A Purine-Sensitive Pathway Regulates Multiple Genes Involved in Axon Regeneration in Goldfish Retinal Ganglion Cells

Abstract

In lower vertebrates, retinal ganglion cells (RGCs) can regenerate their axons and reestablish functional connections after optic nerve injury. We show here that in goldfish RGCs, the effects of several trophic factors converge on a purine-sensitive signaling mechanism that controls axonal outgrowth and the expression of multiple growth-associated proteins. In culture, goldfish RGCs regenerate their axons in response to two molecules secreted by optic nerve glia, axogenesis factor-1 (AF-1) and AF-2, along with ciliary neurotrophic factor. The purine analog 6-thioguanine (6-TG) blocked outgrowth induced by each of these factors. Previous studies in PC12 cells have shown that the effects of 6-TG on neurite outgrowth may be mediated via inhibition of a 47 kDa protein kinase. Growth factor-induced axogenesis in RGCs was accompanied by many of the molecular changes that characterize regenerative growth in vivo, e.g., increased expression of GAP-43 and certain cell surface glycoproteins. 6-TG inhibited all of these changes but not those associated with axotomy per se, e.g., induction of jun family transcription factors, nor did it affect cell survival. Additional studies using RGCs from transgenic zebrafish showed that expression of T␣-1 tubulin is likewise stimulated by AF-1 and blocked by 6-TG. The purine nucleoside inosine had effects opposite to those of 6-TG. Inosine stimulated outgrowth and the characteristic pattern of molecular changes in RGCs and competitively reversed the inhibitory effects of 6-TG. We conclude that axon regeneration and the underlying program of gene expression in goldfish RGCs are mediated via a common, purine-sensitive pathway.

Mst3b, a purine-sensitive Ste20-like protein kinase, regulates axon outgrowth

“…The observation that 6-TG blocks axon outgrowth in primary neurons (9,12) suggested to us that the kinase it inhibits might be widely expressed in the brain. We prepared a high-speed supernatant fraction from neonatal bovine cortex and applied this material to a sulfopropyl-Sepharose column, which was previously shown to yield a partial purification of a 6-TG-sensitive kinase from PC12 pheochromocytoma cells (13).…”

“…Inosine (1 mM) reversed the inhibitory effect of 6-TG. Although by itself inosine has only a modest effect on PC12 cells (19), it induces strong outgrowth in goldfish retinal ganglion cells (7,9), mammalian peripheral ganglionic cells (6), and embryonic rat cortical neurons (see below). Neither 6-TG nor inosine altered cell survival (Fig.…”

“…The purine nucleoside inosine stimulates axon outgrowth in certain types of neurons in culture and in vivo after CNS injury (6)(7)(8)(9)(10). The purine analog 6-thioguanine (6-TG), on the other hand, blocks outgrowth induced by neurotrophic factors (9,11,12), and this effect is paralleled by the inhibition of a previously unidentified 45-50 kDa serine-threonine protein kinase (13).…”

“…The purine analog 6-thioguanine (6-TG), on the other hand, blocks outgrowth induced by neurotrophic factors (9,11,12), and this effect is paralleled by the inhibition of a previously unidentified 45-50 kDa serine-threonine protein kinase (13). Inosine competitively reverses the inhibitory effects of 6-TG on outgrowth (7,9), suggesting that it may act as an agonist of the 6-TG-sensitive kinase. We now identify the purine-sensitive kinase as mammalian Ste20-like protein kinase-3b (Mst3b) and show that it is a key regulator of axon outgrowth.…”

Intrinsic Factors Contributing to Axon Regeneration in the Mammalian Nervous System

“…Application of the purine nucleoside inosine to goldfish neurons potentiates the expression of axonal growth genes and process outgrowth in vitro (Benowitz et al, 1998;Petrausch et al, 2000). Administration of this molecule to adult rats following unilateral corticospinal tract injury (Benowitz et al, 1999) or cortical ischemia (Chen et al, 2002) stimulates vigorous compensatory sprouting of intact axons, accompanied by some functional recovery.…”