BMP-9, without RA, had a transient effect as confirmed by densitometric analysis of bands corresponding to pGSK3 and standardized to that of actin: pGSK3 (Ser9) increased between 0 and 60?min, plateaued and then decreased from 120 to 240?min. and length of neurites and the expression of neuronal markers MAP-2, NeuN and NSE better than did BMP-9. They also promoted differentiation to the cholinergic phenotype more actively than BMP-9, SpBMP-9 being the most effective as shown by increases in intracellular acetylcholine, ChAT and VAchT. Finally, both peptides activated the PI3K/Akt pathway and inhibited GSK3beta, a current AD therapeutic target. BMP-9-derived peptides, especially SpBMP-9, with or without RA, are encouraging molecules that warrant further investigation. Introduction Alzheimers disease (AD) is the most common type of dementia, accounting for about 60% of all cases, affecting over 40 million people worldwide1. However, there is no remedy for AD and the therapies currently available or under investigation have only transient effects and slow disease progression2, 3. Most target only one of the three major hallmarks of AD at a time (cholinergic system dysfunction4, beta amyloid plaque accumulation5 and Tau protein hyperphosphorylation6, 7), although considerable evidence suggests that these hallmarks are all intimately linked8C10. Growth factors (GFs) like neurotrophins (nerve growth factor and brain-derived neurotrophic factor), bone morphogenetic proteins (BMPs) and insulin-like growth factor 2 (IGF-2), which are found in the developing and healthy mature brain, but are dysregulated in AD, seem to prevent the development of the disease. They could take action on several AD hallmarks simultaneously and repair the dysfunctional cell R428 signalling8, 11C16. One subfamily of GFs, the BMPs, may have great potential as they are involved in brain development, maintenance and homeostasis8, 17C19. The BMPs, more than 20 at the last count, were discovered in bone tissue by Urist and Strates in the early 1970s20C22. BMPs transmission in the brain via their type I and type II Serine/Threonine kinase receptors and activate the canonical Smad pathway (Smad 1/5/8), which is usually important in early brain development and neuron Rabbit Polyclonal to NEK5 maturation19, 23, 24. One BMP, BMP-9, may be a encouraging candidate for therapy: it is present in the brain and seems to be linked to the function of cholinergic neurons25, 26. Lopez-Coviella and genes are conserved at the same locus, which suggests that their expressions are coordinated42. We investigated the effect of pBMP-9 and SpBMP-9 around the induction and the maintenance of the cholinergic phenotype since cholinergic dysfunction is usually a major hallmark of AD (Figs?5C7). Open in a separate window Physique 5 Effect of pBMP-9 and SpBMP-9 around the expression of choline acetyltransferase. (A) Merged pictures showing immunostaining for ChAT (FITC, green) and nuclei labelling (Hoechst, blue) of SH-SY5Y cells stimulated for 5d with 0, 0.1, or 1?nM BMP-9, pBMP-9 and SpBMP-9 +/? 10?M RA (Bar?=?100 m). Pictures are representative of at least 2 impartial experiments. (B) Analysis of ChAT fluorescence intensity relative to the nucleus staining was also offered. Results are the means??SEM (***p?0.001). Open in a separate windows Physique 7 Effect of pBMP-9 and SpBMP-9 around the intracellular Ach and AchE. (A) Intracellular Ach in SH-SY5Y cells stimulated with 0, 0.1 or 1?nM BMP-9, pBMP-9 and SpBMP-9 +/? 10?M RA for 3d and 5d (B) AchE activity for SH-SY5Y cells stimulated with 0, 0.1 or 1?nM BMP-9, pBMP-9 and SpBMP-9 +/? 10?M RA for 5d. Results are the means??SEM of at least 4 indie experiments (*p?0.05, **p?0.01, ***p?0.001). Effect of pBMP-9 and SpBMP-9 on choline acetyltransferase The ChAT enzyme responsible for transforming R428 acetyl-co-A and choline to acetylcholine in SH-SY5Y cells incubated with BMP-9 or its derived peptides with or without RA was detected by immunolabelling (Fig.?5A and B). We found ChAT immunostaining in all cell body under all experimental conditions (Fig.?5A). However, the intensity of labelling differed, especially when the cells were stimulated with SpBMP-9 without RA. The CTL without RA experienced the lowest R428 ChAT staining, while cells incubated with 0.1?nM SpBMP-9 had the highest ones as confirmed by the relative fluorescence intensity analysis (p?0.05) (Fig.?5B). Cells stimulated with BMP-9 and pBMP-9 experienced comparable fluorescence intensities as the control, which were lower than that of SpBMP-9-stimulated cells. However, no difference in relative fluorescence intensities was observed when the same assays were run in the presence of RA. Effect of incubation time and pBMP-9 or SpBMP-9 dose on intracellular acetylcholine concentration and VAchT expression and distributions within cells VAchT in the axon terminals plays an important role in the accumulation of Ach prior to its release43. We immunolabelled VAchT to evaluate the effects of SpBMP-9 and pBMP-9 on its expression and distribution in the cells (5d, Fig.?6). The presence of labelled VAchT in small vesicles within the neurites indicates a cholinergic differentiation. Only cells stimulated with 0.1?nM or 1?nM SpBMP-9 (without RA) had VAchT vesicles in their.