PMC

Loss of individual Akt isoforms in vivo does not affect PNS myelination. A, Western blot analysis of the expression of the three Akt isoforms in DRG neurons (Neu), Schwann cells (SC), and nonmyelinated (C) or myelinated (C+C) cocultures. Akt1 and Akt3 are preferentially expressed by neurons, whereas Akt2 is primarily expressed by Schwann cells. n = 3. Data are mean ± SEM. B, Semithin sections of sciatic nerves from 2-month-old mice deficient in each Akt isoform revealed no obvious defects of myelination. Left panels, Magnification of the inset from the semithin section. Right panels (a–f), Representative images of Remak bundles. No abnormalities of myelination or ensheathment were evident at the time point analyzed, although no quantifications were performed. Scale bars: semithin, 100 μm; EM, 2 μm.

Constitutively active Akt promotes CNS and PNS hypermyelination. A, Scheme illustrating the two constitutively active Akt1 constructs driven by the CNP promoter that were used to establish transgenic mice. B, The HA tagged construct was weakly detectable in E13.5 DRGs isolated from transgenic mice and readily detectable in sciatic nerves (SN) from P0.5 until adulthood, with a peak at P10 that correlates with active myelination. C, One-year-old transgenic mice carrying the MyrAkt transgene developed enlarged neural structures. Optic nerves (asterisk indicates the optic chiasm), brains, and sciatic nerves were all enlarged. D, In 2-month-old sciatic nerves, HA was present with variable intensity in Schwann cells. HA was detectable in satellite cells from 2-month-old mouse DRGs without any obvious presence in neurons. Scale bars, 20 μm. E, Four-month-old transgenic brains (MyrAkt) were bigger than WT, with a thicker corpus callosum (blue) and increased MBP staining, evidence of enhanced myelination. Scale bar, 1 mm.

MyrAkt induces Rac1 activity in cultured Schwann cells. A, Western blot showing the controls (GDP, GTPγS, and GST) as well as samples from noninduced (−DOX) and doxycycline-induced (+DOX) Schwann cell pools transfected with the MyrAkt construct. The same −DOX and +DOX samples were loaded into two consecutive lines. B, Quantification of Rac1 and cdc42 levels. Values shown represent active GTPase levels divided by total GTPase levels and corrected for actin, which served as a loading control. All data were normalized to noninduced controls, which were set to 100. Induction of MyrAkt by addition of doxycycline to the culture media increased Rac1 activity significantly (193 ± 25.31, p = 0.01) but not cdc42 (96.82 ± 27.11, p = 0.91). After correction for total cdc42 and loading amounts, the level of active cdc42 in A is not significantly increased. Data are mean ± SEM. Rac1, n = 4 independent sets of experiments; cdc42: n = 3 independent sets of experiments. *p < 0.05.

Nerves from transgenic mice are hypermyelinated. A, At 5 months, sciatic and optic nerves from MyrAkt transgenics were enlarged compared with WT mice, as seen in transverse, toluidine blue-stained sections. Scale bar, 100 μm. B, EM sections show thicker myelin sheaths in 5-month-old MyrAkt and AktDD sciatic nerves. Optic nerves from MyrAkt were also hypermyelinated. Scale bars: sciatic nerve, 10 μm; optic nerve, 2 μm. C, The myelin sheaths in transgenic nerves had increased numbers of myelin lamellae (scale bar, 1 μm) and revealed no change in the periodicity of the myelin lamellae (scale bar, 100 nm). D, g-ratio analysis revealed that hypermyelination was more pronounced in small-caliber axons (0.5–2 μm). E, Average g-ratio in the transgenic dorsal root was lower than in WT (0.62 ± 0.01 vs 0.67 ± 0.01, respectively, p = 0.02). A total of 250–300 axons between 0.5 and 8 μm were measured. This difference was accentuated when only small-caliber axons (0.5–2 μm) were considered (0.52 ± 0.004 vs 0.61 ± 0.01, p = 0.002); n = 3. Data are mean ± SEM. F, The numbers of Schwann cell nuclei were comparable in WT and MyrAkt dorsal roots (DR) (216 ± 9.7 vs 250 ± 29.2, respectively) and ventral roots (VR) (82 ± 5.5 vs 94 ± 8.1, respectively) from 5-month-old animals; n = 4. Data are mean ± SEM. *p < 0.05. **p < 0.01.

Sciatic nerves of MyrAkt transgenic mice exhibit tomacular defects and activation of mTOR. A, Top, Longitudinal electron micrographs of sciatic nerves from 5-month-old WT animals. Asterisk indicates nodes. Arrows indicate clefts. Bottom, Longitudinal electron micrographs of sciatic nerves from transgenic animals reveal myelin infolding at the paranodes (left panel) and tomacula near the clefts (indicated with arrows), resulting in compression of the axon (right panel). Scale bars, 5 μm. B, Example of the different myelin abnormalities observed in sciatic nerves: I, tomacula, showing compression of the axon; II, infolding; and III, outfolding. Scale bars, 5 μm. C, Percentages for each of the major myelin abnormalities found in cross-sections from 3-month-old transgenic sciatic nerves; n = 3. D, Teased fiber from 6-month-old transgenic mouse shows an example of an asymmetric tomacular defect with enlargement in the paranodal region of the left myelin sheath but not the right myelin sheath. Asterisk indicates node. E, Western blot analysis of extracts from nerves from 2-month-old WT and transgenic mice show activation of the mTOR pathway. F, Quantification of Western blots demonstrates that phosphorylated S6RP (pS6RP) is increased in Tg nerves (218.4 ± 25.79, p = 0.01), and the ratio of phosphorylated to total S6RP (rS6RP) is also increased (167.1 ± 16.04, p = 0.014); data were normalized to WT values arbitrarily set to 100. Increased phosphorylation of GSK3b and 4EBP1 was also evident but did not reach statistical significance; n = 3. Data are mean ± SEM. *p < 0.05.

Akt activation promotes myelin formation independently of Krox20 regulation. Purified Schwann cells infected with doxycycline (Dox)-inducible lentiviruses encoding MyrAkt-HA or GFP were cultured in ascorbic acid containing media in the absence of axons. A, GFP induction with Dox did not affect the basal levels of protein for Krox20, P0, and MAG. Basal levels for MAG were not detectable for quantification; n = 3. B, C, Induction of MyrAkt substantially increased P0 protein levels (358.1-, 586.7-, and 1114.5-fold vs 100, average 686.5 ± 223.98, p = 0.058) and significantly increased MAG levels (3541.7-, 9467.8-, and 6717.2-fold vs 100, average 6575.6 ± 1712.19, p = 0.045), as detected by Western blot. Interestingly, a modest but significant reduction of Krox20 was also detected (83.8 ± 5.28, p = 0.037). Western blot quantification data were normalizing by using values from control samples that were arbitrarily set to 100; n = 3. Data are mean ± SEM. D, Krox20 mRNA expression remained unchanged (0.77 ± 0.19, p = 0.27) when Akt was induced with doxycycline. mRNA for P0 and MAG was increased when Akt was induced, although due to the variability of induction (P0 1.7-, 3.5-, and 1.9-fold vs 1, average 2.4 ± 0.57, p = 0.07; and MAG 17.9-, 168.9-, and 42.2-fold vs 1, average 76.3 ± 46.81, p = 0.18) compared with the noninduced, the increase in these markers did not achieve significance by the t test. Real-time PCR data were normalized by using values from control samples that were arbitrarily set to 1. GAPDH was used as a reference; n = 3. Data are mean ± SEM. *p < 0.05.

Rapamycin treatment reduces myelin abnormalities in MyrAkt transgenic mice. A, The phenotype of 3-month-old transgenic mice was largely corrected by treatment with rapamycin for 8 weeks (MyrAkt+Rapa) but not by vehicle (MyrAkt+vehi); no changes were detected in control animals treated with vehicle (WT+Vehi). B, Phosphorylation of S6RP was substantially decreased in the nerves of WT and MyrAkt mice treated with rapamycin. C, Analysis of several parameters is shown for WT mice treated with vehicle and for transgenics (Myr) treated with either vehicle or rapamycin. These include brain weight (WT+Vehi 393 ± 6.3 mg; Myr+Vehi 438 ± 10.2 mg, p = 0.019; Myr+Rapa 371 ± 4.75 mg, p = 0.004), the number of axons per 100 μm² (WT+Vehi 3.4 ± 0.2; Myr+Vehi 2.3 ± 0.07, p = 0.01; Myr+Rapa 3.6 ± 0.18, p = 0.002), and the ratio of myelin abnormalities (i.e., tomacula and myelin infoldings/outfoldings)/total axons counted (WT+Vehi 0.2 ± 0.03% defect; Myr+Vehi 3.6 ± 0.6% defect, p = 0.003; Myr+Rapa 0.67 ± 0.14% defect, p = 0.006); n = 3. More than 3000 axons per n were examined to obtain the percentage of abnormalities. Data are mean ± SEM. D, g-ratios were measured for small myelinated fibers in the dorsal roots of WT mice on vehicle (WT+V), transgenics on Vehicle (Myr+V), and transgenics treated with rapamycin (Myr+R). Quantification of WT+Vehi (0.62 ± 0.006), Myr+Vehi (0.54 ± 0.01, p < 0.001), and Myr+Rapa (0.59 ± 0.02, p = 0.048) demonstrates a significant increase in the g-ratio of transgenic mice treated with rapamycin. A total of 112–175 small-caliber axons (0.5–2 μm) were measured per condition; n = 4. Data are mean ± SEM. E, Rapamycin administration does not prevent the excess of membrane wrapping in the Remak bundles from 3-month-old MyrAkt nerves. Yellow represents Schwann cells. Blue represents axons in the bundle. Scale bar, 1 μm. *p < 0.05. **p < 0.01.

Activated Akt enhances wrapping and sorting but not myelination of axons in Remak Schwann cells. A, EM micrographs from 20-month-old mice reveal aberrant axon-Schwann cell relationships in Remak bundles in transgenic compared with WT mice. Small axons were often surrounded by circumferential layers of uncompacted Schwann cell membranes (AI, shown at higher magnification in inset). Schwann cell membranes surrounding collagen fibers (AII) were also present in the transgenic nerves and were frequently covered by basal lamina. Scale bar, 1 μm. B, In 5-month-old-mice, Schwann cell processes (BI), with associated basal lamina without apparent axonal contact, are present. Arrowheads indicate basal lamina deposition. Schwann cells also formed empty pockets with basal lamina deposited in the interior (BII, inset, arrow). Scale bars, 2 μm. C, Bar graph showing the distribution of the numbers of axons per Schwann cell; Remak bundles with only a few axons (1–5) are increased in the 5-month-old transgenic mice. D, The number of axons per pocket was also decreased in the 5-month-old Tg, suggesting better segregation of axons (one axon per pocket WT 89.12 ± 0.007 vs Tg 96.12 ± 1.17, p = 0.027; 2–10 axons WT 10.59 ± 0.28 vs Tg 3.83 ± 1.22, p = 0.03; 11–20 axons WT 0.28 ± 0.28 vs Tg 0.04 ± 0.04, not significant); n = 2. A total of 100 bundles from sciatic nerves were analyzed per animal. E, Electron micrographs of the sympathetic tract from 20-month-old mice are shown. No myelinated fibers are present, but multilammelar wrapping of axons and collagen fibrils is seen, consistent with a role of Akt in wrapping. Scale bar, 500 nm. F, The numbers of myelinated axons in transgenic dorsal (DR) and ventral (VR) roots at 5 months were not statistically different in MyrAkt versus WT mice; n = 2. D, F, Data are mean ± SEM. *p < 0.05.

Akt inhibition blocks myelin formation in vitro without affecting Krox20 levels. A, Treatment of DRG neuron-Schwann cell cocultures with perifosine (Pf) or LY294002 (LY) blocked myelination. The myelin transcription factors Krox20 and Oct6 continue to be robustly expressed by Schwann cells in the Pf- but not the LY-treated cocultures. n = 5; four coverslips were analyzed for each n. B, Quantification of the blockade of myelination by Pf and LY. The average number of myelin segments per coverslip were as follows: controls, 2195; Pf, 37 (p = 0.0056); and LY, 3 segments (p = 0.005). n = 3; each n represents two coverslips. C, D, Pf-treated cells showed no change in Krox20 protein expression (102.8 ± 12.31, p = 0.83), although a significant decrease in pAkt 473 (40.1 ± 7.77, p = 0.001), MBP (3.1 ± 0.2, p < 0.001), and P0 (12.5 ± 4.49, p < 0.001) was detected. The same markers were also downregulated in LY-treated cocultures (pAkt 26.7 ± 6.84, p < 0.001; MBP 1.6 ± 1.03, p < 0.001; P0 1.69 ± 0.68, p < 0.001), but in this case Krox20 was also decreased (22.1 ± 5.86, p < 0.001) (n = 3, each n represents eight coverslips pooled per condition). Western blot quantification data were normalized using values from control cocultures that were arbitrarily set to 100. E, As detected by real-time PCR, Pf-treated cells showed no changes in Krox20 mRNA (1.27 ± 0.39, p = 0.51) and a significant decrease of mRNA for MBP (0.13 ± 0.03, p < 0.001). A decrease of P0 was also detected, although it did not reach statistical significance (0.62 ± 0.18 p = 0.09). These three markers were significantly downregulated in LY-treated cocultures (Krox20 0.12 ± 0.04, p < 0.001; MBP 0.01 ± 0.003, p < 0.001; P0 0.23 ± 0.06, p < 0.001). Expression data were normalized to control samples that were arbitrarily set as 1; GAPDH expression was used as reference. n = 4; four coverslips were pooled per condition for each n. F, Schwann cell proliferation was not affected by 20 μm of Pf (11.9 ± 0.019 vs Control 11.2 ± 0.02), whereas proliferation was almost absent with 20 μm of LY treatment (0.7 ± 0.002, p = 0.003). n = 4; each n represents three coverslips per condition. G, EM analysis of control, Pf-treated, and LY-treated cultures revealed drug-specific differences. In the presence of Pf, Schwann cells (SC) engaged axons (A) and partially but incompletely sorted them and failed to myelinate. LY-treated Schwann cells completely failed to sort axons. Image depicts representative fields from the cultures shown in A. Scale bars, 1 μm. Data are mean ± SEM. *p < 0.05; **p < 0.01.