CEP-1347 increases ChAT activity in culture and promotes cholinergic neurone survival following fimbria-fornix lesion.

Recent evidence suggests that the activation of the Jun N-terminal kinase (JNK) signal transduction pathway may be important in neuronal responses to stresses such as trophic factor deprivation. Preventing the activation of JNK and expression of c-Jun may, therefore, be neuroprotective. Here, we report that the small molecule CEP-1347, which has been shown to inhibit the JNK signalling pathway, promotes cholinergic activity in cultured embryonic septal neurones. In vivo, we have shown that CEP-1347, administered either by sub-cutaneous (s.c.) injection or by continuous infusion, is partially neuroprotective, for cholinergic neurones in the medial septum, following fimbria-fornix transection. These data suggest that small molecules such as CEP-1347 may have beneficial effects in treating neurodegenerative diseases.

MPTP activates c-Jun NH(2)-terminal kinase (JNK) and its upstream regulatory kinase MKK4 in nigrostriatal neurons in vivo.

The neuropathology of Parkinson's disease is reflected in experimental animals treated with the selective nigrostriatal dopaminergic neurotoxin MPTP. Neurons exposed to MPTP (MPP(+)) express morphological features of apoptosis, although the intracellular pathways that produce this morphology have not been established. The c-Jun NH(2)-terminal kinase (JNK) signaling cascade has been implicated as a mediator of MPTP-induced apoptotic neuronal death based on the ability of CEP-1347/KT-7515, an inhibitor of JNK activation, to attenuate MPTP-induced nigrostriatal dopaminergic degeneration. In these studies, MPTP-mediated activation of the JNK signaling pathway was assessed in the nigrostriatal system of MPTP-treated mice. MPTP elevated levels of phosphorylated JNK and JNK kinase (MKK4; also known as SEK1 or JNKK), by 2.5- and fivefold, respectively. Peak elevations occurred soon after administration of MPTP and coincided with peak CNS levels of MPP(+). Increased MKK4 phosphorylation, but not JNK phosphorylation, was found in the striatum, suggesting that activation of MKK4 occurs in injured dopaminergic terminals. Both JNK and MKK4 phosphorylations were attenuated by pretreatment with l-deprenyl, indicating that these phosphorylation events were mediated by MPP(+). Moreover, CEP-1347/KT-7515 inhibited MPTP-mediated MKK4 and JNK signaling at a dose that attenuates MPTP-induced dopaminergic loss. These data implicate this signaling pathway in MPTP-mediated nigrostriatal dopaminergic death and suggest that it may be activated in the degenerative process in Parkinson's disease.

CEP-1347/KT-7515, an inhibitor of c-jun N-terminal kinase activation, attenuates the 1-methyl-4-phenyl tetrahydropyridine-mediated loss of nigrostriatal dopaminergic neurons In vivo.

We have identified a bis-ethylthiomethyl analog of K-252a, CEP-1347/KT-7515, that promotes neuronal survival in culture and in vivo. The neuronal survival properties of CEP-1347/KT-7515 may be related to its ability to inhibit the activation of c-jun N-terminal kinase, a key kinase in some forms of stress-induced neuronal death and perhaps apoptosis. There is evidence that the selective nigrostriatal dopaminergic neurotoxin, MPTP, produces neuronal apoptosis in culture and in adult mice. Thus, our studies were designed to determine if CEP-1347/KT-7515 could protect dopaminergic neurons from MPTP-mediated neurotoxicity. CEP-1347/KT-7515 was assessed for neuroprotective activity in a low dose MPTP model (20 mg/kg) where there was a 50% loss of striatal dopaminergic terminals in the absence of substantia nigra neuronal loss, and a high dose (40 mg/kg) MPTP model where there was a complete loss of dopaminergic terminals and 80% loss of dopaminergic cell bodies. In the low dose MPTP model, CEP-1347/KT-7515 (0.3 mg/kg/day) attenuated the MPTP-mediated loss of striatal dopaminergic terminals by 50%. In the high dose model, CEP-1347/KT-7515 ameliorated the loss of dopaminergic cell bodies by 50% and partially preserved striatal dopaminergic terminals. CEP-1347/KT-7515 did not inhibit monoamine oxidase B or the dopamine transporter, suggesting that the neuroprotective effects of CEP-1347/KT-7515 occur downstream of the metabolic conversion of MPTP to MPP+ and accumulation of MPP+ into dopaminergic neurons. These data implicate a c-jun N-terminal kinase signaling system in MPTP-mediated dopaminergic degeneration and suggest that CEP-1347/KT-7515 may have potential as a treatment for Parkinson's disease.

Preservation of cholinergic activity and prevention of neuron death by CEP-1347/KT-7515 following excitotoxic injury of the nucleus basalis magnocellularis.

We have identified a class of small organic molecules, derived from the indolocarbazole K-252a, that promote the survival of cultured neurons. However, many of these indolocarbazoles inhibit protein kinase C and neurotrophin-activated tyrosine kinase receptors. These kinase inhibitory activities may limit the utility of these compounds for neurological disorders. A bis-ethyl-thiomethyl analogue of K-252a, CEP-1347/KT-7515, has been identified that lacks protein kinase C and tyrosine kinase receptor inhibitory activities, yet retains the ability to promote survival of cultured neurons, including cholinergic neurons derived from the basal forebrain. In the present studies, CEP-1347/KT-7515 was assessed for neurotrophic activity on basal forebrain neurons of in vivo rats following excitotoxic insult. Ibotenate infusion into the nucleus basalis magnocellularis reduced levels of choline acetyltransferase activity in the cortex, as well as reduced numbers of choline acetyltransferase-immunoreactive and retrogradely (FluoroGold)-labelled cortically-projecting neurons in the nucleus basalis. Systemically administered CEP-1347/KT-7515 attenuated the loss of cortical choline acetyltransferase activity and the loss of the number of choline acetyltransferase-immunoreactive and retrogradely-labelled FluoroGold neurons in the nucleus basalis. Moreover, CEP-1347/KT-7515 ameliorated the loss of cortical choline acetyltransferase if administration was initiated one day, but not seven days post-lesion. Together, these results demonstrate that CEP-1347/KT-7515 protects damaged cortically-projecting basal forebrain neurons from degeneration. Thus, CEP-1347/KT-7515 may have therapeutic potential in neurodegenerative diseases, such as Alzheimer's disease, in which basal forebrain cholinergic neurons degenerate.

High levels of synthesis and local effects of nerve growth factor in the septal region of the adult rat brain.

NGF found in the basal forebrain is believed to be localized to NGF-dependent cholinergic neurons and derived via retrograde axonal transport from NGF-synthesizing target hippocampal and cortical neurons. The basis for this concept of target-derived NGF is the detection of only limited amounts of NGF mRNA in the basal forebrain, despite relatively high NGF levels there. Our work, using a more sensitive and quantitative RNase protection method for detecting relative NGF mRNA levels, suggested, instead, relatively high levels of NGF mRNA synthesis in the septal region of the basal forebrain (BF-S), a region which contained primarily cells that project to the hippocampus. Similar results were obtained in analyses of a larger portion of the basal forebrain, designated "BF," that encompassed cholinergic neurons that project to both the hippocampus and the cortex. The level of NGF mRNA measured in both BF-S and BF was equivalent to approximately 50% of the amount observed in the hippocampus. Furthermore, relative NGF mRNA levels detected in the BF-S, cortex, and hippocampus were shown to be proportional to NGF protein levels quantitated in each region. The detection of relatively high amounts of NGF synthesis in the BF-S was supported in studies demonstrating rapid NGF receptor (Trk) activation in the basal forebrain by exogenous NGF and in experiments showing that NGF mRNA was inducible in the BF-S by 1,25 dihydroxyvitamin D3. The extent of NGF mRNA induction was similar (approximately twofold) in the BF-S, hippocampus, and cortex, suggesting similar regulatory mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Systemic dexamethasone administration increases septal Trk autophosphorylation in adult rats via an induction of nerve growth factor.

Nerve growth factor (NGF) maintains cholinergic neurons in various animals models of neurodegeneration and is thus a potential treatment for certain neurodegenerative disorders such as Alzheimer's disease. Because NGF does not cross the blood-brain barrier, we have proposed elevating endogenous levels of NGF in the central nervous system with small molecules that induce NGF expression, as an alternative strategy. The present studies were conducted to determine whether pharmacologically elevated levels of NGF are sufficient to cause subsequent stimulation of its high affinity receptor, as measured by increased levels of Trk phosphorylation. Dexamethasone (0.5-20 mg/kg, intraperitoneally) caused a time- and dose-dependent increase in NGF mRNA and NGF protein in the hippocampus and septum of adult male Sprague-Dawley rats. Exogenously administered NGF (1 microgram, intracerebroventricularly) led to a rapid (30 min) and transient increase in Trk phosphorylation in the septum, which has high levels of NGF-specific TrkA. Similarly, dexamethasone led to an increase in Trk phosphorylation only within the septum. Dexamethasone-mediated Trk phosphorylation was dose and time dependent, with peak increases being observed 12 hr after injection, concurrently with peak increases in NGF protein. These data demonstrate an increase in activation of the high affinity NGF receptor with a compound that elevates levels of NGF in the central nervous system, and they support the strategy of discovering a pharmacological agent that induces NGF as a method for treating neurodegenerative disorders.

Chronic 1,25-dihydroxyvitamin D3-mediated induction of nerve growth factor mRNA and protein in L929 fibroblasts and in adult rat brain.

We have proposed that elevating levels of nerve growth factor (NGF) in the CNS is a rational strategy for treating certain neurodegenerative disorders. The present studies were conducted to determine: (1) if pharmacologically induced levels of NGF could be sustained for an extended time, and (2) if correlations exist between increases in NGF mRNA and NGF protein in L929 cells and in vivo. Short-term treatment of L929 cells with 1,25-dihydroxyvitamin D3 resulted in a two-fold increase in both NGF mRNA and NGF protein. These increases were sustained for up to 48 h with continuous exposure to 1,25-dihydroxyvitamin D3. In rats, 1,25-dihydroxyvitamin D3 (2.5 nmol; i.c.v.) induced NGF mRNA transiently, with peak two-fold increases observed 4 h post-injection. In contrast to L929 cells, 1,25-dihydroxyvitamin D3 did not elicit an increase in NGF protein after a single administration in vivo. However, consistent with long-term exposure in L929 cells, chronic 6 day infusion of 1,25-dihydroxyvitamin D3 resulted in induction of both NGF mRNA and NGF protein in the brain. These results indicate that 1,25-dihydroxyvitamin D3-mediated NGF induction in cultured L929 cells may predict of NGF induction in vivo, suggesting that L929 cells may have utility in studying underlying mechanisms of NGF induction by 1,25-dihydroxyvitamin D3. On the basis of NGF's ability to increase cholinergic function in animal models of cholinergic degeneration, these results are supportive of a role for NGF inducers as potential drugs for neurodegenerative disorders.

Pharmacological induction of nerve growth factor mRNA in adult rat brain.

Three structurally unrelated compounds, all of which induce nerve growth factor (NGF) in cell culture systems, were assessed for their ability to induce NGF mRNA in adult rat brain using a highly sensitive RNAse protection assay. Interleukin-1 beta (0.5-1 pmol) and 1,25-dihydroxyvitamin D3 (25-25,000 pmol) were extremely potent inducers of NGF mRNA, being respectively at least 50,000 and 4000 times more potent than 4-methylcatechol. These compounds elicited an approximate twofold increase in NGF mRNA in both the hippocampus and cortex, without altering beta-actin mRNA levels after a single intracerebroventricular injection. The duration of NGF induction was dependent on the compound administered. For example, the elevation of NGF mRNA elicited by interleukin-1 beta peaked at 8 h and lasted for at least 24 h. In contrast, the induction of NGF after 1,25-dihydroxyvitamin D3 and 4-methylcatechol administration peaked between 4 and 8 h and was not apparent 24 h after injection. These results demonstrate induction of NGF mRNA in vivo by administration of physiological or pharmacological agents and differentiate these agents by potency and duration of action. Further, these findings indicate that pharmacological induction of NGF may be a viable strategy for the treatment of neurodegenerative disorders such as Alzheimer's disease.

Dopaminergic neurotoxicity of 1-methyl-4-phenylpyridinium analogs in cultured neurons: relationship to the dopamine uptake system and inhibition of mitochondrial respiration.

Several analogs of 1-methyl-4-phenylpyridinium (MPP+) were evaluated for their affinity for the dopamine uptake system and their ability to inhibit NADH dehydrogenase (complex I) of the mitochondrial electron-transport chain. Moreover, these compounds were tested for their ability to cause selective dopaminergic neurotoxicity in cultured mesencephalic neurons. Simultaneous [3H]dopamine and gamma-amino-[14C]butyric acid uptake and immunocytochemical techniques were used as indices of neuronal damage in cultured cells. The compounds that were potent and selective dopaminergic neurotoxins had high affinity for the dopamine transport system, as measured by their ability to cause dopamine release, and were similar to MPP+ in inhibiting mitochondrial respiration. One compound (1-methyl-4-phenylpyrimidinium) had high affinity for the dopamine uptake system but was a weak inhibitor of mitochondrial respiration and, accordingly, was not neurotoxic. The 4'-alkylated analogs of MPP+, which were poor substrates for the dopamine uptake system and extremely potent inhibitors of mitochondrial respiration, caused a nonselective damage of neurons in culture. Analogs that were not substrates for the dopamine carrier and not inhibitors of mitochondrial respiration were not neurotoxic. This study describes the neurotoxicity of a number of analogs of MPP+ and highlights the importance of the dopamine uptake system and the ability to inhibit mitochondrial respiration as critical processes in conferring selectivity and neurotoxicity, respectively, to MPP+ and analogs, for dopaminergic neurons in culture.

1-Methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine-induced toxicity in PC12 cells is enhanced by preventing glycolysis.

The effects of 1-methyl-4-(2'-ethylphenyl)-1,2,3,6-tetrahydropyridine (2'Et-MPTP), 1-methyl-4-(2'-ethylphenyl)pyridinium (2'Et-MPP+), and the classic complex 1 inhibitor, rotenone, on toxicity as well as on rates of glucose use and lactate production were studied using the pheochromocytoma PC12 cell line. PC12 cells are neoplastic in nature and have a high rate of glycolysis accompanied by a large production of lactate and a low use of glucose carbon through the Krebs cycle. 1-Methyl-4-phenylpyridinium (MPP+) and analogues such as 2'Et-MPP+ are actively accumulated by mitochondrial preparations in vitro and block NADH dehydrogenase of complex 1. This blockade results in biochemical sequelae that are ultimately cytotoxic. In this study, untreated PC12 cells used glucose and concomitantly accumulated lactate in a time-dependent manner at all concentrations of glucose studied. Treatment with 50 microM 2'Et-MPP+ or 50 nM rotenone increased both rates significantly, indicating a shift toward increased glycolysis. Cell death caused by the neurotoxins was also time and concentration dependent and markedly enhanced by glucose depletion in the medium. The increase in 2'Et-MPTP-induced toxicity in low glucose-supplemented cells was not due to an increase in pyridinium formation from the tetrahydropyridine, but rather to the lack of glucose for glycolysis. Moreover, inhibition of glycolysis with 2-deoxyglucose or iodoacetic acid also enhanced the lethality of the neurotoxins to the cells. The data in this study provide additional support to the hypothesis that 2'Et-MPP+ or related analogues act to kill cells by inhibiting mitochondrial respiration.(ABSTRACT TRUNCATED AT 250 WORDS)

Inhibition of serotonin release by bombesin-like peptides in rat hypothalamus in vitro.

We investigated the activity of bombesin (BN), neuromedin-C (NM-C) and neuromedin-B (NM-B) on serotonin (5-HT) release and reuptake in rat hypothalamus (HYP) in vitro. BN and NM-C but not NM-B (all 1 microM) decreased K+ evoked 3H-5-HT release from superfused HYP slices by 25%. Bacitracin (BCN, 2 micrograms/ml), a nonspecific peptidase inhibitor, reversed the inhibitory effect of BN on K+ evoked 3H-5-HT release. Phosphoramidon (PAN, 10 microM) an endopeptidase 24.11 inhibitor, abolished the inhibitory effect of BN, but not NM-C, on K+ evoked 3H-5-HT release. The peptidyl dipeptidase A inhibitor enalaprilat (ENP, 10 microM), enhanced both BN and NM-C inhibition of 3H-5-HT release. Bestatin (BST, 10 microM) had no effect on BN or NM-C inhibitory activity on 3H-5-HT release. Neither BN, NM-C nor NM-B affected reuptake of 3H-5-HT into HYP synaptosomes alone or in combination with any of the peptidase inhibitors, nor did these peptides alter the ability of fluoxetine to inhibit 3H-5-HT uptake. These data suggest: a) that BN-like peptides may alter neurotransmission in the HYP by acting presynaptically on the 5-HT release mechanism; b) a similarity in the structural requirements for the BN induced inhibition of 5-HT release and BN evoked thermoregulatory disturbances; and c) that peptidases may selectively augment or reduce pharmacologic activity of BN-like peptides upon CNS administration.