QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

First published online 19 February 2008
doi: 10.1242/jcs.019166

This Article Summary Full Text Full Text (PDF) Supplementary Material Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via Google Scholar Google Scholar Articles by Cosker, K. E. Articles by Eickholt, B. J. Search for Related Content PubMed PubMed Citation Articles by Cosker, K. E. Articles by Eickholt, B. J.

Regulation of PI3K signalling by the phosphatidylinositol transfer protein PITP during axonal extension in hippocampal neurons

¹ MRC Centre for Developmental Neurobiology, King's College London, London, SE1 1UL, UK
² Department of Physiology, University College London, London, WC1E 6JJ, UK
³ Wolfson Centre for Age-Related Diseases, King's College London, London, SE1 1UL, UK

View larger version (113K): [in this window] [in a new window]   Fig. 1. PITP is highly enriched in the adult and developing brain. (A) Comparison of PITP and PITPβ expression in the adult rat. Equal protein amounts of indicated tissue extracts were separated by SDS-PAGE and probed using anti-PITP or anti-PITPβ antibodies. For quantification of PITP and PITPβ expression see Fig. S1 in the supplementary material. Bars show the mean of three independent analyses ± s.e.m. (B) In the adult mouse cerebellum, anti-PITP labelling was detected in the molecular layer and the granule cell layer, whereas low signals were associated with the Purkinje cell layer and the white matter tract. (C) In the P15 mouse cerebellum, PITP was expressed in the molecular layer and in the granule cell layer. In addition, afferent fibres in the developing white matter tract were positive for PITP immunoreactivity. In control experiments performed in parallel, no signal was detected in the presence of control serum (see insert). (D) PITP was broadly expressed in the CA3 cells of the adult hippocampus. A higher magnification of the outlined box in D is shown in E. (E) Particularly enriched labelling was seen in the stratum radiatum. The cell bodies of the pyramidal neurons were devoid of PITP. (F) PITP was broadly expressed in the P15 hippocampus. Scale bars: 200 µm in B; 100 µm in D,F; 50 µm in E. m, molecular layer; g, granule cell layer; pl, Purkinje cell layer; py, pyramidal neurons; sr, stratum radiatum; wm, white matter tract.

View larger version (60K): [in this window] [in a new window]   Fig. 2. Localization of PITP during neuronal maturation in hippocampal neurons. E18 hippocampal neurons were cultured for 2, 5 and 14 days before fixation and antibody labelling. (A) At day 2 (stage 3 neurons), PITP mainly localized to axonal processes (arrowhead). No or little staining was present in the remaining shorter processes clearly visible in the Phalloidin staining visualizing F-actin (red). (B) Line scan of a stage 3 hippocampal neuron demonstrating the relative intensity of PITP fluorescence along each minor neurite (n1-n4) and the axon. (C) Mean relative PITP fluorescence intensity in neurites and axonal processes. Each data point is the mean ± s.e.m., n=7. (D) Percentage of hippocampal neurons that showed PITP fluorescence in all processes (ubiquitous) or in the axon, only. Each data point is the mean ± s.e.m., n=106, *P<0.0001. (E) PITPβ was enriched in the Golgi region of stage 3 hippocampal neurons. (F) At day 5 (stage 4 neurons), anti-PITP antibody labelled both axonal and dendritic processes. (G) At day 14, anti-PITP immunostaining appeared punctate and showed some co-localization with the pre-synaptic marker synaptophysin (red). (H) Hippocampal neurons were cultured for indicated days in vitro (DIV), lysed and 10 µg protein was separated and analysed by western blotting using anti-PITP. Scale bars: 20 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 3. Overexpression of PITP increases axonal growth in hippocampal neurons. (A,B) E18 hippocampal neurons were nucleofected with indicated constructs and cultured for 48 hours. (A) GFP distributed throughout the neuron, whereas (B) PITP-GFP localized to the axons of stage 3 hippocampal neurons. Little signal was detected in immature dendrites (arrowheads); these minor processes, however, were clearly visible in the Phalloidin staining (see insert, red). (C) Representative outlines of hippocampal neurons expressing GFP, PITP-GFP or PITP siRNA. (D) Hippocampal neurons expressing GFP, PITP-GFP or PITPβ-GFP were analysed by immunoblotting using an anti-GFP antibody. (E) Evaluation of process length. Neurites were scored as polarized in the presence of a single, long neurite exhibiting no less than twice the length of the remaining shorter neurites. The length of the longest neurite (axon), and all remaining shorter neurites (immature dendrites) of an individual neuron were measured within each treatment. The asterisk indicates significant increases in axonal length following overexpression of PITP-GFP in comparison with untransfected (WT) or GFP-expressing neurons (*P<0.0001). This effect was specific to the PtdIns transfer activity of PITP, as PITP K61A, a mutant unable to bind and transfer PI, or PITPβ did not induce increased axonal length. All data are the mean ± s.e.m. of at least three independent experiments. Scale bar: 20 µm.

View larger version (32K): [in this window] [in a new window]   Fig. 4. Knockdown of PITP by siRNA decreases axonal growth in hippocampal neurons. E18 hippocampal neurons were nucleofected with indicated constructs or siRNAs, and cultured for 48 hours. (A) PITP siRNA, but not control scrambled siRNA, leads to a reduction in endogenous PITP levels, as validated by western blotting. (B) Knockdown of PITP by siRNA significantly decreased axonal length (*P<0.0001, **P<0.0002). All data are the means ± s.e.m. of at least three independent experiments (n>125). (C) Knockdown of PITP was monitored in each experiment by immunocytochemistry using anti-PITP antibody. Scale bar: 20 µm.

View larger version (25K): [in this window] [in a new window]   Fig. 5. PITP-induced increases in axonal length are mediated by PI3K signalling. Hippocampal neurons were nucleofected with GFP, PITP-GFP or PITP siRNA, and cultured for 48 hours on poly-lysine/laminin or poly-lysine substrates. (A) Neurons expressing PITP-GFP did not exhibit increases in axonal length when cultured on poly-lysine alone. In addition, PI3K inhibition by LY294002 (at 10 µM) antagonized PITP-GFP-mediated increases in axonal length on poly-lysine/laminin substrate. Each data point is the mean ± s.e.m. of at least three independent experiments, n=120. (B) Application of BDNF (50 ng/ml) to hippocampal neurons induces an increase in axonal length, which was partially blocked by LY294002 and fully inhibited by knockdown of PITP by siRNA. Data points show the mean ± s.e.m. of three independent experiments (n>60). (C) Cell lysates of hippocampal neurons expressing GFP, PITP-GFP or PITP siRNA were analysed by western blotting using indicated antibodies. (D) Normalized relative band density of pAkt/Akt and pAkt/actin reveal increased activity of PI3K signalling following overexpression of PITP-GFP (pAkt/actin), n=5. (E) GFP and (F) PITP-GFP expressing hippocampal neurons were fixed after 48 hours and stained using anti-pAkt antibody (red). (G) The relative fluorescence intensity of pAkt labelling in axonal growth cones of PITP-GFP expressing neurons is significantly increased when compared with GFP expressing neurons. Each data point is the mean ± s.e.m. of seven independent experiments (n>120). **P<0.005, ***P<0.0001.

View larger version (16K): [in this window] [in a new window]   Fig. 6. PITP-induced axonal elongation is dependent on Akt/GSK-3/CRMP-2 downstream of PI3K. (A) Schematic representation and signalling relationship of the PI3K/Akt/GSK-3/CRMP-2 pathway previously shown to control axon specification and elongation. In grey, the different mutant constructs and pharmacological inhibitors (LY294002) that were tested in their ability to alter PITP-induced axonal elongation downstream of PI3K signalling. (B) E18 hippocampal neurons were nucleofected with indicated constructs, cultured for 48 hours and analysed by immunoblotting using indicated antibodies. (C) Nucleofection of constitutively active GSK-3β (GSK-3β S9A) with PITP-GFP antagonise PITP-induced increases in axonal length. (D) E18 hippocampal neurons were transfected with indicated constructs by Lipofectamine. Following 48 hours, cultures were fixed and analysed as previously. LY294002 (10 µM) was applied after 8 hours. Data points show the mean ± s.e.m. of at least three independent experiments (n>60), *P<0.02, **P<0.005.