In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 DIV astrocyte cultures pretreated or not with 0.5 mM glutamate for 24 h and than maintained under chronic or acute treatment with 50 μM R(+)enantiomer or raceme alpha-lipoic acid (ALA). GFAP expression significantly increased after (R+)enantiomer acute-treatment and also in glutamate-pretreated ones. Vimentin expression increased after R(+)enantiomer acute-treatment, but it decreased after raceme acute-treatment. Nestin expression drastically increased after acute raceme-treatment in glutamate-pretreated or not cultures, but significantly decreased after (R+)enantiomer acute and chronic-treatments. Cyclin D1 expression increased in raceme acute-treated cultures pretreated with glutamate. MAP-kinase expression slightly increased after (R+)enantiomer acute treatment in glutamate-pretreated or unpretreated ones. These preliminary findings may better clarify antioxidant and metabolic role played by ALA in proliferating and differentiating astrocyte cultures suggesting an interactive cross-talk between glial and neuronal cells, after brain lesions or damages.

Lipoic acid plays a crucial role as antioxidant and metabolic component of enzymes involved in glucose metabolism of different cell types. Choline alphoscerate (α-glyceryl-phosphoryl-choline [αGPC]) is a semisynthetic derivative of phosphatidylcholines representing, among acetilcholine precursors, a cholinergic drug. In the present study, we evaluated the expression of some proliferation and differentiation markers in 15 or 21 DIV astrocyte cultures treated with 50 μM (+)lipoic acid or (+/-)lipoic acid and/or 10 mM αGPC for 24 hr. In addition, we evaluated the possible genoprotective effect by analysis of DNA status detected by alkaline comet assay. The addition of single drugs [(+)lipoic acid, (+/-)lipoic acid, or αGPC] induced an "upward modulation" of the expression of biomarkers used in our study. On the contrary, the cotreatment with either (+)lipoic acid + αGPC or (+/-)lipoic + αGPC surprisingly showed no significant modification or even a downregulation of the above-mentioned biomarkers. This latter finding demonstrated no additional effect after the cotreatment with both drugs with respect to the single treatments alone. Further studies are necessary to clarify the specific mechanism evoked by the processing of these neuroprotective agents in our in vitro models. Finally, these preliminary findings may represent a good tool with which to clarify the antioxidant and metabolic roles played by lipoic acid in proliferating and differentiating astroglial cell cultures, during an interactive cross-talk between glial and neuronal cells, after brain lesions or damage correlated with oxidative stress that may occur in some degenerative diseases.

The aim of the present investigation was to study the effects of choline and choline-containing phospholipids CDP-choline (CDPC) and L-alpha-glyceryl-phosphorylcholine (AGPC) on transglutaminase (TG) activity and expression in primary astrocyte cultures. TG is an important Ca(2+)-dependent protein that represents a normal constituent of nervous systems during fetal stages of development, playing a role in cell signal transduction, differentiation, and apoptosis. Confocal laser scanning microscopy (CLSM) analysis showed an increase of TG activity in astrocyte cultures treated with choline, CDPC, or AGPC at 0.1 microM or 1 microM concentrations. Comparatively, AGPC induced the most conspicuous effects enhancing monodansyl-cadaverine fluorescence both in cytosol and in nuclei, supporting the evidence of the important role played by AGPC throughout differentiation processes tightly correlated to nucleus-cytosol cross- talk during astroglial cells proliferation and development. Western blot analysis showed that in 24h 1 microM AGPC and choline-treated astrocytes increased TG-2, whereas no effect was observed in 24h 1 microM CDP-choline treated astrocytes. Our data suggest a crucial role of choline precursors during different stages of astroglial cell proliferation and differentiation in cultures.

Heme oxygenase-1 (HO-1) plays a crucial role in oxidative stress processes, apoptosis and cell differentiation. Further, some proteins related to cell cycle including cyclins and p21 are important markers of astrocyte cultures. Aim of investigation was to study the effects of cholinergic precursors (choline, CDP-choline, Acetylcholine and α-Glyceril-Phosphorylcholine) on HO-1 and p21 expression during astroglial cell proliferation and differentiation in primary cultures at 14 and 35 days in vitro (DIV) treated for 24 h with choline metabolites. Our results showed a slight reduction of HO-1 expression (data not statistical significant) in astroglial cell cultures treated with CDP-choline at 14 DIV and 35 DIV. On the contrary, ACh and choline induced a significant increase of HO-1 expression in 14 DIV astrocyte cultures. Surprisingly, choline and ACh dramatically reduced HO-1 expression at 35 DIV. A slight decrease not statistical significant was detectable for α-GPC at 14 DIV and particularly significant at 35 DIV. Data concerning p21 expression, a well known protein inhibiting cell cycle, evidenced a significant increase at 14 and 35 DIV after α-GPC treatment. CDP-choline treatment caused a high increase of p21 expression in 14 DIV astrocyte cultures, but no modification at 35 DIV. Instead, ACh treatment induced a marked increment of p21 expression at 35 DIV. Our data suggest that cholinergic precursors modulate HO-1 and p21 expression during astroglial cell proliferation and differentiation in culture and could be considered a tool to study the induced effects of ischemia and hypoxia diseases in some in vitro models to prevent and reduce its effects after treatment with cholinergic drugs.

In this research we aimed to investigate the interactions between growth factors (GFs) and dexamethasone (DEX) on cytoskeletal proteins GFAP and vimentin (VIM) expression under different experimental conditions. Condition I: 24 h pretreatment with bFGF, subsequent 72 h switching in serum-free medium (SFM) and final addition of GFs, alone or by two in the last 24 h, after a prolonged (60 h) DEX treatment. Condition II: 36 h pretreatment with DEX (with bFGF in the last 24 h), followed by SFM for 60 h and final addition for 24 h with growth factors alone or two of them together. Western blot analysis data showed a marked GFAP expression in cultures submitted to Condition I comparing results to untreated or treated controls. VIM expression was instead significantly reduced after GFs addition in the last 24 h of 60 h DEX treatment, respect to control DEX-pretreated ones. Referring data to untreated controls, VIM expression was significantly enhanced after GFs addition. GFAP showed also a significant increase in astrocytes submitted to Condition II, respect to untreated or treated control cultures. VIM expression was up and down regulated under Condition II. Collectively, our findings evidence an interactive dialogue between GFs and DEX in astroglial cultures, co-pretreated with DEX and bFGF, regulating cytoskeletal network under stressful conditions.