Septin/anillin filaments scaffold central nervous system myelin to accelerate nerve conduction

Myelination of axons facilitates rapid impulse propagation in the nervous system. The axon/myelin-unit becomes impaired in myelin-related disorders and upon normal aging. However, the molecular cause of many pathological features, including the frequently observed myelin outfoldings, remained unknown. Using label-free quantitative proteomics, we find that the presence of myelin outfoldings correlates with a loss of cytoskeletal septins in myelin. Regulated by phosphatidylinositol-(4,5)-bisphosphate (PI(4,5)P2)-levels, myelin septins (SEPT2/SEPT4/SEPT7/SEPT8) and the PI(4,5)P2-adaptor anillin form previously unrecognized filaments that extend longitudinally along myelinated axons. By confocal microscopy and immunogold-electron microscopy, these filaments are localized to the non-compacted adaxonal myelin compartment. Genetic disruption of these filaments in Sept8-mutant mice causes myelin outfoldings as a very specific neuropathology. Septin filaments thus serve an important function in scaffolding the axon/myelin-unit, evidently a late stage of myelin maturation. We propose that pathological or aging-associated diminishment of the septin/anillin-scaffold causes myelin outfoldings that impair the normal nerve conduction velocity. DOI: http://dx.doi.org/10.7554/eLife.17119.001


Introduction
Fast nerve conduction is crucial for normal motor, sensory, and cognitive abilities. In vertebrates, rapid ('saltatory') signal propagation is achieved by the myelination of axons. Myelination is largely completed before adulthood, notwithstanding that adult-born oligodendrocytes can assemble new myelin sheaths throughout life (Nave and Werner, 2014). Yet, myelin is one of the most long-lived structures in the CNS (Toyama et al., 2013). With age however, the abundance and dynamics of CNS myelin decreases (Lasiene et al., 2009) while there is an increase in the frequency of pathological redundant myelin sheaths (Peters, 2002;Sturrock, 1976), i.e., local outfoldings of compact myelin with normal-appearing axo-myelinic interface. These myelin outfoldings emerge from the innermost, adaxonal myelin layer, a part of the non-compacted, cytoplasmic channel system through myelin. This compartment of myelin has previously been implicated in glia-axonal metabolic coupling (Nave and Werner, 2014). Analogous focal outfoldings of peripheral myelin are the pathological hallmark of tomaculous neuropathies affecting the PNS. The normal structure of myelin thus requires stabilization, which can fail upon normal aging and in myelin-related disorders. However, the molecular mechanisms that might prevent myelin outfoldings have remained unknown.
A striking variety of myelin-related genes causes -when mutated in human disorders and in animal models -common pathological features including axonopathy, neuroinflammation, hypomyelination, and structural impairments affecting myelin. For example, abundant constituents of CNS myelin such as proteolipid protein (PLP), cyclic nucleotide phosphodiesterase (CNP), and myelin associated glycoprotein (MAG), are not essential for the biogenesis of myelin per se but their deficiency in mice causes complex CNS pathology (Edgar et al., 2009;Griffiths et al., 1998;Li et al., 1994;Montag et al., 1994). The neuropathological profiles observed in these and other myelin mutants are highly overlapping, which has made it difficult to explain distinct aspects of neuropathology by the loss of individual structural myelin proteins. Here, we followed the hypothesis that mutations of single genes can have secondary consequences for the entire protein composition of myelin, which allow elucidating the molecular cause of distinct neuropathological features.
We report the discovery of a previously unrecognized filamentous scaffold in the innermost layer of CNS myelin that extends longitudinally along myelinated axons. This filament is composed of distinct septin monomers (SEPT2/SEPT4/SEPT7/SEPT8) and associated with the adaptor protein anillin (ANLN). The formation of myelin septin filaments is a late stage of myelin maturation, thereby avoiding that the property of septins to rigidify the membranes they are associated with (Gilden and Krummel, 2010;Tooley et al., 2009) could hinder the normal developmental ensheathment of axons. Importantly, this sub-membranous septin/anillin-scaffold (SAS) is required for the normal structure of the axon/myelin unit in vivo as its deficiency causes a specific structural impairment of the myelin sheath, myelin outfoldings, and reduced nerve conduction velocity. These findings were possible by systematic label-free myelin proteome analysis in several models of complex neuropathology.

Neuropathology and myelin proteome analysis in myelin mutants
By quantitative evaluation of electron micrographs ( Figure 1A; Figure 1-figure supplement 1A-E), hypomyelination was a significant feature in Plp1 null and Mag null mice, swellings of the inner tongue of myelin in Mag null and Cnp null mice, axonal spheroids in Plp1 null mice, and degenerated axons in Cnp null mice. Split myelin lamellae (not quantified) were observed in Plp1 null mice. Together, these mouse mutants display distinct but overlapping profiles of neuropathology. Notably, myelin outfoldings ( Figure 1B; Figure 1-figure supplement 1A) were common to all three models, suggesting a common molecular mechanism. eLife digest Normal communication within the brain or between the brain and other parts of the body requires information to flow quickly around the nervous system. This information travels along nerve cells in the form of electrical signals. To speed up the signals, a part of the nerve cell called the axon is frequently wrapped in an electrically insulating sheath made up of a membrane structure called myelin.
The myelin sheath becomes impaired as a result of disease or ageing. In order to understand what might produce these changes, Patzig et al. have used biochemical and microscopy techniques to study mice that had similar defects in their myelin sheaths.
The study reveals that forming a normal myelin sheath around an axon requires a newly identified 'scaffold' made of a group of proteins called the septins. Combining with another protein called anillin, septins assemble into filaments in the myelin sheath. These filaments then knit together into a scaffold that grows lengthways along the myelin-wrapped axon. Without this scaffold, the myelin sheath grew defects known as outfoldings. Axons transmitted electrical signals much more slowly than normal when the septin scaffold was missing from the myelin sheath.
Future studies are needed to understand the factors that control how the septin scaffold forms. This could help to reveal ways of reversing the changes that alter the myelin sheath during ageing and disease.
By subjecting myelin purified from the brains of these models to label-free quantitative mass spectrometry (Distler et al., 2014), we found distinct but overlapping alterations of the myelin proteome ( Figure 1C; Figure 1-source data 1). Notably, the abundance of several septins was reduced in all analyzed mutants ( Figure 1C). Septins have important functions in physiology and cell division (Dolat et al., 2014;Fung et al., 2014), but their role in myelinating cells is unknown Patzig et al., 2014). By mass spectrometry (Figure 1-source data 1), the most abundant septins in wild-type CNS myelin are SEPT2, SEPT4, SEPT7, and SEPT8. The abundance of all four septins was reduced in myelin of all three mutants (Figure 1-figure supplement 1F), as validated by immunoblotting (Figure 1-figure supplement 1G-I). Immunohistochemical analysis of optic nerves confirmed the diminishment of SEPT8 in all three mutants ( Figure 1D). By qRT-PCR, we  could amplify cDNA fragments for Sept2, Sept4, Sept7, and Sept8 from mutant and control corpus callosi with approximately similar efficiency, suggesting that transcriptional regulation is unlikely to cause the loss of myelin septins (Figure 1-figure supplement 1J-M). Together, the presence of myelin outfoldings correlates with loss of myelin septins in three models of complex CNS pathology.
To determine the localization and higher-order structure of myelin septins, we performed immunohistochemistry and confocal microscopy of longitudinal sections of optic nerves and spinal cords. SEPT7 and SEPT8-labelling was found to parallel (but not overlap with) axonal neurofilament labelling ( Figure 2A-C, Video 1), suggesting the presence of longitudinal septin filaments in myelin. We therefore colabelled SEPT8 and a marker for adaxonal myelin (MAG). In cross sections, SEPT8-immunopositive puncta appeared contained within the ring-shaped compartment defined by MAG-immunopositivity ( Figure 2D). Any ring-shaped axon/myelin-unit identified by MAG-labelling exhibited between 0-3 puncta of SEPT8-labeling ( Figure 2E) independent of the axonal diameter ( Figure 2F). Aiming to reveal the exact localization of SEPT8 in the adaxonal cytoplasmic (i.e. non-compacted) compartment of myelin at higher resolution, we used cryo-immuno electron microscopy of optic nerves. Immunogold labelling of SEPT7 and SEPT8 supported the localization in adaxonal myelin ( Figure 2G-H, Figure 2-figure supplement 1A-B). Interestingly, within this compartment SEPT8 immunogold was mostly associated with the innermost membrane of compact myelin ( Figure 2H'-I), in difference to MAG, a transmembrane protein localized to the adaxonal membrane.
Taken together, we have identified a previously unrecognized network of longitudinal septin filaments in the adaxonal non-compact layer of CNS myelin ( Figure 3A). These filaments extend straight or slightly undulating for various lengths up to 20 mm. Septin filaments extend beyond the internodal segments of myelinated axons into the juxtaparanodal regions, as evidenced by occasional proximate-labelling of SEPT8 with Kv1.2 (

Septins in myelin maturation and aging
During oligodendroglial maturation, the abundance of Sept8-mRNA increases, but later than most myelin markers (de Monasterio-Schrader et al., 2012). When we immunoblotted myelin (purified from wild-type mouse brains at P10, P15, P21, P28, representing ongoing myelin maturation), the abundance of SEPT4, SEPT7, and SEPT8 increased with age whereas the abundance of the classical myelin markers PLP and myelin basic protein (MBP) did not change ( Figure 3B). When co-immunolabelling SEPT8 and MAG in optic nerves (ages P15, P21, P28), MAG was readily detectable at all examined ages ( Figure 3C). Conversely, SEPT8 was detectable at P28 but not at P15 or P21 ( Figure 3C). Thus, the assembly of myelin septins represents a late stage of myelin maturation coinciding with a developmentally decreased frequency of myelin outfoldings ( Figure 3D). Considering the increase of myelin outfoldings in the aging brain ( [Peters, 2002;Sturrock, 1976] and Figure 3D), we also assessed the abundance of septins (SEPT2, SEPT4, SEPT7, SEPT8) in myelin purified from wild-type mouse brains at two years of age. Interestingly, their abundance was decreased compared to myelin from young adult brains ( Figure 3E), though not as strongly as in myelin of Mag null , Cnp null and Plp1 null mice. Together, the developmental assembly of myelin septins represents a late stage of myelin maturation correlating with a reduced frequency of myelin outfoldings. On the other hand, the increase of myelin outfoldings with normal aging coincides with a reduced abundance of myelin septins.

Association of myelin septins with anillin
The mRNA abundance profile of Sept8 during oligodendroglial maturation coincides with that of Anln (de Monasterio-Schrader et al., 2012), which encodes the pleckstrin homology (PH) domaincontaining protein anillin (ANLN) (Menon et al., 2014;Oegema et al., 2000) that can serve as an adaptor to recruit septins onto membranes, at least in yeast (Liu et al., 2012). We have thus tested whether anillin is also associated with septins in myelin. When co-immunolabelling SEPT8 and ANLN in the optic nerves of mature wild-type mice, ANLN was readily detectable and co-localized with SEPT8 ( Figure 4A). Importantly, ANLN immunolabelling was strongly reduced in Mag null , Cnp null and Plp1 null mice, similar to that of SEPT8 ( Figure 4B). The diminishment of ANLN was confirmed by immunoblotting of myelin purified from the brains of these mutants ( Figure 4C). Together, these data imply that myelin septin filaments are associated with anillin.

Mice lacking a myelin septin/anillin filament
To examine whether a causal relationship exists between myelin septins and myelin outfoldings, we generated mouse mutants by gene targeting ( Conditional and constitutive Sept8-mutants were born at expected frequencies, and their major myelinated fibre tracts formed normally as judged by light microscopic examination of histochemically stained myelin ( Figure 6A). Electron microscopy did not reveal abnormalities of developmental myelination ( Figure 6B), the Quantification of immunogold labelling of SEPT8 and MAG relative to the innermost membrane layer of compact myelin (type 1) and the adaxonal myelin membrane (type 2). Note that SEPT8 immunogold labelling is associated with the innermost membrane layer of compact myelin while MAG labelling is associated with the adaxonal myelin membrane. Mean ± SEM. Analysis of 3 animals; two-tailed paired t-test; SEPT8 **p=0.002, MAG, *p=0.02. DOI: 10.7554/eLife.17119.006 The following figure supplement is available for figure 2: Note that all experimental data support a model that myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) assemble as longitudinal filaments in the non-compact adaxonal compartment of myelin, in which they underlie the innermost membrane of compact myelin. (B) Immunoblotting indicates that the abundance of several septins (SEPT4, SEPT7, SEPT8) increases with age in myelin purified from wild-type brains at P10, P15, P21, and P28, reflecting the maturation of myelin. Note that the abundance of the classical myelin markers MBP and PLP is unaltered. (C) Immunolabelling of WT optic nerves detects SEPT8 (green) at P28 but not at P15 or P21. Note that the myelin marker MAG (red) is detectable at all time points, reflecting that the optic nerve is largely myelinated by P14. Images are representative of three experiments. (D) Quantitative evaluation of electron micrographs of WT optic nerves indicates that the frequency of axon/myelin-units with myelin outfoldings declines between P15 and P75 (i.e. with myelin maturation) and is strongly increased at 2 years of age (i.e with normal aging). Mean ± SEM. n=4 animals were analyzed. One-way ANOVA with Tukey's multiple comparison test; P15 vs. P75, *p=0.044; P15 vs. 2 yrs, ***p<0.001, P75 vs. 2 yrs, ***p<0.001. (E) Immunoblotting indicates that the abundance of myelin septins is decreased in myelin purified from mice at the age of two years compared to P75, reflecting normal aging. Blot is representative of 3 animals per age. ATPase-alpha1 (ATP1A1) served as a control. DOI: 10.7554/eLife.17119.009 percentage of myelinated axons ( Figure 6C), and myelin thickness ( Figure 6D).
Next, we examined if SEPT8-deficiency affects the protein composition of myelin. By label-free quantitative mass spectrometry, septins and ANLN were almost undetectable in myelin purified from Sept8 null/null mice whereas the abundance of all conventional myelin markers was unaltered compared to littermate controls ( Figure 6E; Figure 1-source data 1). Loss of myelin septins in Sept8-mutant mice was validated by immunoblotting ( Figure 6F; Figure 6-figure supplement 1E) and immunohistochemistry ( Figure 6-figure supplement 1F,G). By immunoblotting, abundance and phosphorylation of Akt and Erk were similar in Sept8 null/null and control corpus callosi (data not shown). By qRT-PCR, cDNA-fragments for Sept2, Sept4, and Sept7, were amplified with about equal efficiency from mutant and control corpus callosi ( Figure 6-figure supplement 1D), implying that the loss of myelin septins (secondary to SEPT8-deficiency) is a posttranscriptional event. Together, these results strongly suggest that SEPT2, SEPT4, SEPT7, and ANLN are degraded if not stabilized by their incorporation into a myelin septin filament, which requires SEPT8 in its core oligomer.  When analyzing mature Sept8-mutants by electron microscopy, myelin outfoldings were obvious ( Figure 7A). However, myelin outfoldings were a significant feature only in adult Sept8-mutants, but not at the developmental age of P15 ( Figure 7B). We did not observe hypomyelination ( Figure 6C . Thus, the deficiency of myelin septin filaments very specifically caused myelin outfoldings but not complex neuropathology, in marked difference to the lack of CNP, MAG, PLP, or oligodendroglial PTEN. It is noteworthy that myelin outfoldings per se do not cause neuroinflammation. To elucidate the dynamics of the degradation of the septin/anillin scaffold in mature myelin, we have induced recombination of the Sept8 gene by injecting Tamoxifen into adult Sept8 flox/flox ;Plp Cre-ERT2 mice (Figure 7-figure supplement 2A), thereby preventing the assembly of new myelin septin core oligomers in myelinating cells. Sept8 flox/flox mice subjected to Tamoxifen injections served as controls. By immunoblot, the abundance of myelin septins (SEPT2, SEPT4, SEPT7, SEPT8) was about halved in myelin purified from the brains of Sept8 flox/flox ;Plp Cre-ERT2 mice 4 weeks post Tamoxifen injection (pti) and greatly reduced 8 weeks pti (Figure 7-figure supplement 2B), reflecting the degradation of myelin septin filaments. Using the same model we also tested whether depleting the septin/anillin-scaffold causes myelin outfoldings when induced after developmental myelination has been completed. Indeed, myelin outfoldings were evident 18 weeks pti according to electron microscopic analysis (Figure 7-figure supplement 2C,D). Thus, continuous replenishment of the myelin septin scaffold is required to prevent the formation of myelin outfoldings.

Analysis of nerve conduction velocity
To test whether the septin/anillin-scaffold of myelin is relevant for CNS function in vivo, we measured nerve conduction in the spinal cord of Sept8 null/null and Sept8 flox/flox ;Cnp Cre/WT mice at various ages. Indeed, conduction velocity was reduced by about 20% in Sept8-mutants ( Figure 7E). Considering

Discussion
Myelin outfoldings are frequently observed during developmental myelination, and these membranes have been interpreted as a reservoir for the elongation of axon/myelin-units during body growth (Cullen and Webster, 1979;Rosenbluth, 1966;Snaidero et al., 2014). Our data reveal that the assembly of septin filaments is a late stage of myelin maturation. By forming a scaffold underlying the innermost membrane layer of compact myelin, septins prevent outfoldings of the entire stack of compact myelin layers in the adult CNS. Septin filaments assemble by associating with membranes (Bridges et al., 2014) involving the interactions of particular septin monomers and the adaptor protein anillin with PI(4,5)P 2 (Bertin et al., 2010;Liu et al., 2012;Zhang et al., 1999), a recruitment mechanism conserved between the yeast cleavage furrow and mammalian myelin. As submembraneous filaments, septins can provide structure and rigidity to the membranes they are associated with (Gilden and Krummel, 2010;Spiliotis and Gladfelter, 2012;Tooley et al., 2009). The adaxonal myelin compartment is regularly spaced, and the cytoplasmic surfaces of the flanking membranes are in close proximity but not compacted. Adaxonal myelin differs from compact myelin by lacking MBP and instead containing oligodendroglial cytoplasm. In our model ( Figure 7F), the septin/anillin scaffold stabilizes compact myelin by associating with its innermost membrane surface. This architecture counteracts hydrostatic pressure and lateral membrane flow (Gilden and Krummel, 2010), which in the absence of adhesive forces would push compact myelin outward, e.g. as a consequence of the net growth of individual myelin membranes or the slow but lifelong intercalation of new myelin segments between existing sheaths (Young et al., 2013). High PI(3,4,5)P 3 -levels trigger the active net growth of myelin sheaths and thus myelin thickness via the Akt/mTOR pathway (Chan et al., 2014;Goebbels et al., 2010;Macklin, 2010) whereas high PI(4,5)P 2 -levels are required to recruit the septin/anillin-scaffold of myelin. Loss of septins in the myelin sheath is likely owing to dysregulated filament assembly or stability, which in addition to membrane-phosphoinositides and ANLN may also involve other regulatory factors (Joberty et al., 2001). Several mechanisms are thus feasible how mutations affecting structural myelin proteins may cause the post-transcriptional decrease of septins. For example, PLP is required for a normal myelin lipid composition  and CNP prevents premature closure of the cytoplasmic channels through myelin (Snaidero et al., 2014), which probably provide transport routes for molecules and vesicles into the adaxonal myelin layer (Nave and Werner, 2014). It is plausible that both phenomena may directly or indirectly affect the septin/anillin-scaffold that prevents the formation of myelin outfoldings.
The axon/myelin-unit comprises various stabilizing molecules. Recently, a cytoskeletal actin/spectrin-lattice has been discovered that underlies the axonal membrane along its entire length (Xu et al., 2013). Moreover, neuronal and glial Ig-CAMs interact along the axon-myelin interface (Kinter et al., 2012). We note that at the sites of pathological myelin outfoldings, the axo-myelinic membrane apposition is not disrupted. However, the lack of myelin septins causes the entire stack of compacted myelin to focally detach from the adaxonal oligodendroglial membrane and to 'fold out'. This reflects strong adhesive forces between adjacent layers of compact myelin mediated by myelin proteins such as MBP. The non-compact compartments of myelin are considered as cytoplasmic routes enabling the axon-supportive function of oligodendrocytes (Nave and Werner, 2014). We conclude that the septin/anillin-scaffold reported here stabilizes mature CNS myelin while allowing diffusion and transport processes in the non-compact adaxonal myelin layer to take place.

Mouse models
Plp1 null , Cnp null , Mag null , and Pten flox/flox ;Cnp Cre/WT mice were described previously (Goebbels et al., 2010;Klugmann et al., 1997;Lappe-Siefke et al., 2003;Montag et al., 1994). To homogenize the genetic background, the lines were backcrossed to the C57BL/6N strain for >10 generations before breeding the experimental animals for the present study. Genotyping of the Plp1 allele was by genomic PCR with primers 1864 ( Embryonic stem cells (ES) harbouring an engineered allele of the Sept8 gene were acquired from the European Conditional Mouse Mutagenesis Program (Eucomm). ES were microinjected into blastocysts derived from FVB mice, and embryos were transferred to pseudo-pregnant foster mothers, yielding 6 chimeric males. For ES clone EPD0060_3_G04, germline transmission was achieved upon breeding with C57BL/6N-females, yielding mice harbouring the Sept8 lacZ-neo allele. The lacZ/neo cassette was excised in vivo upon interbreeding with mice expressing FLIP recombinase (129S4/SvJae-Sor-Gt(ROSA)26Sortm1(FLP1)Dym/J; backcrossed into C57BL/6N), yielding mice carrying a Sept8 flox allele. To inactivate expression of SEPT8 whole-body-wide, exon 2 of the Sept8 gene was excised in vivo upon interbreeding with mice expressing Cre recombinase under control of the adenoviral EIIA promoter (Holzenberger et al., 2000) (backcrossed into C57BL/6N), yielding mice carrying a Sept8null allele. When heterozygous Sept8 null mice were interbred to obtain homozygous mutants, those were born at Mendelian frequency. For simplicity, Sept8 null/null mice are also termed knockout ('KO'). To inactivate expression of SEPT8 in myelinating cells, exon 2 was excised in vivo upon appropriate interbreedings of Sept8 flox mice with mice expressing Cre recombinase under control of the Cnp promoter (Lappe-Siefke et al., 2003). For simplicity, Sept8 flox/flox ;Cnp Cre/WT mice are also termed conditional mutant ('COND'). Corresponding control mice (genotypes Sept8 WT/WT ;Cnp WT/WT , Sept8flox/WT ;Cnp WT/WT , and Sept8 flox/flox ;Cnp WT/WT ) are labelled 'WT 'throughout. For Tamoxifen-induced inactivation of SEPT8 expression in myelinating cells, Sept8 flox mice were interbred with mice expressing Cre-ERT2 in myelinating cells under control of the Plp-promoter (Leone et al., 2003). Sept8 flox/flox ;Plp Cre-ERT2 (inducible conditional mutant; Sept8 icKO) and control Sept8 flox/flox mice were injected with 1 mg Tamoxifen per day beginning at 8 weeks of age for five consecutive days, then after a pause of two days again for five consecutive days.
Routine genotyping of the Sept8 allele was by PCR with sense primer P1 (5'-GAAGCAGCCA TA-GAGGAGATCC; binding 5 'of the first loxP-site) in combination with antisense primers P2 (5'-GGTG-GCTTTG AACTTGCTATCC; binding the segment flanked by loxP-sites), and P3 (5'-CAGGCAGATG TATATGCAGCAG; binding to the lacZ cassette) or P4 (5'-CAGGCAGATG TATATGCAGCAG; binding 3 'of the third loxP site). The PCR shown in Figure 6-figure supplement 1B was performed with P1, P2, and P3. All experimental animals were progeny of heterozygous parents to facilitate that mutant animals were analyzed together with littermate controls as far as possible. Mice were kept in the mouse facility of the Max-Planck-Institute of Experimental Medicine with a 12 hr light/ dark cycle and 2-6 animals per cage. All experiments were approved by the local Animal Care and Use Committee in agreement with the German Animal Protection Law.

Electron microscopy (EM)
Immungold labelling of cryosections was performed as described . Optic nerves dissected from male WT (C57Bl/6N) mice at P75 were used. Antibodies were specific for SEPT8 (ProteinTech Group 11769-1-AP, 1:50) (yielding similar results as a previously published antibody (Ihara et al., 2003) kindly provided by M. Kinoshita, 1:50), SEPT7 (IBL18991, 1:50), or MAG (Chemicon MAB1567, 1:50). To quantitatively assess the exact protein localization within the adaxonal non-compact myelin layer, all gold particles located to this compartment on the micrographs (a minimum of 200 gold particles per animal) were assigned to one of two categories as schematically depicted in Figure 2H': associated with the innermost membrane layer of compact myelin (type 1) or associated with the adaxonal myelin membrane (type 2).
Preparation of samples for transmission electron microscopy by high pressure freezing and freeze substitution was performed essentially as described . Briefly, optic nerves were dissected and placed into aluminum specimen carriers with an indentation of 0.2 mm. The remaining space was covered with 20% PVP (Sigma) in PBS, and the sample was cryofixed using a HPM100 high-pressure freezer (Leica). Freeze substitution was carried out in a Leica AFS (Leica, Vienna, Austria) as follows: samples were initially kept in tannic acid (0.1% in acetone) at À90˚C for 100 hr, washed with acetone (4 Â 30 min, À90˚C) and then transferred into OsO 4 (EMS; 2%) and uranyl acetate (SPI Chem, 0.1%) in acetone at À90˚C. The temperature was raised from À90 to À20˚C in increments of 5˚C/hr, then kept unaltered at À20˚C for 16 hr, and then raised to +4˚C in increments of 10˚C/hr. The samples were then washed with acetone (3 Â 30 min at 4˚C), allowed to adjust to room temperature, and finally transferred into Epon (Serva) (25%, 50%, and 75% Epon in acetone for 1-2 hr each, 90% Epon in acetone for 18 hr, 100% Epon for 4 hr). The samples were placed in an embedding mold and polymerized (60˚C, 24 hr). Preparation of samples for transmission electron microscopy by conventional aldehyde fixation was performed as described . Briefly, mice were perfused with 2,5% glutaraldehyde and 4% formaldehyde in phosphate buffer containing 0.5% NaCl. Spinal cord samples, optic nerves, and brain samples were postfixed in 1% OsO 4 in 0.1 M phosphate buffer and embedded in epoxy resin (Serva). Ultrathin sections (50 nm) were cut using a Leica Ultracut S ultramicrotome (Leica) and contrasted with an aqueous solution of 4% uranyl acetate (SPI Chem) followed by lead citrate. The samples were examined in a LEO 912AB Omega transmission electron microscope (Zeiss). Pictures were taken with an on-axis 2048 Â 2048-CCD-camera (TRS).
For assessment of pathology, mice were analyzed at postnatal day 75 (P75) unless indicated otherwise; 3-6 male mice were used per genotype and age. 10-15 randomly selected, non-overlapping images were taken per optic nerve at 7000 Â magnification (1 field=220 mm). Electron micrographs were analyzed using ImageJ (Fiji). To quantify axonal pathology and the proportion of nonmyelinated axons, all axons on the micrographs (a minimum of 700 axons per animal) were assigned to one of five categories: healthy-appearing myelinated axons, healthy-appearing nonmyelinated axons, axons with myelin comprising a swollen adaxonal compartment (inner tongue), axonal spheroids, and degenerated axons. Axons were counted as myelinated if ensheathed by at least one complete layer of compacted myelin. Axonal changes were identified by mild signs of pathology including invaginations of adaxonal myelin membrane into the axon. Degenerated axons were identified by tubovesicular structures and amorphous cytoplasm. The area occupied by myelin outfoldings was quantified by a point counting method (Edgar et al., 2009). Briefly, a regular grid of 0.25 mm 2 was placed on the images. The number of intercepts coinciding with myelin outfoldings was related to the evaluated area. When quantifying myelinated axons, axonal pathology, axonal diameters, and myelin outfoldings, to compare Cnp null , Mag null , and Plp1 null mice with WT mice significance was determined using GraphPad Prism 6.0. To compare SEPT8 mutant mice with WT littermates, significance was determined using GraphPad Prism 6.0. The g-ratio was calculated as the ratio between the axonal Feret diameter and the Feret diameter of the corresponding myelin sheath. To determine g-ratios, a regular grid was placed on the images for randomization. All axons crossed by the grating were assessed, yielding a minimum of 100 myelinated axons per animal. The g-ratio was assessed using GraphPad Prism 6.0. All quantifications were performed blinded to the genotype.

Immunohistochemistry
Immunohistochemistry to determine neuropathology on sections of paraffin-embedded brains and spinal cords was essentially as described . Antibodies were specific for MAC3 (1:400; Pharmingen) or glial fibrillary acidic protein (GFAP; 1:200; NovoCastra). Images were captured at 20x magnification using a bright-field light microscope (Zeiss AxioImager Z1) coupled to a Zeiss AxioCam MRc camera controlled by Zeiss ZEN 1.0 software and processed using ImageJ 1.46 and Adobe Photoshop. Mice were 12 months old; sections from 4-6 male mice per genotype including the mean of both fimbriae were quantified. To quantify white matter area immunopositive for MAC3 or GFAP, the hippocampal fimbria was selected and analyzed using an ImageJ plugin for semiautomated analysis. Data were normalized to wild-type levels. All quantifications were performed blinded to the genotype. Statistical analysis was performed using GraphPad Prism 6.0.
Silver impregnation of myelin on histological sections was as described (Gallyas, 1979). Images were captured at 10x magnification (Zeiss AxioImager Z1) and stitched using Zeiss Zen2011. Figure 6A shows sections from mice of the indicated genotypes at one year of age.
To visualize cells with Sept8 gene activity, we performed lacZ immunohistochemistry on the brains of heterozygous Sept8 lacZ mice. Mice were perfused with 4% PFA. Vibratome sections of 100 mm thickness (Leica VT 1000S) were incubated at 37˚C with X-gal solution (1.2 mg X-gal per ml, 5 mM potassium ferricyanide, 5 mM potassium ferrocyanide, and 2 mM MgCl 2 in PBS). After the sections had incubated for about 2 hr in the dark, they were rinsed with PBS, dried, and mounted using Aqua-Poly/Mount (Polysciences). Images of the whole brain were captured at 10x magnification (Zeiss AxioImager Z1) and stitched (Zeiss Zen2011); the cerebellum was imaged at 40x magnification.
The number of SEPT8-puncta (in Figure 2D-F) was determined using Fiji. Axonal diameter was determined using a threshold for the neurofilament immunolabelling signal and the particle analyzer plugin. SEPT8-punctae per myelinated axon as identified by the MAG-immunopositive ring were counted in WT at P75 (n=4 animals, 1 section each). Per animal, 6 random confocal micrographs of spinal cord white matter with a size of 2500 mm 2 per micrograph were quantified, yielding about 100 axons per animal. Statistical analysis was performed using GraphPad Prism 6.0. For the quantifications of nodal density, the frequency of occurrence of two CASPR-immunopositive paranodes was analyzed using Fiji. CASPR-immunopositivity was converted using a threshold and counted using ITNC plugin (n=3 animals per genotype, 1 section each, 8 random confocal micrographs of spinal cord white matter with a size of 2500 mm 2 per micrograph). Statistical analysis was performed using GraphPad Prism 6.0.

Myelin purification
A light-weight membrane fraction enriched for myelin was purified from mouse brains by sucrose density centrifugation and osmotic shocks as described (Jahn et al., 2013). For immunoblot analyses of myelin during development and aging, male wild-type (C57Bl/6N) mice of the indicated ages were used. For proteome or immunoblot analyses of mutant mice and their respective wild-type littermates, male mice at the age of 75 days were used. Protein concentrations were determined using the DC protein assay (BioRad).

Proteome analysis
Differential quantitative myelin proteome analyses were based on previously described label-free, gel-free procedures (Distler et al., 2014;Jahn et al., 2013;Patzig et al., 2011). Briefly, brains were dissected from three male Cnp null , Mag null , Plp1 null , or Sept8 null/null mice at the age of 75 days and three respective control littermates, and myelin was biochemically purified (see above). Per sample, 25 mg of total myelin protein was precipitated using the ProteoExtract Protein Precipitation Kit (Calbiochem), solubilized in 50 mM NH 4 HCO 3 with 0.1% Rapigest (Waters) (10 min, 85˚C), reduced with 5 mM DTT (45 min, 56˚C), and alkylated with 15 mM iodoacetamide (45 min, room temperature) in the dark. Solubilized proteins were digested with 0.5 mg of sequencing grade trypsin (Promega) for 16 hr (37˚C). After digestion, Rapigest was hydrolyzed by adding 10 mM HCl, the resulting precipitate was removed by centrifugation (13,000 g, 15 min, 4˚C), and the supernatant was transferred into an autosampler vial. Data for the analysis of myelin purified from Cnp null , Plp1 null , and Sept8 nul/ nulll mice and the respective littermate controls were obtained using separation of tryptic peptides by nanoscale ultraperformance liquid chromatography (nanoUPLC) coupled to a quadrupole time of flight (QTOF) Premier mass spectrometer (Waters) with an alternating low and elevated (E) energy mode of acquisition (MS E ) (UPLC-MS E ). Data for the analysis of myelin purified from Mag null mice and littermate controls were obtained using a Synapt G2S mass spectrometer (Waters). For each biological replicate, 2-4 technical replicates were measured. The continuum LC-MS E data were processed and searched using the IDENTITYE-algorithm of ProteinLynx Global Server (PLGS) version 2.3 (Waters). Protein identifications were assigned by searching the Uni-ProtKB/Swiss-Prot Protein Knowledgebase Release 52.3 for mouse proteins (12,920 entries) supplemented with known possible contaminants (porcine trypsin, human keratins) using the precursor and fragmentation data afforded by LC-MS acquisition as described previously (Patzig et al., 2011). Mass tolerances for peptide and fragment ions were set at 15 and 30 ppm, respectively. Peptide identifications were restricted to tryptic peptides with no more than one missed cleavage. Carboxyamidomethylation of Cys was set as fixed modification, and oxidation of Met, acetylation of protein N termini, and deamidation of Asn and Gln were searched as variable modifications. For a valid protein identification, the following criteria had to be met: at least two peptides detected together with at least seven fragments. The false-positive rate for protein identification was set to 1% based on search of a 5x randomized database, which was generated automatically using PLGS 2.3 by reversing the sequence of each entry. By using the replication rate as a filter, the false-positive rate was further minimized, as false-positive identifications do not tend to replicate across injections due to their random nature. By requiring a protein identification to be made in at least three technical replicates, the effective false-positive rate was lowered to <0.2%. For label-free absolute quantification of protein abundance by mass spectrometry based on the TOP3-method (Silva et al., 2006), data were post-processed using ISO-Quant software (Distler et al., 2014). The relative abundance of a protein in myelin was accepted as altered if both, significant according to unpaired two-tailed t-test and exceeding a threshold of 25%. the stimulation electrode (cathode) and the recording electrode was measured in situ by using a thin cotton thread. fü r Verbraucherschutz und Lebensmittelsicherheit (LAVES); permit numbers 33.9-42502-04-11/0418 and -14/1677.