(a) Developmental expression of endogenous MKP-1 in the cortex. (b) Knockdown efficiency of short hairpin RNAs (shRNA#3-7; –, empty vector) against MKP-1. (c) Representative images of individual cortical layer II–III neurons from in utero electroporated brains expressing control (GFP), mkp-1::IRES-GFP (mkp-1::GFP) or shRNA#7 (shRNA#7::GFP) plasmids. Arrows highlight differences in dendritic development. (d) Summary of the morphological effects of MKP-1 gain and loss-of-function on layer II/III cortical neurons. (e) Traces of axon terminals within the contralateral cortical layers of transfected brains. Primary (purple), secondary (blue), tertiary (yellow) and quartenary (green) branches from axons (red) are highlighted. (f–h) Representative images of sections from P7 brains electroporated at E15.5 with control (GFP) (f), mkp-1::IRES-GFP (mkp-1::GFP) (g) or shRNA #7 (shRNA#7::GFP) (h) plasmids. Red lines indicate the boundary between white matter (WM) and lateral striatum (str). Data from MKP-1 (n=23), GFP (n=14) and shRNA#7 (n=11) animals were collected from at least 3 independent experiments. Scale bars, 200 µm. (i,j) Number of axonal branches within the contralateral cortical layers were expressed as mean ± s.e.m.. Data were collected from individual axons expressing GFP (n=8), mkp-1::GFP (n=11), shRNA#7::GFP (n=16), shRNA#6::GFP (n=10) from at least 5 different brains and at least 3 independent electroporations per condition. Significant differences are indicated as follows: p<0.05, t-test *, MKP1 vs. GFP; †, GFP vs. shRNA#7; ‡, shRNA#7 vs. shRNA#6.

Dissociated cortical neurons were cultured for 7 days or the indicated DIV before pharmacological treatments. MKP-1 protein is highly inducible by BDNF (50 ng/ml) at any stage in culture (a) both in FACS sorted GFP-positive interneurons and GFP-negative excitatory neurons (b), as well as by depolarization (c) using 25 mM KCl for 3 hours. (c,d) Induction was prevented when TrkB signaling (TrkB.Fc, 100 ng/ml; K252a, 100 nM), the Erk pathway (U0126, 10 µM) or gene expression (CHX, 20 µg/ml; ActD, 5 µM) were pharmacologically blocked. (e,f) Treatment of inhibitory or excitatory neurons FACS sorted from transgenic mice expressing GFP in all cortical interneurons (e) and mixed cortical neuron cultures (f) with BDNF (50 ng/ml) or 45 mM KCl for 2 h leads to a decrease in pJNK immunoreactivity. Results from 4 independent experiments were quantified, normalized to JNK and expressed as percentile of control GFP cells (mean ± s.e.m., *, t-test p=0.004).

(a) Timeline of the experimental procedure used to manipulate axonal branching in vitro. (b) Number of primary axonal branches expressed as mean ± s.e.m. GFP/TrkB.Fc is significantly different from MKP-1 (***, p=0.0004, n=6 experiments), MKP-1^ASA (**, p=0.0046, n=3 experiments) and GFP/BDNF (***, p<0.0001, n=5 experiments) using a t-test. (c,e) Images (c) and quantification (e) of the number of primary branches in GFP-transfected cortical neurons derived from mkp-1^−/− mice and their wildtype littermates. Results from 3 mkp-1^+/+ and 6 mkp-1^−/− embryos were expressed as mean±s.e.m. The number of cells analyzed is indicated in the bars. BDNF induces branching in wildtype neurons only (wildtype, TrkB.Fc vs BDNF: p=0.0036; mkp-1^−/−, N.S, not significant, TrkB.Fc vs BDNF: p=0.547 using a t-test). Scale bar, 50 µm. (d) Scheme summarizing the effects on branching. Cultured neurons grow axons and branch (black outline). This branching can be enhanced by BDNF or KCl (red lines) in wildtype neurons, however, mkp-1 null neurons fail to increase arborization in response to stimulation. (f) While incubation with BDNF for 3 h decreases pJNK levels in wildtype neurons (see also Fig. 3e,f), it fails to do so in mkp-1^−/− cells.

(a,b) Localization of MKP-1 in primary cortical neurons. (a) Cytosolic (C) and nuclear (N) extracts were analyzed for endogenous MKP-1. (b) Transfected Flag-MKP-1. Scale bar, 20 µm. (c) Cortical neurons were infected with wildtype MKP-1, MKP-1^ASA or GFP between DIV1–5 and analyzed by Western blot. (d) Representative images of MKP-1, MKP-1^ASA or GFP expressing neurons. Arrows indicate the longest neurite. Scale bar, 40 µm. (e) Length of the longest neurite per cell shown as mean ± s.e.m. Numbers in bars indicate the number of cells analyzed. ***, t-test p<0.0001, n=3 independent experiments. (f) Rescue of axon growth by a MKP1-insensitive JNK mutant (JNK^R69S/D326N) when co-transfected with MKP-1. ***, t-test, p<0.001, n=4 independent experiments. (g) MKP-1 overexpression from DIV1–5 in cortical neurons affected the phosphorylation of stathmins, expressed as percentile of control GFP cells (mean ± s.e.m.; *, p<0.015, MKP1 vs. GFP and ***, p<0.001, MKP1^ASA vs. GFP using t-test; n=3 independent experiments). Mono (1p) and multi (4p) phospho-isoforms are active and inactive, respectively. (h) Tubulin tyrosination and acetylation were analyzed to assess microtubule stability. Results are expressed as percentile of control (mean ± s.e.m., n=3 independent experiments). Significant differences are as follows: MKP-1 vs. GFP (**, tyr, p=0.03; **, ace, p=0.006; ***, de-tyr, p=0.0001) and MKP-1^ASA vs. GFP (***, tyr, p=0.001; **, ace, p=0.004; ***, de-tyr, p<0.0001) using t-test.

(a) A 5-min pulse treatment with 50 ng/ml BDNF induces transient expression of MKP-1. Arc is shown for comparison. MKP-1 protein levels were expressed as fold induction compared to pre-stimulation (mean + s.d., n=3). MG132 was added just after the BDNF pulse for the indicated times. (b) Scheme of the tet^OFF system composed of 2 plasmids used to uncouple MKP-1 expression from BDNF signaling. (c) MKP-1 protein turnover was rapid but prolonged by BDNF-induced phosphorylation in 293-TrkB cells when MKP-1 induction was uncoupled from BDNF signaling. Addition of doxycyclin (DOX) turned off MKP-1 expression. The * indicates that the sample was not treated with BDNF. (d) Schematic representation of the setup used to physically separate axons from the somatodendritic compartment. (e) Representative Western blots of the material scraped from both sides of the filter membrane. Pre-treatment of the total side with K252a, TrkB.Fc or U0126 exerted distinct effects on retrograde BDNF signaling and MKP-1 induction. Tau and MAP2 are markers of the axons and somatodendritic compartments, respectively. (f) To uncouple MKP-1^ASA induction from BDNF signaling we used a tet^ON system where DOX induced MKP-1 in cortical neurons infected at DIV1–5. 1p and 4p indicate the mono- and multi-phosphorylated forms of STMN1, respectively.