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

First published online September 22, 2005
doi: 10.1242/10.1242/jcs.02572

This Article Summary Full Text Full Text (PDF) 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 HighWire Citing Articles via Google Scholar Google Scholar Articles by Lallemend, F. Articles by Malgrange, B. Search for Related Content PubMed PubMed Citation Articles by Lallemend, F. Articles by Malgrange, B. Social Bookmarking                         What's this?

Activation of protein kinase CßI constitutes a new neurotrophic pathway for deafferented spiral ganglion neurons

¹ Research Centre for Cellular and Molecular Neurobiology, Developmental Neurobiology Unit, University of Liège, Av. de l'Hopital 1 (B36), 4000 Liège, Belgium
² Department of Neurology, University of Liège, Av. de l'Hopital 13 (B35), Sart-Tilman, 4000 Liège, Belgium
³ Department of Otorhinolaryngology, University of Liège, Av. de l'Hopital 13 (B35), Sart-Tilman, 4000 Liège, Belgium

View larger version (64K): [in a new window]   Fig. 1. cPKC expression and cellular distribution in the spiral ganglion (SG). (A) mRNA levels of cPKCs in P5 rat brain and SG. Semi-quantitative RT-PCR was performed using primers specific for PKC, PKCßI, PKCßII, PKC and ß-actin genes. Densitometric analysis was performed and results are expressed as the ratio of cPKC gene/ß-actin in arbitrary units (au). (B) Immunoblotting for PKC, PKCßI, PKCßII and PKC from P5 rat SG or adult rat hippocampus total protein extracts. All four isoforms of cPKCs are recognised by their specific antibody at the expected molecular weight of 80 kDa in hippocampus protein extracts, whereas only PKC and PKCßI isoforms are present in SG extracts. Each western blot was repeated on at least three different occasions, and with protein extracts from distinct SG samples. (C) Representative confocal micrographs of P5 rat SG sections selected in the medial cochlear turn and double stained for ßIII tubulin (TUJ1, neuronal marker) and different cPKC isoforms detected with the antibodies used in western blot experiments. Anti-PKC antibody reveals a ubiquitous expression towards SG cells whereas anti-PKCßI antibody stains SGNs exclusively. No PKCßII or PKC were detected in SG sections. The experiments were performed in triplicate and repeated on at least three independent occasions, and observations were made in all cochlear turns, with similar results. Bar, 40 µm.

View larger version (30K): [in a new window]   Fig. 2. Specific neuronal-PKCßI activation can rescue SGNs from apoptosis induced by TFD. (A) SGNs deprived of trophic factors were cultured for 24 hours in the presence or absence of increasing concentrations of PMA. The neurons were fixed and immunostained with anti-ßIII tubulin antibody (TUJ1), as described in Materials and Methods, and the remaining number of viable neurons as a percentage of neurons in the untreated control condition was determined. All TUJ1-positive neurons were included in these counts, regardless of neurite length. Each such determination was performed in triplicate and repeated in three independent experiments. The mean value is shown in the figure; error bars in this and all subsequent figures indicate standard deviation (n=9; *P<0.05; ***P<0.001). (B) SGN cultures were treated or not with 100 nM PMA and neuronal apoptosis was determined by coupling the TUNEL bioassay to TUJ1 immunostaining 15 hours after completion of the spiral ganglia dissection. One hundred percent TUNEL-positive neurons was defined as the number of neurons presenting a TUNEL stain per total number of neurons in control cultures (n=6; ***P<0.001). The inset shows a representative confocal micrograph of TUJ1 (red)/TUNEL (green) double-stained P5 SGNs in control condition (-PMA) in which three out of five neurons are dying (arrows). Bar, 20 µm. (C) SGNs were cultured for 24 hours in the presence of 100 nM PMA and increasing concentrations of GF109203X, a specific inhibitor of PKC with high affinity for conventional isoforms. Cells were fixed and neuron viability was quantified, as determined in A (n=10; **P<0.01; ***P<0.001). (D) SGNs were incubated for 24 hours with 100 nM PMA (as a positive control) or increasing concentrations of the PMA inactive analogue, 4-PMA. Neuronal cell counts were obtained as described in A (n=6; ***P<0.001). (E) Schematic representation of the neuroprotection paradigm performed in F. (F) SGNs were cultured in the presence or absence of PMA or PDBu (each at 100 nM) in conditions presented in E. SGNs were deprived of trophic factors for 4 hours. The neurons were then subjected to a PMA or PDBu treatment for 30 minutes or 20 hours. For each medium change, wells were rinsed three times in definite control medium, i.e. without trophic factor, to ensure the total elimination of agents from wells. Neuron viability was measured as determined in A (n=6-12; *P<0.05). (G,H) Representative confocal micrographs of expression and cellular redistribution of PKC and PKCßI in SG cells before and after stimulation with a 30 minute PMA treatment. SG cells were cultured for 4 hours in a definite control medium. At this time, cultures were treated with 100 nM PMA or vehicle (control condition) for 30 minutes. Next, the cells were fixed and immunostained with TUJ1 and PKC or PKCßI antibodies. In H, PKCßI is uniformly located exclusively in the cytoplasm of neurons in untreated cultures, whereas a 30 minute treatment with PMA induces its redistribution to the cell membrane (arrowheads). Insets show two TUJ1-positive neurons at higher magnification with a cytoplasmic (control condition) or a membranous (with PMA) PKCßI staining. The experiments were performed in duplicate and repeated on at least three different occasions, with similar results. Bars, 50 µm.

View larger version (41K): [in a new window]   Fig. 3. Non-neuronal cells do not mediate PKC-activator-induced neuroprotection. (A) Schematic representation of the neuroprotection paradigm performed in B. (B) SGNs were cultured in the presence or absence of PMA or PDBu (each at 100 nM) on 96-hour SG cultures activated or not by PMA or PDBu for 30 minutes (see conditions presented in A). The number of viable neurons in the new 24-hour cultures as a percentage of neurons in the untreated control condition (a) was determined (n=6; ***P<0.001). (C) SG cells were cultured in medium containing BrdU and either vehicle or one of the tested compounds (100 nM PMA, 10% FBS, 10 µM AraC). After 15 or 24 hours, cells were fixed and processed for immunocytochemistry and propidium iodide staining to identify nuclei. The proliferative level in culture was evaluated as the number of BrdU-positive cells per total cell number in the culture. Each determination was performed in duplicate, and repeated in three independent experiments. The mean value is shown (n=6; ns, not significant; **P<0.01; ***P<0.001). (D) SG cells were cultured for 24 hours in the presence or absence of 100 nM PMA and specific anti-proliferative agents, i.e. AraC (10 µM) and FUDR (20 µM). Average numbers of TUJ1-positive SGNs under different experimental conditions were counted, as previously described in Fig. 2A (n=9; ***P<0.001). (E) AraC was used to block proliferation in culture of SGNs in the presence or absence of 100 nM PMA and, after 72 hours, cells were fixed and neuronal viability was expressed as the percentage of neurons present in the untreated control condition (n=6; ns, not significant; ***P<0.001).

View larger version (33K): [in a new window]   Fig. 4. MEK/ERK and PI3K/Akt, but not NF-B, are key components in the PMA-induced survival of SGNs. SGNs were treated for 24 hours with or without PMA at 100 nM and increasing concentrations of U0126 (U), LY294002 (LY) or 2 µM BAY 11-7082 (BAY) or 1 mM sulfasalazine (SZ) to reduce, respectively, the enzymatic activity of MEK, PI3K or NF-B complex. Neuronal survival was quantified, as determined in Fig. 2A (n=8; *P<0.05; **P<0.01).

View larger version (26K): [in a new window]   Fig. 5. PKC activation increases neurite outgrowth in SGN cultures. (A) Representative photomicrographs of TUJ1-stained SGNs in 24 hour culture treated without (CTR) or with 100 nM PMA (PMA). Arrows in the control condition show aneuritic neurons usually found in 24 hour control cultures. Bar, 100 µm. (B) SGNs were treated for 24 hours with or without increasing concentrations of PMA and in combination or not with the specific PKC inhibitor, i.e. GF (1 µM). Subsequently, cells were fixed and immunostained with TUJ1 antibody and the neuritic index in each condition as a percentage of the neuritic index in the untreated control condition was determined (n=6; ***P<0.001).

View larger version (17K): [in a new window]   Fig. 6. The survival-promoting action of neurotrophins in SGNs depends on PI3K and MEK/ERK signalling but is independent of PKC activity. (A) SGNs were cultured in the presence of BDNF or NT3 (each at 20 ng/ml) plus LY294002 or U0126 (each at 20 µM). Cells were fixed 24 hours later and the remaining number of viable neurons were counted, as determined in Fig. 2A (n=6; ***P<0.001). (B) SGNs were treated with 100 nM PMA, NT3 or BDNF (each at 20 ng/ml) in the presence or absence of 1 µM GF109203X to block the enzymatic activity of PKC. 24 hours later, cells were fixed and neuronal survival was scored, as described in Fig. 2A (n=9; ns, not significant; ***P<0.001).

View larger version (36K): [in a new window]   Fig. 7. PKC activation and neurotrophins synergistically enhance survival of SGNs. SGNs were cultured for 96 hours in the presence of different combinations of PMA (P, 100 nM), BDNF (B, 100 ng/ml) and NT3 (N, 100 ng/ml). Cells were fixed and neuronal viability in the different experimental conditions was expressed as a percentage of the average number of TUJ1-positive neurons in PMA condition. Black bars represent measurements observed in experimental conditions and grey bars represent theoretical values calculated by considering the experimental survival effect of each factor alone (n=15; ns, not significant; ***P<0.001).

View larger version (22K): [in a new window]   Fig. 8. Bryostatin 1, a non-tumour-promoting PKC activator, rescues SGNs from cell death. (A) SGNs were deprived of trophic factor in the presence of increasing concentrations of bryo or 100 nM PMA for 24 hours. Neurons were fixed and the remaining number of viable neurons were counted, as determined in Fig. 2A (n=6; **P<0.01; ***P<0.001). The inset shows a representative micrograph of TUJ1-stained SGNs in a 24 hour culture treated with bryo (10 nM). Bryo treatment strongly enhanced neurite outgrowth, in comparison with the control condition in Fig. 5B. Bar, 80 µm. (B) Representative photomicrographs of PKCßI membrane redistribution in SGNs upon 30 minute bryo exposure. SGNs were cultured for 4 hours in control medium. Next, cultures were treated for 30 minutes with 10 nM bryo. Cells were then fixed and immunostained with PKCßI and TUJ1 antibodies; nuclear staining with TO-PRO-3 was used to appreciate cell density. Before the bryo treatment, PKCßI had a uniform cytoplasmic distribution, as seen in the control condition (Fig. 2H). The experiments were performed in duplicate and repeated on two different occasions, with similar results. Bar, 40 µm. (C) SGNs were treated with 100 nM PMA or 10 nM bryo in the presence or absence of 1 µM GF109203X. After 24 hours, cells were fixed and neuronal viability was examined, as described in Fig. 2A (n=6; ***P<0.001).

View larger version (13K): [in a new window]   Fig. 9. PKC activation increases survival of adult SGNs. Adult SGNs were cultured in the presence or absence of bryo (10 nM) for 24 hours. Numbers of viable neurons in cultures were counted, as determined previously in Fig. 2A (n=6; ***P<0.001).

View larger version (22K): [in a new window]   Fig. 10. A model showing the multiple actions of PKC activators (PMA/bryostatin) and neurotrophins (BDNF/NT3) on intracellular signalling pathways that lead to protection and neuritogenesis of SGNs after deafferentation in vitro. Upon activation of PKCßI and TrkB/TrkC, both the PI3K/Akt and MAPK/ERK pathways are required for protection and neurite extension of SGNs, as demonstrated by using specific inhibitors of PKC (GF109203X, Gö6976, LY333531), PI3K (LY294002) and MEK (U0126). The combination of neurotrophins with PMA treatment leads to a synergistic action on SGN survival. PYK2 represents an intermediary kinase, which could be involved in the signal pathway linking PKCßI activity to PI3K signalling (see Discussion for details).

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter