Neuronal cyclic AMP controls the developmental loss in ability of axons to regenerate

Abstract

Unlike neonatal axons, mammalian adult axons do not regenerate after injury. Likewise, myelin, a major factor in preventing regeneration in the adult, inhibits regeneration from older but not younger neurons. Identification of the molecular events responsible for this developmental loss of regenerative capacity is believed key to devising strategies to encourage regeneration in adults after injury. Here, we report that the endogenous levels of the cyclic nucleotide, cAMP, are dramatically higher in young neurons in which axonal growth is promoted both by myelin in general and by a specific myelin component, myelin-associated glycoprotein (MAG), than in the same types of neurons that, when older, are inhibited by myelin-MAG. Inhibiting a downstream effector of cAMP [protein kinase A (PKA)] prevents myelin-MAG promotion from young neurons, and elevating cAMP blocks myelin-MAG inhibition of neurite outgrowth in older neurons. Importantly, developmental plasticity of spinal tract axons in neonatal rat pups in vivo is dramatically reduced by inhibition of PKA. Thus, the switch from promotion to inhibition by myelin-MAG, which marks the developmental loss of regenerative capacity, is mediated by a developmentally regulated decrease in endogenous neuronal cAMP levels.

Fig. 1.

Developmental changes in neurite outgrowth of DRG neurons on myelin correlate with changes in endogenous levels of cAMP. Dissociated DRG neurons from P1–P22 animals, as indicated, were cultured overnight on a substrate of purified rat CNS myelin or poly-l-lysine; afterward, they were fixed and immunostained for GAP43. In each experiment, at each DRG age, the mean length of the longest GAP43-positive neurite for 180–200 neurons was measured (±SEM) for at least three separate experiments. Results are presented as percentage of P1 neurite length (A). To measure cAMP, 2 × 10⁵ dissociated DRG neurons from P1–P22 animals, as indicated, were plated into each well, and cAMP was measured using a competitive immunoassay. Results are the mean (±SE) of at least three experiments, each performed in sextuplicate (B). DRG neurons were removed from P0 and P5 animals, fixed immediately, and immunostained for cAMP (C). Scale bar, 50 μm.

Fig. 2.

Developmental changes in neurite outgrowth of DRG neurons on myelin and MAG are dependent on changes in endogenous levels of cAMP. Dissociated DRG neurons from P1 or P5 animals, as indicated, were cultured overnight on a substrate of purified rat CNS myelin (A, B), MAG-expressing cells (striped bars), or control cells (black bar) (C). Where indicated, 200 nm KT5720 (+KT), 20 μmRp-cAMP (+Rp), or 1 μm KT5823 (+PKG Inh.) was added during culture; afterward, they were fixed and immunostained for GAP43. Scale bar (shown in A), 10 μm. In each experiment, at each DRG age, the mean length of the longest GAP43-positive neurite for 180–200 neurons was measured (±SEM) for at least three separate experiments. For neurons grown on myelin, results are presented as percentage of P1 neurite length in the absence of cyclic nucleotides (B). For neurons grown on cells, results are presented as percentage of neurite length on control cells in the absence of cyclic nucleotides (C).

Fig. 3.

Developmental changes in neurite outgrowth of RG neurons on myelin correlate with, and are dependent on, changes in endogenous levels of cAMP. Dissociated RG cells from E18 or P5 animals were measured for cAMP (1 × 10⁶cells per well) using a competitive immunoassay. Results are the mean (±SE) of at least three experiments, each performed in sextuplicate (A). E18 or P5 RG neurons were cultured overnight on a substrate of purified myelin (B,C). Where indicated, 200 nm KT5720 (+KT), 20 μm Rp-cAMP (+Rp), 1 μm KT5823 (+PKG Inh.), 1 mm db-cAMP, 50 μm Sp-cAMP, or 1 mm db-cGMP was added during culture as described in Figure 2, after which the neurons were fixed and immunostained for GAP43. Scale bar, 10 μm. Neurite length was measured for 180–200 neurons (±SEM) in each experiment, and results are from at least three experiments. Results are presented as percentage of E18 neurite length, in the absence of cyclic nucleotides. C, Stippled bars represent E18 neurons; white bars represent P5 neurons. *p < 0.05.

Fig. 4.

Developmental changes in neurite outgrowth of RG neurons on MAG are dependent on changes in endogenous levels of cAMP. Dissociated RG neurons from E18 (A) or P5 (B) animals were cultured overnight on a substrate of MAG-expressing cells (striped bars) or control cells (black bars). Where indicated, 200 nm KT5720 (+KT), 20 μmRp-cAMP (+Rp), 1 μm KT5823 (+PKG Inh.), 1 mm db-cAMP, 50 μm Sp-cAMP (+Sp), or 1 mm db-cGMP was added during culture; afterward, they were fixed and immunostained for GAP43. In each experiment, the mean length of the longest GAP43-positive neurite for 180–200 neurons was measured (±SEM) for at least three separate experiments. Results are presented as a percentage of neurite length on control cells in the absence of cyclic nucleotides. **p < 0.001.

Fig. 5.

Developmental changes in neurite outgrowth of raphespinal neurons on MAG and myelin are dependent on changes in endogenous levels of cAMP. Raphe nucleus neurons were dissociated from P0–P1 and P4–P8 rat pups and grown on either MAG-expressing (striped bars) or control CHO cells (black bars) (A, B) or purified myelin (C). Where indicated, H89 (2 μm), KT5720 (200 nm), db-cAMP (1 mm), Sp-cAMP (50 μm), or inhibitors–agonists of cGMP were added. Results represent the average neurite length of 180–200 GAP43-positive neurites per experiment from three separate experiments. For neurons grown on myelin, results are presented as percentage of P0–P1 neurite length, and for neurons grown on cells, results are presented as a percentage of neurite length on control cells, each in the absence of cyclic nucleotides. D, Dissociated raphe cells from P0–P1 or P3–8 animals were measured for cAMP (2 × 10⁵ cells per well) using a competitive immunoassay. Results are the mean (±SE) of at least three experiments, each performed in sextuplicate. *p< 0.05; **p < 0.001.

Fig. 6.

Regeneration of neonatal spinal axonsin vivo is cAMP-dependent. P2–3 rat pups were subjected to overhemisection lesion of their spinal cords at T6. Embryonic spinal cord transplants were placed into the lesion site without (A, B) or with H89 (0.5 mm) (C, D). Corticospinal axons were labeled anterogradely with BDA, and animals were killed at 4 weeks after transplantation. Dashed lines (A,C) indicate the approximate border between host and transplant (TP) tissue, and the boxed areas in A and C are shown at higher magnification in B and D, respectively. Corticospinal axon growth within the transplant is dramatically decreased in the presence of H89. Arrows inC indicate the few axons that regenerated into the TP in the H89-treated animal. A similar decrease in axonal growth by H89 was also seen for serotonergic axons (data not shown). Scale bars, 100 μm.