
For analysis of myelination, immunohistochemistry on Schwann cell/DRG neuron co-cultures was performed as follows: cells were fixed for 15 min in 4% paraformaldehyde, permeabilized for 15 min in ice-cold methanol at 2208C, blocked for 20 min with 10% normal goat serum (Dako), 1% BSA (Sigma) and then incubated with primary antibody for 1 h. After extensive washing, the coverslips where incubated with the secondary antibody for 30 min, washed and mounted. For double immunostaining with anti-NF-H and anti-MBP antibody, the coverslips were blocked with 1% BSA, 10% NGS for 20 min on ice, and primary antibodies were incubated overnight at 48C. For LAMP1 staining, cells were permeabilized using 0.1% saponin after fixation.

Analysis of myelination
To quantify the amount of myelin, the number of MBP-positive segments in each explant/coverslip was assessed. As myelination is also a function of the amount of neurites/axons and of the Schwann cell number in the culture, the network of NF-H-positive filaments and the number of Schwann cells (DAPI) were also evaluated in each explant. Using a fluorescence microscope, at least 5-10 fields per cover were randomly acquired and MBP-positive myelinated fibres were counted per field. Average (among 5-10 fields) of MBP fibres was calculated per cover and statistical analysis was performed on different covers per condition/per experiment (SEM) in at least three different experiments. In the case of cholesterol treatment, the entire DRG was also reconstructed to evaluate the difference in the area occupied by myelinated segments in treated versus untreated explants. MBP-positive fibres and vacuolated organelles were acquired using TCS SP5 laser-scanning confocal (Leica) or Olympus BX (Olympus Optical) fluorescent microscope, and Zeiss Axiovert S100 TV2 with Hamamatsu OrcaII-ER. P0-LAMP1 co-localization analysis was performed using DeltaVision-Olympus IX70 with DeltaVision RT Deconvolution System.

Imaging and statistical analysis
Micrographs were acquired using a digital camera (Leica F300), and figures were prepared using Adobe Photoshop, version 7.0 and 8.0 (Adobe Systems). Statistical analysis was performed using the Student's t-test; two tails, unequal variance and a ¼ 0.005 were used. Error bars in the graphs represent SEM.

SUPPLEMENTARY MATERIAL
Supplementary Material is available at HMG online.