Adult neuronal regeneration induced by transgenic integrin expression

Abstract

In a variety of adult CNS injury models, embryonic neurons exhibit superior regenerative performance when compared with adult neurons. It is unknown how young neurons extend axons in the injured adult brain, in which adult neurons fail to regenerate. This study shows that cultured adult neurons do not adapt to conditions that are characteristic of the injured adult CNS: low levels of growth-promoting molecules and the presence of inhibitory proteoglycans. In contrast, young neurons readily adapt to these same conditions, and adaptation is accompanied by an increase in the expression of receptors for growth-promoting molecules (receptors of the integrin family). Surprisingly, the regenerative performance of adult neurons can be restored to that of young neurons by gene transfer-mediated expression of a single alpha-integrin.

Fig. 1.

Adult neurons do not extend neurites on weakly growth-promoting or inhibitory substrata. a, Phase micrographs of adult and early postnatal (P2–P3) sensory neurons cultured 20 hr on substrata containing low levels of fibronectin (FN1), low levels of laminin (LM1), or the inhibitory proteoglycan aggrecan in combination with high levels of laminin (PG/LM20). Early postnatal neurons extend numerous neurites on these substrata, whereas adult neurite extension is quite limited. b, Early postnatal neurons (solid bars) extend neurites on all substrata tested. Outgrowth of adult neurons (open bars) is poor even on high levels of laminin (LM20) and fibronectin (FN20) and is further compromised on weakly growth-promoting (LM1, FN1) and proteoglycan-containing (PG/LM) substrata. Data from at least three independent experiments are shown. *Adult outgrowth on LM20 substrata is significantly better than adult outgrowth on either LM1 or PG/LM substrata (p < 0.025;t test). All postnatal conditions are significantly different from adults (p < 0.00001; t test).

Fig. 2.

Integrin expression can be increased in adult neurons by adenovirus-mediated gene transfer. a, Fluorescent micrographs of adult neurons in culture 72 hr after infection with adenoviral constructs expressing integrin α subunits. Live cultures were stained with antibodies that recognize both the transgenic and endogenous integrin subunit (for α₁) or the transgenic subunit specifically (for α₅). b, Western blot analysis of surface integrin protein expressed in adult neurons after infection. Neurons were cultured on low levels of laminin or fibronectin and infected as indicated. Surface-biotinylated protein was immunoprecipitated with antibodies that recognize both endogenous and transgenic integrin-containing receptors. The total surface expression of integrin α₁- and α₅-containing receptors is increased in integrin-infected cells, whereas integrin expression in β-gal-infected neurons does not respond to weakly growth-promoting substrata and remains low.

Fig. 3.

Outgrowth of adult neurons on poor substrata can be greatly improved by increasing integrin expression.a, Phase micrographs of adult neurons overexpressing either a laminin receptor (α₁Infected) or a fibronectin receptor (α₅ Infected). Outgrowth of adult neurons is specifically improved on substrata containing the ligand for the overexpressed receptor. b, The growth of integrin-infected neurons as a percentage of increase over controls (infected with β-gal). Neurons overexpressing α₅ (solid bars) and α₁ (open bars) show improved outgrowth specifically on substrata containing ligand for the overexpressed receptor. Data from at least four independent experiments are shown for all conditions. *Significantly different from the other conditions on the same substratum (p ≤ 0.0001;t test).

Fig. 4.

The outgrowth of adult neurons after manipulation of integrin expression is comparable with that of early postnatal neurons. Adult neurons were infected with adenoviral constructs containing integrin α₁ or β-galactosidase and were cultured for 72 hr to ensure strong expression of the transgenes. Postnatal day zero rat neurons were cultured in parallel for 72 hr without infection. Neurons were removed from the plates and recultured on LM1 substrata for 6 hr. Cultures were fixed, and the number of neurites per cell and the percentage of cells with neurites were determined for at least 100 cells from four independent experiments. *The β-galactosidase-infected adult neurons are significantly different from both P0 and α₁-expressing adult neurons (p < 0.0001; t test).