Neurite Elongation In Neurons Research Articles

Altered Cytoskeletal Composition and Delayed Neurite Elongation in tau45–230-Expressing Hippocampal Neurons

Calpain-mediated tau cleavage into the neurotoxic tau45–230 fragment plays an important role in Alzheimer's disease (AD). This tau fragment accumulates mainly in the cytoplasm of degenerating neurons. However, subcellular localization studies indicated that a pool of tau45–230 associates with the cytoskeleton in hippocampal neurons. In the present study, we assessed whether such localization could underlie tau45–230 neurotoxic effects. Quantitative Western blot analysis showed decreased levels of full-length tau bound to microtubules in tau45–230-expressing hippocampal neurons when compared to controls. In addition, the presence of this tau fragment induced a transient increase in tyrosinated tubulin, a marker of unstable microtubules, followed by a significant decrease in the levels of this tubulin isoform. The data obtained also showed a significant reduction in actin filaments in tau45–230-expressing neurons. These changes in microtubules and actin filaments correlated with delayed neurite elongation and axonal differentiation in the presence of this tau fragment. Together, these results suggest that tau45–230 could exert its toxic effects, at least in part, by modifying the composition of the neuronal cytoskeleton and impairing neurite elongation in neurons undergoing degeneration.

MARK2 Rescues Nogo-66-Induced Inhibition of Neurite Outgrowth via Regulating Microtubule-Associated Proteins in Neurons In Vitro.

The ability of neurons in the adult mammalian central nervous system (CNS) to regenerate after injury is limited by inhibitors in CNS myelin. Nogo-66 is the most important myelin inhibitor but the mechanisms of Nogo-66 inhibition of neurite outgrowth remain poorly understood. Particularly, the relationship between Nogo-66 and microtubule-affinity regulating kinase 2 (MARK2) has not been examined. This study investigated the role of MARK2 in Nogo-66 inhibition and the function of MARK2 in neurite elongation in neurons in vitro. MARK2 and phosphorylated MARK2 at Ser212 (p-Ser212) alterations in Neuro 2a cells were assessed at different Nogo-66 exposure times; the relationships between MARK2 and microtubule-associated proteins (MAPs) were determined via the overexpression or interference of MARK2. Our study reports that Nogo-66 inhibited the expression of total MARK2 but also reduced Ser212 phosphorylation of MARK2, whereas levels of MAP1-b and tau varied depending on MARK2 overexpression or reduced expression. Furthermore, MARK2 increased the proportion of tyrosinated α-tubulin, thereby disrupting the stability of tubulin, most likely affecting axonal growth. In line with these results, overexpression of MARK2 promoted neurite elongation and therefore is able to rescue the inhibitory effect of Nogo-66 on neurite growth. In conclusion, the intracellular PKB/MARK2/MAPs/α-tubulin pathway appears to be essential for neurite elongation in neurons in vitro. These results suggest a critical role for MARK2 in overcoming Nogo-66-induced inhibition of axon outgrowth in neurons. Pharmacological activators of MARK2 may be applicable to promote successful axonal outgrowth following many types of CNS injuries.

Proteome profile changes in SH-SY5y neuronal cells after treatment with neurotrophic factors

Artemin, one of glial cell line-derived neurotrophic factors (GDNFs) supports sensory neuron. Although a role of artemin and GDNF as neurite outgrowth regulators in early neuron development has been suggested, the immediate effects of artemin and GDNF on neuronal cells have not been elucidated. Here, we investigated artemin and GDNF actions on the neuronal cell proteome. To identify immediate-early protein changes by artemin and GDNF in neuronal cells, we used a differential proteomics approach in SH-SY5y human neuronal cells treated with artemin or GDNF for 1 h. Eleven proteins that changed after both artemin and GDNF treatment were identified using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization time-of-flight tandem mass spectroscopy. The calcium ion-binding chaperone calreticulin and calcium/calmodulin-binding nuclear matrix protein matrin 3 showed common quantitative differences after both artemin and GDNF treatment. Cytoskeletal proteins also showed quantitative profile differences, which are functionally relevant to cytoskeletal rearrangement leading to the neurite elongation in neurons. These protein changes were detected in neuronal cells without accompanying changes in mRNA levels. These results suggest that immediate changes induced by artemin and GDNF are related to cytoskeletal protein level changes without transcriptional regulation.

Effects of nerve growth factor and heart cell conditioned medium on neurite regeneration of aged sympathetic neurons in culture

The effects of nerve growth factor (NGF) and heart-cell-conditioned medium (HCM) on the neurite regeneration of aged sympathetic neurons were investigated in culture. Investigation of HCM was carried out by two different methods: one was the use of whole HCM on collagen substratum, which reflected component(s) effective in solution (HCM-S); the other was the use of polyornithine (PORN)-binding component(s) (P-HCM). Superior cervical ganglion neurons prepared from male mice from 6 to 30 months of age were cultured in MEM-10% FCS on collagen or gelatin-PORN substratum for 3 days. The number of neurons with neurites and the length of neurites were quantified as neurite production and elongation, respectively. Neuronal survival was not affected by addition of NGF, HCM-S or P-HCM. Neurite production of early adult neurons was enhanced by NGF, HCM-S or P-HCM. In contrast, neurite production of aged neurons was enhanced by only HCM-S, but not NGF or P-HCM. HCM-S did not promote neurite elongation in neurons at any age. Neurite elongation of early adult neurons was enhanced by NGF or P-HCM. Neurite elongation of aged neurons was enhanced by P-HCM. However, responsiveness of NGF for neurite elongation varied according to substrata. No age-related difference was found in neurite production and elongation in the absence of NGF, HCM-S or P-HCM. These results indicate that responsiveness of aged sympathetic neurons is various in different growth factors.