glycogen synthase kinase-3 ( gsk-3 ) , an evolutionarily conserved ubiquitous serine  threonine kinase consisting of  and  isoforms , is a multifaceted protein with diverse cellular and neurophysiological functions .
the main structural difference between gsk-3 and gsk-3 isoforms lies in the n- and c - terminal regions , while their sequences within the kinase domain are highly homologous .
a growing body of evidence indicates that gsk-3 is pro - apoptotic and that its dysfunction may be linked to the pathophysiology of mood disorders , schizophrenia , diabetes , and various neurological / neurodegenerative diseases , among others ( for review , meijer et al . , 2004 ;
, 2007 ; chiu and chuang , 2010 ; li and jope , 2010 ) .
gsk-3 inhibition has attracted widespread attention as one of the critical therapeutic targets whereby lithium exerts its effects on mood stabilization , neurogenesis , neurotrophicity , neuroprotection , anti - inflammation , and others ( for review , rowe and chuang , 2004 ; rowe et al .
, 2007 ; beurel et al . , 2010 ) . pharmacological inhibition or gene knockout / knockdown of this kinase mimics the anti - depressant and anti - manic effects of lithium observed in rodent models ( gould et al . , 2004 ; kaidanovich - beilin et al
, 2004 , 2009 ; obrien et al . , 2004 ; rosa et al . , 2008 ; omata et al . , 2011 ) .
the activities of gsk-3 are negatively regulated by phosphorylation of gsk-3 at ser21 and gsk-3 at ser9 .
gsk-3 can be inhibited by lithium through direct binding to the atp - dependent magnesium - sensitive catalytic site of the enzyme ( klein and melton , 1996 ; stambolic et al .
, 1996 ) , and/or indirectly through enhanced serine phosphorylation of gsk-3 isoforms by multiple mechanisms ( figure 1 ) .
lithium has been shown to enhance gsk-3 serine phosphorylation by activation of protein kinase a ( pka ; jope , 1999 ; liang et al . , 2008 ) , or phosphatidylinositol 3-kinase ( pi3-kinase)-dependent akt ( chalecka - franaszek and chuang , 1999 ) and protein kinase c- ( kirshenboim et al . , 2004
it has also been reported that lithium can disrupt the -arrestin-2pp2a  akt complex that dephosphorylates / inactivates akt , thereby enhancing gsk-3 serine phosphorylation ( beaulieu et al . , 2005 ) .
moreover , it has been proposed that lithium can interrupt auto - regulation of gsk-3 via disinhibition of the inhibitory action of inhibitor-2 complex on protein phosphatase-1 ( pp-1 ; zhang et al . , 2003 ) .
this article reviews the findings supporting the role of gsk-3 inhibition in mediating lithium s neuroprotective effects against excitotoxicity in both cultured neurons and animal models of ischemic stroke .
lithium , a competitive inhibitor of magnesium , directly inhibits atp  magnesium - dependent catalytic activity of gsk-3 .
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 -arrestin-2 ( arr2)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 .
we have designed isoform - specific small interfering rnas ( sirnas ) to distinguish the functional and regulatory differences between the two gsk-3 isoforms in rat cerebral cortical neuronal cultures ( liang and chuang , 2007 ) .
transfection with sirna for gsk-3 or gsk-3 or with dominant - negative mutants specific for either isoform produced almost complete protection against glutamate - induced , n - methyl - d - aspartate ( nmda ) receptor - mediated excitotoxicity .
the sirna - induced neuroprotection was associated with enhanced n - terminal phosphorylation in both gsk-3 isoforms .
moreover , transfection with  or  isoform - specific dominant - negative mutants of gsk-3 mimicked lithium - induced neuroprotection against glutamate excitotoxicity .
these results strongly suggest that both gsk-3 and  are involved in glutamate - induced neuronal death and that both isoforms are the initial targets of lithium - elicited neuroprotection .
gsk-3 has also been implicated in neuronal development , maturation / differentiation , and aging in the mammalian cns ( spittaels et al . , 2002 ; kim et al .
substrates phosphorylated by gsk-3 include metabolic , signaling , and structural proteins as well as transcription factors .
it is known that inhibition of gsk-3 results in activation , and sometimes suppression , of an array of transcription factors ( for review , grimes and jope , 2001 ; jope and roh , 2006 ; chiu and chuang , 2010 ) . among the long list of transcription factors regulated by gsk-3
are cyclic amp response element binding protein ( creb ) , nuclear factor-b ( nf-b ) , activating protein-1 ( ap-1 ) , heat - shock factor-1 ( hsf-1 ) , -catenin , t - cell factor ( tcf)/lymphoid enhancer factor ( lef ) , and p53 .
dysfunction of gsk-3-mediated phosphorylation of transcription factors is believed to relate with the pathophysiology of various pathological conditions ( for review , chiu and chuang , 2010 ) .
we found that gsk-3 silencing activated camp response element ( cre)- and nf-b - responsive transcription more robustly than gsk-3 silencing ( liang and chuang , 2006 ) .
dna array further identified two novel gsk-3-regulated transcription factors , early growth response-1 ( egr-1 ) and smad3/4 , both of which play important roles in growth , differentiation , survival , and plasticity of brain cells ( harada et al .
, 2001 ; derynck and zhang , 2003 ; lee and kim , 2004 ; droguett et al . , 2010 ) . specifically , the binding activity of egr-1 was down - regulated by sirna for gsk-3 , but was up - regulated by sirna for gsk-3 ( liang and chuang , 2006 ) . by using sirnas or dominant - negative mutants specific to gsk-3 isoforms , inhibition of gsk-3
the differential roles of gsk-3 isoforms are further supported by the opposite effects of gsk-3 and  sirnas on the protein levels of plasminogen activator inhibitor type-1 ( pai-1 ) , a smad3/4-regulated gene product .
these results demonstrate that selective silencing or inhibition of the two gsk-3 isoforms could produce different and sometimes opposite effects on the regulation of certain transcription factors including novel gsk-3 targets ( liang and chuang , 2006 ) .
for example , the disruption of gsk-3 in mice is embryonic lethal , despite the normal expression of gsk-3 , indicating that the presence of  isoform can not compensate for the loss of  isoform ( hoeflich et al . , 2000 ) .
transfection and sirna studies suggested that gsk-3 inhibition decreased the processing of -amyloid ( a ) precursor protein to form a140 and a142 , while gsk-3 appeared to have a lesser role ( phiel et al . , 2003 ) .
in addition , gsk-3 , but not gsk-3 , is required for interferon--induced activation of signal transducer and activator of transcription-3 ( beurel and jope , 2009 ) .
together , these findings underscore important similarities and differences between the roles of gsk-3 isoforms  and  in cell survival as well as transcription , and suggest that the development of isoform - specific inhibitors may be essential for therapeutic intervention of gsk-3-related neuropathological conditions .
we have also explored lithium s effects on smad3/4-dependent transcriptional activity and the underlying mechanisms .
smad3/4 is a down - stream mediator of the signaling pathway triggered by transforming growth factor- ( tgf- ) , and plays a prominent role in regulating the expression of proteins involved in neuronal survival , differentiation , and synaptic plasticity ( for review , gomes et al . , 2005 ) .
treating cultured cortical neurons with therapeutically relevant concentrations of lithium significantly decreased smad3/4-dependent transactivation and protein levels of pai-1 , a tgf--responsive smad3/4-dependent gene product ( liang et al . , 2008 ) .
of particular relevance to the therapeutic efficacy of lithium , pai-1 has been implicated in the etiology and progression of neurodegenerative diseases and mood disorders ( for review , pawlak et al . , 2003 ;
lithium s effects on smad3/4 likely result from cross - talk of signaling pathways between camp / pka and pi3-kinase / akt / gsk-3. we have shown that lithium - induced smad3/4 suppression involved gsk-3 inhibition through the activation of pka and cell survival factor akt followed by the phosphorylation of gsk-3 at ser9 and creb at ser133 ( liang et al . , 2008 ) .
creb binding protein ( cbp ) and p300 are known to be co - activators of creb .
our data further demonstrated that over - expression of p300 , but not cbp , completely antagonized lithium - induced reduction of pai-1 promoter activity .
a series of experimental data support the notion that , in smad3/4 signaling , the inhibitory effects of lithium are due to complex formation of activated creb and p300 , which results in limited interactions of p300 with the transcription factors / smad complexes . this in turn prevents efficient smad3/4-dependent transcription of smad3/4-dependent genes such as pai-1 and p21 ( figure 2 ) .
transcriptional activations triggered by stimulation of cell surface tgf- and bdnf receptors are mediated by smad3/4- and pi3-kinase / akt - dependent pathways , respectively .
lithium treatment - induced inhibition of gsk-3 , 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--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 .
lithium - induced neuroprotection against glutamate excitotoxicity was first noted in rodent primary neuronal cultures of cerebellar granule cells ( cgcs ) , cerebral cortical neurons , and hippocampal neurons ( nonaka et al . , 1998 ) . this experimental paradigm was selected because glutamate - related excitotoxicity has been implicated in many neurodegenerative diseases including stroke ( for review , chuang , 2004 ; chiu and chuang , 2010 ) .
our pioneering studies have shown that glutamate - induced , nmda receptor - mediated excitotoxicity was robustly reduced by extended lithium chloride pretreatment ( 57  days ) in cultured rat cgcs and cortical neurons , partly via inhibition of nmda receptor - mediated calcium influx ( nonaka et al . , 1998 ; hashimoto et al . , 2002a ) . moreover ,
these effects of lithium were likely due to the attenuation of constitutive phosphorylation at tyr1472 of the nr2b subunit of nmda receptors , possibly as a result of inhibiting src tyrosine kinase ( hashimoto et al . , 2002a , 2003 ) .
although glutamate - induced excitotoxicity in cultured cortical neurons was blocked by treatment with either lithium or mk-801 ( an nmda receptor antagonist ) , the src kinase inhibitor su6656 only partially diminished this toxicity ( hashimoto et al . , 2003 ) , suggesting that other components are involved . in cgcs , lithium - induced neuroprotection against glutamate excitotoxicity was associated with up - regulation of the anti - apoptotic protein bcl-2 , down - regulation of the pro - apoptotic proteins p53 and bax , and suppressed release of cytochrome c from mitochondria ( chen and chuang , 1999 ) , whereas the involvement of gsk-3 in the regulation of nmda signaling by lithium treatment is currently unclear and requires further investigations .
cyclin - dependent kinase 5 ( cdk5 ) also regulates signaling mediated by nmda receptors , either directly through phosphorylation of the nr2b subunit or indirectly through phosphorylation of psd-95 ( morabito et al . , 2004 ; zhang et al . , 2008
when it binds to p25 ( the product of calpain - mediated cleavage of p35 ) , cdk5 becomes pro - apoptotic and its activity is dysregulated ( lee et al .
, p25 accumulation was observed in neurons in response to glutamate or oxidative stress , and also in the brains of several animal models of neurodegenerative diseases . sustained activation of cdk5 in neurons has been implicated in many neurodegenerative diseases ( cruz and tsai , 2004 ; dhariwala and rajadhyaksha , 2008 ) . in cultured rat cgcs ,
lithium pretreatment prevented colchicine - induced apoptosis and associated increase in cdk5 expression and fragmentation of p35 into p25 ( jorda et al .
. additionally , pretreatment with lithium also attenuated intracellular calcium increase , calpain activity , cdk5 activation , and cellular death in primary cultured hippocampal neurons and rat striatum following the treatment of 3-nitropropionic acid ( crespo - biel et al . , 2009 ) , a succinate dehydrogenase inhibitor ( for review , brouillet et al . , 1999 ) .
therefore , lithium - induced inhibition of calpain and cdk5 activation may also contribute to protection against glutamate excitotoxicity .
prior to changes in gene expression , lithium rapidly and transiently activated the cell survival pi3-kinase and its down - stream target , akt-1 , through phosphorylation at ser473 , thereby reversing glutamate - induced inactivation of this signaling pathway in cgcs ( chalecka - franaszek and chuang , 1999 ) .
activated akt is known to affect several anti - apoptotic targets including bcl-2 associated death promoter ( bad ) , creb , members of the forkhead family , and procaspase-9 ( for review , neri et al . , 2002 ; nicholson and anderson , 2002 ; huang and reichardt , 2003 ) .
in addition , lithium also triggered ser21 phosphorylation of the  isoform of gsk-3 ( and hence resulted in inhibition ) , and this effect was prevented by a pi3-kinase inhibitor ( chalecka - franaszek and chuang , 1999 ) .
another signaling pathway affected by lithium is the mitogen - activated protein ( map ) kinase pathway .
one of the down - stream targets of map kinase is creb , a transcription factor that is involved in learning and memory , and promotes the expression of bcl-2 as well as brain - derived neurotrophic factor ( bdnf ; for review , finkbeiner , 2000 ) . in cgcs , toxic concentrations of glutamate - induced an nmda receptor - dependent decrease in creb phosphorylation at ser133 and creb - driven transcriptional activity ( kopnisky et al . , 2003 ) .
concurrent with its neuroprotective effects , long - term ( but not acute ) lithium treatment suppressed glutamate - induced dephosphorylation of creb .
we also found that glutamate rapidly activated c - jun - n - terminal kinase ( jnk ) and p38 kinase in cgcs , resulting in a robust increase in ap-1 binding ( chen et al . , 2003a ) .
these two kinases are also activated by a variety of apoptotic insults ( for review , mielke and herdegen , 2000 ) , and ap-1 has been known to be activated by different stress factors as well .
experiments using lithium and curcumin , a selective ap-1 inhibitor , suggest that nmda receptor - mediated apoptotic death requires concerted action of jnk and p38 to enhance ap-1 binding , and that lithium s neuroprotection is mediated , at least in part , by suppressing the jnk and p38 kinase pathways .
as one of the major neurotrophins , bdnf is essential for cortical development , synaptic plasticity , and neural survival , and is likely a key mediator of the clinical efficacy of anti - depressants and anxiolytic drugs ( for review , woo and lu , 2006 ) .
the notion that bdnf plays a key role in neuronal survival is supported by our observation that bdnf and neurotrophin-4 ( nt-4 ) , but not nt-3 , completely protected immature cgcs from apoptosis induced by cytosine arabinoside ( leeds et al . , 2005 ) .
it was first reported that chronic treatment with lithium increased the expression of bdnf in the rat brain ( fukumoto et al . , 2001 ) , and we have documented that bdnf protein levels were increased in cortical neurons following lithium treatment ( hashimoto et al . , 2002b ) .
we hypothesized that this bdnf up - regulation and subsequent activation of its receptor trkb might play a critical role in mediating the neuroprotective effects of lithium . in confirmation of this hypothesis
, we found that lithium s neuroprotection against glutamate excitotoxicity was blocked by a trkb inhibitor , k252a , or by a neutralizing antibody against bdnf , and was mimicked by exogenous bdnf in rat cortical neurons .
in addition , lithium increased intracellular levels of bdnf and this was followed by activation of trkb .
furthermore , lithium - induced neuroprotection was prevented in cortical neurons from heterozygous ( + / ) or homozygous ( / ) bdnf knockout mice ( hashimoto et al . , 2002b ) .
rodent bdnf has a complex genomic structure that makes it an ideal target for multiple and complex regulation .
we found that treatment of rat cortical neurons with therapeutic concentrations of lithium ( e.g. , 1  mm ) caused a significant increase in the levels of bdnf exon iv - containing mrna , while levels of exon i , ii , or vi - containing mrna remained unchanged ( yasuda et al . , 2009 ) .
it is known that exon iv - containing bdnf transcripts are expressed in response to kcl - induced depolarization in rat cortical neurons ( tao et al . , 2002 ) .
this transcriptional activation requires utilization of the promoter region 80  bp up - stream from the transcription initiation site of exon iv - containing three calcium responsive elements ( cares ; chen et al . , 2003b ) .
we generated various bdnf promoter iv deletion constructs to investigate whether lithium treatment causes an increase in bdnf promoter iv activity , and , if so , which region of promoter iv confers the sensitivity to this drug .
we identified that the drug - induced up - regulation of exon iv - containing bdnf transcript was associated with a significant increase in the activity of bdnf promoter iv and total bdnf protein . to our surprise , the lithium - responsive element(s ) in promoter iv resides in a region up - stream from the cares responsible for depolarization - induced bdnf induction ( 170 to 704  bp ) .
moreover , activation of bdnf promoter iv occurred in cortical neurons depolarized with kcl and depletion of these three cares failed to abolish lithium - induced activation .
importantly , we found that lithium - induced activation of promoter iv was mimicked by pharmacological inhibitors of gsk-3 ( sb216763 , sb415286 , inhibitor i , and inhibitor vii ) or by transfection with specific sirna for gsk-3 or gsk-3. additionally , their dominant - negative mutants also mimicked lithium - induced activation of promoter iv .
these results demonstrate that gsk-3 is the initial target of lithium to selectively activate bdnf promoter iv and that bdnf induction by lithium involves a novel responsive region in promoter iv of the bdnf gene .
lithium - induced , gsk-3-dependent bdnf promoter iv activation could be a part of the molecular mechanisms underlying its neuroprotective effects and as such , possibly accounts for the therapeutic actions in bipolar patients .
it should be noted that in addition to lithium , other gsk-3 inhibitors have been shown to almost completely block glutamate - induced excitotoxicity in rat cortical neuronal cultures ( liang and chuang , 2007 ) .
these include atp - competitive inhibitors , sb216763 and sb415286 , and atp - non - competitive inhibitors , inhibitor i and vii .
as mentioned in the preceding section , glutamate - induced death of cortical neurons was mitigated by silencing of gsk-3 and/or  , or both isoforms , or inhibition of gsk-3 activity via transfection with dominant - negative mutants of gsk-3/ isoforms ( liang and chuang , 2007 ) .
studies from other laboratories also supported the roles of gsk-3 inhibition in protecting neurons from glutamate neurotoxicity .
for example , stimulation of nmda receptors in cultured rat hippocampal or cortical neurons activated gsk-3 by pp-1-mediated serine dephosphorylation of gsk-3 ( szatmari et al . , 2005 ) .
treatment of primary rat cortical neurons with -amino-3-hydroxy-5-methyl-4-isoxazole propionate ( ampa ) , lithium or sb216763 blocked glutamate - induced caspase-3 activation and excitotoxicity , and the protective effects of ampa required pi3-kinase  akt - dependent serine phosphorylation of gsk-3 ( nishimoto et al . , 2008 ) .
further , in organotypic cultures of chick embryo spinal cord , lithium prevented kainate - induced excitotoxic death of motoneurons by targeting gsk-3 , and this neuroprotection was associated with cytopathological changes ( caldero et al . , 2010 ) .
stroke is the third leading cause of death in the united states and a major global cause of serious long - term disability in adults .
ischemic strokes represent approximately 87% of all cases , while the rest are hemorrhagic strokes ( roger et al . , 2011 ) .
in addition to physical deficits , stroke victims also suffer from vascular depression and dementia , both of which are difficult to treat with conventional medicine .
it is becoming clear that there is a substantial increase in extracellular glutamate in the brain following cerebral ischemia , and that a significant portion of ischemia - induced brain damage is mediated by over - stimulation of nmda receptors .
shortly after ischemia , the interruption of cerebral blood flow depletes oxygen and glucose and subsequently prevents atp production .
inadequate atp supply will cause the malfunction of atp - dependent ion pumps and alter the ion concentration gradient across the neuronal membranes .
the resulting failure to transport glutamate leads to an accumulation of glutamate in the extracellular space and over - stimulates nmda receptors , which leads to a toxic influx of calcium and in turn drives the activation of damaging calcium - mediated intracellular enzymes .
this cascade of events ultimately results in mitochondrial failure , production of reactive oxygen species , neuroinflammation , and cell necrosis and apoptosis ( allen and bayraktutan , 2009 ; deb et al . , 2010 ) .
gsk-3 has been strongly implicated in the neuronal cell death caused by cerebral ischemic insult .
one study in rats subjected to transient middle cerebral artery occlusion ( mcao ) demonstrated a rapid increase in the expression of cytoplasmic and nuclear gsk-3 protein in ipsilateral lamina i , ii , v , and vi in young rat brains , whereas in lamina v and vi in old rat brains ( sasaki et al .
although the phosphorylation status of gsk-3 was not mentioned , these findings implicate a role of gsk-3 in cerebral ischemic injury .
it is well known that gsk-3 can be phosphorylated at serine and tyrosine residues in which ser9 phosphorylation renders it inactive , while tyr216 phosphorylation is necessary for its functional activity ( hughes et al . , 1993 ) .
discrepancies exist in the literature regarding changes of ser9 and tyr216 phosphorylation levels following cerebral ischemia .
the phosphorylation levels of gsk-3 at ser9 and akt at ser473 were reported to be markedly enhanced in the vulnerable hippocampal ca1 region , but not in the ischemia - resistant ca3 region in rats subjected to transient global cerebral ischemia , while there was no change in levels of tyr216 phosphorylation or total gsk-3 ( endo et al . , 2006 ) .
levels of gsk-3 ser9 phosphorylation were also increased shortly after permanent focal cerebral ischemia and decreased to basal levels or even lower 24  h after ischemic onset ( sasaki et al . , 2006 ; gao et al . , 2008
however , it has also been reported that transient focal cerebral ischemia in rats caused an increase in gsk-3 tyr216 phosphorylation in degenerating cortical neurons with no alteration in ser9 phosphorylation ( bhat et al . , 2000 ) .
this discrepancy may stem , in part , from the difference of ischemic severity and ischemic models across various studies .
it appears that transient focal cerebral ischemia tends to activate gsk-3 and subsequently to induce apoptotic cell death .
in contrast , gsk-3 is inactivated shortly after permanent focal cerebral ischemia or global cerebral ischemia , which in turn may promote survival of vulnerable neurons .
ischemia both gsk-3 and gsk-3 mediated the expression of a lethal protein , neuronal pentraxin 1 ( russell et al . , 2011 ) . in light of these findings
the beneficial effects of lithium in rodent cerebral ischemic models demonstrated by us and others support this notion . in an initial study ,
long - term lithium pretreatment at therapeutically relevant doses decreased brain infarct volume , reduced apoptotic cell death and improved behavioral performance after permanent cerebral ischemia - induced by mcao ( nonaka and chuang , 1998 ; xu et al . , 2003 ) . in a subsequent study , we demonstrated that subcutaneous injection of rats with lithium at therapeutic doses ( e.g. , 0.5 and 1.0  meq / kg ) after the onset of transient mcao markedly decreased infarct volume , reduced tunel - positive dna damage , and suppressed neurological deficits measured by sensory , motor , and reflex tests ( ren et al . , 2003 ) .
the time window for these beneficial effects was at least 3  h after the onset of ischemia .
heat - shock protein 70 ( hsp70 ) , a well - established cytoprotective factor against apoptosis , was induced in the ischemic penumbra where neuronal recovery takes place .
post - insult treatment with lithium increased the dna binding activity of hsf-1 to the heat - shock element , superinducing hsp70 which inhibits brain ischemia - induced apoptosis ( ren et al . , 2003 ) .
lithium - elicited gsk-3 inhibition is likely associated with hsf-1 activation and hsp70 induction ( bijur and jope , 2000 ) .
notably , post - insult lithium treatment mitigated apoptosis and brain damage by preventing gsk-3 and erk dephosphorylation , suppressing calpain and caspase-3 activation , and inhibiting mitochondrial release of cytochrome c and apoptosis - inducing factor in a neonatal hypoxic 
these findings suggest that lithium - induced gsk-3 inhibition contributes to its anti - apoptotic effects under ischemic conditions .
in addition , it was found that lithium pretreatment largely suppressed ischemia - induced exploratory behavioral changes and memory impairments in gerbils after global cerebral ischemia ( bian et al . , 2007 ) .
these behavioral benefits were associated with an increase in the number of viable cells and a decrease in apoptotic cells in the ca1 hippocampal area of ischemic gerbils . moreover , lithium - induced neuroprotection in the ischemic brain was accompanied by down - regulation of pro - apoptotic p53 in the ca1 , and up - regulation of anti - apoptotic bcl-2 and hsp70 , both of which are targets of gsk-3 .
it is likely that lithium protection against ischemia - induced injury involves multiple mechanisms . in the rat hippocampus ,
lithium was reported to inhibit ischemia - induced nmda receptor hyperactivation by inhibiting nmda subunit 2a tyrosine phosphorylation and its interactions with src and fyn through psd-95 ( ma and zhang , 2003 ) .
lithium also attenuated hypoxia - induced serine dephosphorylation of gsk-3 and  in the mouse brain ( roh et al . , 2005 ) .
additionally , in organotypic cultures of rat hippocampus subjected to oxygen and glucose deprivation , lithium showed neuroprotection in conjunction with hsp27 activation ( cimarosti et al . , 2001 ) .
post - ischemic inflammation is a dynamic process involving a complicated set of interactions between inflammatory cells and molecules ( iadecola and alexander , 2001 ) . a recent study documented the anti - inflammatory effects of lithium in a neonatal rat hypoxic  ischemic model .
post - insult lithium treatment significantly reduced total tissue loss following hypoxia  ischemia , and this beneficial effect of lithium was associated with inhibiting microglia activation and attenuating levels of pro - inflammatory cytokines or chemokines , such as interleukin-1 and chemokine ligand 2 ( li et al . , 2011 ) .
hsp70 over - expression can inactivate the key inflammatory transcription factor nf-b by stabilizing the nf-b - ib complex , and thereby preventing nuclear translocation of activated nf-b subunits in a mouse mcao model ( zheng et al . , 2008 ) . besides anti - inflammation
, lithium also increased proliferation and differentiation of hippocampal neural progenitor cells in both non - ischemic and ischemic brains without altering the relative levels of neuronal and astrocytic differentiation , and this effect lasted at least 7  weeks after hypoxia  ischemia in neonatal rats ( li et al . ,
in line with this finding , chronic lithium pretreatment was found to increase the generation and survival of newborn cells in the hippocampal dentate gyrus , and did not affect the neuronal or astrocytic differentiation of these newborn cells in a transient four - vessel occlusion model ( yan et al .
erk1/2 phosphorylation following ischemia was enhanced by lithium treatment , while erk1/2 inhibitor u0126 prevented the effects of lithium in increasing brdu - positive cells and improving spatial learning and memory ( yan et al . , 2007 ) .
in fact , chronic lithium treatment has been demonstrated to increase activity in the mek / erk pathway in vivo , and lithium s neuroprotection has been suggested to depend on the induction of this signaling pathway ( einat et al .
in addition , it was reported that activation of erk associates with and phosphorylates gsk-3 at the thr43 residue , which primes this kinase for its subsequent phosphorylation at ser9 by p90rsk , resulting in inactivation of gsk-3 and up - regulation of -catenin ( ding et al . , 2005 ) .
therefore , lithium might affect gsk-3 phosphorylation through the mek / erk pathway , which in turn inhibits this kinase via rsk .
gsk-3 is also negatively regulated by the wnt/-catenin signaling pathway and , accordingly , activating the canonical wnt pathway has been shown to contribute to adult hippocampal neural progenitor cell proliferation triggered by lithium treatment ( wexler et al . , 2008 ) . in a collaborative study ,
the neurohemodynamic aspects of recovery induced by delayed chronic lithium treatment were assessed using functional magnetic resonance imaging ( mri ; kim et al . , 2008 ) .
rats were subjected to transient mcao and then injected with lithium ( licl , 1  meq / kg , s.c . )
this delayed lithium injection was followed by daily injections , and on day 15 , an mri scan was performed to monitor changes in blood oxygen level dependence ( bold ) and functional cerebral blood volume ( fcbv ) responses using electric stimulation of forelimbs .
the mean activated volume ratio and total activation magnitude ratio between ipsilateral and contralateral cortices for both bold and fcbv were significantly higher in the lithium - treated than in the saline - treated rats .
the lithium - induced increase in fcbv in the peri - infarct regions suggests a possible vascular transformation .
indeed , the size and distribution of immunohistochemical staining of cd31 , a microvasculature marker , were enhanced by lithium treatment in the peri - infarct regions .
co - localized with cd31 , the tissue staining of matrix metalloproteinase-9 ( mmp-9 ) was also much more pronounced following lithium treatment , suggesting mmp-9-dependent neurovascular remodeling in the recovering brain area .
moreover , treatment of cultured rat brain endothelial cells with lithium in a follow - up study was also found to increase the protein levels of vascular endothelial growth factor ( vegf ) via a mechanism involving the pi3-kinase and gsk-3 signaling pathways ( guo et al . ,
since vegf has been linked to angiogenesis , neurogenesis , and neuroprotection ( for review , fan and yang , 2007 ) , vegf over - expression may contribute to lithium s ability to promote neurovascular remodeling and to induce functional recovery after ischemic stroke .
ample evidence supports the therapeutic potential of mesenchymal stem cells ( mscs ) in several human diseases including stroke .
however , it is increasingly recognized that the effectiveness of msc transplantation is limited by their poor migration toward disease target sites such as ischemic brain regions . in a recent study
, we investigated whether treatment of mscs with lithium and another mood stabilizing drug , valproic acid ( vpa ) , would enhance cell migration ( tsai et al . , 2010 ) .
we found that treatment of mscs with lithium ( 2.5  mm for 1  day ) selectively elevated the transcript and protein levels of mmp-9 and its enzymatic activity .
these effects were mimicked by pharmacological inhibition or gene silencing of gsk-3. lithium treatment also potentiated stromal cell - derived factor-1 ( sdf-1)-dependent msc migration across the extracellular matrix , which was suppressed by two mmp-9 inhibitors , doxycycline and gm6001 .
short - term ( 3  h ) exposure of mscs to a relatively high concentration ( 2.5  mm ) of vpa markedly increased the transcript and protein levels of cxc chemokine receptor 4 ( cxcr4 ) .
vpa - induced cxcr4 expression required its ability to inhibit histone deacetylases ( hdacs ) , including the hdac1 isoform , and involved histone hyperacetylation at the cxcr4 gene promoter .
vpa treatment enhanced sdf-1-mediated msc migration , which was completely blocked by amd3100 , a cxcr4 antagonist .
notably , combining lithium and vpa treatment further increased msc migration , and the additive enhancement of migration was completely blocked by the co - presence of amd3100 and gm6001 .
our results suggest that lithium and vpa stimulate msc migration through distinct targets and mediators : gsk-3mmp-9 and hdac  cxcr4 , respectively ( tsai et al . , 2010 ) .
in a follow - up in vivo study , mscs were primed with lithium and/or vpa and then injected into the tail vein of transient mcao rats 24  h after ischemic onset . priming with lithium or vpa increased the number of mscs homing to the cerebral infarcted regions such as the cortex and striatum 2  weeks after transplantation , and co - priming with lithium and vpa further enhanced this migratory effect ( tsai et al . , 2011 ) .
mcao rats receiving lithium- and/or vpa - primed mscs showed improved functional recovery , reduced infarct volume , and enhanced angiogenesis in the infarcted penumbra regions .
these beneficial effects of lithium and vpa priming were reversed by pharmacological inhibition of mmp-9 and cxcr4 , respectively , suggesting that these effects were likely mediated by lithium - induced mmp-9 up - regulation and vpa - induced cxcr4 over - expression .
together , these findings raise the potential utility of using mscs primed with inhibitors of gsk-3 and hdac to enhance the migration and homing capacity for transplantation into stroke victims .
in addition to lithium , other pharmacological gsk-3 inhibitors have been shown to exert neuroprotective effects against cerebral ischemia by various groups .
a specific gsk-3 inhibitor , chir025 , was demonstrated to protect cultured hippocampal neurons from glutamate excitotoxicity and to attenuate death of cortical neurons following oxygen 
glucose deprivation , an in vitro model of cerebral ischemia ( kelly et al . , 2004 ) .
moreover , chir025 reduced infarct size in focal cerebral ischemic rats , but did not affect tunel - positive neurons or caspase-3/9 activities , although bcl-2 expression was increased .
gsk-3 enzymatic activity was markedly elevated after transient mcao in rats , and this gsk-3 activation was blocked by jugular vein injection of gsk-3 inhibitor viii ( koh et al . , 2008 ) .
pre- or post- ( up to 2  h ) mcao injection with inhibitor viii also reduced blood glucose levels , infarct size , caspase-3 activity , and water content in the ipsilateral brain hemisphere .
furthermore , ischemia - induced inflammation - related signals such as cox-2 over - expression and neutrophil infiltration were alleviated by this gsk-3 inhibitor .
prophylactic or therapeutic administration of a gsk-3 inhibitor tdzd-8 reduced infarct volume and cerebral injury in the rat hippocampus after transient ischemia ( collino et al . , 2008 ) .
this was accompanied by suppression of ischemia - induced oxidative stress , apoptosis , and neuroinflammation .
delayed treatment with compound i , a gsk-3 and cdk inhibitor , decreased tunel - positive cells in the ipsilateral hippocampus and striatum of adult ( but not juvenile ) mice subjected to hypoxic 
these neuroprotective effects of compound i were associated with long - lasting functional recovery . finally , gsk-3 inhibition by sb216763 counteracted oxygen 
glucose deprivation - induced mitochondrial biogenesis impairment and reduced mitochondrial reactive oxygen species generation in primary cortical neurons ( valerio et al . , 2011 ) .
when systematically administrated to permanent mcao mice , sb216763 decreased infarct volume and restored the loss of mitochondrial dna , thus supporting a novel role of gsk-3 inhibitors in stimulating the renewal of functional mitochondria following ischemic stroke .
a growing body of evidence supports that lithium , a mood stabilizer used to treat bipolar disorder , has neuroprotective properties in both cellular and in vivo experimental settings .
one of the major targets of lithium is gsk-3 , a serine / threonine kinase implicated in the pathogenesis of diverse cns disorders .
lithium inhibits gsk-3 activity by direct binding to the enzyme or indirectly by enhancing serine phosphorylation of both  and  isoforms through multiple mechanisms .
lithium has been used as a prototype drug to seek evidence for the involvement of gsk-3 inhibition in lithium - induced protection against excitotoxicity in cultured neurons and animal models of cerebral ischemic stroke .
lithium at therapeutically relevant concentrations robustly protected primary brain neurons from glutamate - induced , nmda receptor - mediated excitotoxicity .
the neuroprotective effects of lithium were associated with gsk-3 inhibition , and were mimicked by other pharmacological gsk-3 inhibitors , by silencing gsk-3 and/or  isoforms , or by expression of isoform - specific dominant - negative mutants .
these results support the roles of gsk-3 inhibition in lithium - elicited protection against excitotoxicity .
lithium rapidly activated the cell survival pi3-kinase  akt signaling pathway to enhance gsk-3 serine phosphorylation and to block glutamate - induced akt inactivation as well as apoptosis .
lithium also caused an increase in the expression of cytoprotective bcl-2 and suppressed glutamate - induced up - regulation of pro - apoptotic p53 and bax , resulting in blocking cytochrome c release from mitochondria .
bdnf promoter iv was selectively activated by gsk-3 inhibition using lithium or other drugs or through gene silencing / inactivation of either isoform . this effect on
however , there is a gap in the understanding of how gsk-3 inhibition causes an increase in bdnf promoter activity .
in addition , lithium s neuroprotective effects were associated with inhibition of nmda receptor - mediated calcium influx and suppression of p38/jnk and ap-1 activation , thus reducing apoptosis .
this effect appears to stem from inhibition of src / fyn kinase to suppress nr2b tyr1472 phosphorylation of the receptor .
it remains to be explored as to whether this lithium - induced action on nmda receptors is related to gsk-3 inhibition .
the potential roles of these other targets in mediating the neuroprotective effects of this drug also deserve future investigation .
it is well known that glutamate overflow and nmda receptor hyper - stimulation are early events following cerebral ischemia . in rodent
ischemic models , pre- or post - insult treatment with therapeutic doses of lithium decreased infarct volume , caspase-3 activity and apoptotic cells in the injured brain .
the beneficial time window of lithium is at least 3  h after the ischemic onset .
up - regulation of hsp70 and bcl-2 as well as down - regulation of p53 likely contributed to the protective effects of lithium in the ischemic conditions , thus supporting similar underlying neuroprotective mechanisms in the excitotoxic cellular models and animal models of ischemic stroke .
limited data suggested that lithium might also display anti - inflammatory effects by inhibiting ischemia - induced microglia activation and pro - inflammatory factors release .
delayed and chronic injections of lithium improved functional mri responses such as increases in bold and fcbv .
indeed , lithium was found to induce two pro - angiogenic factors , mmp-9 and vegf in a gsk-3-dependent manner .
lithium has also been reported to stimulate erk1/2 activity and to enhance proliferation of hippocampal neural progenitor cells and memory performance after ischemia . finally , lithium promoted migration of mscs in vitro by up - regulation of mmp-9 through gsk-3 inhibition and this migratory effect was potentiated by co - treatment with vpa , another mood stabilizer .
notably , transplantation of lithium  vpa co - primed mscs into ischemic rats markedly increased msc migration to the injured brain regions , decreased infarct size and improved the neurological performance .
lithium - induced stem cell migration , neurogenesis , and angiogenesis all likely contribute to functional recovery .
figure 3 illustrates proposed molecular events leading to lithium - induced beneficial effects following cerebral ischemia .
it should be noted that several other gsk-3 inhibitors have also been reported to exert beneficial effects in rodent ischemic models and their actions were accompanied by suppression of ischemia - increased gsk-3 activity .
accordingly , gsk-3 inhibitors have therapeutic potential to treat stroke and other excitotoxicity - related neurodegenerative diseases .
lithium has been used in bipolar patients over 60  years and its clinical profiles are well understood .
therefore , lithium is a prime candidate for use in clinical trials of new therapies for stroke victims .
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-b .
counteraction of gsk-3 inhibition of vegf and mmp-9 by lithium enhances angiogenesis and neurovascular remodeling .
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 .
the authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest .
 the mood stabilizer lithium inhibits glycogen synthase kinase-3 ( gsk-3 ) directly or indirectly by enhancing serine phosphorylation of both  and  isoforms . 
 lithium robustly protected primary brain neurons from glutamate - induced excitotoxicity ; these actions were mimicked by other gsk-3 inhibitors or silencing / inhibiting gsk-3 and/or  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-3 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 . 
Introduction
Regulation and Function of GSK-3 Isoforms
Involvement of GSK-3 and Other Molecules in Lithium-Elicited Neuroprotection Against Glutamate-Induced Excitotoxicity in Cellular Models
Neuroprotective Effects of Lithium in Preclinical Models of Ischemic Stroke
Conclusion
Conflict of Interest Statement