Cytoskeleton involvement in lithium-induced SH-SY5Y neuritogenesis and the role of glycogen synthase kinase 3ß.

Lithium modulates signals impacting on the cytoskeleton, a dynamic system contributing to neural plasticity at multiple levels. In this study, SH-SY5Y human neuronal cells were cultured in the absence (C) or in presence (Li) of a 0.5 mM Li2CO3 (i.e. 1 mM lithium ion) for 25-50 weeks. We investigated the effect of this treatment on (1) morphological changes of cells observed using Hemalun eosin staining assay, (2) cytoskeletal changes by indirect immunofluorescence (IIF) staining of microtubules (α-tubulin) and heavy neurofilaments subunits (NF-H) and by measuring the expression rate changes of genes coding for receptor for activated C kinase (RACK1), casein kinase2 (CK2) and thymosine beta-10 using cDNA arrays technology, (3) cell adhesion properties by IIF staining of ß-catenin protein. Besides, we have tried to understand the molecular mechanism of lithium action that triggers changes in cytoskeleton and neurites outgrowth. Thus, we examined the effect of this treatment on glycogen synthase kinase 3 (GSK3) expression and activity using western blotting of GSK3 and phosphorylated ß-catenin, a downstream GSK3 target protein. Our results showed that lithium treatment reduces axon length, increases axonal spreading, enhances neurites growth and neurites branching with an increase of growth cone size. Moreover, genes coding for CK2 and thymosine beta-10 were significantly up-regulated, however, that coding for RACK1 was down-regulated. The most interesting result in this work is that mechanism underlying lithium action was not related to the inhibition of GSK3 activity. In fact, neither expression rate nor activity of this protein was changed.

Chronic neuroprotective effects of low concentration lithium on SH-SY5Y cells: possible involvement of stress proteins and gene expression.

To investigate the molecular mechanism underlying the neuroprotective effect of lithium on cells, in this study, we exposed SH-SY5Y cells to 0.5 mmol/L lithium carbonate (Li2CO2) for 25-50 weeks and then detected the expression levels of some neurobiology related genes and post-translational modifications of stress proteins in SH-SY5Y cells. cDNA arrays showed that pyruvate kinase 2 (PKM2) and calmodulin 3 (CaM 3) expression levels were significantly down-regulated, phosphatase protein PP2A expression was lightly down-regulated, and casein kinase II (CK2), threonine/tyrosine phosphatase 7 (PYST2), and dopamine beta-hydroxylase (DBH) expression levels were significantly up-regulated. Besides, western blot analysis of stress proteins (HSP27, HSP70, GRP78 and GRP94) showed an over-expression of two proteins: a 105 kDa protein which is a hyper-phosphorylated isoform of GRP94, and a 108 kDa protein which is a phosphorylated tetramer of HSP27. These results suggest that the neuroprotective effects of lithium are likely related to gene expressions and post-translational modifications of proteins cited above.