G. W. Perry

Protein Synthesis and Axonal Transport During Nerve Regeneration

Abstract— Protein synthesis and axonal transport have been studied in regenerating peripheral nerves. Sciatic nerves of bullfrogs were unilaterally crushed or cut. The animals were killed 1, 2, or 4 weeks later, and 8th and 9th dorsal root ganglia removed together with sciatic nerves and dorsal roots. The ganglia were selectively labeled in vitro with [35S]‐methionine. Labeled proteins, in dorsal root ganglia and rapidly transported to ligatures placed on the sciatic nerves and dorsal roots, were analyzed by two‐dimensional polyacryl‐amide gel electrophoresis. Qualitative analysis of protein patterns revealed no totally new proteins synthesized or rapidly transported in regenerating nerves. However, quantitative comparison of regenerating and contralateral control nerves revealed significant differences in abundance for some of the proteins synthesized in dorsal root ganglia, and for a few of the rapidly transported proteins. Quantitative analysis of rapidly transported proteins in both the peripheral processes (spinal nerves) and central processes (dorsal roots) revealed similar changes despite the fact that the roots were undamaged. The overall lack of drastic changes seen in protein synthesis and transport suggests that the neuron in its program of normal maintenance synthesizes and supplies most of the materials required for axon regrowth.

Changes in Protein Content of Goldfish Optic Nerve During Degeneration and Regeneration Following Nerve Crush

Abstract: After the goldfish optic nerve was crushed, the total amount of protein in the nerve decreased by about 45% within 1 week as the axons degenerated, began to recover between 2 and 5 weeks as axonal regeneration occurred, and had returned to nearly normal by 12 weeks. Corresponding changes in the relative amounts of some individual proteins were investigated by separating the proteins by two‐dimensional gel electrophoresis and performing a quantitative analysis of the Coomassie Brilliant Blue staining patterns of the gels. In addition, labelling patterns showing incorporation of [3H]proline into individual proteins were examined to differentiate between locally synthesized proteins (presumably produced mainly by the glial cells) and axonal proteins carried by fast or slow axonal transport. Some prominent nerve proteins, ON1 and ON2 (50–55 kD, pI ∼6), decreased to almost undetectable levels and then reappeared with a time course corresponding to the changes in total protein content of the nerve. Similar changes were seen in a protein we have designated NF (∼130 kD, pI ∼5.2). These three proteins, which were labelled in association with slow axonal transport, may be neurofilament constituents. Large decreases following optic nerve crush were also seen in the relative amounts of α‐ and β‐tubulin, which suggests that they are localized mainly in the optic axons rather than the glial cells. Another group of proteins, W2, W3, and W4 (35–45 kD, pI 6.5–7.0), which showed a somewhat slower time course of disappearance and were intensely labelled in the local synthesis pattern, may be associated with myelin. A small number of proteins increased in relative amount following nerve crush. These included some, P1 and P2 (35–40 kD, pIs 6.1–6.2) and NT (∼50 kD, pI ∼5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (∼45 kD, pI ∼4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (∼60 and 65 kD respectively, pIs 4.5–5.0), that are carried mainly by slow axonal transport. Other proteins, including actin, that showed no net changes in relative amount (but presumably changed in absolute amount in direct proportion to the changes in total protein content of the nerve), are apparently distributed in both the neuronal and nonneuronal compartments of the nerve.

Protein Synthesis and Rapid Axonal Transport During Regrowth of Dorsal Root Axons

Damage to the sciatic nerve produces significant changes in the relative synthesis rates of some proteins in dorsal root ganglia and in the amounts of some fast axonally transported proteins in both the sciatic nerve and dorsal roots. We have now analyzed protein synthesis and axonal transport after cutting the other branch of dorsal root ganglia neurons, the dorsal roots. Two to three weeks after cutting the dorsal roots, [35S]methionine was used to label proteins in the dorsal root ganglia in vitro. Proteins synthesized in the dorsal root ganglia and transported along the sciatic nerve were analyzed on two‐dimensional gels. All of the proteins previously observed to change after sciatic nerve damage were included in this study. No significant changes inproteins synthesized in dorsal root ganglia or rapidly transported along the sciatic nerve were detected. Axon regrowth from cut dorsal roots was observed by light and electron microscopy. Either the response to dorsal root damage is too small to be detected by our methods or changes in protein synthesis and fast axonal transport are not necessary for axon regrowth. When such changes do occur they may still aid in regrowth or be necessary for later stages in regeneration.

Role of fast axonal transport in regeneration of goldfish optic axons.

Publisher Summary This chapter provides evidence that the goldfish optic tectum contains factors that can influence axonal outgrowth and possibly other aspects of optic nerve regeneration. It investigates the possible role of the tectum by determining the effects of removing the lobe of the tectum to which the regenerating axons normally project. The increased supply of material that is generated by goldfish retinal ganglion cells during regeneration is essential for the maintenance of axonal outgrowth. Although the normal level of transport is apparently adequate for the initiation of outgrowth and the first few days of elongation, a critical point is reached in about 8 days when this supply is no longer sufficient. The cell body reaction in these neurons involves the increased production of axonally transported proteins, but not necessarily the production of new proteins. The presence of the optic tectum influences not only the production of axonally transported proteins, but also their disposition in new axon segments. This influence is not effective to a significant degree, until after the regenerating axons have reached the tectum.

Effects of a conditioning lesion on bullfrog sciatic nerve regeneration: Analysis of fast axonally transported proteins

We have shown that bullfrog sciatic nerves respond to a conditioning lesion similarly to goldfish optic nerve and rat or mouse sciatic nerve; that is, following a crush the rate of regeneration is faster in nerves that have received a conditioning lesion compared to nerves that have not. Also, damaged nerve fibres show initial growth or sprouting earlier in a previously conditioned nerve compared to nerves that have not received a prior conditioning lesion. We have not detected changes in the transport of fast axonally transported proteins with the conditioning lesion paradigm, other than those changes seen in regenerating nerves after receiving a single lesion. However, more label was present in a few fast axonally transported proteins at the lesion site in conditioned nerves compared to non-conditioned nerves, and this difference is not apparently due to increased transport. It seems that changes in fast axonally transported proteins probably do not contribute directly to the mechanism underlying the conditioning lesion effect of higher out growth rates, although some of the fast transported proteins may be involved in functions, possibly at the growing tip of damaged fibres, which promote or result from the conditioning effect.

Distribution of GFAP+ astrocytes in adult and neonatal rat brain.

Astrocytes can proliferate as a result of trauma to the brain, such as occurs in a variety of diseases. Understanding the normal distribution of astrocytes is necessary before the extent of astrogliosis can be clearly determined. However, little is known about the normal distribution of GFAP+ astrocytes especially during development. This study examined distribution of GFAP+ astrocytes in regions of the cortex, cerebellum, and brainstem of adult and rat pup brains by immunocytochemistry using antibodies against GFAP. The findings showed a differential distribution of GFAP+ astrocytes in the rat brain. A paucity of GFAP expression was found in most regions of the normal adult rat brainstem, whereas GFAP+ astrocytes were abundantly distributed in all areas of the cortex and cerebellum. A similar regional heterogeneity in the distribution of GFAP+ astrocytes was seen in the neonatal rat brain. These findings suggest that the development of the differential pattern of GFAP+ astrocytes seen in the rat brain does not occur postnatally, but instead is present at birth and appears to be determined during fetal development.

A comparison of fluorographic methods for the detection of 35S-labeled proteins in polyacrylamide gels

Eight different methods of fluorographic enhancement of sensitivity to 35S decay after gel electrophoresis were compared. Using Kodak X-Omat AR X-ray film, we found that some fluors were about equivalent to 2,5-diphenyloxazole/dimethyl sulfoxide embedding, whereas several other fluors were not quite as effective, but still were significantly more sensitive than control autoradiography. The most sensitive procedures can yield a detectable darkening of film with less than 1 dpm/mm2 of 35S after a 1-week exposure.

Differential Regulation of Fast Axonally Transported Proteins During the Early Response of Rat Retinal Ganglion Cells to Axotomy

Abstract: We have found that the early response of axotomized rat retinal ganglion cells is characterized by the differential regulation of a number of fast axonally transported proteins. The abundance of 23 radiolabeled fast transported proteins was analyzed at 2 and 5 days after axotomy using two‐dimensional gel electrophoresis. Corresponding changes in retinal GAP‐43 mRNA were measured using northern analysis. Within 2 days of injury, >40% of the transported proteins analyzed, including GAP‐43, showed increased labeling above control levels. Approximately 13% of transported proteins decreased below control levels, whereas the remainder did not change. Five days after axotomy, only GAP‐43 and another fast transported protein, C3, continued to sustain measurable increased labeling above control levels; all previously elevated proteins appeared to have been down‐regulated by this time, which corresponds to the onset of cell death. These differential changes were accompanied by parallel increases in GAP‐43 mRNA. These results suggest that the molecular changes within rat retinal ganglion cells are differentially regulated within two stages subsequent to damage, initial regenerative growth followed by cell death.

Sudden infant death syndrome: a theory.

A hypothesis, and supporting evidence, is presented for the sudden infant death syndrome (SIDS). Our model is as follows. Fetal hemoglobin levels are abnormally elevated in SIDS infants, which contributes to a state of chronic hypoxia. Chronic hypoxia produces pronounced depressive effects on the respiratory system during slow wave sleep (a state of normal respiratory depression). These depressive effects are particularly manifest during that period of development in which slow wave sleep begins to occupy a very large percentage of total sleep time in infants -2-4 mo of age. A downward spiral is initiated in slow wave sleep such that hypoxia-induced decreases in ventilation produce more extreme hypoxia leading to further respiratory depression and ultimately death due to a cessation of respiration.

On the identification of α- and β-tubulin subunits

Abstract: Confusion appears to have arisen in the literature regarding the designation of α‐and β‐tubulin in polyacrylamide gels. The presence or absence of 8 M‐urea in sodium dodecyl sulfate (SDS) polyacrylamide gels leads to different patterns for unalkylated tubulin subunits (and other proteins), making difficult the designation of the α and β subunits by original definition using electrophoretic mobility in the molecular weight dimension. The specific biochemical property of posttranslational tyrosylation of the α subunit has been used to identify further this subunit. Under all conditions tested, the β subunit has been found to be more acidic than the α subunit, with isoelectric point differences that agree with theoretical and published values. If the tubulin subunits are reduced and alkylated, the β subunit migrates more rapidly in SDS polyacrylamide gels, with or without urea present. However, unalkylated tubulin subunits can comigrate or even reverse their relative mobility if 8 M‐urea‐SDS polyacrylamide gels are used for subunit separation. The results also confirm the earlier reports that the post‐translational tyrosylation of protein appears exclusively restricted to α‐tubulin and can be demonstrated in an in vivo situation. In addition, the results suggest that only the α2 subunit of tubulin is tyrosylated.