Neurons undergoing apoptosis can be rescued by trophic factors that simultaneously increase the activity of extracellular signal-regulated kinase (ERK) and decrease c-Jun N-terminal kinase (JNK) and p38. We identified a molecule, CEP-1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNK1 activity. Cultured rat embryonic motoneurons, in the absence of trophic factor, began to die 24-48 hr after plating. During the first 24 hr ERK1 activity was unchanged, whereas JNK1 activity increased fourfold. CEP-1347 completely rescued motoneurons for at least 72 hr with an EC50 of 20 +/- 2 nM. CEP-1347 did not alter ERK1 activity but rapidly inhibited JNK1 activation. The IC50 of CEP-1347 for JNK1 activation was the same as the EC50 for motoneuron survival. Inhibition of JNK1 activation by CEP-1347 was not selective to motoneurons. CEP-1347 also inhibited JNK1 activity in Cos7 cells under conditions of ultraviolet irradiation, osmotic shock, and inhibition of glycosylation. Inhibition by CEP-1347 of the JNK1 signaling pathway appeared to be selective, because CEP-1347 did not inhibit p38-regulated mitogen-activated protein kinase-activated protein kinase-2 (MAPKAP2) activity in Cos7 cells subjected to osmotic shock. The direct molecular target of CEP-1347 was not JNK1, because CEP-1347 did not inhibit JNK1 activity in Cos7 cells cotransfected with MEKK1 and JNK1 cDNA constructs. This is the first demonstration of a small organic molecule that promotes motoneuron survival and that simultaneously inhibits the JNK1 signaling cascade.

No abstract

Molecular cloning has identified two vesicular monoamine transporters (VMATs), one expressed in non-neural cells of the periphery (VMAT1) and the other by multiple monoamine cell populations in the brain (VMAT2). Functional analysis has previously shown that VMAT2 has a higher affinity than VMAT1 for monoamine neurotransmitters as well as the inhibitor tetrabenazine. The analysis of chimeric transporters has also identified two major regions required for the high affinity interactions of VMAT2 with these ligands. We have now used site-directed mutagenesis to identify the individual residues responsible for these differences. Focusing on the region that spans transmembrane domains 9 through 12, we have replaced VMAT2 residues with the corresponding residues from VMAT1. Many residues in this region had no effect on the recognition of these ligands, but substitution of Tyr-434 with Phe and Asp-461 with Asn reduced the affinity for tetrabenazine, histamine, and serotonin. Although the ability to affect recognition of multiple ligands suggests a general structural role for these residues, the mutations did not affect dopamine recognition, indicating a more specific role, possibly in recognition of the ring nitrogen that occurs in tetrabenazine, histamine, and serotonin but not dopamine. The mutation K446Q reduced the affinity of VMAT2 for tetrabenazine and serotonin but not histamine, whereas F464Y reduced serotonin affinity and perhaps histamine recognition but not tetrabenazine sensitivity, providing more evidence for specificity. Interestingly, the V max of both VMATs for dopamine exceeded that for serotonin by 3-5-fold, indicating a difference in the speed of packaging of these two neurotransmitters. We also found that VMAT1 has a higher affinity for tryptamine than VMAT2. This mutually exclusive interaction with serotonin and tryptamine also suggests a physiological rationale for the existence of two VMATs. Surprisingly, the residue responsible for this difference, Tyr-434, also accounts for the higher affinity interaction of VMAT2 with tetrabenazine, histamine, and serotonin. Interestingly, replacement of Tyr-434 with alanine increases the affinity of VMAT2 for both serotonin and dopamine and reduces the rate of dopamine transport.

Cell survival, death, and stress signals are transduced from the cell surface to the cytoplasm and nucleus via a cascade of phosphorylation events involving the mitogen-activated protein kinase (MAPK) family. We compared the distribution of p42 mitogen-activated protein kinase (p42MAPK) and its activator MAPK or ERK kinase (MEK1; involved in transduction of growth and differentiation signals), with c-Jun N-terminal kinase (JNK1) and its activator MEK4 (involved in transduction of stress and death signals) in the adult rat central nervous system. All four kinases were present in the cytoplasm, dendrites, and axons of neurons. The presence of p42MAPK and JNK1 in dendrites and axons, as well as in cell bodies, suggests a role for these kinases in phosphorylation and regulation of cytoplasmic targets. A high degree of correspondence was found between the regional distribution of MEK1 and p42MAPK. Immunostaining for MEK1 and p42MAPK was intense in olfactory structures, neocortex, hippocampus, striatum, midline, and interlaminar thalamic nuclei, hypothalamus, brainstem, Purkinje cells, and spinal cord. In addition to neurons, p42MAPK was also present in oligodendrocytes. Whereas MEK4 was ubiquitously distributed, JNK1 was more selective. Immunostaining for MEK4 and JNK1 was intense in the olfactory bulb, lower cortical layers, the cholinergic basal forebrain, most nuclei of the thalamus, medial habenula, and cranial motor nuclei. The distribution of MEK1 and p42MAPK proteins only partially overlapped with that of MEK4 and JNK1. This suggests that the growth/differentiation and death/stress pathways affected by these kinases may not necessarily act to counterbalance each other in response to extracellular stimuli. The differential distribution of these kinases may control the specificity of neuronal function to extracellular signals.

No abstract