More interestingly, the increases in MT stability correlate with decreases in neuronal plasticity, and both occur during aging and in some neurodegenerative
diseases. Therefore, learning about stable MT fragments, which are unique to neurons, is crucial for understanding normal axonal development and neuronal differentiation; this may also aid in identifying novel therapeutic targets for neurodegeneration and regeneration. The existence of a stable, biochemically distinct fraction of axonal tubulin was demonstrated some years ago (Brady et al., 1984; Sahenk and Brady, 1987). When preparing MTs from brain extracts, a substantial amount Dinaciclib concentration of tubulin remains in the pellet following low-temperature depolymerization. This fraction is termed cold-insoluble, or cold-stable tubulin. A more extensive differential extraction using cold and Ca2+ extractions to produce labile, cold-stable, and cold/Ca2+-stable fractions was developed (Figure 1A). The cold/Ca2+ fraction was enriched in axons. Using axonal transport to metabolically label MTs in rat optic nerve, the cold/Ca2+-stable tubulin fraction (P2) was examined by 2D-PAGE. A striking difference was found between tubulins in soluble and those in stable MTs: some tubulins in P2 exhibited a significant basic shift during isoelectric focusing (IEF) (Brady et al., 1984). This suggested that tubulins in stable MTs were biochemically distinct from those in
cold-labile MTs. Specifically, cold-stable Pexidartinib research buy MTs contained very tubulins significantly more basic than predicted from sequence or observed in cold-cycled MTs. Stability of MTs has been related to differences in MAPs, specific tubulin isotypes, and posttranslational modifications, but no factor has been identified that is sufficient to make MTs stable to depolymerization by cold or elevated Ca2+. MAPs stabilize cycled MTs in vitro (Chapin and Bulinski, 1992), but the increase in stability is modest and MAPs partition with both stable and labile MTs (Brady et al., 1984). Similarly, detyrosination and acetylation of α-tubulin correlate with MT stability in many systems (Bulinski
et al., 1988), but in vitro these modifications confer no measurable change in MT stability (Maruta et al., 1986; Webster et al., 1990) and are found in all cell types. Specific tubulin isotypes may contribute to MT stability (Falconer et al., 1994), but none partitions specifically with stable MTs, and again, differences in stability are modest. The native pI values for highly conserved tubulin isoforms all fall within a narrow range (pI = 5.5–5.6 for mouse α-tubulins, pI = 4.8–4.9 for mouse β2–6 tubulins and pI = 5.6 for β1 tubulin). MAPs do not associate with tubulin in IEF gels or change the charge on tubulins. Thus, no known tubulin isotype or modification can account for both the basic shift and exceptional stability of P2 tubulins, suggesting a novel posttranslational modification.