Axon Outgrowth Is Regulated by an Intracellular Purine-sensitive Mechanism in Retinal Ganglion Cells

Abstract

Although purinergic compounds are widely involved in the intra-and intercellular communication of the nervous system, little is known of their involvement in the growth and regeneration of neuronal connections. In dissociated cultures, the addition of adenosine or guanosine in the low micromolar range induced goldfish retinal ganglion cells to extend lengthy neurites and express the growth-associated protein GAP-43. These effects were highly specific and did not reflect conversion of the nucleosides to their nucleotide derivatives; pyrimidines, purine nucleotides, and membrane-permeable, nonhydrolyzable cyclic nucleotide analogs were all inactive. The activity of adenosine required its conversion to inosine, because inhibitors of adenosine deaminase rendered adenosine inactive. Exogenously applied inosine and guanosine act directly upon an intracellular target, which may coincide with a kinase described in PC12 cells. In support of this, the effects of the purine nucleosides were blocked with purine transport inhibitors and were inhibited competitively with the purine analog 6-thioguanine (6-TG). In PC12 cells, others have shown that 6-TG blocks nerve growth factor-induced neurite outgrowth and selectively inhibits the activity of protein kinase N, a partially characterized, nerve growth factor-inducible serine-threonine kinase. In both goldfish and rat retinal ganglion cells, 6-TG completely blocked outgrowth induced by other growth factors, and this inhibition was reversed with inosine. These results suggest that axon outgrowth in central nervous system neurons critically involves an intracellular purine-sensitive mechanism.Under normal circumstances, neurons in the adult mammalian central nervous system fail to regenerate axons injured by stroke or trauma, resulting in long lasting disabilities in sensory, motor, or cognitive functions. However, many central nervous system neurons have the potential to regrow their axons if exposed to appropriate growth factors and/or if inhibitory influences that normally prevail in the mature central nervous system are suppressed (1-3). To identify factors that foster axonal regrowth in the central nervous system, we have turned to the classic model of optic nerve regeneration in lower vertebrates. Following injury to the optic nerve, retinal ganglion cells (RGCs) 1 in fish and amphibia re-extend axons that synapse upon appropriate central targets within 1-2 months (4). In a dissociated cell culture model of this system, two factors secreted by optic nerve glia were found to be responsible for inducing axon outgrowth from goldfish RGCs (5, 6). The more potent of these, axogenesis factor-1 (AF-1), has a molecular mass Ͻ1 kDa and stimulates lengthy axonal outgrowth without altering cell survival. AF-2 is a larger polypeptide that exerts a significant but less pronounced effect than AF-1. The specificity of these effects is demonstrated by the inactivity of a host of polypeptide growth factors or low molecular mass differentiation factors in this system (5).In the present st...

Secondary degeneration reduced by inosine after spinal cord injury in rats

“…[13][14][15][16][17][18][19][20][21] These effects kindle the hope for its use in SCI to conquer secondary degeneration. The aim of the present study was to investigate the possible neuroprotective and ameliorating effects and the time window of inosine in an experimental spinal cord clip compression injury model in adult rats.…”

How does an axon grow?

“…As axon guidance molecules activate the same growth cone mechanisms discussed above, it is not surprising that their effects may overlap with effects on elongation as well. Finally, other extracellular signals as simple as purine nucleotides may be critical to induce axon growth, although the mechanism of this activity is still mysterious (Benowitz et al 1998). Therefore peptide trophic factors appear to most strongly induce axon outgrowth, but adhesion molecules can greatly potentiate this growth, and molecules thought to be primarily involved in growth cone guidance may also have some elongation promoting activity.…”

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

“…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 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.…”

“…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).…”