Functions of JNK stresskinases in neuronal apoptosis and differentiation

The c-Jun N-terminal kinases (JNKs), a subfamily of the mitogen-activated protein kinases (MAPKs), are considered as essential signalling molecules for neurodegeneration in the mammalian brain. However, they also have physiological functions such as in development, differentiation, proliferation and neuroregeneration. The aims of the present study were to determine the physiological and pathological roles of JNK isoforms in PC12 cells and in primary neurons. 6-OHDA enhanced total JNK activity in the cytoplasm, nucleus and at the mitochondria in PC12 cells. Inhibition of JNKs by SP600125 or transfection with dominant negative JNK2 (dnJNK2) but not dnJNK1, substantially attenuated 6-OHDA-induced cell death. JNK2 translocated into the nucleus and mitochondria following 6-OHDA and mediates cytochrome c release and caspase-3 activation. In primary neurons JNKs also mediate death. Glutamate and 6-OHDA induced death and caspase-3 activation in primary hippocampal and cortical neurons, respectively. Inhibition of JNKs with SP600125 rescued these otherwise dying cells and inhibited caspase-3 activation, indicating JNK-mediated death. Besides neurodegenerative and pro-apoptotic functions, the family of JNKs has also been implicated in neuronal differentiation and development. Therefore the role of JNKs in neuronal differentiation were also analysed in this study. Inhibition of JNKs by SP600125 prevented neurite elongation in primary hippocampal neurons. These results provide evidence that JNKs have apoptotic effects after an excitotoxic stimulus but in parallel to their apoptotic effects they have also physiological functions on neurite formation and elongation in vitro during neuritogenesis

The JNK Inhibitor XG-102 Protects against TNBS-Induced Colitis

The c-Jun N-terminal kinase (JNK)-inhibiting peptide D-JNKI-1, syn. XG-102 was tested for its therapeutic potential in acute inflammatory bowel disease (IBD) in mice. Rectal instillation of the chemical irritant trinitrobenzene sulfonic acid (TNBS) provoked a dramatic acute inflammation in the colon of 7–9 weeks old mice. Coincident subcutaneous application of 100 µg/kg XG-102 significantly reduced the loss of body weight, rectal bleeding and diarrhoea. After 72 h, the end of the study, the colon was removed and immuno-histochemically analysed. XG-102 significantly reduced (i) pathological changes such as ulceration or crypt deformation, (ii) immune cell pathology such as infiltration and presence of CD3- and CD68-positive cells, (iii) the production of tumor necrosis factor (TNF)-α in colon tissue cultures from TNBS-treated mice, (iv) expression of Bim, Bax, FasL, p53, and activation of caspase 3, (v) complexation of JNK2 and Bim, and (vi) expression and activation of the JNK substrate and transcription factor c-Jun. A single application of subcutaneous XG-102 was at least as effective or even better depending on the outcome parameter as the daily oral application of sulfasalazine used for treatment of IBD

c-Jun and phospho-c-Jun.

<p>Western blot analysis of c-Jun and phospho-c-Jun in colon homogenates from untreated controls (co) and 12 h or 24 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Hematoxylin and eosin (H&E) scores.

<p>Hematoxylin and eosin (H&E) scores from distal (A) and medial (B) colon. For the tissue damage score (hatched bars), the scores of ulcer, crypts and submucosa were summed-up for each individual animal, and the mean±SEM was calculated for each group. The mean±SEM of the infiltration score (grey bar) is separately shown. ***, ** = p<0.001 and p<0.01 for all groups compared with TNBS group.</p

JNK2-Bim co-precipitation.

<p>JNK1 and JNK2 immunoprecipitates (IP) from colon tissue homogenates were analyzed by Western blotting with an anti-Bim antibody. Pounceau staining demonstrated equal loading (data not shown).</p

Representative CD3 and CD68 immunofluorescence.

<p>Representative CD3 (left) and CD68 (right) immunofluorescence of the distal colon from normal mice, trinitrobenzene sulfonic acid (TNBS) administration and treatment with sc. 100 µg/kg XG-102.</p

Apoptosis.

<p>Western blot analysis of (A) caspase-3 and cleaved caspase-3, (B) Bax and Bim, and (C) FasL and p53 from colon extracts of untreated controls (co) 12 h, 24 h and 72 h following trinitrobenzene sulfonic acid (TNBS) administration without or with XG-102 (100 µg/kg sc.). These blots are representative of 3 independent experiments.</p

Production of TNF-α.

<p>TNF-α release (pg/ml) into the supernatant of organic colon culture from normal mice, following trinitrobenzene sulfonic acid (TNBS) only, and treatment with sc. 100 µg/kg XG-102. ** = p<0.01 for all groups compared with TNBS group.</p

Tissue damage scores.

<p>Tissue damage scores.</p