Figure 7.
Inhibition of CaMKK by STO-609 inhibits neurite outgrowth and causes axonal growth cone collapse. A, High-density cultures of rat hippocampal neurons were transfected on day 2 with plasmid encoding soluble EGFP alone (Control) or in combination with STO-609-insensitive CaMKKL233F or caCaMKI. Neurons were cultured for 12 hr to allow for expression of the transfected protein and then treated without (i.e., vehicle) or with 2.6 μm STO-609 for 2 d. Neurons were then fixed and imaged. Quantification of total neurite length is shown for 30 cells per condition. Statistical analysis compares neurite length of kinase-expressing (CaMKKL233F or caCaMKI) versus control cells in the absence or presence of STO-609. *p values of <0.01 (t test). B, C, Inhibition of CaMKK by STO-609 results in growth cone collapse. Low-density cultures of 3-d-old hippocampal neurons were treated with 2.6 μm of STO-609 and then fixed and stained for filamentous actin (phalloidin; red) and microtubules (green). Randomly selected axonal growth cones (80–100 per condition) were scored as normal (C, left panel) or collapsed (C, right panel) by an investigator without knowledge of the treatment conditions. The bar graph (B) summarizes the percentage of growth cones that were collapsed as a function of time after addition of STO-609 (± SE of percentage). After 90 min in STO-609 the percentage of collapsed growth cones quadrupled. Scale bar: C,10 μm. D, Inhibition of CaMKK by STO-609 suppresses growth cone motility. At 5, 60, or 90 min after addition of 2.6 μm STO-609, axonal growth cone motility was assessed in an individual neuron by acquiring phase-contrast images every 5 sec for a period of 5 min. Single images of these time-lapse recordings show a change from a spread growth cone morphology with lamellar veils extending between filopodia (left) to a reduced growth cone size at 60 min (middle) and 90 min (right), with fewer and smaller lamellar extensions. The time-lapse movie available as supplemental material shows that STO-609 treatment markedly inhibited the motility of this growth cone after 60 and 90 min of treatment. Growth cones of untreated sister cultures showed no reduction of growth cone motility over a period of 120 min (data not shown).