Enhanced neuronal differentiation of pheochromocytoma 12 cells on polydopamine-modified surface.

Since pheochromocytoma 12 (PC12) cells have the ability of neuronal differentiation upon nerve growth factor (NGF) treatment, they are a good model for studying the neuronal differentiation. Establishing a strong adhesion of PC12 cells to the culture substrate may increase neuronal differentiation, and the use of L-3,4-dihydroxyphenylalanine (L-DOPA), which is responsible for the adhesive property of mussel adhesive proteins (MAPs), is a feasible strategy for such strong adhesion. We hypothesized that a polydopamine-modified surface can promote PC12 cell adhesion and subsequent neuronal differentiation. We examined whether polydopamine-modified surface promotes PC12 cell adhesion, and further evaluated the neuronal differentiation of these cells. The polydopamine modification enhanced the cell adhesion and viability, and also promoted the neuronal differentiation of NGF-stimulated PC12 cells, as evidenced by the elongation of neurites and expression of neuronal differentiation markers, by increasing the activation of NGF/Trk-Rho GTPase signal pathway. Our findings will help develop an improved strategy for functionalizing biomaterial substrates for less-adhesive cells including neural cells.

Enhanced nerve growth factor efficiency in neural cell culture by immobilization on the culture substrate.

Nerve growth factor (NGF) immobilization on a culture substrate may dramatically reduce the amount of NGF required for pheochromocytoma (PC12) cell culture. Coverslips on which NGF had been immobilized, or with NGF added to the culture medium daily, were used to culture PC12 cells. We examined the effects of adding 5, 10, or 100 ng of NGF to cultures daily, and compared them to the effects of immobilizing 5, 10, or 100 ng of NGF on culture substrates in a single dose. Cultures with 10 or 5 ng NGF added daily showed dramatically decreased cell viability, mitochondrial metabolic activity, and neuronal differentiation compared to cultures with 100 ng NGF added daily, while also exhibiting increased apoptosis. In contrast, a single dose of 100 ng immobilized NGF yielded results similar to 100 ng NGF added daily (total: 300 ng over 3 days), and 10 or 5 ng immobilized NGF showed far better results than 10 or 5 ng NGF added daily. These results demonstrate that NGF immobilization can dramatically reduce the amount of NGF required in neuronal cell culture.

p62 protects SH-SY5Y neuroblastoma cells against H2O2-induced injury through the PDK1/Akt pathway.

The p62 protein has been identified as a major component of the protein aggregations associated with neurodegenerative disease. Oxidative insult has also been identified as a principal cause of neurodegenerative disease. Thus, in the present study, we investigated the potential role of p62 in oxidative stress-induced cell death in SH-SY5Y human neuroblastoma cells. The results indicated that H(2)O(2) treatment induced p62 expression in SH-SY5Y cells. In addition, p62 showed neuroprotective effects against H(2)O(2)-induced cell death in differentiated SH-SY5Y cells. p62 expression prolonged Akt phosphorylation during the later stages of H(2)O(2)-induced cell death. Furthermore, coexpression of p62 and wild-type PDK1, the upstream kinase of Akt, further increased Akt phosphorylation and cell viability, whereas the expression of kinase-defective PDK1 reversed the cytoprotective effects of p62 under oxidative stress. Overexpression of p62 led to the dissociation of PDK1 from the 14-3-3theta protein, which is thought to be a negative regulator of PDK1 kinase activity. These findings suggest a mechanism that involves the p62-mediated modulation of the interaction between signaling molecules and results in cell survival.

Shp2 is involved in neuronal differentiation of hippocampal precursor cells.

HiB5 is a multipotent hippocampal stem cell line whose differentiation into cells of a neuronal phenotype is promoted by neurotrophic factors such as PDGF and brain-derived neurotrophic factor (BDNF). We examined the potential role of Src homology 2 (SH2)-containing protein tyrosine phosphatase (Shp2) in this differentiation process. We found that Shp2 became tyrosine phosphorylated following PDGF treatment. Wild-type Shp2 enhanced the expression of neurofilament, synapsin I and PSD95 by PDGF and BDNF, whereas their expression was attenuated by the catalytically inactive mutants Shp2C/S and Shp2DeltaP. Formation of dendritic spine-like structures increased with wild-type Shp2, but diminished with Shp2C/S and Shp2DeltaP. PSD95, localized in the post-synaptic density region of dendritic spines, PDGFRbeta and TrkB were co-immunoprecipitated with Shp2 antibodies. These results suggest that Shp2 plays a positive role in mediating PDGF- and BDNF-activated signaling which promotes the formation of dendritic spines.

Distinct effect of neurotrophins delivered simultaneously by an adenoviral vector on neurite outgrowth of neural precursor cells from different regions of the brain.

For many years, it has been demonstrated that neurotrophins regulate the adult nervous system, implicating their potential as therapeutic agents for the treatment of neurodegenerative diseases. We generated adenoviral vectors encoding brain-derived neutotrophin factor (BDNF) and neurotrophin-3 (NT3) and tested either separately or together for the ability to induce differentiation of neuronal precursor cells with two different origins. Separate transduction of adenovirus delivering BDNF (BDNF-Ad) or NT3 (NT3-Ad) induced the neuronal differentiation in hippocampal and cortical precursor cells. NT3-Ad infected cells extended short neurites, whereas BDNFAd infected cells had longer neurites. In the early differentiation of hippocampal precursor cells, simultaneous infection of BDNFAd and NT3-Ad promoted further differentiation and neurite elongation compared with the separate infection of each virus. In contrast, simultaneous infection did not show the synergistic effect in the cortical precursor cells, suggesting that the neurotrophins play distinct roles in different regions of the brain. However, the numbers of neurites and spines per differentiated cells were markedly increased in cortical as well as hippocampal precursor cells, indicating the promotion of efficient neurite elongation and formation of dendritic spine, when BDNF-Ad and NT3-Ad were co-infected. These results suggest more studies in the effect of a combinatorial use of neurotrophins on different sites of brain need to be carried out to develop gene therapy protocols for neurodegenerative diseases.

Berberine promotes axonal regeneration in injured nerves of the peripheral nervous system.

Berberine, an isoquinoline alkaloid component of Coptidis Rhizoma (goldenthread) extract, has been reported to have therapeutic potential for central nervous system disorders such as Alzheimer's disease, cerebral ischemia, and schizophrenia. We have previously shown that berberine promotes the survival and differentiation of hippocampal precursor cells. In a memory-impaired rat model induced by ibotenic acid injection, the survival of pyramidal and granular cells was greatly increased in the hippocampus by berberine administration. In the present study, we investigated the effects of berberine on neurite outgrowth in the SH-SY5Y neuronal cell line and axonal regeneration in the rat peripheral nervous system (PNS). Berberine enhanced neurite extension in differentiating SH-SY5Y cells at concentrations of 0.25-3 µg/mL. In an injury model of the rat sciatic nerve, we examined the neuroregenerative effects of berberine on axonal remyelination by using immunohistochemical analysis. Four weeks after berberine administration (20 mg/kg i.p. once per day for 1 week), the thickness of remyelinated axons improved approximately 1.4-fold in the distal stump of the injury site. Taken together, these results indicate that berberine promotes neurite extension and axonal regeneration in injured nerves of the PNS.

The effect of the controlled release of nerve growth factor from collagen gel on the efficiency of neural cell culture.

In this study, we tested the hypothesis that the amount of nerve growth factor (NGF) required for pheochromocytoma (PC12) cell culture can be dramatically reduced by controlled release of NGF from a collagen gel coating on the culture surface. Cells were cultured on collagen gels loaded with various amounts of NGF. As a control, PC12 cells were cultured on collagen gels with daily addition of various amounts of NGF to the culture medium. After an initial 12h burst, NGF was steadily released from the gels for 4 days. Apoptotic activity and cell viability were determined using terminal uridine nick end labeling and the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, respectively. Neuronal differentiation was determined using immunocytochemistry and Western blot analysis. Compared to 100 ng NGF daily addition (300 ng over 3 days), 1 ng NGF daily addition showed dramatically decreased cell viability and neuronal differentiation and increased apoptotic activity. In contrast, collagen gels loaded with 10 ng NGF yielded cell viability, apoptotic activity, and neuronal differentiation similar to those of culture with 100 ng NGF daily addition. Our method reduced the amount of NGF required for PC12 cell culture to 1/3th of that used in daily addition without affecting cell viability, apoptosis, or differentiation. This method could economize large-scale culture of stem cells by reducing the amount of costly growth factors needed.