The JIP1 Scaffold Protein Regulates Axonal Development in Cortical Neurons

Summary The development of neuronal polarity is essential for the determination of neuron connectivity and for correct brain function. The c-Jun N-terminal kinase (JNK)-interacting protein-1 (JIP1) is highly expressed in neurons and has previously been characterized as a regulator of JNK signaling. JIP1 has been shown to localize to neurites in various neuronal models, but the functional significance of this localization is not fully understood [1–4]. JIP1 is also a cargo of the motor protein kinesin-1, which is important for axonal transport [2, 4]. Here we demonstrate that before primary cortical neurons become polarized, JIP1 specifically localizes to a single neurite and that after axonal specification, it accumulates in the emerging axon. JIP1 is necessary for normal axonal development and promotes axonal growth dependent upon its binding to kinesin-1 and via a newly described interaction with the c-Abl tyrosine kinase. JIP1 associates with and is phosphorylated by c-Abl, and the mutation of the c-Abl phosphorylation site on JIP1 abrogates its ability to promote axonal growth. JIP1 is therefore an important regulator of axonal development and is a key target of c-Abl-dependent pathways that control axonal growth.


Immunofluorescence and Image Analysis
Cortical neurons were fixed with 4% paraformaldheyde, and the following antibodies were used: anti-JIP1 (1:100, B7, Santa Cruz, sc-25267), anti-JIP1 (1:100) [S3], anti-MAP (1:1000, Sigma), and anti-Tau-1 (1:100, Chemicon). Secondary antibodies were Alexa 488 or 594-labeled goat anti-mouse (isotype specific) or anti-rabbit (Invitrogen). Images were obtained with an Olympus BX51 microscope attached to a CCD Coolsnap-ES camera (Photometrics, United Kingdom). For the measurement of axons, neurons were fixed at 3-4 days in vitro (d.i.v.), and images from random fields for each condition were taken from at least six independent neuronal preparations. An axon was defined as a neurite that was longer than 80 mm and at least three times the length of other neurites (in parallel, axons were also identified as neurites with JIP1-positive tips). Axon length was measured with Image J (http://rsb.info.nih.gov/ij/), and data are expressed as percent of respective controls (w100 axons measured for each condition from six to eight independent experiments). The average length of axons in control groups was 282 mm 6 22 (mean 6 standard error of the mean [SEM]). Dendrite length was also measured in control and JIP1 siRNA experiments, and data were expressed as the mean 6 SEM from five to seven independent experiments and w300 dendrites for each condition. Average dendrite length in control groups was 48 mm 6 0.85. Unlike the effect observed on axons, the length of dendrites was not modified by the JIP1 siRNA (47.7 mm 6 0.82). Statistical significance was determined via one-way ANOVA, with post hoc Tukey's test for comparison between groups. Values of p < 0.05 (*) were taken to be statistically significant, (**) indicates values of p < 0.001.

Immunoprecipitations and GST Pull-Downs
Cells were washed once with ice-cold phosphate-buffered saline and then lysed in buffer containing 20 mM Tris, pH 7.4, 137 mM NaCl, 25 mM b-glycero-phosphate, 2 mM sodium pyrophosphate, 2 mM EDTA, 1% Triton X-100, 10% glycerol, 1 mM phenylmethylsulfonyl fluoride, 1 mM sodium orthovanadate, and 5 mg/ml leupeptin. Lysates were centrifuged at 14,000 3 g for 10 min to remove insoluble material. Epitope-tagged proteins were immunoprecipitated from lysates by incubation with anti-T7 tag antibody (Novagen) and protein A-Sepharose beads (Sigma) for 3 hr at 4 C. GST proteins were isolated by incubation of lysates with glutathione-Sepharose 4B (Amersham Biosciences). Beads were washed three times in lysis buffer, Figure S2. Relocalization of JIP1 after Induction of Supernumerary Axons and the Disruption of Cytoskeleton Dynamics The formation of axon-dendrite polarity can be affected by a variety of experimental manipulations [S6-S10]. In particular, it has recently been demonstrated that inhibition of glycogen synthase-3b activity resulted in the formation of multiple axons [S11]. (A) At 2 d.i.v., the inhibition of GSK-3 activity (1 mM SB415286, 24 hr) in cortical neurons induced JIP1 localization to multiple neurites. (B) At 5 d.i.v.. staining with the axonal marker Tau-1 (red) confirmed the generation of supernumerary axons after GSK-3 inhibition. Under these conditions, JIP-1 (green) localized to all of the Tau-1-positive neurites. Scale bars represent 20 mm. (C) Neuronal polarization has also been shown to depend on microtubule-dynamics changes, which are necessary to maintain the differences between the initial axonal segment and the dendrites and to allow the development of polarized axonal transport [S9]. Inhibition of microtubule dynamics with taxol (1 mM, 24 hr) promoted JIP1 (red) localization to multiple neurites. Scale bar represents 20 mm. (D) Higher-magnification analysis of axons showing JIP1 localization to central areas of axonal growth cones after short-term disruption of microtubule dynamics with taxol (10 mM, 2 hr). Scale bars represent 10 mm. (E) The depletion of actin filaments with cytochalasin D (Cyt-D, 1 mM, 2 hr) promotes a drastic change in axonal growth-cone structure, with a significant increase in JIP1-enriched microtubule protrusions. Scale bars represent 10 mm. Together, these results confirm the specific localization of JIP1 to axons and its preferential association with dynamic microtubules capable of protruding into peripheral growth-cone areas.

Figure S3. JIP1 Knockdown Affects Axonal Branch Formation
Axonal growth is required for development of correct neuron connectivity, a process which also involves the extension of branching processes either by bifurcation or interstitial development. It has been previously suggested that the extension of growth cones and axonal branches may involve similar signaling cues and cytoskeletal reorganization [S12, S13]. (A) Cortical neurons were transfected with JIP1 siRNA 24 hr after being plated, and 72 hr later the neurons were evaluated according to an index of 0 to >5 branching points. In this protocol, a small increase in the number of neurons with no axonal branches was observed.
(B and C) In developmentally delayed cortical neurons (see Supplemental Experimental Procedures) where a significant knockdown of JIP1 protein levels by siRNA has occurred prior to axonal differentiation, there was a substantial increase in the percent of neurons with no axonal branches and a decrease in the distance from the cell body to the first branching point.    Neurosci. 27, 8604-8615. S11. Jiang, H., Guo, W., Liang, X., and Rao, Y. (2005). Both the establishment and the maintenance of neuronal polarity require active mechanisms: critical roles of GSK-3b and its upstream regulators. Cell 120, 123-135. S12. Kalil, K., Szebenyi, G., and Dent, E.W. (2000). Common mechanisms underlying growth cone guidance and axon branching. J. Neurobiol. 44, 145-158. S13. Dent, E.W., Barnes, A.M., Tang, F., and Kalil, K. (2004). Netrin-1 and semaphorin 3A promote or inhibit cortical axon branching, respectively, by reorganization of the cytoskeleton. J. Neurosci. 24, 3002-3012. (B) Similar to wild-type JIP1, the ectopic expression of the human JIP1-R160G/P161G mutant increased axonal growth and rescued the decreased axonal growth caused by the JIP1 siRNA. Data are expressed as percent of respective controls (mean 6 standard error of the mean [SEM], from five independent experiments and w100 axons measured for each condition). Statistical significance was determined via one-way ANOVA, with post hoc Tukey's test for comparison between groups. Values of p < 0.05 (*) were taken to be statistically significant, (**) indicates values of p < 0.001. Figure S6. The Contribution of Arg to JIP1-Mediated Axonal Growth (A) Arg kinase phosphorylates JIP1 at Tyr278 in vitro. Constructs expressing T7-JIP1 and T7-JIP1-Y278F were introduced into COS-7 cells with or without an Arg-expressing vector. JIP1-containing complexes were immunoprecipitated with anti-T7 tag antibody, and the presence of Tyr-phosphorylated JIP1 was analyzed with phosphotyrosinespecific antibodies. (B) Immunoblot for Arg protein levels after transfection with Arg siRNA.
(C) Measurement of axon length in neurons transfected with Arg siRNA plus or minus a construct expressing myc-tagged human JIP1. The knockdown of Arg protein levels did not affect basal axonal growth, but produced a small decrease in the JIP1-dependent increase in axon length. Data are expressed as percent of respective controls (mean 6 SEM, from five independent experiments and w100 axons measured for each condition). Statistical significance was determined via one-way ANOVA, with post hoc Tukey's test for comparison between groups. Values of p < 0.05 (*) were taken to be statistically significant, (**) indicates values of p < 0.001.