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.     

Pharmacokinetic interactions of CEP-1347 and atazanavir in HIV-infected patients

CEP-1347 is a potent inhibitor of mixed lineage kinase (MLK), which was investigated for ameliorating HIV-associated neurocognitive disorders. CEP-1347 and atazanavir pharmacokinetics were determined when CEP-1347 50 mg twice daily was administered to HIV-infected patients (n?=?20) receiving combination antiretroviral therapy including atazanavir and ritonavir (ATV/RTV, 300/100 mg) once daily continuously. Co-administration of CEP-1347 and ATV/RTV resulted with significant changes in pharmacokinetics of ATV but not RTV. Specifically, an increase in ATV accumulation ratio of 15 % (p?=?0.007) and a prolongation of T _1/2 from 12.7 to 15.9 h (p?=?0.002) were observed. The results suggested that co-administration of CEP-1347 with ATV/RTV in HIV-infected patients might result in limited impact on ATV but not on RTV pharmacokinetics.     

The safety and tolerability of a mixed lineage kinase inhibitor (CEP-1347) in PD

CEP-1347 is an inhibitor of members of the mixed lineage kinase family, key signals triggering apoptotic neuronal death. The authors performed a randomized, blinded, placebo-controlled study assessing the safety, tolerability, pharmacokinetics, and acute symptomatic effects of CEP-1347 in 30 patients with Parkinson's disease (PD). In this short-term study, CEP-1347 was safe and well tolerated. It had no acute effect on parkinsonian symptoms or levodopa pharmacokinetics, making it well suited for larger and longer studies of its potential to modify the course of PD.     

CEP-1347 reduces mutant huntingtin-associated neurotoxicity and restores BDNF levels in R6/2 mice

Huntington's disease (HD) is a devastating neurodegenerative disorder caused by an expanded polyglutamine repeat within the protein Huntingtin (Htt). We previously reported that mutant Htt expression activates the ERK1/2 and JNK pathways [Apostol, B.L., Illes, K., Pallos, J., Bodai, L., Wu, J., Strand, A., Schweitzer, E.S., Olson, J.M., Kazantsev, A., Marsh, J.L., Thompson, L.M., 2006. Mutant huntingtin alters MAPK signaling pathways in PC12 and striatal cells: ERK1/2 protects against mutant huntingtin-associated toxicity. Hum. Mol. Genet. 15, 273-285]. Chemical and genetic modulation of these pathways promotes cell survival and death, respectively. Here we test the ability of two closely related compounds, CEP-11004 and CEP-1347, which inhibit Mixed Lineage Kinases (MLKs) and are neuroprotective, to suppress mutant Htt-mediated pathogenesis in multiple model systems. CEP-11004/CEP-1347 treatment significantly decreased toxicity in mutant Htt-expressing cells that evoke a strong JNK response. However, suppression of cellular dysfunction in cell lines that exhibit only mild Htt-associated toxicity and little JNK activation was associated with activation of ERK1/2. These compounds also reduced neurotoxicity in immortalized striatal neurons from mutant knock-in mice and Drosophila expressing a mutant Htt fragment. Finally, CEP-1347 improved motor performance in R6/2 mice and restored expression of BDNF, a critical neurotrophic factor that is reduced in HD. These studies suggest a novel therapeutic approach for a currently untreatable neurodegenerative disease, HD, via CEP-1347 up-regulation of BDNF.     

Riluzole promotes cell survival and neurite outgrowth in rat sensory neurones in vitro

This study explored the effects of riluzole administration on cell survival and neurite growth in adult and neonatal rat dorsal root ganglion (DRG) neurones in vitro. Neuronal survival was assessed by comparing numbers of remaining neurones in vehicle- and riluzole-treated cultures. A single dose of 0.1 microm riluzole was sufficient to promote neuronal survival in neonatal DRG cultures, whereas repeated riluzole administration was necessary in adult cultures. However, a single administration of riluzole was sufficient to induce neuritogenesis, promote neurite branching and enhance neurite outgrowth in both neonatal and adult DRG cultures. The effects of a single dose of riluzole on adult DRG neurones after peripheral nerve or dorsal root injury were also studied in vitro at 48 h. For both types of injury, riluzole enhanced neurite outgrowth in terms of number, length and branch pattern significantly more on the injured side as compared with the contralateral side. No effect was seen on cell survival. The results suggest that, in addition to its cell survival effects, riluzole has novel growth-promoting effects on sensory neurones in vitro and that riluzole may offer a new way to promote sensory afferent regeneration following peripheral injury.     

A rapid method for determination of cell survival in primary neuronal DRG cultures

A simple and rapid enzyme-linked immunosorbent assay (ELISA) has been developed to provide an alternative to cell counting to detect increases in cell survival in primary neuronal cultures. This sensitive assay has the advantage of being less time consuming and labour intensive than cell counting, can be used to quantify cell survival and is more accurate than estimation methods of counting. The ELISA uses an antibody raised to GAP-43, a growth-associated protein which is strongly expressed by developing and regenerating neurones. The effects of nerve growth factor (NGF), neurotrophin-3 (NT-3) and brain-derived neurotrophic factor (BDNF) on GAP-43 immunoreactivity in dissociated primary cultures of rat and chick dorsal root ganglia have been compared to results obtained by cell counting. Data show that human NGF produced the greatest increase in GAP-43-immunoreactive neurones in both species; this increase in immunoreactivity correlated well with the increased survival shown by cell count data. Results prove that the ELISA can also be used to accurately detect small changes in cell survival as seen with NT-3 and BDNF, or potentiation of the effects obtained with the trophic factor NT-3. In conclusion, this ELISA may be a useful tool to detect neurotrophic effects of unknown agents or novel neurotrophins.     

Role of NGF in spared DRG following partial dorsal rhizotomy in cats

Neuroplasticity occurs in the spinal cord in response to lesions, but less is known about the underlying mechanism. This investigation explored the role of intrinsic NGF in axonal sprouting of dorsal root ganglia (DRG) in cats subjected to unilateral removal of L1–L5, L7–S2 DRG, but leaving the L6 DRG (spared DRG) undamaged. The expression of mRNA and protein for NGF and TrkA increased significantly by using in situ hybridization histochemistry and immunohistochemistry. ELISA assay showed that the level of NGF was up-regulated in the spared DRG, compared to the control side. In vitro studies showed that cultured neurons prepared from DRG explants of cats that received partial ganglionectomy had greater neurite growth compared to those prepared from untreated controls, and that such increase in neurite was not observed in explants from cats that received partial ganglionectomy and NGF antibody treatment. Taken together, the present findings provided crucial evidence that NGF in DRG might be involved in axonal sprouting in deafferentated cats.     

NGF rescues DRG neurons in vitro from oxidative damage produced by hemodialyzers

Using dorsal root ganglion neurons (DRG), in vitro, we studied the effects of nerve growth factor (NGF) on a toxin extracted from ethylene oxide (EO) sterilized hemodialyzers. Tissue culture medium passed through dialyzers produced beading of DRG axons that was inhibited by increasing the concentration of NGF from 3.5 to 10 ng/ml. The antioxidant enzymes, catalase and glutathione peroxidase (GPx), prevented neurite beading while superoxide dismutase (SOD) alone did not. 3-amino-1,2,4-triazole (Az), an inhibitor of catalase blocked the protective effects of catalase and NGF. 1,3 bis[chloromethyl]-1-nitrosourea (BCNU) inhibits glutathione reductase, and reduces intracellular glutathione levels; it blocked the protective effects of NGF. Dialyzer treated medium was found to have increased peroxide content. In parallel experiments, NGF protected DRG neurons from hydrogen peroxide (H(2)O(2)) toxicity that was inhibited by Az and BCNU. NGF was also shown to upregulate glutathione in DRG neurons. We propose that EO gas used in the sterilization of hemodialyzers is responsible for the neurotoxicity and is most likely due to oxidative damage in DRG neurons. NGF protects DRG from this toxin by upregulating antioxidants such as catalase, GPx and GSH.     

Interferon beta promotes survival in primary astrocytes through phosphatidylinositol 3-kinase

Although interferon-beta (IFN-beta) has been demonstrated to be effective in the treatment of multiple sclerosis (MS) patients, the mechanism(s) underlying its beneficial effects has not been uncovered yet. Until now, most of the effort in the study of the relevant mechanisms of IFN-beta has dealt with its ability to modulate the immune response. Only recently, it has been proposed that the beneficial effects of IFN-beta in MS patients could depend on its ability to modulate astrocyte function. In the present work, we have found that IFN-beta treatment promotes the survival of astrocytes through stimulation of the phosphatidylinositol 3-kinase (PI-3K)/Akt pathway. We propose that the beneficial effects of IFN-beta in MS therapy may depend, at least in part, on its capacity to protect astrocytes against the apoptotic cell death that occurs in the early steps of the pathogenesis of MS.     

Neurokinins inhibit low threshold inactivating K currents in capsaicin responsive DRG neurons

Neurokinins (NK) released from terminals of dorsal root ganglion (DRG) neurons may control firing of these neurons by an autofeedback mechanism. In this study we used patch clamp recording techniques to determine if NKs alter excitability of rat L4-S3 DRG neurons by modulating K⁺ currents. In capsaicin (CAPS)-responsive phasic neurons substance P (SP) lowered action potential (AP) threshold and increased the number of APs elicited by depolarizing current pulses. SP and a selective NK₂ agonist, [βAla⁸]-neurokinin A (4–10) also inhibited low threshold inactivating K⁺ currents isolated by blocking non-inactivating currents with a combination of high TEA, (−) verapamil and nifedipine. Currents recorded under these conditions were heteropodatoxin-sensitive (Kv4 blocker) and α-dendrotoxin-insensitive (Kv1.1 and Kv1.2 blocker). SP and NKA elicited a > 10 mV positive shift of the voltage dependence of activation of the low threshold currents. This effect was absent in CAPS-unresponsive neurons. The effect of SP or NKA on K⁺ currents in CAPS-responsive phasic neurons was fully reversed by an NK₂ receptor antagonist (MEN10376) but only partially reversed by a PKC inhibitor (bisindolylmaleimide). An NK₁ selective agonist ([Sar⁹, Met¹¹]-substance P) or direct activation of PKC with phorbol 12,13-dibutyrate, did not change firing in CAPS-responsive neurons, but did inhibit various types of K⁺ currents that activated over a wide range of voltages. These data suggest that the excitability of CAPS-responsive phasic afferent neurons is increased by activation of NK₂ receptors and that this is due in part to inhibition and a positive voltage shift in the activation of heteropodatoxin-sensitive Kv4 channels.     

Class 1 heparin binding growth factor promotes the differentiation but not the survival of ciliary neurones in vivo.

Molecules isolated from target tissues of various neuronal populations have been shown to enable the survival of those innervating neurones. A molecule derived from bovine heart which can support the survival of parasympathetic neurones of the chick embryo ciliary ganglion in vitro has been sequenced and identified as a member of the class 1 heparin binding growth factors (HBGF-1). When injected into developing chick embryos during the period of naturally occurring cell death in the ciliary ganglion, HBGF-1 failed to rescue those neurones which normally die. There was, however, a premature increase in the activity of choline acetyltransferase within the ganglion and a shift to the right in the size distribution of the neurones. We conclude that HBGF-1 may promote the differentiation of some subpopulation of neurones of the ciliary ganglion of the chick embryo but does not act in vivo as a survival molecule.     

Inflammation-induced hyperexcitability of nociceptive gastrointestinal DRG neurones: the role of voltage-gated ion channels

Gastrointestinal (GI) inflammation modulates the intrinsic properties of nociceptive dorsal root ganglia neurones, which innervate the GI tract and these changes are important in the genesis of abdominal pain and visceral hyperalgesia neurones exhibit hyperexcitability characterized by a decreased threshold for activation and increased firing rate, and changes in voltages-gated Na(+) and K(+) channels play a major role in this plasticity. This review highlights emerging evidence that specific subsets of channels and signalling pathways are involved and their potential to provide novel selective therapeutics targets for the treatment of abdominal pain.     

In sympathetic but not sensory neurones, phosphoinositide-3 kinase is important for NGF-dependent survival and the retrograde transport of I-βNGF

The way in which the same ligands and receptors have different functional effects in different cell types must depend on subtle differences in the second messenger cascades. Sensory and sympathetic neurones both retrogradely transport nerve growth factor (NGF) and depend on NGF for their developmental survival. NGF binding to the high affinity tyrosine kinase (TrkA) receptors initiates second messenger signalling cascades, one of which includes the activation of phosphoinositide-3 kinase (P13-kinase). We demonstrate that 100-fold higher concentrations of the P13-kinase inhibitor. Wortmannin, are required to inhibit the survival effects and retrograde axonal transport of NGF in sensory neurones than in sympathetic neurones. Similarly, although less potently than Wortmannin, the P13-kinase inhibitor LY294002 required a 10-fold higher concentration to inhibit the survival effects of NGF in sensory than in sympathetic neurones. Inhibitors of other second messengers, including staurosporine, pertussis and cholera toxins, failed to have an effect on the transport of the NGF receptor complex in both cell types. Also, Wortmannin did not affect the structural integrity of the sympathetic nerve terminals. As P13-kinase is present in both neuronal populations, this suggests that the Wortmannin sensitive isoform of P13-kinase (p110) is essential in sympathetic neurones both for survival and for NGF-TrkA receptor complex trafficking. As sensory neurones also depend on NGF for their developmental survival and endocytose and retrogradely transport the NGF-TrkA receptor complex, this population of neurones may either recruit a different isoform of P13-kinase or utilize P13-kinase independent signalling pathways for these cellular functions.     

ATP-gated ion channel expression in primary auditory neurones

Extracellular ATP acts via ionotropic P2X receptors to mediate fast neurotransmission in the central and autonomic nervous systems. Recent data, including identification of P2X2 receptor mRNA expression by spiral ganglion neurones, suggests that purinergic signalling may influence auditory neurotransmission via ATP-gated ion channels assembled from these subunits. Expression of the P2X2 receptor was localized to the region of the spiral ganglion neurone synapses with the inner hair cells using a P2X2 receptor specific antiserum. Whole-cell patch clamping of neurones cultured from post-natal day 3-5 spiral ganglia demonstrated a heterogeneity of ATP-activated conductances, consistent with the functional expression of P2X2 receptor subunit isoforms along with possible co-expression of additional P2X receptor subunits. These data provide substantive support for a purinergic transmission element at the peripheral auditory synapse.     

A rapid method for semi-quantitative analysis of neurite outgrowth from chick DRG explants using image analysis

Neurite outgrowth from dorsal root ganglion (DRG) explants is a method of evaluating neurotrophic activity of growth factors and neurotrophin mimetics. The drawbacks to this approach are the difficulties in quantifying the response. Neurite counts are time consuming and labour intensive, and the accuracy is often questionable due to branching and fasciculation of the neurites. We report here a method of semi-quantitative analysis of neurite outgrowth from chick DRG explants, using image analysis to quantify the area occupied by neurites emanating from the ganglion. This method is rapid, takes into account both the length and number of neurites, and is unaffected by neurite fasciculation or branching. Primary explants of chick DRGs were treated with the neurotrophins nerve growth factor (NGF) or neurotrophin-3 (NT-3) and with the compound K252a. K252b was tested for potentiation of the response to NT-3. The results show a dose dependent outgrowth of neurites from explants treated with NGF, NT-3 and K252a, and potentiation of the NT-3 response by K252b. These responses were quantified by neurite area quantification using image analysis. We conclude that neurite area measurement using image analysis provides a robust means of evaluating neurotrophic activity of growth factors and neurotrophin mimetics in vitro.     

Reduced NGF secretion by Schwann cells under the high glucose condition decreases neurite outgrowth of DRG neurons

Background

Schwann cells (SCs) have been supposed to play prominent roles in axonal regeneration under various diseases. Here, to evaluate the direct interaction between SCs and dorsal root ganglion (DRG) neurons under a diabetic condition, the effects of Schwann cell-conditioned media on neurite outgrowth of DRG neurons were investigated.

Methods

Immortalized mouse Schwann cells (IMS) were cultured under 5.5 mM glucose (NG) or 30 mM glucose (HG) conditions for 4 days. IMS-conditioned media (IMS-media) were added to the culture media of neurons isolated from 8-week-old DDY mice. Neurons were cultured for 48 h with or without mouse recombinant NGF (mrNGF) or nerve growth factor (NGF) neutralizing antibody. The concentrations of NGF in IMS-media by ELISA and neurite outgrowth by a computed image analysis system were evaluated.

Results

Neurite outgrowth was significantly enhanced by IMS-media (IMS-media (–): 177 ± 177 µm, IMS-media (+): 1648 ± 726). The neurite outgrowth cultured with IMS-media obtained under the HG condition was significantly reduced compared with that under the NG condition (NG: 1474 ± 652, HG: 734 ± 331). The NGF concentrations were significantly lower in IMS-media under the HG condition than in those under the NG condition. The accelerated neurite outgrowth by IMS-media was inhibited by NGF neutralizing antibody.

Conclusions

These results suggest that SCs play important roles in neurite outgrowth of DRG neurons, and that the decreased NGF secretion by SCs under the diabetic condition would cause a defect of axonal regeneration, resulting in the development of diabetic neuropathy.