Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy - PubMed (original) (raw)

Induction of postnatal schwann cell death by the low-affinity neurotrophin receptor in vitro and after axotomy

D E Syroid et al. J Neurosci. 2000.

Abstract

Schwann cells express the low-affinity neurotrophin receptor (p75), but no role for either the neurotrophins or their cognate receptors in Schwann cell development has been established. We have found that Schwann cells isolated from postnatal day 1 (P1) or P2 mice that were p75-deficient exhibited potentiated survival compared to wild-type cells after growth factor and serum withdrawal. There was, however, no disparity in the survival of p75-deficient and wild-type Schwann cells isolated at embryonic day 15, suggesting that the death-inducing effects of p75 are developmentally regulated. A comparable degree of cell death was also observed in the sciatic nerves of both wild-type and p75-deficient mice at P1. However, 24 hr after axotomy, there was a 13-fold increase in the percentage of apoptotic nuclei in the distal nerve stumps of the transected sciatic nerves of neonatal wild-type but not p75-deficient mice. The expression of both the p75 and nerve growth factor (NGF) genes was upregulated after axotomy in neonatal wild-type nerves. Collectively, these results suggest that NGF-mediated activation of p75 is likely to be an important mediator of Schwann cell apoptosis in the context of peripheral nerve injury.

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Figures

Fig. 1.

Fig. 1.

Postnatal Schwann cells isolated from p75-deficient mice exhibit a survival advantage in vitro, in comparison to wild-type cells, after growth factor withdrawal. Shown in A is a graphical representation of survival of postnatal cells using an MTT assay, from a representative experiment of three independent assessments, with five separate wells assessed daily over a 3 d period. Shown in B and_C_ are photomicrographs of cultures of postnatal wild-type (B) and p75-deficient (C) cells grown in serum-free conditions for 48 hr demonstrating a large number of crenated wild-type cells (arrowheads), whereas the majority of p75-deficient cells maintain a bipolar morphology. Scale bar, 10 μm.

Fig. 2.

Fig. 2.

The p75 and NGF genes are expressed by postnatal wild-type Schwann cells after growth factor withdrawal, but the trkA gene is expressed at barely detectable levels. cDNA samples generated from purified wild-type Schwann cells were subjected to PCR. The expected 172 bp p75 (lane 2) and 409 bp NGF (lane 4) fragments were amplified from the sample. The expected 320 bp (lane 6) trkA fragment was also amplified, although at a very low level. This contrasted with the robust amplification of the expected 320 bp trkA fragment in a cDNA sample derived from PC12 cells (lane 8). Negative controls were obtained by subjecting reagents to PCR reaction conditions without template cDNA (p75 primers, _lane 3;_NGF primers, lane 5; trkA primers, lane 7), and there was no amplification of the relevant bands. Lane 1 represents the ΦX174_Hae_III DNA ladder.

Fig. 3.

Fig. 3.

Schwann cell apoptosis is upregulated in the axotomized nerves of P1 wild-type mice in comparison to that exhibited in the axotomized nerves of p75-deficient mice. The sciatic nerves of P1 mice were axotomized, and the distal stumps were harvested 24 hr later. The sectioned nerves were assessed using DAPI staining to identify nuclei (A, C) and by TUNEL (B, D). A significantly increased number and percentage of condensed and TUNEL-positive nuclei were present in the wild-type nerves (A, B) in comparison to the number observed in nerves isolated from p75-deficient mice (C, D). Scale bar, 10 μm.

Fig. 4.

Fig. 4.

Expression of NGF and p75 mRNA is upregulated in rat sciatic nerve during Wallerian degeneration. Unilateral transections of P1 and adult rat sciatic nerves were performed, and ribonuclease protection analyses were performed using total RNA (1 μg per hybridization reaction) isolated from both the intact contralateral nerve (CL) and the transected distal nerve stump (T), 24 hr after transection, or using 10 μg of tRNA as a negative control, as indicated. NGF expression (A) and p75 expression (B) in the degenerating neonatal nerve is upregulated by ∼3-fold and 14-fold, respectively, relative to that in the contralateral control nerve. NGF and p75 expression is also upregulated in degenerating adult nerves, but the level of expression only reaches that found in the intact P1 nerve. The major 484 bp protected NGF RNA fragment and the 452 bp protected p75 RNA fragment are indicated. Autoradiographic exposure was for 66 hr. The bottom panels in A and_B_ show the relative GAPDH expression from corresponding cohybridization reactions using the GAPDH RNA probe. Autoradiographic exposure was for 20 hr.

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