(Рис.1.)  |  Fig. 1. EGFR kinase activity is required for myelin inhibition. (A to D) Quantitation of the effect of EGFR inhibitors on neurite outgrowth from p7-9 CGNs on myelin (A), Nogo-66 (B), MAG (C), or PDL (D). Data represent mean neurite lengths derived from at least three individual experiments, representing a total of about 500 neurons per condition. AG1478 and PD168393 treatments significantly enhanced neurite outgrowth on inhibitors [(A) to (C)] but not on PDL (D). The concentrations of each drug are as follows: AG1288: 1 and 10 µM; AG1478: 10 and 100 nM; PD168393: 10 and 100 nM. Statistical analyses were performed by analysis of variance (ANOVA) [myelin (A): F = 64.8, df = 7, P < 0.0001; Nogo-66 (B): F = 20.5, df = 7, P < 0.001; MAG (C): F = 21.91, df =7, P < 0.0001; PDL (D): F = 2.102, df = 6, P = 0.1767), followed by Dunnett's posttest to compare outgrowth on each inhibitor with or without drug treatments. Both AG1478 and PD168393 treatments on inhibitors, but not AG1288, were significant when compared with no treatment on inhibitors alone (P < 0.001, except for 10 nM PD168393 on MAG, where P < 0.05) [(A) to (C)]. None of the three drug treatments showed a significant change in outgrowth on PDL alone (D). Error bars show mean + SEM. (E and F) Effects of PD168393 on retinal neurite outgrowth inhibition by myelin. Retinal explants from P5-6 mice were cultured within a collagen matrix with and without myelin (10 µg/ml) in the presence or absence of PD168393 (10 nM) for 72 hours. Explants were fixed and stained with antibodies to tubulin (E). Scale bar, 200 µm. The mean total neurite lengths (+SEM, N = 10) are shown in (F). PD168393 significantly promoted outgrowth of explants when compared to myelin alone (*, Student's t test, P < 0.0001). (G) Wild-type (wtEGFR) and kinase-deficient EGFR (kdEGFR) HSV-infected CGNs were stimulated with 1-ng/ml EGF for 5 min and the lysates were immunoblotted with an antibody against pTyr1173 EGFR (pEGFR) and reprobed with antibodies to EGFR. (H) DRGs were infected with HSV viruses and plated on control and myelin substrates. Average neurite lengths from three different experiments (at least 100 neurons from each experiment) were obtained as above. kdEGFR significantly promoted DRG outgrowth on myelin when compared with wtEGFR (*, Student's t test, P < 0.01). Error bars show means ± SEM


(Рис.2.)  |  EGFR activation by myelin inhibitors. (A) Serum-starved CGNs were stimulated with 5 nM AP–Nogo-66 for durations indicated. Lysates were blotted with an antibody to pEGFR, stripped, and reblotted with an antibody to EGFR, as in Fig. 1. (B) CGNs were stimulated with AP (5 nM), Semaphorin3A (Sem, 100 ng/ml), AP–Nogo-66 (N66, 5 nM), AP-OMgp (OM, 5 nM), and EGF (1 ng/ml) for 4 min, and the lysates were probed as above. (C) Nogo-66 activates ERK1/2 MAPKs in an EGFR-dependent manner. CGNs were preincubated (30 min) with AG1478 or PD16933 (both at 100 nM) and stimulated with Nogo-66 for 10 min. Lysates were blotted with antibodies against phospho-ERK1/2 and ERK1/2. (D) NgR-dependent EGFR activation. CGNs were infected with lentiviruses expressing full-length (FL NgR) or dominant-negative NgR (DN NgR) (10, 12) and stimulated with AP–Nogo-66 as in (B). The lysates were blotted with an antibody to pEGFR and an antibody to NgR. (E and F) CGNs were preincubated with AG1478 (100 nM), EGTA (3 mM), BAPTA-AM (5 µM), Go6976 (100 nM), TAPI-0 (1 µM), PTX (100 ng/ml), PP2 (10 nM), and GM6001 (100 nM) and stimulated with AP–Nogo-66 (5 nM) (E) or EGF (1 ng/ml) (F) for 5 min


(Рис.3.)  |  Requirement of EGFR activity for CSPGs and Nogo-66 inhibition but not Semaphorin3A repulsion. (A) Retinal explants were grown in collagen gels with and without CSPGs (200 ng/ml, Chemicon) and PD168393 (100 nM) for 3 days, fixed and stained with antibodies to tubulin. Scale bar, 200 µm. (B) Average total neurite lengths of retinal explants in the conditions described in (A). PD168393 significantly increased neurite length of explants when compared with CSPGs alone (*, Student's t test P = 0.0015). Error bars show means + SEM. (C) CSPGs activate EGFR in a Ca2+-dependent manner. Serum-starved CGNs were stimulated with CSPGs for 5 min with and without 3 mM EGTA and the lysates were blotted. (D and E) Growth cone collapse after 30-min addition of AP–Nogo-66 ( FPRIVATE "TYPE=PICT;ALT=~" 5 nM) and Semaphorin3A (Sema3A) (100 ng/ml) to embryonic day 13 (E13) chick DRG cultures infected with or without HSVs expressing wtEGFR or kdEGFR. Representative growth cone morphology is shown in (D) and the average percentage of collapsed growth cones + SEM from quadruplicate experiments in (E). kdEGFR significantly decreased growth cone collapse in response to Nogo-66 when compared with wtEGFR (ANOVA, F = 298, df = 8, P < 0.001, using Dunnett's posttest). (F and G) EGFR inhibitors did not affect the repulsive activity of Semaphorin3A. E14 rat DRGs were embedded in collagen adjacent to 293 cells expressing Sema3A. AG1288 (10 µM) and PD168393 (PD, 100 nM) were added at the same time, and cells were grown for 48 hours. Neurites were visualized by anti-tubulin staining. Representative images are shown in (F) and the quantitation from quadruplicate experiments in (G). Repulsive effects were quantified by comparing the ratio of proximal to distal neurite length (P/D) with respect to the 293 cell aggregates. No significant differences between AG1288 or PD168393-treated groups and their controls were detected by Student's t test. Error bars in (G) show means + SEM


(Рис.4.)  |  PD168393 promotes optic nerve regeneration. (A to D) Representative images of optic nerves stained with antibodies to GAP43 from control [(A) and (C)] or PD168393-treated [(B) and (D)] mice. The injury site was identified visually and with lectin staining (marked by C). The images (C) and (D) are magnified views of the postcrush area. (C) The control nerve where few GAP-43 fibers are evident. (D) Numerous regenerating fibers, including some that have turned (filled triangle) as well as those that are straight (open triangle). Scale bar, 100 µm in (A) and (B) and 50 µm in (C) and (D). (E) Quantitation of regenerating fibers in control and PD168393-treated mice. Fibers were counted at 250-µm intervals from the crush site from three nonconsecutive sections and the number of fibers at a given distance was calculated as (27). There is a significant difference between DMSO and PD168393 treatment groups by ANOVA (F = 63, df = 1, P < 0.001, n = 6 for each group), with Bonferroni posttests at each distance indicating a significant difference between DMSO and PD168393 treatment at 250 µm (P < 0.001) and 500 µm (P < 0.01). Error bars show means ± SEM. (F and G) Anti-tubulin stained RGCs in control and PD168393-treated retinas. Note large axon bundle (filled triangle) and retinal ganglion cell (open triangle) in (F). Scale bar, 10 µm. (G) Quantitation of surviving RGCs in control and PD168393-treated mice revealed no difference in survival between the two groups (Student's t test, P = 0.349). The results are plotted against the results from noncrushed controls. Error bars show means + SEM

статья

Отсутствие регенерации аксонов во взрослом мозге млекопитающих частично обусловлено ингибиторным эффектом молекул, ассоциирующихся с миелином ЦНС и глиальными рубцами. Сигнальные механизмы, лежащие в основе этого явления пока неясны. Koprivica с соавт. показали, что подавление регенерации опосредуется активацией epidermal growth factor receptor (EGFR) и что EGFR ингибиторы способствуют восстановлению поврежденных волокон зрительного нерва.

Потенциальный ингибиторный эффект миелина и глиального рубца на рост аксона можно довольно просто исследовать в культуре клеток. Когда нейроны растут в присутствии либо миелина, либо активного компонента глиального рубца – хондроитин сульфат протеогликана (chondroitin sulphate proteoglycans - CSPGs), они имеют слабую арборизацию аксонов в сравнении с соответствующими контрольными клетками. Этот относительно простой метод исследования позволил авторам скринировать около 400 хорошо охарактеризованных молекул по их способности реверсировать ингибиторную активность миелина и CSPGs.

Удивительно то, что несколько EGFR киназных ингибиторов, включая AG1478, PD168393 и Tarceva усиливают у крыс рост аксонов в разобщенных гранулярных нейронах мозжечка и dorsal root ganglion (DRG) нейроны растут в присутствии либо миелина, либо CSPGs. Кроме того, рост аксонов DRG нейронов, экспрессирующих доминантно-негативную форму EGFR был недолго блокирован миелином или CSPGs. Это указывает на то, что EGFR киназная активность может участвовать в подавлении регенерации.

Авторы изучили эффект миелина на EGFR фосфорилирование в гранулярных нейронах мозжечка, растущих некоторое время в среде лишенной сыворотки. Как Nogo66, так и миелиновый гликопротеин олигодендроцитов - myelin-associated inhibitory proteins – инициировали быстрое фофорилирование EGFR. Такого эффекта не наблюдали в нейронах, сверхэкспрессирующих доминантно-негативную мутантную форму NgR, обычного лиганд-связывающего компонента рецепторного комплекса, указывая на то, что NgR или функциональные гомологи, возможно, нужны для активации EGFR.

Действительно ли EGFR ингибиторы оказывают действие на репарацию нерва in vivo? Для проверки этой гипотезы авторы буквально размельчили зрительный нерв у взрослой мыши и использовали гель, содержащий PD168393, накладывая его на поврежденный участок сразу после нарушения целостности нерва. Через две недели был выявлен значительный рост аксонов у животных, обработанных ингибитором EGFR, и девятикратное увеличение числа регенерирующих волокон 0.25 μm в нарушенном участке по сравнению с контролем.

Эти находки имеют важное терапевтическое значение для больных со спинальными нарушениями и повреждениями мозга. Поскольку один из EGFR ингибиторов - Tarceva – был одобрен US Federal Drug Administration для лечения рака легкого, то можно ожидать, что в скором времени начнутся клинические испытания этого препарата и в восстановлении поврежденных нервов.

См. также:

Filbin, M. T. Myelin-associated inhibitors of axonal regeneration in the adult mammalian CNS. Nature Rev. Neurosci. 4, 703–713 (2003) He’s laboratory