Neuregulin-1 type III determines the ensheathment fate of axons

Abstract

The signals that determine whether axons are ensheathed or myelinated by Schwann cells have long been elusive. We now report that threshold levels of neuregulin-1 (NRG1) type III on axons determine their ensheathment fate. Ensheathed axons express low levels whereas myelinated fibers express high levels of NRG1 type III. Sensory neurons from NRG1 type III deficient mice are poorly ensheathed and fail to myelinate; lentiviral-mediated expression of NRG1 type III rescues these defects. Expression also converts the normally unmyelinated axons of sympathetic neurons to myelination. Nerve fibers of mice haploinsufficient for NRG1 type III are disproportionately unmyelinated, aberrantly ensheathed, and hypomyelinated, with reduced conduction velocities. Type III is the sole NRG1 isoform retained at the axon surface and activates PI 3-kinase, which is required for Schwann cell myelination. These results indicate that levels of NRG1 type III, independent of axon diameter, provide a key instructive signal that determines the ensheathment fate of axons.

Figure 1. NRG1 Type III Is Essential for Schwann Cell Ensheathment and Myelination

(A) Rat Schwann cells were maintained in vitro for 3 weeks with sensory neurons obtained from wt (Aa–Ac) or NRG1 type III^−/− (Ad–Af) mice, fixed, and stained for the myelin protein P0 (Aa and Ad), neurofilament (Ab and Ae), and cell nuclei (Ac and Af). Numerous myelin segments are evident in wt cultures; none form in NRG1 type III^−/− cultures. Scale bar, 100 μm. (B) Cocultures of wt, NRG1 type III^+/−, and NRG1 type III^−/− neurons and rat Schwann cells were maintained in myelinating conditions for 8, 14, or 21 days; extracts prepared from these cocultures were probed for P0, MBP, and Oct-6. All proteins are detected in extracts of wt cocultures at both day 14 and day 21, are present at reduced levels in extracts of NRG1 type III^+/− cocultures, and are absent in extracts from NRG1 type III^−/− cocultures. Oct-6 is detectable at day 14 in wt and NRG1 type III^+/− with longer exposures (data not shown). (C) Schwann cells were maintained with wt and NRG1 type III^−/− neurons for 15 days in myelinating conditions and stained for Oct-6 (fluorescein), MBP (rhodamine), and with the Hoechst nuclear dye (blue). Oct-6 is expressed in Schwann cell nuclei in the wt cocultures just prior to the onset of myelination; no expression was observed in Schwann cells cocultured with NRG1 type III^−/− neurons. Scale bar, 50 μm.

Figure 2. Ultrastructure of Schwann Cell Association with Wt and NRG1 Type III^−/− Neurites

Electron micrographs of cocultures of Schwann cells and wt (A, C, and E) and NRG1 type III^−/− (B, D, and F) neurons maintained for 60 days. Myelinated axons (MA) are common in wt cocultures. Interruptions in the surrounding myelin sheaths represent typical non-compacted specializations of the myelin sheath; a thinly myelinated axon is also indicated (star) in panel (E). Smaller axons are not myelinated, but are consistently ensheathed (arrowheads, [C and E]). Despite apposition of Schwann cell processes, most axons in NRG1 type III^−/− cocultures lack any ensheathment or are incompletely ensheathed (asterisks). No myelin is detectable in any of the NRG1 type III^−/− cocultures, and many large diameter axons (asterisks, [F]) remain unsorted; one fully ensheathed fiber is apparent ([F], arrowhead). Scale bars, 1 μm (E and F), 2 μm (A, B, D, and E). Schwann cell nuclei are visible in many of the micrographs.

Figure 3. Forced Expression of NRG1 Type III Rescues the Myelination Defect of NRG1 Type III^−/− Neurons and Results in Hypermyelination

(A) Detergent lysates of wt, NRG1 type III^+/−, and NRG1 type III^−/− DRG neurons were fractionated by SDS PAGE and blotted with an antibody to the “a-tail” C-terminal epitope. The 135 kDa band corresponding to the full-length NRG1 type III pro-protein (indicated with an arrow) is present in wt, reduced in NRG1 type III^+/−, and completely missing in NRG1 type III^−/− lysates; a cleaved NRG1 band of ∼65 kDa is present in all extracts. (B) Detergent lysates of cocultures of wt or NRG1 type III^−/− neurons infected (Vs#1) or not with a lentiviral construct driving expression of an HA-tagged NRG1 type III construct were probed with antibodies to NRG1 and P0. (C) NRG1 type III expression by infected NRG1 type III^−/− neurons was detected by live staining for the HA epitope (rhodamine); neurons were double stained for the intermediate filament protein peripherin (fluorescein). Scale bar, 40 μm. (D) Rescue of myelination of NRG1 type III^−/− neurons by expression of NRG1 type III is shown in cocultures double stained for MBP (rhodamine) and neurofilament (fluorescein). Scale bar, 40 μm. (E) A fiber with hypermyelinated segments (indicated with white arrows) is illustrated. Scale bar, 40 μm. (F) Quantitation of hypermyelination showing the mean ± SEM. The thickest myelin segments in the infected NRG1 type III^−/− cocultures were approximately twice the thickness of those in uninfected wt cocultures (p < 0.0001).

Figure 4. NRG1 Type III Expression Induces Myelination

(A) Detergent lysates of SCG, NGF-dependent, and BDNF/NT3-dependent DRG neurons were fractionated by SDS PAGE and blotted with an antibody to the “a-tail” C-terminal epitope. The 135 kDa and ∼65 kDa are present in all extracts although levels vary considerably between these populations of neurons (B) SCG neurons were infected with two separate lentiviral stocks to drive expression of NRG1type III. Detergent extracts were prepared and blotted for NRG1 as shown; the blot is slightly overexposed by comparison to panel (A) to better show the increase in NRG1 levels. (C) Cocultures of Schwann cells and control (left) or SCG neurons infected with NRG1 type III lentivirus were stained for MBP (rhodamine) and neurofilament (fluorescein). Scale bar, 100 μm. (D) Quantitation of the number of myelin segments in control and infected SCG cocultures from two separate experiments (four coverslips/condition/experiment) showing the mean ± SEM. The increase in the number of myelin segments is statistically significant (**p = 0.006, ***p = 0.0005).

Figure 5. Type III Is the Sole Isoform on the Axon Surface and Regulates PI 3-Kinase Activity

(A) Cultures of wt, NRG1 type III^+/−, and NRG1 type III^−/− neurons were incubated with erbB2/3-Fc and then fixed and visualized with rhodamine-conjugated anti-human Fc antibodies (Aa–Ac); neurofilament staining from the corresponding fields is shown (Ad–Af). ErbB2/3-Fc binds strongly to wt (Aa), modestly to NRG1 type III^+/− (Ab), and not at all to NRG1 type III^−/− neurons (Ac). Scale bar, 40 μm. (B) Membranes prepared from wt, NRG1 type III^+/−, and NRG1 type III^−/− neurons were centrifuged onto Schwann cell cultures, and after 20 min, lysates were prepared, blotted, and probed for phospho-Akt, total Akt, and phospho-erk as shown. (C) NRG levels were assayed in culture media from wt, NRG1 type III^+/−, and NRG1 type III^−/− neurons by measuring erbB receptor phosphorylation in L6 myotubes. A representative Western blot revealing erbB receptor phosphorylation is shown. Following the phosphotyrosine (pY) Western blot, the membrane was stripped and reprobed for total erbB. The ratio of pY to total erbB is a quantitative measure of the amount of NRG in the media. Results from two separate experiments are shown below (mean ± SD). (D) Rat DRG neurons were nucleofected with HA-tagged NRG1 type III (Da–Dc) or type I (Dd–Di) constructs were stained for HA (Da, Dd, and Dg) and peripherin (Db, De, and Dh); merged images (Dc, Df, and Di) are also shown. NRG1 type III is expressed at the axon surface (Da), whereas type I NRG1 is detectable at the surface of neurons treated for 48 hr with the MMP inhibitor GM 6001 (Dg) but not in untreated cultures (Dd). Scale bar, 40 μm.

Figure 6. NRG1 Type III Induces Myelination as a Juxtacrine Signal from Axons

(A) Schwann cells were grown with wt and NRG1 type III^−/− neurons in myelinating conditions for 14 days without (control) or with the addition of 1 nM or 10 nM of recombinant, soluble extracellular domain of type III (sECD). Cultures were fixed and stained for MBP (rhodamine) and cell nuclei (Hoechst, blue). Numerous myelin segments are present in wt cocultures but none in NRG1 type III^−/− cocultures, even when supplemented with sECD. (B) Schwann cells were plated onto control CHO cells (Ba and Bb) or CHO cells that stably express NRG1 type III (Bc and Bd). After 14 days, Schwann cells were stained for S100 (fluorescein, [Ba and Bc]) and MBP (rhodamine, [Bb and Bd]). There was a marked increase in the number of Schwann cells grown on the CHO-III cell monolayer but no MBP expression was detected. Scale bars, 100 μm.

Figure 7. NRG1 Type III^+/− Mice Are Hypomyelinated and Aberrantly Ensheathed

(A) Extracts were prepared from sciatic nerve at the postnatal time points shown and fractionated by SDS-PAGE, blotted, and probed with antibodies NRG1 and the transcription factors Oct-6 and Krox-20. (B) Semiquantitative analysis of PNS myelin protein levels was performed by probing Western blots with ¹²⁵I protein A and quantitating by PhosphorImager analysis. Myelin protein levels are normalized to actin as indicated; the means (±SEM) from two different experiments are shown. (C) Electron micrographs of Remak bundles in sciatic nerves from wt (left panels) and NRG1 type III^+/− (right panels) adult mice show altered axonal segregation; insets are shown at higher magnification in the lower panels. In the wt mice, axons (A) are fully ensheathed by Schwann cell processes (Sc), whereas NRG1 type III^+/− axons frequently directly appose each other without intervening Schwann cells processes. All of the profiles seen in the inset from the NRG1 type III^+/− mice are axons, a number of which are flattened or elongated in cross-section. Scale bar, 1 μm. (D) The number of axons per Ramak bundle for wt and NRG1 type III^+/− sciatic nerves were binned into separate groups and are shown as a percentage of the total axons. Over 120 bundles for each genotype were counted (n = 2 mice of each genotype). The maximum number of axons/bundle in the wt mice was 34; in NRG1 type III^+/− mice, bundles containing up to 60 unmyelinated fibers were observed (p < 0.0001). (E) The percentages of axons in Schwann cell pockets in Remak bundles were binned into four categories: a single axon/pocket; 2–10 axons/pocket; 11–20 axons/pocket, and 21–35 axons/pocket. Over 800 unmyelinated axons for each genotype (n = 2) were counted. While almost all axons are present in individual Schwann cell pockets in wt nerves, the number in NRG1 type III^+/− nerves is significantly reduced (**p = 0.006). The maximum number of axons per pocket in the wt nerves was 7 versus 35 in the NRG1 type III^+/− nerves (***p < 0.0001).

Figure 8. Schematic Summary of Abnormalities in Nerves with Altered NRG1 Type III Levels

Alterations in axon ensheathment and myelination as a function of NRG1 type III levels are illustrated based on in vivo and in vitro analysis. In wt mice, unmyelinated axons in the PNS are segregated into separate pockets of Remak bundles and myelinated axons are fully wrapped. In NRG1 type III^+/− mice, a greater proportion of axons remain unmyelinated and are frequently fasciculated together in Remak bundles, sometimes with distorted cross-sectional profiles; myelinated axons have thinner sheaths. In the absence of NRG1type III, Schwann cells fail to ensheath axons and even large axons remain unsegregated and unmyelinated.