TrkB Agonist LM22A-4 Increases Oligodendroglial Populations During Myelin Repair in the Corpus Callosum.

The neurotrophin, brain-derived neurotrophic factor (BDNF) promotes central nervous system (CNS) myelination during development and after injury. This is achieved via activation of oligodendrocyte-expressed tropomyosin-related kinase (Trk) B receptors. However, while administration of BDNF has shown beneficial effects, BDNF itself has a poor pharmacokinetic profile. Here, we compare two TrkB-targeted BDNF-mimetics, the structural-mimetic, tricyclic dimeric peptide-6 (TDP6) and the non-peptide small molecule TrkB agonist LM22A-4 in a cuprizone model of central demyelination in female mice. Both mimetics promoted remyelination, increasing myelin sheath thickness and oligodendrocyte densities after 1-week recovery. Importantly, LM22A-4 exerts these effects in an oligodendroglial TrkB-dependent manner. However, analysis of TrkB signaling by LM22A-4 suggests rather than direct activation of TrkB, LM22A-4 exerts its effects via indirect transactivation of Trk receptors. Overall, these studies support the therapeutic strategy to selectively targeting TrkB activation to promote remyelination in the brain.

Role of JNK, MEK and adenylyl cyclase signalling in speed and directionality of enteric neural crest-derived cells.

BACKGROUND: Cells derived from the neural crest colonize the developing gut and give rise to the enteric nervous system. The rate at which the ENCC population advances along the bowel will be affected by both the speed and directionality of individual ENCCs. The aim of the study was to use time-lapse imaging and pharmacological activators and inhibitors to examine the role of several intracellular signalling pathways in both the speed and the directionality of individual enteric neural crest-derived cells in intact explants of E12.5 mouse gut. Drugs that activate or inhibit intracellular components proposed to be involved in GDNF-RET and EDN3-ETB signalling in ENCCs were used. FINDINGS: Pharmacological inhibition of JNK significantly reduced ENCC speed but did not affect ENCC directionality. MEK inhibition did not affect ENCC speed or directionality. Pharmacological activation of adenylyl cyclase or PKA (a downstream cAMP-dependent kinase) resulted in a significant decrease in ENCC speed and an increase in caudal directionality of ENCCs. In addition, adenylyl cyclase activation also resulted in reduced cell-cell contact between ENCCs, however this was not observed following PKA activation, suggesting that the effects of cAMP on adhesion are not mediated by PKA. CONCLUSIONS: JNK is required for normal ENCC migration speed, but not directionality, while cAMP signalling appears to regulate ENCC migration speed, directionality and adhesion. Collectively, our data demonstrate that intracellular signalling pathways can differentially affect the speed and directionality of migrating ENCCs.

Mitochondrial uncoupling reveals a novel therapeutic opportunity for p53-defective cancers.

There are considerable challenges in directly targeting the mutant p53 protein, given the large heterogeneity of p53 mutations in the clinic. An alternative approach is to exploit the altered fitness of cells imposed by loss-of-wild-type p53. Here we identify niclosamide through a HTS screen for compounds selectively killing p53-deficient cells. Niclosamide impairs the growth of p53-deficient cells and of p53 mutant patient-derived ovarian xenografts. Metabolome profiling reveals that niclosamide induces mitochondrial uncoupling, which renders mutant p53 cells susceptible to mitochondrial-dependent apoptosis through preferential accumulation of arachidonic acid (AA), and represents a first-in-class inhibitor of p53 mutant tumors. Wild-type p53 evades the cytotoxicity by promoting the transcriptional induction of two key lipid oxygenation genes, ALOX5 and ALOX12B, which catalyzes the dioxygenation and breakdown of AA. Therefore, we propose a new paradigm for targeting cancers defective in the p53 pathway, by exploiting their vulnerability to niclosamide-induced mitochondrial uncoupling.

Targeting TrkB with a Brain-Derived Neurotrophic Factor Mimetic Promotes Myelin Repair in the Brain.

Methods to promote myelin regeneration in response to central myelin loss are essential to prevent the progression of clinical disability in demyelinating diseases. The neurotrophin brain-derived neurotrophic factor (BDNF) is known to promote myelination during development via oligodendrocyte expressed TrkB receptors. Here, we use a structural mimetic of BDNF to promote myelin regeneration in a preclinical mouse model of central demyelination. In female mice, we show that selective targeting of TrkB with the BDNF-mimetic enhances remyelination, increasing oligodendrocyte differentiation, the frequency of myelinated axons, and myelin sheath thickness after a demyelinating insult. Treatment with exogenous BDNF exerted an attenuated effect, increasing myelin sheath thickness only. Further, following conditional deletion of TrkB from premyelinating oligodendrocytes, we show the effects of the BDNF-mimetic on oligodendrocyte differentiation and remyelination are lost, indicating these are dependent on oligodendrocyte expression of TrkB. Overall, these studies demonstrate that targeting oligodendrocyte TrkB promotes in vivo remyelination in the brain.SIGNIFICANCE STATEMENT Novel strategies to promote myelin regeneration are required to prevent progressive neurodegeneration and clinical disability in patients with central demyelinating disease. Here, we test whether selectively targeting the TrkB receptor on the myelin-producing oligodendrocytes, can promote remyelination in the brain. Using a structural mimetic of its native ligand, BDNF, we show that stimulation of TrkB enhances remyelination, increasing oligodendrocyte differentiation, the frequency of myelinated axons and thickness of the myelin sheath following a demyelinating insult. Further, we show that these effects are dependent on the phosphorylation of oligodendrocyte expressed TrkB receptors in vivo Overall, we demonstrate that selective targeting of TrkB has therapeutic potential to promote remyelination in the brain.

The role of activated MEK-ERK pathway in quercetin-induced growth inhibition and apoptosis in A549 lung cancer cells.

Dietary phytochemicals have been shown to be protective against various types of cancers. However, the precise underlying protective mechanisms are poorly understood. In the present study, we report that treatment of A549 cells with quercetin resulted in a dose-dependent reduction in cell viability and DNA synthesis with the rate of apoptosis equivalent to 1.2 +/- 0.8, 6.3 +/- 0.9, 16.5 +/- 1.5, 36.4 +/- 2.6 and 42.5 +/- 5.8% on treatment with 0.1% dimethylsulfoxide, 14.5, 29.0, 43.5 and 58.0 micro M quercetin, respectively. Concomitantly, quercetin treatments led to a 1.1-, 1.1-, 2.5- and 3.5-fold increase in Bax. Similar elevations were also observed in Bad, which increased 1.1-, 2.1-, 2.2- and 2.3-fold, respectively, as compared with control. While Bcl-2 was decreased by 30%, Bcl-x(L) was elevated in a dose-dependent fashion. Quercetin also induced the cleavage of caspase-3, caspase-7 and PARP (poly ADP-ribose polymerase). While Akt-1 and phosphorylated Akt-1 were inhibited, the extracellular signal-regulated kinase (ERK) was phosphorylated following quercetin treatment in a dose-dependent fashion. Phosphorylation of ERK and c-Jun occurred at 3 h and was sustained over 14 h. Phosphorylation of MEK1/2 was increased in concordance with ERK activation. Quercetin-induced phosphorylation of c-Jun N-terminal kinase (JNK) and cleavage of caspase-3 occurred 6 h after quercetin exposure and before cleavage of caspase-7 and PARP was detected. Inhibition of MEK1/2 but not PI-3 kinase, p38 kinase or JNK abolished quercetin-induced phosphorylation of c-Jun, cleavage of caspase-3 and -7, cleavage of PARP and apoptosis. Inhibition of caspase activation completely blocked quercetin-induced apoptosis. Expression of constitutively activated MEK1 in A549 cells led to activation of caspase-3 and apoptosis. The results suggest that in addition to inactivation of Akt-1 and alteration in the expression of the Bcl-2 family of proteins, activation of MEK-ERK is required for quercetin-induced apoptosis in A549 lung carcinoma cells.

Molecular mass distribution of sodium alginate by high-performance size-exclusion chromatography.

A sensitive high-performance size-exclusion chromatography (HPSEC) method with simple UV detection was developed for the molecular mass analysis of sodium alginate. It was used to evaluate alginates of varying molecular mass and the results were compared with the viscosity measurements. This HPSEC method was sensitive to serve as the stability indicating method for alginate after storage under different conditions. The information of relative molecular mass distribution of alginate was provided with reference to pullulan molecular mass standards. The comparison of the HPSEC chromatograms of alginate, pullulan and dextran revealed the effect of chemical composition of a polysaccharide and its effect on apparent molecular mass distribution.