Loss of prohibitin 2 in Schwann cells dysregulates key transcription factors controlling developmental myelination

Abstract

Schwann cells are critical for the proper development and function of the peripheral nervous system (PNS), where they form a collaborative relationship with axons. Past studies highlighted that a pair of proteins called the prohibitins play major roles in Schwann cell biology. Prohibitins are ubiquitously expressed and versatile proteins. We have previously shown that while prohibitins play a crucial role in Schwann cell mitochondria for long-term myelin maintenance and axon health, they may also be present at the Schwann cell-axon interface during development. Here, we expand on this, showing that drug-mediated modulation of prohibitins in vitro disrupts myelination and confirming that Schwann cell-specific ablation of prohibitin 2 (Phb2) in vivo results in severe defects in radial sorting and myelination. We show in vivo that Phb2-null Schwann cells cannot effectively proliferate and the transcription factors EGR2 (KROX20), POU3F1 (OCT6), and POU3F2 (BRN2), necessary for proper Schwann cell maturation, are dysregulated. Schwann cell-specific deletion of Jun, a transcription factor associated with negative regulation of myelination, confers partial rescue of the developmental defect seen in mice lacking Schwann cell Phb2. Finally, we identify a pool of candidate PHB2 interactors that change their interaction with PHB2 depending on neuronal signals, and thus are potential mediators of PHB2-associated developmental defects. This work develops our understanding of Schwann cell biology, revealing that Phb2 may modulate the timely expression of transcription factors necessary for proper PNS development, and proposing candidates that may play a role in PHB2-mediated integration of axon signals in the Schwann cell.

Keywords: BAP32; BAP37; BRN2; KROX20; OCT6; REA; c‐JUN.

FIGURE 1. PHB2-SCKO nerves display developmental defects as early as post-natal day (P)5.

Representative cross sections from the sciatic nerves of mice at P5 (a), P10 (b) and P20 (c). In controls, dark staining of the myelin sheath rings around axons is evident at all ages. In PHB2-SCKO animals there are far fewer myelinated axons and at P20 many large sized axons remain in unsorted bundles. Quantification of the number of myelinated axons per nerve cross section is shown in (d) and the number of improperly sorted bundles per nerve cross section in P20 mice in (e). N = 3–4 animals per genotype and each data point represents an individual animal. Data are presented as mean ± SEM and statistical significance, as calculated by unpaired t-test and in (d) corrected for multiple comparisons using the Holm-Šídák method, indicated as follows: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001. Scale bar = 10 μm.

FIGURE 2. AKT and mTORC1 signaling is elevated at P5 in PHB2-SCKO nerves.

(a) Quantification (upper panel) of western blots (lower panels) probing for total ERK, phosphorylated ERK (p-ERK), and β-tubulin (loading control) in sciatic nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (b) Quantification (upper panel) of western blots (lower panels) probing for total AKT, phosphorylated AKT (p-AKT), and β-tubulin (loading control) in sciatic nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (c) Quantification (upper panel) of western blots (lower panels) probing for total S6RP, phosphorylated S6RP (p-S6RP), and β-tubulin (loading control) in sciatic nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (d) Quantification (upper panel) of western blots (lower panels) probing for total 4E-BP1, phosphorylated 4E-BP1 (p-4E-BP1), and β-tubulin (loading control) in sciatic nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. Each data point represents an individual animal, except for in the P1 and P5 peripheral nerve lysates, when multiple nerves from animals with the same genotype were pooled, and each data point represents an independent pool of nerves. Expression of the protein of interest was first calculated relative to the loading control β-tubulin and then phosphorylated relative to total levels, as total and phosphorylated protein levels were probed for on different membranes. Expression was then normalized to the average relative expression of the controls, for each age. In some cases the same membrane was probed sequentially for multiple signaling proteins. Uncropped blots are shown in Figure S7. Data are presented as mean ± SEM and statistical significance, calculated by unpaired t-test and corrected for multiple comparisons using the Holm-Šídák method, indicated as follows: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 3. Key Schwann cell transcription factors are dysregulated in PHB2-SCKO nerves.

(a) Representative western blot showing EGR2 and β-tubulin (loading control) levels in peripheral nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (b) Quantification of EGR2 levels from western blots. (c) Representative images showing immunohistochemistry of EGR2 (green) in P5 control and PHB2-SCKO longitudinal sciatic nerve sections. DAPI is shown in blue. (d) Quantification of the number of EGR2 positive nuclei (as a percentage of total nuclei) shown in (C). (e) Representative western blot showing POU3F1 (OCT6) and β-tubulin (loading control) levels in peripheral nerve lysate from P1, P5, P10 and P20 control and PHB2-SCKO animals. (f) Quantification of the POU3F1 levels from western blots as per (E). (g) Representative images showing immunohistochemistry of POU3F1 (green) in P5 control and PHB2-SCKO sciatic nerve longitudinal sections. DAPI is shown in blue. (h) Quantification of the number of POU3F1 positive nuclei (as a percentage of total nuclei) shown in (g). (i) Representative western blot showing POU3F2 (BRN2) and β-tubulin (loading control) levels in peripheral nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (j) Quantification of the POU3F2 levels from western blots as per (l). Each data point represents an individual animal, except for in the P1 and P5 peripheral nerve lysates, when multiple nerves from animals with the same genotype were pooled, and each data point represents an independent pool of nerves. Expression of the protein of interest was calculated relative to the loading control β-tubulin then normalized to the average relative expression of the controls, for each age. Data are presented as mean ± SEM. In (b), (f), and (j) statistical significance was calculated by unpaired t-test and corrected for multiple comparisons using the Holm-Šídák method. In (d) and (h) statistical significance was calculated by unpaired t-test. Statistical significance is represented on the graphs as follows: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 4. Schwann cells in PHB2-SCKO nerves do not proliferate effectively.

(a) Representative western blot showing SOX10 and β-tubulin (loading control) levels in peripheral nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (b) Quantification of SOX10 levels from western blots. Expression of SOX10 was calculated relative to the loading control β-tubulin then normalized to the average relative expression of the controls, for each age. (c) Representative images showing immunohistochemistry of SOX10 (green) and proliferation marker MKI67 (red) in P5 control and PHB2-SCKO sciatic nerve longitudinal sections. DAPI is shown in blue. Arrows indicate proliferating Schwann cells (SOX10 and MKI67 positive). Arrow heads indicate Schwann cells not proliferating (SOX10 positive, MKI67 negative). Scale bar = 100 μm. (d) Quantification of the number of SOX10 positive nuclei (as a percentage of total nuclei) shown in (c). (e) Quantification of the number of proliferating Schwann cells, calculated as the number of MKI67 positive nuclei as a percentage of SOX10 positive nuclei, as shown in (c). (f) Representative images showing TUNEL staining (red) in P5 control and PHB2-SCKO sciatic nerve longitudinal sections. DAPI is shown in blue. Arrows indicate TUNEL positive cells dying by apoptosis. Scale bar = 50 μm. (g) Quantification of the number of TUNEL positive nuclei (as a percentage of total DAPI positive nuclei). Each data point represents an individual animal, except for in the P1 and P5 peripheral nerve lysates, when multiple nerves from animals with the same genotype were pooled, and each data point represents an independent pool of nerves. Data are presented as mean ± SEM. In (b) statistical significance was calculated by unpaired t-test and corrected for multiple comparisons using the Holm-Šídák method. In (d), (e) and (g) statistical significance was calculated by unpaired t-test. Statistical significance is represented on the graphs as follows: *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 5. JUN-SCKO confers a partial rescue of the developmental defect in PHB2-SCKO animals.

(a) Representative western blot showing JUN and β-tubulin (loading control) levels in peripheral nerve lysate from P1, P5, P10, and P20 control and PHB2-SCKO animals. (b) Quantification of the JUN levels from western blots as per. (a) Expression of JUN was calculated relative to the loading control β-tubulin then normalized to the average relative expression of the controls, for each age. (c) Representative images showing immunohistochemistry of JUN (green) in P5 control and PHB2-SCKO sciatic nerve longitudinal sections. DAPI is shown in blue. (d) Quantification of the number of JUN positive nuclei (as a percentage of total nuclei) shown in (c). (e) Representative western blot showing JUN and β-tubulin (loading control) levels in peripheral nerve lysate from P20 control, PHB2-SCKO, PHB2-SCKO; JUN-SCHET, PHB2-SCKO; JUN-SCKO and JUN-SCKO animals. (f) Quantification of JUN levels from western blots. Expression of JUN was calculated relative to the loading control β-tubulin then normalized to the average relative expression of a control reference that was run on every blot. (g) Representative cross sections from P20 sciatic nerves. Scale bar = 10 μm. (h) Quantification of the number of sorted axons in a 1:1 relationship with Schwann cells from a whole nerve cross section. (i) Quantification of the number of myelinated fibers in a whole nerve cross section. (j) Quantification of the number of amyelinated fibers in a whole nerve cross section. (k) Percentage of sorted axons that are myelinated (block color) or amyelinated (diagonal stripes). (l) Quantification of the number of improperly sorted bundles in a whole nerve cross section. (m) Average g-ratios calculated from P20 sciatic nerve electron micrographs (representative images shown in Figure S4). (n) Distribution of g-ratio size relative to axon diameter. For each animal the g-ratios of 50 random axons were measured, except for in the case of two mutant animals whereby the g-ratio could only be measured from 34 axons each. Each data point represents an individual animal, except for in the P1 and P5 peripheral nerve lysates, when multiple nerves from animals with the same genotype were pooled, and each data point represents an independent pool of nerves and in (n), where each data point represents an individual axon. Data are presented as mean ± SEM. In (b) statistical significance was calculated by unpaired t-test and corrected for multiple comparisons using the Holm-Šídák method. In (d) statistical significance was calculated by unpaired t-test. In (f) and (h–m) statistical significance was calculated by one-way ANOVA: (f) F (4, 23) = 8.076, p < 0.001, (h) F (2, 15) = 96.16, p < 0.001, (i) F (2, 15) = 100.7. p < 0.001, (j) F (2, 15) = 31.40, p < 0.001, (k) myelinated axons F (2, 15) = 23.45, p < 0.001, amyelinated axons F (2,15) = 23.45, (l) F (2, 15) = 40.52, p < 0.001, (m) F (2, 16) = 11.65, p < 0.001 and the statistical significance indicated on the graphs was determined by Tukey’s multiple comparisons tests. *p < 0.05, **p ≤ 0.01, ***p ≤ 0.001.

FIGURE 6. The PHB2 interactome in the Schwann cell changes depending on neuronal signals.

(a) Schematic demonstrating the experiment protocol. Primary rat Schwann cells were transfected with PHB2-turboID and plated alone or with primary rat dorsal root ganglia (DRG) neurons, with or without biotin supplementation, for 2 h. Biotinylated proteins were harvested using streptavidin-affinity purification (AP) and analyzed using LC–MS. (b) Proteins identified by LC–MS which interacted with PHB2-turboID to a greater extent when Schwann cells were plated with neurons, compared to when Schwann cells were plated alone. The proteins listed were enriched in their PHB2-turboID interactions in three independent replicates, and the relative total ion chromatograph (TIC) count intensity compared to when Schwann cells were plated alone is indicated by color. Red panels indicate a protein that did not interact at all with PHB2-turboID in the Schwann cell alone condition, but was identified as a PHB2-turboID interactor when neurons were present. (c) Proteins identified by LC–MS which decreased interactions with PHB2-turboID when Schwann cells were plated with neurons, compared to when Schwann cells were plated alone. The proteins listed decreased their PHB2-turboID interaction in three independent replicates, with the relative TIC count intensity of each protein in the Schwann cells plated with neurons condition compared to Schwann cells alone, indicated by the color of the box. Candidates who interacted with PHB2-turboID only if neurons were absent are indicated by black panels. ^† AHNAK fragment.

FIGURE 7. A working model for PHB2’s role in Schwann cell and peripheral nerve development.

(a) We hypothesize that PHB2 plays a role at the Schwann cell plasma membrane in response to axonal signals by facilitating cell–cell contacts and/or promoting the cytoskeletal rearrangements required for Schwann cells to radially sort axons. As a result, yet unidentified signaling mechanisms promote Schwann cell proliferation and the transcription of pro-myelination transcription factors POU3F1, POU3F2, and EGR2, in order to allow proper peripheral nerve development. (b) Schwann cells lacking PHB2 may be unable to respond to axonal signals during the development of the peripheral nerve due to dysfunctional cell–cell contacts and/or a failure of cytoskeletal rearrangement. Through AKT/mTORC1 and/or other unidentified signaling pathways this may signal to the nucleus to prevent Schwann cell division, cause inhibition of pro-myelinating transcription factors including POU3F1, POU3F2, and EGR2 and cause an increase in the negative regulator of myelination, JUN. In turn, high levels of JUN inhibit EGR2, and low levels of EGR2 mean JUN is not inhibited. As a result, PHB2-null Schwann cells fail to radially sort and myelinate the axons in the developing peripheral nerve.