GSK-3 as a Target for Lithium-Induced Neuroprotection Against Excitotoxicity in Neuronal Cultures and Animal Models of Ischemic Stroke

Abstract

The mood stabilizer lithium inhibits glycogen synthase kinase-3 (GSK-3) directly or indirectly by enhancing serine phosphorylation of both α and β isoforms. Lithium robustly protected primary brain neurons from glutamate-induced excitotoxicity; these actions were mimicked by other GSK-3 inhibitors or silencing/inhibiting GSK-3α and/or β isoforms. Lithium rapidly activated Akt to enhance GSK-3 serine phosphorylation and to block glutamate-induced Akt inactivation. Lithium also up-regulated Bcl-2 and suppressed glutamate-induced p53 and Bax. Induction of brain-derived neurotrophic factor (BDNF) was required for lithium's neuroprotection to occur. BDNF promoter IV was activated by GSK-3 inhibition using lithium or other drugs, or through gene silencing/inactivation of either isoform. Further, lithium's neuroprotective effects were associated with inhibition of NMDA receptor-mediated calcium influx and down-stream signaling. In rodent ischemic models, post-insult treatment with lithium decreased infarct volume, ameliorated neurological deficits, and improved functional recovery. Up-regulation of heat-shock protein 70 and Bcl-2 as well as down-regulation of p53 likely contributed to lithium's protective effects. Delayed treatment with lithium improved functional MRI responses, which was accompanied by enhanced angiogenesis. Two GSK-3-regulated pro-angiogenic factors, matrix metalloproteinase-9 (MMP-9) and vascular endothelial growth factor were induced by lithium. Finally, lithium promoted migration of mesenchymal stem cells (MSCs) by up-regulation of MMP-9 through GSK-3β inhibition. Notably, transplantation of lithium-primed MSCs into ischemic rats enhanced MSC migration to the injured brain regions and improved the neurological performance. Several other GSK-3 inhibitors have also been reported to be beneficial in rodent ischemic models. Together, GSK-3 inhibition is a rational strategy to combat ischemic stroke and other excitotoxicity-related brain disorders.

Keywords: cerebral ischemia; excitotoxicity; glycogen synthase kinase-3; lithium; mesenchymal stem cells.

Figure 1

Inhibitory regulation of GSK-3 by lithium. Lithium negatively regulates the constitutively activated GSK-3 activity through multiple mechanisms. Lithium, a competitive inhibitor of magnesium, directly inhibits ATP–magnesium-dependent catalytic activity of GSK-3. The activity of GSK-3 is also reduced by phosphorylation at a specific serine residue. Lithium can indirectly increase this serine phosphorylation of GSK-3 through PI3-kinase-mediated phosphorylation/activation of Akt, PI3-kinase-mediated activation of PKC, and cAMP-dependent activation of PKA. Lithium can also increase the serine phosphorylation of GSK-3 by disrupting the β-arrestin-2 (βArr2)–PP2A–Akt complex that dephosphorylates and inactivates Akt. In addition, by disinhibiting the inhibitory action of inhibitor-2 (I-2) on protein phosphatase-l (PP-1) that dephosphorylates GSK-3 at serine residues, lithium’s direct inhibition of GSK-3 interrupts this auto-regulation of GSK-3 and further decreases GSK-3 activity. Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections. Dashed lines represent pathways with reduced activity as a result of lithium treatment. I-2, inhibitor-2; PP-1, protein phosphatase-l. (Modified from Chiu and Chuang, 2010).

Figure 2

Negative regulation of Smad3/4-dependent transcription by lithium. Transcriptional activations triggered by stimulation of cell surface TGF-β and BDNF receptors are mediated by Smad3/4- and PI3-kinase/Akt-dependent pathways, respectively. Lithium treatment-induced inhibition of GSK-3β, directly and indirectly via cAMP-dependent activation of PKA as well as BDNF-stimulated activation of PI3-kinase/Akt pathways, potentiates BDNF-induced phosphorylation/activation of CREB. This in turn increases CRE-mediated transactivation and expression of survival factors such as BDNF and Bcl-2. Enhanced gene transcription triggered by BDNF, via sequestration of transcriptional co-activator p300, suppresses Smad3/4-dependent transactivation and subsequently decreases the expression of TGF-β-responsive genes, PAI-1, and p21. Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections. Dashed lines represent pathways with reduced activity as a result of lithium treatment. CRE, cAMP response element. (Modified from Liang et al., 2008).

Figure 3

Proposed lithium’s neuroprotective effects against cerebral ischemia. The neuroprotective effects of lithium against cerebral ischemia are proposed to result from its interactions with cell survival and apoptotic machinery. A significant portion of brain damage following cerebral ischemia is caused by an increase in extracellular glutamate and subsequent over-stimulation of NMDA receptor-mediated toxic increase in intracellular calcium. This signaling pathway plays a critical role in mediating glutamate-induced caspase activation and apoptosis. Lithium at therapeutically relevant concentrations inhibits NMDA receptor-mediated calcium influx, which in turn decreases subsequent activation of JNK, p38 kinase, and transcription factor AP-1. Inhibition of intracellular calcium increase also attenuates the activity of calpain and calpain-mediated activation of pro-apoptotic Cdk5/p25 kinase. On the other hand, lithium can directly and indirectly reduce the activity of constitutively activated GSK-3 by multiple mechanisms, leading to disinhibition of several transcription factors, such as CREB and HSF-1, and resulting in induction of major cytoprotective proteins such as BDNF, VEGF, MMP-9, HSP70, and Bcl-2. A decrease in GSK-3 activity further reduces the activity of pro-apoptotic protein p53 and its downregulating effect on Bcl-2. BDNF, via activating its cell surface receptor and the down-stream ERK and PI3-kinase/Akt pathways, induces neuroprotective effects in part by inhibiting GSK-3 and stimulating CREB. Induction of BDNF is an early and essential step for neuroprotection and is involved in lithium-induced neurogenesis. In addition, superinduction of HSP70 by lithium treatment not only inhibits brain ischemia-induced apoptosis, but also contributes to the anti-inflammatory effects of lithium through inactivation of NF-κB. Counteraction of GSK-3 inhibition of VEGF and MMP-9 by lithium enhances angiogenesis and neurovascular remodeling. MMP-9 is also a key molecule involved in potentiating MSCs migration by lithium. Improvement in transplanted MSCs migration toward ischemic sites might increase neurogenesis as well. Taken together, these effects of lithium in reducing apoptosis, suppressing inflammation, enhancing angiogenesis and neurogenesis, contribute to behavioral improvement and functional recovery after ischemia. Lines with solid arrows represent stimulatory connections; lines with flattened ends represent inhibitory connections. Dashed lines represent pathways with reduced activity as a result of lithium treatment. NMDA-R, NMDA receptor.