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BMC Neuroscience

Open AccesResearch article

Glutamate-induced apoptosis in neuronal cells is mediated viacaspase-dependent and independent mechanisms involving calpain

and caspase-3 proteases as well as apoptosis inducing factor (AIF)and this process is inhibited by equine estrogensYueMei Zhang and Bhagu R Bhavnani*

Address: Department of Obstetrics and Gynecology, University of Toronto, Institute of Medical Sciences, University of Toronto, Department ofObstetrics and Gynecology, St. Michael's Hospital, Toronto, Ontario, Canada

Email: YueMei Zhang - [email protected]; Bhagu R Bhavnani* - [email protected]

* Corresponding author

Abstract

Background: Glutamate, a major excitatory amino acid neurotransmitter, causes apoptotic neuronal cell

death at high concentrations. Our previous studies have shown that depending on the neuronal cell type,

glutamate-induced apoptotic cell death was associated with regulation of genes such as Bcl-2, Bax, and/orcaspase-3 and mitochondrial cytochrome c. To further delineate the intracellular mechanisms, we have

investigated the role of calpain, an important calcium-dependent protease thought to be involved inapoptosis along with mitochondrial apoptosis inducing factor (AIF) and caspase-3 in primary cortical cells

and a mouse hippocampal cell line HT22.

Results: Glutamate-induced apoptotic cell death in neuronal cells was associated with characteristic DNA

fragmentation, morphological changes, activation of calpain and caspase-3 as well as the upregulation and/

or translocation of AIF from mitochondria into cytosol and nuclei. Our results reveal that primary cortical

cells and HT22 cells display different patterns of regulation of these genes/proteins. In primary cortical

cells, glutamate induces activation of calpain, caspase-3 and translocation of AIF from mitochondria tocytosol and nuclei. In contrast, in HT22 cells, only the activation of calpain and upregulation and

translocation of AIF occurred. In both cell types, these processes were inhibited/reversed by 17-estradiol

and 8,17-estradiol with the latter being more potent.

Conclusion: Depending upon the neuronal cell type, at least two mechanisms are involved in glutamate-

induced apoptosis: a caspase-3-dependent pathway and a caspase-independent pathway involving calpainand AIF. Since HT22 cells lack caspase-3, glutamate-induced apoptosis is mediated via the caspase-

independent pathway in this cell line. Kinetics of this apoptotic pathway further indicate that calpain rather

than caspase-3, plays a critical role in the glutamate-induced apoptosis. Our studies further indicate thatglutamate- induced changes of these proteins can be inhibited by estrogens, with 8,17-estradiol, a novel

equine estrogen being more potent than 17-estradiol. To our knowledge, this is the first demonstration

that glutamate-induced apoptosis involves regulation of multiple apoptotic effectors that can be inhibited

by estrogens. Whether these observations can help in the development of novel therapeutic approaches

for the prevention of neurodegenerative diseases with estrogens and calpain inhibitors remains to be

investigated.

Published: 15 June 2006

BMC Neuroscience 2006, 7:49 doi:10.1186/1471-2202-7-49

Received: 07 December 2005

Accepted: 15 June 2006

This article is available from: http://www.biomedcentral.com/1471-2202/7/49

2006 Zhang and Bhavnani; licensee BioMed Central Ltd.This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0),which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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BMC Neuroscience 2006, 7:49 http://www.biomedcentral.com/1471-2202/7/49


BackgroundHigh concentrations (mM) of the excitatory neurotrans-mitter glutamate can accumulate in the brain and arethought to be involved in the etiology of a number of neu-rodegenerative disorders including Alzheimer's disease

[1,2]. A number of invitro studies indicate that at highconcentrations, glutamate is a potent neurotoxin capableof destroying neurons by apoptosis [3,4]. We and othershave previously reported that glutamate induces charac-teristic oligonucleosomal DNA fragmentation (DNA lad-der) and apoptotic cell death by up and down-regulationof Bax and Bcl-2, in a stable mouse hippocampal neuronalcell line HT22 which lacks caspase-3, the primary activatorof apoptotic DNA fragmentation [5]. In contrast, in pri-mary cortical cells, glutamate-induced cell death involvesupregulation of caspase-3 and its activation via a caspase-dependent pathway involving mitochondrial signaling[6]. Glutamate-induced DNA fragmentation observed in

HT22 cells implies that regulatory factors other than cas-pase-3 are involved in the apoptotic process in these cells.

Recent studies demonstrate that calpain, a calcium-dependent protease, and apoptosis inducing factor (AIF)can play an important role in apoptotic cell death via acaspase-independent apoptotic pathway [7-11]. Gluta-mate toxicity appears to involve a rapid Ca2+ influx intoneurons and these high levels of intracellular Ca2+ arecytotoxic [12,13]. Ca2+ can activate several key enzymes,including nitric oxide synthase (NOS) and proteases suchas calpains and can also result in mitochondrial dysfunc-tion [12,14]. Furthermore, a reduction in mitochondrial

transmembrane potential has been reported to accom-pany AIF release and early apoptosis [15,16]. AIF is a ubiq-uitously expressed flavoprotein with significanthomology to bacterial oxidoreductases and has NADHoxidase activity [17]. Following induction of apoptosis,

AIF translocates from the outer mitochondrial membraneto the cytosol and the nucleus, resulting in the inductionof nuclear chromatin condensation and large molecular

weight DNA fragmentation in a caspase-independentmanner [18,19]. Proteases such as caspases, calpains andgranzyme B [20-22], have been reported to play a criticalrole in mediating apoptosis, especially the key modulatorcaspase-3. Similarly, calpains have been implicated in

apoptosis in response to hypoxia and irradiation exposurein neuronal and non-neuronal cells [23]. Calpain is a cal-cium-dependent papain-like neutral cysteine protease,

which is widely distributed in neurons [23,24]. A numberof subcellular targets have been identified as substrates forcalpain cleavage, including spectrin, microtubules-associ-ated protein (MAP), tau and neurofilaments, however, theprecise physiological role of calpain remains obscure [24].

Activation of calpain is triggered by an elevation of cyto-plasmic free Ca2+ concentration which results in the cleav-age of various proteins and culminates in cell death [25].

Activation of calpain is an early event in the onset of apop-tosis in thymocytes therefore inhibitors of calpain canreduce this process of cell death [26,27]. Calpain is alsoimplicated in neuronal cell death associated with cerebralischemia and other neuronal insults [28,29].

A number of studies have demonstrated that estrogens arepotent antioxidants that can inhibit some of the neuro-toxic effects of oxidative stress [4,30,31]. We and othershave reported that estrogens can increase cell survival andattenuate invitrocell death induced by potential Alzhe-imer's disease-related insults, such as exposure to oxidizedLDL and glutamate in a hippocampal cell line and pri-mary cortical neurons [32-34]. The mechanism underly-ing neuroprotection by estrogens against glutamateoxidized neurotoxicity is not fully understood. Recently

we have reported that neuroprotection of estrogensagainst glutamate cytotoxicity is associated with modula-

tion of the expression of Bcl-2 family of genes and cas-pase-3 proteins, inhibition of cytochrome c release frommitochondria into the cytosol and prevention of DNAfragmentation [5,6]. In the present study, we selected twocell types, primary cortical cells and hippocampal cell lineHT22, to investigate the roles of calpain, apoptosis induc-ing factor (AIF) and caspase-3 in glutamate-induced apop-tosis and its prevention by estrogens. The estrogenschosen for this study were 17-estradiol (17-E2) and

8,17-estradiol (8, 17-E2), the latter being the morepotent antioxidant [35,36].

Results

Expression of ERand ERmRNA in rat cortical cellsIt has been reported that the HT22 cell line lacks estrogenreceptors [4]. In order to ascertain expression and relativedistribution of estrogen receptors ER or ER in rat corti-cal cells, we performed RT-PCR and Western blot analysis(Figure 1). The results revealed that freshly isolated neuro-nal cortical cells or cells after 78 days in culture expressedboth ER and ER with the expression of ER beinghigher than that of ER. Both subtypes of ER mRNAs andthe corresponding proteins were found in the adult ratuterus (positive control), with ER levels higher thanthose of ER. This is in keeping with a previous report[41].

Anti-apoptotic effects of protease inhibitors of calpain and

caspase-3 in mouse hippocampal cell line (HT22) and

primary cortical cells

We have previously reported that glutamate treatmentinduces apoptotic cell death in HT22 cells and primarycortical cells in a time and dose-dependent manner [5,6].

We had shown that the commitment point to this deathwas ~6 hours after initiation of 1 mM glutamate exposurein primary cortical cells and for ~8 hour exposure with 78 mM glutamate in HT22 cells. Based on these prelimi-
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nary data, all subsequent experiments were carried outwith a 6 hour treatment with 1 mM glutamate in primary

cortical cells and 8 hours with 78 mM glutamate in HT22cells.

Effects of calpain and caspase inhibitors on glutamate-

induced cell death

(a) In mouse hippocampal cell line

Treatment of HT22 cells with 8 mM glutamate was initi-ated in the presence or absence of various concentrationsof calpain I inhibitor (ALLN) or PD150606 for up to 8hours. The results are depicted in Figures 2 A and 2B.Glutamate (8 mM) alone caused a significant increase(50%) in cell death compared to control, while calpaininhibitors, either ALLN or PD150606, reduced glutamate-

induced cell death in a dose-dependent manner. ALLN(12.550 M) reduced cell death by 5070% (Figure 2A).Similarly, 50 to 100 M of PD150606 decreased cell deathby 70100% (Figure 2B). However, when cells weretreated at a lower glutamate concentration (34 mM) fora longer period (16 hours) however, cell death increasedby 40 to 50%, and this cell death was prevented by 12.550 M ALLN (data not shown). In the absence of gluta-mate, neither inhibitor had an effect on cell viability. Theeffect of calpain inhibitors on glutamate-induced apop-totic cell death was also associated with characteristic

morphological changes (Figure 3). After an 8 hour incuba-tion with 8 mM glutamate, degenerated and dead cells

were clearly visible in the culture (Figure 3B); the cellularextensions (dendrites) seen in untreated HT22 cells (Fig-ure 3A) were retracted and the cells appeared rounded andhad detached. In the presence of calpain inhibitors, 25 M

ALLN or 100 M PD150606, most cells survived and hadthe appearance of normal untreated cells (Figure 3C and3D). Only a small number of dead cells were visible in thepresence of 25 M ALLN (Figure 3C). In contrast, caspaseinhibitors, either the specific caspase-3 inhibitor z-DEVD(Figure 3E) or a pancaspase inhibitor z-VAD (Figure 3F) ata concentration of 100 M, failed to inhibit cell deathinduced by 8 mM glutamate. This cell death was furtherconfirmed by using LDH release assay (data not shown).

(b) In primary cultures of rat cortical neuronal cells

To determine the role of calpain and caspase-3 in primarycortical cells, cells were treated with 1 mM glutamate inthe presence or absence of calpain inhibitor (12.5 M

ALLN) or specific caspase-3 inhibitor (50 M z-DEVD) for6 hours (Figure 4). Glutamate treatment alone inducedone fold increase (50%) in LDH release or cell death com-pared to control. Caspase-3 inhibitor z-DEVD signifi-cantly inhibited glutamate-induced cell death by 60%.Similarly, the presence of calpain inhibitor ALLN resulted

Expression of estrogen receptors (ER and ER) mRNA and protein in rat neuronal cortical cellsFigure 1Expression of estrogen receptors (ER and ER) mRNA and protein in rat neuronal cortical cells. Rat corticalcells were cultured for 78 days and total RNA and protein were extracted. RT-PCR and Western blot analysis were carriedout as described under "Methods". Rat uterus was used as a positive control.

Rat Tissue 7 days Procedureuterus Cortex Control

G3PDH

ER

ER

Rat Tissue 7 days 8 daysuterus Cortex

mRNA

expression

Protein

expression
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in a 70% reduction in (1 mM) glutamate -induced celldeath. These data indicate that both caspase and calpain-mediated apoptosis were involved in glutamate-inducedcell death in primary cortical cells. However, the addition

of a combination of two protease inhibitors did not resultin a greater decrease in LDH release or cell death, suggest-ing that calpain and caspase inhibitors has no synergeticeffects on glutamate-induced cell death. Protection byboth inhibitors was also detectable by characteristic mor-phological changes (Figure 5). Thus after a 6 hour incuba-tion with 1 mM glutamate, degenerated and dead cells

were clearly visible in the culture (Figure 5B), the cellularextensions (dendrites) seen in untreated cortical cells (Fig-ure 5A) were retracted, and cells appeared rounded. In thepresence of calpain inhibitors (12.5 M ALLN) or caspase-

3 specific inhibitor (50 M z-DEVD), dendrites wereretained and cells had the appearance of untreated normalcells (Figures 5C and 5D), however, some degeneratedand dead cells were also visible. Activation of caspase-3

was also detected with a highly specific active caspase-3

antibody raised against amino acids 163 to 175 (p18 sub-unit) of murine caspase-3 (data not shown). No change incaspase-3 activity was found in HT22 exposed to variousconcentrations of glutamate for up to 8 hours, while acti-

vation of caspase-3 significantly increased in glutamate-treated cells in primary cortical cells as observed previ-ously by us [6].

Effects of calpain and caspase inhibitors on glutamate-

induced DNA fragmentation

To further investigate the effects of calpain and caspase-3inhibitors on glutamate-induced programmed cell death(apoptosis), their effects on glutamate-induced character-

istic DNA fragmentation were determined. HT22 cellswere harvested after 8 hours or 16 hours of glutamate (8mM) exposure in the presence or absence of calpaininhibitors or caspase inhibitors. Total DNA was extracted,purified and subjected to agarose gel electrophoresis. Theresults indicate that untreated cells did not display anyDNA fragmentation (Figures 6A and 6B, Lane 1). In con-trast, (8 mM) glutamate treatment resulted in characteris-tic DNA fragmentation or laddering (Figures 6A, Lane 2)that was associated with an increase in cell death. Addi-tion of optimal doses of calpain inhibitors, 100 MPD150606 or 25 M ALLN, reduced or blocked gluta-mate-induced DNA fragmentation, (Figure 6 A, Lanes 3

and 5). In contrast, in the presence of caspase inhibitors,(8 mM) glutamate-induced DNA fragmentation (Figure6B, Lane 4) was not prevented by either the caspase-3 spe-cific inhibitor, 100 M z-DEVD (Figure 6B, Lane 5) or 100M z-VAD (Figure 6B, Lane 6) for up to 16 hours. In theabsence of glutamate, none of the inhibitors alone (Fig-ures 6A and 6B) had any effect on DNA fragmentation.

Determination of caspase and calpain activation

Activation of calpain and caspase-3 after glutamate expo-sure in the primary cortical cells and HT22 cells wasassessed by Western blot analysis by examining proteoly-sis of-fodrin (-II-spectrin) utilizing an epitope-specific

antibody directed against calpain or caspase-3 cleavagesite of-fodrin. Cleavage of-fodrin leading to formationof 150/145 kDa breakdown fragments is a well-recog-nized marker for the calpain-generated protein break-down product (BDP) and formation of 120 kDa fragmentfor the caspase-3 specific (BDP). Antibodies raised againstthis epitope of-fodrin have been shown previously to bespecific for calpain-cleaved -fodrin breakdown products145 kDa or/and 150 KDa (145/150 kDa BDPs) and cas-pase-produced -fodrin breakdown products 120 KDa(120 KDa BDP) [21,42]. The presence of-fodrin BDPs

Effect of various concentrations of calpain inhibitors, ALLNand PD150606, on glutamate-induced cell death in HT22cellsFigure 2Effect of various concentrations of calpain inhibitors,

ALLN and PD150606, on glutamate-induced celldeath in HT22 cells. HT22 cells were treated with 8 mMglutamate in the presence or absence of 150 M ALLN (A)and 1100 M PD150606 (B). Cell death was assessed bymeasuring LDH release after 8 hrs of treatment *P< 0.05compared to control; P< 0.05 compared to glutamatealone.

0

25

50

75

100

*

*

*

*

*

A

Control Glu Alone8 mM

1 12.5 25 50 ALLNalone

Glutamate + ALLN (M)

LDHRe

lease

(%M

ax

imum

)

0

25

50

75

100

*

* **

Glutamate + PD150606 (M)Control Glu Alone

8 mM

1 25 50 100 PD alone

LDHRe

lease

(%M

ax

imum

) B
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Effect of calpain and caspase inhibitors on glutamate toxicity in HT22 cells as assessed by phase contrast microscopy (20)Figure 3Effect of calpain and caspase inhibitors on glutamate toxicity in HT22 cells as assessed by phase contrastmicroscopy (20). HT22 cells were treated with 8 mM glutamate in the presence or absence of calpain inhibitors, 25 MALLN and 100 M PD150606, and caspase inhibitors, 100 M z-DEVD and 100 M z-VAD, for 8 hrs. Normal cells aredepicted by arrows and degenerated cells by arrowheads. A = untreated control cells; B = glutamate alone; C = glutamate +ALLN; D = glutamate + PD150606; E = glutamate + z-DEVD; F = glutamate + z-VAD.

A B

DC

FE


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145/150 and BDP 120 would suggest that calpain and cas-pase-3 are activated under apoptotic conditions [43].

In HT22 cells, untreated cells contained the 240 KDa

intact-fodrin band and following treatment with gluta-mate, the 240 KDa band was cleaved into 145 KDa BDP,but not the -fodrin 120 KDa BDP in this cell line. Asshown in Figure 7A, glutamate induced calpain activity ina time-dependent manner. Thus, calpain activity began toincrease at 6 hours after 78 mM glutamate exposure,

which preceded glutamate-induced LDH release andapoptotic cell death, and was robustly activated by 78hours. After a 1416 hour treatment, degradation ofnative -fodrin was observed and BDPs were less noticea-ble. The addition of calpain inhibitors (100 MPD150606 or 25 M ALLN) completely blocked orreduced glutamate-induced formation of 145 kDa BDPs

(Figure 7B), which is consistent with the observation thatthey protect against glutamate-induced DNA fragmenta-tion. These data further demonstrate that calpain proteaseis indeed activated and mediates glutamate-inducedapoptotic cell death via a caspase-independent pathway inHT22 cells.

In contrast to HT22 cells, cleavage of-fodrin (240 kDa)protein into 145 kDa and 120 kDa BDPs was found inuntreated cells (78 days of culture) (Figure 8A and 8B).Similarly, glutamate treatment resulted in an increase in

the activity of calpain and caspase-3 in a time-dependentmanner. Formation of 145 kDa BDPs by calpain began toincrease earlier (0.5 hour) than the caspase-mediated 120kDa BDP (3 hours), with greater accumulation of bothbreakdown products (145 kDa and 120 kDa) after 6 hours

(Figure 8 A). To further investigate the -fodrin break-down patterns produced by calpain or caspase-3, theeffects of calpain and caspase inhibitors on glutamate-induced cleavage of-fodrin were investigated by immu-noblotting. The presence of calpain inhibitor 25 M ALLNreduced the formation of BDPs 145 kDa in primary corti-cal cells exposed to glutamate, but it had no effects on 120kDa BDP120 (Figure 9). However, the addition of cas-pase-3 specific inhibitor 50 M z-DEVD completely inhib-ited the formation of 120 kDa BDP, but had no effect onthe formation of 145 kDa BDPs (Figure 9).

We have previously reported that primary cortical cells in

culture for longer than 2 days display characteristic DNAfragmentation. To further detect whether cellular mecha-nisms mediating this apoptotic process involve activationof these proteases, cleavage of-fodrin was measured byusing Western blot analysis with cells from fresh tissue,day 0, day 1, day 3, days 7 and 8 in culture (Figure 8B).Calpain-mediated cleavage of-fodrin protein into 145KDa BDPs could be seen on day 0 of culture (dissociatedcells prior to culture) prior to the initiation of DNA frag-mentation (2 days). The activation further increased up to7 days. Caspase-3-dependent 120 KDa BDP was detecta-ble only after 7 days of culture. These data indicate thatapoptosis occurs during the development of the neuronal

cells (increasing days in culture) and is also associatedwith calpain and caspase-3 activation. The results furtherindicate that activation of calpain precedes the presence ofapoptosis characterized by DNA laddering and caspase-3activity and suggest that calpain protease may be the firstprotease in mediating neuronal apoptosis.

Effect of estrogens on the activation of calpain and

caspase-3

We have previously reported that estrogens, 17-estradiol(17-E2) and

8,17-estradiol (8,17-E2), protect againstglutamate-induced cell death in a dose-dependent man-ner. HT22 cells were treated with 7 mM glutamate for 7

8 hours in the presence or absence of 1 M and 10 M17-E2 or 8,17-E2. Cell death was measured by LDHrelease assay and the results are summarized in Figure10 A. Both 17-E2 and

8,17-E2 inhibited glutamate-induced cell death, with 8,17-E2 being more potent.

Thus, in the presence of 17-E2, only 10 M 17-E2 couldsignificantly reduce cell death compared to glutamatealone (Figure 10A). However, the presence of8,17-E2, 1M or 10 M, completely inhibited cell death andreturned the release of LDH to control levels. In contrast,in primary cortical cells exposed to 1 mM glutamate for 6

Effect of calpain and caspase inhibitors on glutamate-inducedcell death in primary cortical cellsFigure 4Effect of calpain and caspase inhibitors on glutamate-induced cell death in primary cortical cells. Primary

cortical cells isolated from 18 day old fetal rat brains werecultured for 78 days and treated with 1 mM glutamate aloneor in the presence of the optimal concentration of 12.5 Mcalpain inhibitor ALLN and 50 M z-DEVD caspase-3 inhibi-tor. LDH release (% maximum) was measured after 6 hrs oftreatment *P< 0.05 compared to control; P< 0.05 com-pared to glutamate alone.

0

10

20

30

*

**

*

+ + + +

+ + +

+ + +

Glutamate (1 mM)

ALLN (12.5 M)DEVD (50 M)

LDHRelease(%Maximum)
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hrs, 1 M or 10 M 17-E2 or8,17-E2 could completely

protect against cell death (Figure 10B). Following meas-urement of LDH, inhibition by estrogens of glutamate-induced cleavage of-fodrin in both cell types was deter-mined by Western blot analysis. In HT22 cells, the pres-ence of 10 M 17-E2 and 1 M

8,17-E2 in 7 mMglutamate for 8 hours significantly decreased glutamate-induced cleavage of-fodrin into 145 KDa BDP by 21%and 51%, respectively, compared to glutamate alone (Fig-ure 11A). However, 10 M 8,17-E2 totally inhibited theformation of 145 KDa BDP. Moreover, in primary corticalcells exposed to glutamate (1 mM) in the presence of

estrogens (1 M and 10 M 17-E2 or8,17-E2) for 6

hours, estrogens not only inhibited calpain-mediated for-mation of 145 KDa BDP, but also reduced caspase-3-mediated formation of 120 KDa BDP (Figure 11B). Thus,1 M 17-E2 significantly reduced breakdown products of145 and 120 KDa compared to glutamate alone. Simi-larly, 10 M 17-E2 and 1 M

8,17-E2 could completelyprevent activation of calpain and caspase-3 proteases andtotally block cleavage of-fodrin. These data indicate thatestrogens protect against glutamate-induced apoptoticcell death by inhibiting both calpain and caspase-3 activ-ity in neuronal cells, with 8,17-E2 being more potent.

Effect of calpain and caspase inhibitors on glutamate-induced cell death in primary cortical cells as assessed by phase contrastmicroscopy (20)Figure 5Effect of calpain and caspase inhibitors on glutamate-induced cell death in primary cortical cells as assessed byphase contrast microscopy (20). Primary cortical cells were isolated and treated as described under Figure 4. Normalcells are depicted by arrows and degenerated cells by arrowheads. A = untreated control cells; B = glutamate lone; C = gluta-mate + ALLN; D = glutamate + z-DEVD.

A B

C D
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Effects of glutamate and estrogens on protein levels of

apoptosis inducing factor (AIF) in different cell fractions

from two cell types

Following the determination of calpain and caspase-3protease's involvement in apoptotic cell death, regulationof mitochondrial apoptotic effector AIF in cells undergo-ing apoptosis was determined in primary cortical cells andHT22 cells. Protein levels in cytosol, mitochondria, nucleiand whole cell lysates were detected by Western blot anal-

ysis. In cortical cells, treatment with glutamate (1 mM)resulted in an increase in AIF protein levels in the cytosoland nuclei in a time dependent-manner (Figure 12A).Concomitant with these changes, the levels of AIF proteindecreased in the mitochondria and no changes were

found in whole cell lysates. The elevation of cytosolic andnuclear AIF and its reduction in the mitochondria wereassociated with an increase in cell death. These results

indicate that AIF protein is released or translocates frommitochondria into cytosol and nuclei in primary corticalcells undergoing apoptotic cell death following exposureto glutamate. However, exposure of HT22 cells to gluta-mate (78 mM) enhanced AIF protein levels in all subcel-lular fractions of cells in a time-dependent manner that isalso associated with glutamate-induced cell death. Asshown in Figure 12B, AIF protein levels were increased notonly in the cytosol and nuclei but also in the mitochon-dria and whole cell lysates. These data demonstrate thatglutamate can upregulate AIF protein levels in HT22 cells.

Effect of calpain and caspase inhibitors on glutamate-induced DNA fragmentation (DNA ladder) in HT22 cellsFigure 6Effect of calpain and caspase inhibitors on glutamate-induced DNA fragmentation (DNA ladder) in HT22 cells.HT22 cells were treated with 8 mM glutamate in the presence or absence of calpain inhibitors 25 M ALLN and 100 M

PD150606, and caspase inhibitors 100 M z-DEVD and 100 M z-VAD. A: HT22 cells were treated with glutamate in the pres-ence of calpain inhibitors for 8 hrs. Lane 1 = untreated control cells; Lane 2 = glutamate alone; Lane 3 = glutamate +PD150606; Lane 4 = PD150606 alone; Lane 5 = glutamate + ALLN; Lane 6 = ALLN alone. B: HT22 cells were treated withglutamate in the presence of caspase inhibitors for 16 hrs. Lane 1 = untreated control cells; Lane 2 = z-DEVD alone; Lane 3 =z-VAD alone; Lane 4 = glutamate alone; Lane 5 = glutamate + z-DEVD; Lane 6 = glutamate + z-VAD; M: 100 bp DNA marker.Gel electrophoresis was carried out as described under "Methods" and the gels were stained with ethidium bromide.

M 1 2 3 4 5 6 M 1 2 3 4 5 6

A B
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Glutamate-induced the cleavage of-fodrin in HT22 cells and inhibition by calpain inhibitors as detected by Western blot anal-ysisFigure 7Glutamate-induced the cleavage of-fodrin in HT22 cells and inhibition by calpain inhibitors as detected byWestern blot analysis . A: HT22 cells were treated with 78 mM glutamate for the indicated times. Control sample showedintact -fodrin protein (240 kDa). Accumulation of calpain-mediated 145 kDa BDP was detected after 6 hrs of glutamate treat-ment, but was not detectable after 24 hrs of treatment. There is no evidence showing caspase-3-produced 120 kDa BDP. B:HT22 cells were treated with 78 mM glutamate in the presence of calpain inhibitors, 100 M PD150606 and 25 M ALLN, for8 hrs. Both calpain inhibitors could block or reduce calpain-mediated 145 kDa BDP. Treatment with calpain inhibitors alonehad no effect on proteolysis of-fodrin. Each experiment was repeated 3 times and a representative example of the Westernblot is shown.

A

-Fodrin240 KDa

145 KDa

240 KDa

145 KDa

B

Glutamate (7-8 mM)

Calpain InhibitorsPD150606 ALLN

Actin

120 KDa

Control 6 hrs 7 hrs 8 hrs 14 hrs

-Fodrin

Actin

120 KDa

Glutamate

(7-8 mM)

+ + + 100 M 100 M 25 M 25 M


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Glutamate-induced the cleavage of-fodrin in primary cortical cellsFigure 8Glutamate-induced the cleavage of-fodrin in primary cortical cells. A: primary cortical cells isolated from 18 dayold fetal rat brains were cultured for 78 days and then cells were treated with 1 mM glutamate for the indicated times. West-ern blot analysis showed that calpain-mediated 145 kDa BDP and caspase-3-mediated 120 kDa BDP could be detected inuntreated control cells. Accumulation of 145 kDa BDP was evident at 0.5 hr earlier than that of 120 kDa BDP, with greateraccumulation of both breakdown products occurring at 6 hrs. B: primary cortical cells were cultured for the indicated days andthe cleavage of-fodrin was detected by Western blot. Tissue sample only contained the intact 240 kDa -fodrin protein butno cleavage products. Cleavage of the 240 kDa -fodrin protein into 145 kDa BDP began at day 0 of culture (dissociated cellsprior to seeding on plates) and the 120 kDa BDP of-fodrin was only found after 7 days of culture. Each experiment wasrepeated 3 times and a representative example of the Western blot is shown.

Control 0.5 hrs 1.5 hrs 3 hrs 6 hrs

A

-Fodrin

Actin

Glutamate

(1 mM)

240 KDa

145 KDa

120 KDa

Tissue 0 1 3 7 8

240 KDa

145 KDa120 KDa

-Fodrin

Actin

Culture of Primary Cortical Cells (days)

B

Time of Glutamate Treatment


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Effect of calpain and caspase-3 inhibitors on glutamate-induced cleavage of-fodrin in primary cortical cellsFigure 9Effect of calpain and caspase-3 inhibitors on glutamate-induced cleavage of-fodrin in primary cortical cells.Primary cortical cells isolated from 18 day old fetal rat brains were cultured for 78 days, and then cells were treated with 1mM glutamate alone or in the presence of calpain inhibitor 12.5 M ALLN and caspase-3 inhibitor 50 M z-DEVD for 6 hrs. (I):representative Western blots showing calpain and caspase-3- mediated proteolysis of-fodrin and inhibition by protease inhib-itors. (II): semi-quantitative analysis of calpain and caspase-3-mediated proteolysis of-fodrin. Accumulation of breakdownproducts was assessed by Western blot analysis as described under "Methods". Actin was used as a loading control. The barsdepict densitometric analysis of Western blots from at least three experiments ( SEM) *P< 0.05 compared to control; P

glutamate-induced apoptosis

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