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 approximately 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 (approximately 130 kD, pI approximately 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 alpha- and beta-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 (approximately 50 kD, pI approximately 5.5), that appeared to be synthesized by the glial cells. Increases were also seen in one axonal protein, B (approximately 45 kD, pI approximately 4.5), that is carried by fast axonal transport, as well as in two axonal proteins, HA1 and HA2 (approximately 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.

Isolation of cDNA clones encoding RICH: a protein induced during goldfish optic nerve regeneration with homology to mammalian 2',3'-cyclic-nucleotide 3'-phosphodiesterases.

“…Particular interest has been placed in identifying proteins that are induced in the ganglion neuronal cell during regeneration in this system (14,(16)(17)(18)(19)(20)(21). Some of these proteins might prove regulatory and essential for the regeneration process.…”

“…It is known that both retinal RNA (9)(10)(11)(12) and protein (13)(14)(15) turnover increase following optic nerve axotomy. Specific retinal growthassociated proteins (GAPs) appear to be induced (16)(17)(18)(19)(20)(21), and there is a marked increase in the synthesis of constitutive cytoskeletal components, such as tubulin (14,17). Among the induced proteins, an acidic protein doublet (p68/70; pl 4.8-4.9) was detected by the in vivo labeling of newly synthesized proteins with radioactive precursors (17).…”

Expression of glial fibrillary acidic protein (GFAP) in goldfish optic nerve following injury

“…In an attempt to unravel the molecular events associated with this regeneration (Grafstein and Murray, 1969), particular attention has been focused on the role of certain axonally transported proteins, designated "growth-associated proteins," which are transported within the fast phase of axonal transport and which show a marked increase in synthesis during regeneration (Benowitz and Lewis, 1983; Heacock and Agranoff, 1982;Perrone-Bizzozero and Benowitz, 1987). In addition, the role of certain intermediate filament proteins has also been investigated (Perry et al, 1985;Quitschke and Schechter, 1983a). These proteins are either transported along the regenerating axons within the slow phase of axonal transport or are present within the environment surrounding these axons (Deaton and Freeman, 1983;Jones et al, 1986;Perry et al, 1987;Quitschke and Schechter, 1984) and have been considered to play a role in axonal growth and plasticity of the system.…”

Characterization of a goldfish antigen during development and regeneration of the visual system

“…Ganglion cells respond to nerve injury by increases in perikaryal size (Murray & Grafstein, 1969), polyribosome content (Murry & Forman, 1971;Whitnall & Grafstein, 1983), RNA content (Murray, 1973), and protein synthesis (Heacock & Agranoff, 1976;Burrell et al, 1978;Perry et al, 1987). Proteins with the same characteristics as GAP-43 have since been reported in regenerating optic nerves of the goldfish (Benowitz et al, 1981;Heacock & Agranoff, 1982;Benowitz & Lewis, 1983;Perry et al, 1985). These proteins have been referred to as growth association proteins (GAPs), an example of which is the 43 kD protein from toad optic nerves, which increased during optic nerve regeneration (Skene & Willard, 1981).…”

“…These studies point to a role for laminin in optic nerve regeneration (Hopkins et al, 1985 studies have shown a response by non-neuronal cells during regeneration that includes an increased release of proteins during regeneration (Arenander & DeVellis, 1981; Perry et al, 1985;Schwartz et al, 1985;Freeman et al, 1986;Bawnik et al, 1987). These studies point to a role for laminin in optic nerve regeneration (Hopkins et al, 1985 studies have shown a response by non-neuronal cells during regeneration that includes an increased release of proteins during regeneration (Arenander & DeVellis, 1981; Perry et al, 1985;Schwartz et al, 1985;Freeman et al, 1986;Bawnik et al, 1987).…”