Target Regulation of Axotomy‐Sensitive Proteins

Abstract

Large changes in the production of certain proteins often follow axotomy. How the cell body is signaled to make these changes, or terminate them after regeneration is finished, is unclear. This issue was addressed by studying an axotomized giant identified neuron, the giant cerebral neuron of the sea slug Aplysia, both in vivo and in culture. One week after axon crush in vivo, there were increases of 1.5-18-fold in the 5-h incorporation of [35S]methionine into seven proteins identified by two-dimensional gel electrophoresis. There were decreases of five- to 28-fold in the labeling of four other proteins. An axotomized giant cerebral neuron grows vigorously when placed in culture and forms chemical synapses with appropriate target cells while continuing unabated growth. The labeling of two of the proteins that up-regulate after axotomy in vivo was suppressed by the presence of target cells in culture. For one of the proteins, this effect was also produced by membranes of target cells, but not by medium conditioned by exposure to target cells. These results are consistent with the idea that loss of membrane-membrane contact with target cells (or its restoration) is involved in the initiation (or termination) of the up-regulation of certain proteins after axotomy.

Retrograde axonal transport of locally synthesized proteins, e.g., actin and heat shock protein 70, in regenerating adult frog sciatic sensory axons

“…The lesion could also discontinue the retrograde transport of one or several target derived factors, e.g., nerve growth factor (for review see Harper and Thoenen, 1980;Thoenen and Barde, 1980) or other such factors derived from Schwann cells or other cells resident in the nerve (Varon et al, 1981). In addition, the disruption of a membrane to membrane contact could account for this target disconnection signal as proposed by Savage et al (1990). As an alternative to this negative signalling, the lesion might trigger the production of one or several factors at the site of injury, which could be taken up by the injured neuron and be retrogradely transported to the cell body to provoke the metabolic changes needed in the regeneration process.…”

Early Changes in Nuclear Proteins Following Axotomy

“…> 1,000 Ci/mmoI (ICN); three ganglia/500 pCi [35S]methionine/0.5 ml of hemolymph]. Ganglia were then rinsed with label-free supplemented Leibowitz-15 medium (SL-15) (Savage et d., 1990) and pinned in a dish containing SL-I5 at 0-4°C. Polyvinylpyrrolidone was added to the medium (0.5% final concentration) to minimize loss of soluble nuclear proteins due to swelling of extruded nuclei (Paine et al, 1983).…”

“…Seven to 10 nuclei, or four to five whole cells, were collected in a tube containing 10-15 p1 of Laemmli sample buffer (Laemmli, 1970). Preparation of samples for, and running of, two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis (2-D SDSPAGE), with isoelectric focusing in the first dimension and 7.5% acrylamide in the second dimension, were performed as previously described (Savage et d., 1990). This technique resolved proteins in the apparent PI range of approximately 4.5-7.8 and the apparent molecular mass range of approximately 25-200 kDa.…”

“…We have addressed this issue using giant identified neurons from the central nervous system of the marine slug, Aplysia californica. Like other neurons (Perry and Wilson, 1981;Skene and Willard, 1981), these display changes in a small group of proteins after axotomy (Savage et al, 1990). They are attractive experimental subjects here, because the neurons are so large that the nuclei can be manually dissected and cleaned.…”

Sequestration of cAMP response element-binding proteins by transcription factor decoys causes collateral elaboration of regenerating Aplysia motor neuron axons

“…Collateral elaboration is thought to be a key event in the navigation of axons to the proper target during axonal regeneration as well as during the development of the CNS (3,4). This process appears to involve alterations in gene expression and protein synthesis (5)(6)(7)(8)(9)(10)(11). However, the genomic programs that mediate collateral formation are yet to be identified.…”