TLQP-21, a neuroendocrine VGF-derived peptide, preventscerebellar granule cells death induced by serum and potassiumdeprivation

Cinzia Severini,* Maria Teresa Ciotti,* Laura Biondini,* Stefania Quaresima,*Anna Maria Rinaldi,� Andrea Levi,* Claudio Frank� and Roberta Possenti*,�

*Institute of Neurobiology and Molecular Medicine, CNR, Rome, Italy

�Department of Neuroscience, University of Rome ‘‘Tor Vergata’’, Rome, Italy

�Istituto Superiore di Sanita, Viale Regina Elena, Rome, Italy

Vgf, originally identified as a nerve growth factor responsivegene (Levi et al. 1985; Salton 1991), is expressed in neuronswithin the central and peripheral nervous system and invarious endocrine cells. In the rat brain, VGF is abundant inthe cortex, hypothalamus, hippocampus, cerebellum andolfactory system and present in other brain areas in differentamounts (reviewed in Salton et al. 2000; Levi et al. 2004).

Vgf encodes for a 617 amino acid protein in rat and mouse,and 615 in human. Specific prohormone convertases PC1/3and PC2 cleave the precursor protein bearing a number ofpeptides that are stored in dense core granules and secretedthrough the regulated pathway (Trani et al. 2002; Alder et al.2003). More than 10 different VGF peptides have beendetected in rat brain, bovine hypophysis, and human CSF(Liu et al. 1994; Stark et al. 2001; Trani et al. 2002; Carretteet al. 2003). In the rat cerebellum, the expression pattern ofVGF mRNA levels were measured during the post-natalperiod of morphological and functional development both in

developing and adult animals (Salton 1991; Snyder andSalton 1998; Snyder et al. 1998), and the processing of VGFprotein to smaller polypeptides deriving from the C-terminal

Received June 12, 2007; revised manuscript received August 2, 2007;accepted September 14, 2007.Address correspondence and reprint requests to Dr Cinzia Severini,

CNR, Institute of Neurobiology and Molecular Medicine, CNR, Via delFosso di Fiorano, 65, 00143 Rome, Italy.E-mail: c.severinii@inmm.cnr.itAbbreviations used: [Ca2+]i, intracellular calcium concentration; Act-

D, actynomicine D; Akt, serine/threonine protein kinase; BME, basalEagle’s medium; CGCs, cerebellar granule cells; DIV, days in vitro; Erk,extracellular signal-regulated kinase; IGF-1, insulin-like growth factor-1;JNK, c-jun N-terminal kinase; MAPs, microtubule-associated proteins;MEK, MAPK ERK kinase; PAGE, polyacrylamide gel electrophoresis;PBS, phosphate-buffered saline; PKC, protein kinase C; S-K5, serumand potassium deprivation; UO126, 1-4-diamino-2-3-dicyano-1-4-bis(2-aminophynyltio) butadiene.

Abstract

Different VGF peptides derived from Vgf, originally identified

as a nerve growth factor responsive gene, have been

detected in neurons within the central and peripheral nervous

system and in various endocrine cells. In the current study, we

have evaluated the ability of TLQP-21, a VGF-derived pep-

tide, to protect, in a dose- and time-dependent manner, pri-

mary cultures of rat cerebellar granule cells (CGCs) from

serum and potassium deprivation-induced cell death. We

demonstrated that TLQP-21 increased survival of CGCs

by decreasing the degree of apoptosis as assessed by cell

viability and DNA fragmentation. Moreover, TLQP-21 signifi-

cantly activated extracellular signal-regulated kinase 1/2,

serine/threonine protein kinase, and c-jun N-terminal kinase

phosphorylation, while decreased the extent of protein kinase

C phosphorylation, as demonstrated by western blot analysis.

In addition, TLQP-21 induced significant increase in intra-

cellular calcium (as measured by fura-2AM) in about 60% of

the recorded neurons. Taken together, the present results

demonstrate that TLQP-21 promotes the survival of CGCs via

pathways involving, within few minutes, modulation of kinases

associated with CGCs survival, and by increasing intracellular

calcium which can contribute to the neuroprotective effect of

the peptide.

Keywords: cerebellar granule cells, neuronal death, TLQP-

21, Vgf.

J. Neurochem. (2008) 104, 534–544.

d JOURNAL OF NEUROCHEMISTRY | 2008 | 104 | 534–544 doi: 10.1111/j.1471-4159.2007.05068.x

534 Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

region was previously investigated in rat cerebellar granulecells (CGCs) (Trani et al. 1995).

Recently, we identified and characterized in rat brainextracts a new VGF-derived peptide designated as TLQP-21,which spans from residue 556 to residue 576 of the precursorsequence. This peptide has been reported to be one of themajor peptide fragments present in the brain of the rat(Bartolomucci et al. 2006) and to exert biological activityboth in vivo (Bartolomucci et al. 2006; Jethwa et al. 2007)and in vitro (inducing contraction of gastric fundus strips,unpublished work).

In vitro cultures of newborn rat CGCs represent one ofthe most widely used experimental model to study neuronalcell death because of the high degree of cellular homo-geneity, allowing the study of the effect of substances thatcan modulate this process. CGCs survive and differentiatein vitro in the presence of depolarizing concentrations ofKCl (25 mmol/L), but undergo apoptosis when cultured inphysiological low KCl (5 mmol/L) conditions (Gallo et al.1987; D’Mello et al. 1993). The mechanism of action ofdepolarizing concentrations of KCl remains controversial.Generally, it is believed that depolarization involves a lowincrease in intracellular Ca2+ concentration (D’Mello et al.1993; Galli et al. 1995) and MAPK activation (Rosen et al.1994).

In view of the finding reporting the physiological presenceof small polypeptides deriving from processing of theC-terminal region of VGF protein in CGCs, we investigatedwhether TLQP-21 can regulate the viability of these neurons.

Our results demonstrate that: (i) TLQP-21 protects CGCsfrom apoptosis induced by serum and potassium withdrawalconditions, (ii) neuroprotective action of TLQP-21 is depen-dent on the modulation of extracellular signal-regulatedkinase (Erk) 1/2, serine/threonine protein kinase (Akt),protein kinase C (PKC) and c-jun N-terminal kinase (JNK)kinases, and (iii) neuroprotective action is mediated byintracellular calcium increase.

Materials and methods

ChemicalsRabbit polyclonal antibodies (anti-phospho-Akt), anti-Akt, anti-

phospho-Erk1/2, anti-Erk1/2, anti-PKC, anti-JNK, and anti-phos-

pho-JNK, were from Cell Signaling technology (Beverly, MA,

USA). Donkey anti-rabbit peroxidase-coupled secondary antibodies

were from Amersham-GE Healthcare (Arlington Heights, IL, USA).

Monoclonal antibodies against microtubule-associated proteins

(MAPs) from Chemicon (Temecula, CA, USA) and Tetramethyl

Rhodamine Iso-Thiocyanate conjugated secondary antibodies from

Sigma (St Louis, MO, USA).

TLQP-21 (TLQPPASSRRRHFHHALPPAR) and the scrambled

control peptide Scr-21 (LRPSHTRPAHQSFARPLHRPA) were

synthesized by PRIMM (Milan, Italy). The other reagents were

from Sigma Aldrich.

Primary cultures of cerebellar granule cellsThe regional animal Ethics committee approved the experiments.

Cultures enriched in granule neurons were obtained from dissociated

cerebella of 8-day-old Wistar rats (P8) as previously described by

Levi et al. (1984). Cells were plated in basal Eagle’s medium

(BME; Gibco, Milan, Italy) supplemented with 10% fetal calf serum

(Gibco), 25 mmol/L KCl, 2 mmol/L glutamine (Gibco) and 100 lg/mL gentamycin (Gibco) in 24-well clusters, (NUNC, Roskilde,

Denmark), previously coated with poly-L-Lysine. CGCs were plated

at a constant density of 0.5 · 106 cells/well in BME.

1b-Arabinofuranosylcytosine (10 lmol/L) was added to the

culture medium 18–20 h after plating to prevent proliferation of

non-neuronal cells. After 6 days ‘in vitro’ (DIV), extracellular KClwas lowered from 25 to 5 mmol/L for neuronal cell death.

Cultured neurons were washed (2X) with serum-free BME

containing 5 mmol/L KCl incubated in the same medium up to

48 h (D’Mello et al. 1993). Neuronal viability was assessed by

counting the number of intact nuclei according to the method

described by Volonte et al. (1994). The culture medium was

removed and replaced with 0.5 mL of a detergent containing

lysing solution (0.5% ethylhexadecyldimethylammonium bromide,

0.28% acetic acid, 0.5% Triton X-100, 3 mmol/L NaCl, 2 mmol/L

MgCl2, in phosphate-buffered saline (PBS), pH 7.4 diluted 1/10).

After 2 min, the cells were collected and the solution consisted of

a uniform suspension of single intact and nuclei that were then

quantified by counting in a hemocytometer (Fortuna, Wertheim,

Germany). Broken or damaged nuclei were not included in the

counts. Alternatively, CGCs were fixed in 4% paraformaldehyde

and permeabilized with 0.2% Triton X-100 in Tris–HCl 0.1 mol/L,

pH 7.4 for 5¢ and then incubated with Hoechst 33258

(0.25 lg/mL) for 5 min at 20�C. After washing with PBS, the

percentage of shrunken and condensed nuclei was assessed.

To verify the effect of TLQP-21 on neuronal cell death, at 7 or 6

DIV CGCs were incubated for 24 or 48 h, respectively, with the

peptide tested and simultaneously exposed to KCl 5 mmol/L.

Counting intact nuclei as previously described assessed cell

viability.

ImmunocytochemistryCerebellar granule cells were fixed and permeabilized with 3.5%

formaldehyde, 70% ethanol and 5% acetic acid for 15 min at 20�Cand fixed with 4% (w/v in PBS) paraformaldehyde for 15 min at

20�C. Fixed cells were washed in PBS, pH 7.4, and permeabilized

with 0.2% Triton X-100-Tris–HCl, pH 7.4, for 5 min. The

coverslips were treated with a monoclonal antibody against MAPs

1 : 200 overnight at 4�C in a moist chamber, rinsed in PBS, and

stained with Tetramethyl Rhodamine Iso-Thiocyanate conjugated

secondary antibodies for 30 min at 20�C.To visualize nuclear morphology, cells were incubated for 5 min

with 1 lg/mL of the DNA dye Hoechst 33258 and then visualized

by a Laica fluorescent photomicroscope (Wetzlar, Germany).

Cell death ELISA assayTo detect the enrichment of histone-associated DNA fragments

(mononucleosomes and oligonucleosomes) in cytoplasmic fractions

of CGCs, the samples were processed and analyzed with the Cell

Death Detection ELISAPLUS kit (Roche, Mannheim, Germany).

After plating at the density of 0.5 · 106 cells/well in 24-well clusters,

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544

TLQP-21 neuroprotection of cerebellar granule cells | 535

CGCs were treated with TLQP-21 (1 lmol/L) or insulin-like

growth factor-1 (IGF-1) (25 ng/mL) and simultaneously exposed

to KCl 5 mmol/L. After 12 h, cells were analyzed into 96-well plates

at 1 · 103 cells/well density, according to the kit instructions. The

absorbance was measured with a Victor 3V 1420 multilabel counter

(Perkin Elmer, Turku, Finland). The bound anti-DNA-peroxidase

was quantified by using the substrate 2,2¢-azino-di-(3-ethyl) benz-isothiazoline sulfonate measured at an absorbance of 405 nm.

Background absorbance values, determined by measuring samples

of cell lysates and reaction buffer, were subtracted from the

sample absorbance values. The enrichment of histone-associated

DNA fragments was calculated considering the values found in

K25-treated cells as one.

Western blottingFor whole-cell lysates, the culture medium was discarded, the

neurons washed twice with ice-cold PBS, and lysed in lysis buffer

[50 mmol/L Tris–HCl (pH 7.5), 150 mmol/L NaCl, 10 mmol/L

EDTA, 10 mmol/L EGTA, 50 lmol/L NaF, 2.5 lmol/L sodium

pyrophosphate, 1 lmol/L beta-glycerophosphate, 1 lmol/L

Na3VO4, 1X protease inhibitor mixture, Sigma]. Equivalent

amounts of cell extracts (5 · 105 cells) were mixed with sodium

dodecyl sulfate-reducing sample buffer according to Invitrogen Nu

polyacrylamide gel electrophoresis (PAGE) (Invitrogen, Carlsbad,

CA, USA). Following heating at 70�C for 10 min, proteins were

subjected to sodium dodecyl sulfate–PAGE (Nu PAGE) and

transferred electrophoretically to polyvinylidene difluoride mem-

brane (Amersham). After staining with Ponceau S to verify

uniformity of protein loads/transfer, the membranes were analyzed

for immunoreactivity. Incubation with primary antibodies was

performed overnight at 4�C (rabbit anti-Erk1/2/p-Erk1/2, anti-Akt/

p-Akt, anti-JNK/p-JNK, p-a-PKC, 1 : 1000) and then membranes

were incubated with donkey anti-rabbit peroxidase-coupled second-

ary antibodies (1 : 5000) for 1 h at 20�C. Immunoreactivity was

developed by enhanced chemiluminescence system (Amersham). To

normalize loaded sample, the blots were stripped (Restore western

blot Stripping; Pierce, Rockford, IL, USA) and reprobed with an

antibody against a-tubulin (Sigma, 1/10.000, overnight at 4�C). Foranalysis of the western blotting data, densitometric analysis was

performed using Scan Analysis software for Mc Intosh (Biosoft,

Ferguson, MO, USA).

Fura-2AM experimentsOptical fluorimetric recordings with fura-2AM were used to

evaluate the intracellular calcium concentration ([Ca2+]i). Fura-

2AM stock solutions were obtained by adding 50 lg of fura-2AM to

50 lL of 75% dimethylsulfoxide plus 25% pluronic acid. Cells were

bathed for 60 min at 20�C in 1 mL of extracellular solution

(in mmol/L: 125 NaCl, one KCl, five CaCl2, one MgCl2, eight

glucose, and 20 HEPES, pH 7.35) with a final fura concentration of

5 lmol/L. This solution was then removed and replaced with

extracellular solution, and the dishes were quickly placed on the

microscope stage. To measure fluorescence changes, an Hamamatsu

(Shizouka, Japan) Argus 50 computerized analysis system was

used, recording every 6 s the ratio between the values of light

intensity at 340 and 380 nm stimulation (Frank et al. 2004). Thisprogram allowed analyzing all the selected cells as digital data,

subsequently used for statistical analysis.

Data analysisAnalyses of data from treated cultures and the corresponding control

condition were performed by one-way ANOVA for repeated measures

followed byDunnett’s test formultiple comparisons. Data are reported

as mean ± SEM, n being the number of independent experiments.

The significance level was set at p < 0.05 (*) and p < 0.01 (**).

Results

Action of TLQP-21 on CGCs deathAs previously reported, when switched from a culturemedium containing high depolarizing K+ concentration

12-rcS5K52K T 12-rcS5K12-PQL T 12-PQL

48 h24 h

** **

Via

ble

cells

(% o

f con

trol

)

(a)

(b)

0.01 0.1 0.3 1 3 10 IGF-1Act-DK5K250

100

75

50

25

0

100

75

50

25

* ****

****

**

TLQP-21 (µmol/L)

Via

ble

cel

ls(%

of

con

tro

l)

Fig. 1 Activity of TLQP-21 on cerebellar granule cells (CGCs) viabil-

ity. (a) Extracellular KCl was lowered from 25 (K25) to 5 mmol/L (K5)

for apoptosis induction and simultaneously neurons were treated with

TLQP-21 at different doses, or with insulin-like growth factor-1 (IGF-1)

(25 ng/mL) or with actynomicine D (Act-D) (1 lg/mL) and incubated

for 48 h. TLQP-21 significantly and dose dependently protected CGCs

when compared with K5 value. The highest neuroprotective effect was

evident at 1 lmol/L TLQP-21 while IGF-1 and Act-D completely pre-

vented CGCs death. (b) The neuroprotective effect of TLQP-21 was

significant after 24 h and the maximal effect was obtained after 48 h

incubation time when compared with the corresponding K5 value,

while no effect was observed at any time incubating CGCs with Scr-21

(1 lmol/L). Data represent means (±SEM) from at least five inde-

pendent experiments. Statistically significant differences were calcu-

lated by one-way ANOVA for repeated measures followed by Dunnett’s

test, analyzing data from treated cultures and corresponding control

(K5) (*p < 0.05; **p < 0.01).

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

536 | C. Severini et al.

(25 mmol/L) to one containing a low even though morephysiological K+ concentration (5 mmol/L), CGCs degener-ate and die, prevalently by apoptosis (D’Mello et al. 1993).A dose–response curve was carried out following 48 hincubation with TLQP-21. As shown in Fig. 1a, treatmentwith 0.01, 0.1, 0.3, 1, 3, and 10 lmol/L TLQP-21 caused anincrease in cell viability of 3.2 ± 2.3%, 6.3 ± 2.7%, 14.4 ±3.0%, 23.5 ± 2.9%, 22.8 ± 3.4% and 21.4 ± 1.6%, respec-tively. Thus, the concentration of 1 lmol/L was used in allsubsequent experiments. No effect was observed using thescrambled peptide (Scr-21) at concentrations up to 10 lmol/L (data not shown). As positive controls, we used two well-established protective factors for CGCs following K+

withdrawal, IGF-1 and actynomicine D (Act-D) (D’Melloet al. 1993; Galli et al. 1995). A complete preservation ofcell viability occurred in the presence of both IGF-1(63 ± 1.3% increase at 25 ng/mL) and Act-D (60 ± 1.8%increase at 1 lg/mL).

Moreover, time-dependent response effects, incubatingCGCs with 1 lmol/L TLQP-21 simultaneously the apoptosisinduction, showed that the neuroprotective effect wassignificant after 24 h of treatment in K5 conditions (19.6 ±3.1%), while a maximal action was obtained after 48 h(24.7 ± 2.7%). On the contrary, no effect was observed usingScr-21 at the same concentration (Fig. 1b).

HoechstsPAM

52K

5K

12-PQLT+

1-FGI+

5K

5K

Fig. 2 TLQP-21 neuroprotective activity.

Cerebellar granule cells (CGCs) were

incubated with TLQP-21 (1 lmol/L) or

insulin-like growth factor-1 (IGF-1) (25 ng/

mL) and simultaneously exposed to KCl

5 mmol/L. On the left, CGCs morphological

analysis assessed upon 48 h of apoptosis

by immunofluorescence using an antibody

against the microtubule-associated proteins

(MAPs). On the right, Hoechst 33258

staining showing intact and condensed

apoptotic nuclei (2.6X magnification). Val-

ues corresponding to apoptotic nuclei as

percentages of total nuclei were: K25: 11%

(16/142), K5: 67% (98/154), K5 + TLQP-

21: 34% (55/160), K5 + IGF-1: 18% (19/

105). Scale bar: 25 lm.

D-tcA1-FGI12-PQLT5K52K

0

1

2

3

4

5

6

7

8

9

**

*

**En

rich

men

t o

f D

NA

frag

men

tati

on

5K

Fig. 3 Effect of TLQP-21 on cerebellar granule cells (CGCs) DNA

fragmentation. CGCs were treated with TLQP-21 (1 lmol/L) or insulin-

like growth factor-1 (IGF-1) (25 ng/mL) or actynomicine D (Act-D)

(1 lg/mL) and simultaneously exposed to KCl 5 mmol/L. DNA frag-

mentation was determined 12 h after the addition of the drugs and

detected by the ELISA method. Data were expressed as relative DNA

fragmentation enrichment. Data represent means (±SEM) from six

independent experiments. Statistically significant differences were

calculated by one-way ANOVA for repeated measures followed by

Dunnett’s test, analyzing data from treated cultures and corresponding

controls (K5) (*p < 0.05; **p < 0.01).

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544

TLQP-21 neuroprotection of cerebellar granule cells | 537

Morphological analysis, by immunofluorescence methodusing an antibody against the MAPs, indicated that in S-K5conditions CGCs resulted severely damaged and showedmorphological alterations associated with apoptosis, dis-playing severe neuronal soma shrinkage and axonalretraction. On the contrary, CGCs incubated for 48 h withTLQP-21 or IGF-1 showed a sustained neurites network(Fig. 2).

Reported data were also confirmed by cell viabilityquantification by Hoechst staining, indicating the samepercentage obtained counting intact nuclei (Fig. 2).

Effect of TLQP-21 on internucleosomal DNAfragmentationNeuronal death is known to be accompanied by the charac-teristic features of apoptosis, such as DNA fragmentation(Nagata et al. 2003). To determine whether a TLQP-21neuroprotective effect is attributable to an inhibition ofapoptosis, the levels of DNA fragmentation were mea-

sured by ELISA. As shown in Fig. 3, the DNA fragmen-tation levels were drastically increased in K5 samples.At 1 lmol/L, TLQP-21 showed about 23.4% rate ofinhibition against K5-induced DNA fragmentation, whileboth IGF-1 (25 ng/mL) and Act-D (1 lg/mL) com-pletely inhibited the DNA fragmentation induced by K5conditions.

Neuroprotection by TLQP-21 was MAPK/Erk1/2 and AktdependentAs MAPK/Erk1/2 and Akt signaling pathways are two well-studied survival-promoting signaling mechanisms in neurons(Gunn-Moore et al. 1997; Bonni et al. 1999; Yuan andYankner 2000), we investigated the involvement of thesekinases in the neuroprotective effect of TLQP-21.

We used as positive control IGF-1, the most potent anti-apoptotic factor for CGCs by modulating the MAPK/Erk1/2and especially the Akt pathway (D’Mello et al. 1997; Dattaet al. 1997; Dudek et al. 1997).

12-rcS(a)′06′03′51′5C

1-FGI

2/1krE-p

12-PQLT

2/1krE

′06′03′51′5C

Protein signal intensity Protein signal intensity

6030155K5 6030155K50123456

* * *

TLQP-21 1 µmol/L IGF (25ng/mL)

P-E

rk 1

/2 le

vel

P-E

rk 1

/2 le

vel

012345

**

**

****

**

2/1krE-p

2/1krE

48 h24 h12-PQLT1FGI5K52K 12-PQLT1FGI5K52K

(b)

nilubTu

′06′03′51′5C

2/1krE-p

5K52K

h6h4h3h2

2/1krE-p

(min incubation time) (min incubation time)

Fig. 4 Action of TLQP-21 on extracellular signal-regulated kinase

(Erk) 1/2 phosphorylation during cerebellar granule cells (CGCs) death

induced by potassium and serum deprivation. CGCs were switched in

S-K5 for different times. Cells were lysed and processed for immu-

noblotting. Western blot analyses were performed using an antibody

specific for the phosphorylated (active) forms of Erk1/2 (p-Erk).

(a) CGCs were treated, in K5 conditions, with TLQP-21 (1 lmol/L) or

insulin-like growth factor-1 (IGF-1) (25 ng/mL) for different times (from

5 to 60 min). The same blots were stripped and reprobed with an

antibody against total Erk1/2 (Erk). The action of Scr-21 (1 lmol/L) on

Erk1/2 phosphorylation after different incubation times is also shown.

Quantitative analysis of Erk1/2 phosphorylation is depicted below the

respective blots and was determined by band densitometric analysis.

The densitometric values from phosphorylated Erk1/2 were normal-

ized using total Erk1/2 as internal controls. The ratio of the band

intensities was calculated considering the values found in K5 (C)

treated cells as one. Data represent means (±SEM) from four inde-

pendent experiments. Statistically significant differences were calcu-

lated by one-way ANOVA for repeated measures followed by Dunnett’s

test, analyzing data from treated cultures and corresponding controls

(K5) (*p < 0.05; **p < 0.01). (b) Representative western blots from at

least three experiments showing Erk1/2 phosphorylation of CGCs

treated with TLQP-21 (1 lmol/L) or IGF-1 (25 ng/mL) in K5 conditions

for 24 and 48 h. The same blots were stripped and reprobed with an

antibody against a-tubulin to assess the amount of proteins present in

each lane (lower panel).

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

538 | C. Severini et al.

Cerebellar granule cells were treated for different timeswith K5 or K5 medium containing TLQP-21 (1 lmol/L)or IGF-1 (25 ng/mL). Quantitative analysis of Erk1/2 andAkt phosphorylation of different experiments was deter-mined by densitometric analysis and the densitometricvalues from phosphorylated Erk1/2 and Akt were normal-ized using total Erk1/2 and Akt as internal controls. Theratio of the band intensities was calculated considering thevalues found in K5-treated cells as one. Preliminaryexperiments were carried out to evaluate the mostfavorable time at which such signal transduction pathwayswere activated without modification in the phosphorylationbecause of K5 conditions. As a significant increase inErk1/2 phosphorylation can be detected from 3 h ofstarvation time, earlier times of incubation were used(Fig. 4a).

As shown in Fig. 4a, TLQP-21 significantly increasedlevels of phosphorylated Erk1/2 and this effect was remark-able within 15 min after the peptide administration. The

same result was obtained using IGF-1 and the highestphosphorylation was found at the same time (15 min). Inaddition, using Scr-21 at 1 lmol/L concentration, no effectwas observed at any time.

Relative high amounts of phosphorylated Erk1/2 wererevealed after 24 and 48 h but these values correlate withcorresponding high levels of total MAPKs and a-tubulin,because of an increase of viable cells (Fig. 4b).

To confirm the involvement of MAPK-Erk1/2 pathway inthe mechanism of action of TLQP-21, CGCs were treatedwith the MAPK ERK kinase (MEK) inhibitor 1-4-diamino-2-3-dicyano-1-4-bis (2-aminophynyltio) butadiene (UO126)at concentrations of 10 lmol/L. UO126 was added at 6 DIV1 h before or simultaneously the induction of apoptosis. Asthe inhibitor showed the same effect in all cases, the firstprotocol described was used for further experiments. Asshown in Fig. 5, UO126 completely prevented the increasedErk1/2 phosphorylation exerted by both TLQP-21 andIGF-1, while treatment with MEK inhibitor alone did notreduce the intensity of p-Erk immunolabeling, when com-pared with K5 culture values.

Additionally, as shown in Fig. 5, both 5 and 10 lmol/L ofUO126 completely prevented the neuroprotective activityexerted by TLQP21 1 lmol/L in K5 conditions, while aweak effect was observed on neuronal viability treatingCGCs with UO126 alone. Conversely, the same inhibitor wasunable to antagonize the neuroprotection carried out by IGF-1. To assess the effect of the MEK inhibitor on healthyneurons, equivalent experiments were carried out in K25conditions showing that the drug did not influence survival ofCGCs at different doses.

Similarly, Akt phosphorylation was found to be increasedafter 15 min of treatment with TLQP-21, while phosphory-lation of Akt increased further after that time using IGF-1(Fig. 6a). After 24 and 48 h of incubation, IGF-1 treatmentincreased Akt phosphorylation while TLQP-21 did notmodify the amount of phosphorylated Akt, although totalAkt and a-tubulin were found to be increased in both cases(Fig. 6b).

Neuroprotection by TLQP-21 was protein kinase C and JNKdependentProtein kinase C is known to play a role in various neuronalevents, including differentiation and neuroprotection (Singhet al. 1994; Zirrgiebel et al. 1995).

We performed western blots using cell lysates obtainedfrom various time points after K+ deprivation and probedthem with a phospho-specific PKC or a phospho-specific JNKantibody. Quantitative analysis of PKC phosphorylation ofdifferent experiments was determined by densitometry anal-ysis and the densitometric values from phosphorylated PKCand JNK were normalized using a-tubulin as internal control.The ratio of the band intensities was calculated consideringthe values found in K5-treated cells as one.

2/1KRE-p621OU+

K25

U01

26 5

U01

26 1

0

K5

U01

26 5

U01

26 1

0

K5

U01

26 5

U01

26 1

0

K5

U01

26 5

U01

26 1

00

100

75

50

25

12-PQLT 1-FGI

***

**

Via

ble

cel

ls (

% o

f co

ntr

ol)

′06′03′51′5C

1-FGI′06′03′51′5C

12-PQLT

Fig. 5 Effect of 1-4-diamino-2-3-dicyano-1-4-bis (2-aminophynyltio)

butadiene (UO126). Representative western blots from at least three

experiments showing extracellular signal-regulated kinase (Erk) 1/2

phosphorylation of cerebellar granule cells (CGCs) in K5 conditions

treated with TLQP-21 (1 lmol/L) or insulin-like growth factor-1 (IGF-1)

(25 ng/mL) in the presence of U0126 (10 lmol/L), for different times

(from 5 to 60 min) (upper panels). CGCs in K25 and K5 conditions

were treated with UO126, a specific MAPK kinases inhibitor, at dif-

ferent doses (5 and 10 lmol/L) alone or in the presence of TLQP-21

(1 lmol/L) or IGF-1 (25 ng/mL) and incubated for 48 h. U0126 treat-

ment induced a complete inhibitory effect on the TLQP-21-promoted

neuroprotection at the concentration of 10 lmol/L while this inhibitor

did not affect IGF-1 neuroprotection (lower panel). Data represent

means (±SEM) from four independent experiments. Statistically sig-

nificant differences were calculated by one-way ANOVA for repeated

measures followed by Dunnett’s test, analyzing data from treated

cultures and corresponding control (K5) (*p < 0.05; **p < 0.01).

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544

TLQP-21 neuroprotection of cerebellar granule cells | 539

As previously demonstrated, different JNK isoformscoexist in CGCs, the phosphorylation of some of which isreportedly involved in apoptosis of these neurons (Coffeyet al. 2002).

Four major isoforms were revealed in K5 conditions bythe use of the antibody against total JNK (Cell Signaling,data not shown), and using the antibody against phospho-JNK (Cell Signaling), three phosphorylated isoforms of 54–46 and 44 kDa were showed (Fig. 7).

As shown in Fig. 7, western blot analysis indicated that intime-course experiments, a significant but transient decreasein PKC phosphorylation was obtained after 15 min, timecorresponding to the maximal activity of both Erk1/2 andAkt. Similar results were obtained using IGF-1.

Conversely, TLQP-21 treatment induced a significantincrease of one isoform of phospho-JNK (the lower bandcorresponding to 44 kDa) after 15 min, similarly to IGF-1that, however, exerted a more prolonged effect.

TLQP-21 increased intracellular calcium as measured byfura-2AMTo investigate whether neuroprotective effect of TLQP-21was associated with an increase of intracellular calcium,we performed experiments to explore whether TLQP-21application was able to modify calcium levels in CGCs inculture.

Preliminary experiments were carried out to assess theTLQP-21 concentration able to induce a statistically signif-icant increase of [Ca2+]i, resulting 10 lmol/L the mosteffective concentration.

Ten lmol/L of TLQP-21 induced a significant increase in[Ca2+]i in a subpopulation of CGCs corresponding to about60% of the analyzed cells (value obtained analyzing morethan 150 cells), while Scr-21, applied at the same concen-tration was fully inactive, as shown in Fig. 8a.

TLQP-21 induced, in responsive neurons a marked rise inthe fura-2AM ratio, from 1 to 1.42 ± 0.08 (value obtained

tkA-p

tkA

12-PQLT

′06′03′51′5C′06′03′51′5C

1-FGI

tkA-p

tkA

48 h24 h12-PQLT1-FGI5K52K 12-PQLT1-FGI5K52K

Protein signal intensity Protein signal intensity

6030155K5 6030155K50 0.0

2.5

5.0

7.5

1

2

3

TLQP-21 1 µmol/L IGF (min incubation time)

P-A

kt 1

/2 le

vel

P-A

kt 1

/2 le

vel

*

**

**

**

** **

nilubTu

(a)

(b)

(min incubation time)

Fig. 6 Action of TLQP-21 on serine/threonine protein kinase (Akt)

phosphorylation during S-K5 cerebellar granule cells (CGCs) death.

(a) CGCs were switched to S-K5 alone or in the presence of TLQP-21

(1 lmol/L) or insulin-like growth factor-1 (IGF-1) (25 ng/mL) for dif-

ferent times (from 5 to 60 min). Cells were lysed and processed for

immunoblotting. Western blot analyses were performed using an

antibody specific for the phosphorylated (active) form of Akt (p-Akt)

(upper panel). The same blot was stripped and reprobed with an

antibody against total Akt (lower panel). Quantitative analysis of Akt

phosphorylation is depicted below the respective blots and was

determined by band densitometric analysis. The densitometric values

from phosphorylated Akt were normalized using total Akt as internal

controls. The ratio of the band intensities was calculated considering

the values found in K5-treated cells (C) as one. (b) Representative

western blots from at least three experiments showing Akt phos-

phorylation of CGCs treated with TLQP-21 (1 lmol/L) or IGF-1 (25 ng/

mL) in K5 conditions for 24 and 48 h. The same blots were stripped

and reprobed with an antibody against a-tubulin to assess the amount

of proteins present in each lane (lower panel). Data represent means

(±SEM) from four independent experiments. Statistically significant

differences were calculated by one-way ANOVA for repeated measures

followed by Dunnett’s test, analyzing data from treated cultures and

corresponding controls (K5) (*p < 0.05; **p < 0.01).

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

540 | C. Severini et al.

calculating 1 min of exposure) while the [Ca2+]i innon-responding neurons remained constant. Figure 8a showsan average of 40 reactive cells, Fig. 8b shows the [Ca2+]i of asingle responsive cell while Fig. 8c the [Ca2+]i of a single notresponsive cell. The effect of this peptide was compared withthe increase in the fura-2AM ratio produced by application ofIGF-1 (25 ng/mL), exerted by every cell tested (from 1 to1.78 ± 0.12). Figure 8d shows a representative trace of the[Ca2+]i induced by increasing concentrations of TLQP-21(1, 3, and 10 lmol/L) and by IGF-1 (25 ng/mL). At the endof each experiment, CGCs reactivity was tested triggeringthe calcium entry by high potassium-dependent (KCl,56 mmol/L) membrane depolarization, a no specific modelbased on a diffused neuronal depolarization (from 1 to1.92 ± 0.14).

Discussion

This study provides the first evidence that TLQP-21, anaturally occurring VGF-derived peptide, protects a subpop-ulation of CGCs against apoptosis induced by serum and K+

deprivation. The molecular mechanism of TLQP-21 neuro-protection is shown to involve MAPK/Erk1/2, Akt, PKCkinases and JNK. In addition, TLQP-21 is able to increaseintracellular calcium in the responsive neurons.

Although TLQP-21 was identified in brain tissue extracts,its regional localization and its concentration within the CNS,as well as the identification of its receptor are not known yet.

In the current study, we showed that TLQP-21 increasessurvival of CGCs against death induced by serum and K+

deprivation in a dose-dependent manner, as demonstrated bycell viability data.

As this peptide was able to decrease the DNA fragmen-tation, as revealed by ELISA experiments, it is reasonable tobelieve that TLQP-21 acts as an anti-apoptotic factor.

Concerning underlying mechanisms of TLQP-21 neuro-protective effect we first showed, by western blot analyses,the up-regulation of MAPK–Erk1/2 pathway, as treatmentwith TLQP-21 positively modulated the Erk1/2 phosphor-ylation at early times of incubation, in a way comparablewith IGF-1, the most potent anti-apoptotic factor in thissystem.

p-P

KC

leve

l

**

**

**

**

*

nilubuT

KNJ-p

CKP-p

12-PQLT′06′03′51′5C′06′03′51′5C

1-FGI

Protein signal intensity Protein signal intensity

Protein signal intensity Protein signal intensity

6030155K5

6030155K5

6030155K5

6030155K5

0.0

0.5

1.0

1.5p

-PK

C le

vel

1.5

2.0

2.5

3.0

0.0

0.5

1.0

p-P

KC

leve

l 1.5

2.0

0.0

0.5

1.0

p-P

KC

leve

l

0.0

0.5

1.0

1.5

TLQP-21 1 µmol/L (min incubation time)

TLQP-21 1 µmol/L (min incubation time)

IGF (25 ng/mL)(min incubation time)

IGF (25 ng/mL)(min incubation time)

Fig. 7 TLQP-21 activity on protein kinase

C (PKC) and c-jun N-terminal kinase (JNK)

phosphorylation during S-K5 cerebellar

granule cells (CGCs) death. CGCs were

switched to S-K5 alone or in the presence

of TLQP-21 (1 lmol/L) or insulin-like growth

factor-1 (IGF-1) (25 ng/mL) for different

times (from 5 to 60 min). Cells were lysed

and processed for immunoblotting. Western

blot analyses were performed using an

antibody specific for the phosphorylated

PKC forms (p-PKC) and for the phosphor-

ylated JNK forms (p-JNK). Quantitative

analysis of PKC and JNK phosphorylation

was depicted below the respective blots

and was determined by band densitometry

analysis. The densitometric values from

phosphorylated PKC and JNK were nor-

malized using a-tubulin as internal control.

The ratio of the band intensities was cal-

culated considering the values found in

K5-treated cells (C) as one. Data represent

means (±SEM) from four independent

experiments. Statistically significant differ-

ences were calculated by one-way ANOVA

for repeated measures followed by

Dunnett’s test, analyzing data from treated

cultures and corresponding controls (K5)

(*p < 0.05; **p < 0.01).

� 2007 The AuthorsJournal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544

TLQP-21 neuroprotection of cerebellar granule cells | 541

The MAPK–Erk1/2 pathway is known to be importantfor neuronal survival in different systems (Villalba et al.1997; Bonni et al. 1999; Obara et al. 2007). In particular,neurotrophin-mediated protection of neurons in vitro and invivo is mediated via the Erk pathway, resulting in phosphor-ylation of Erk1/2 and the phosphoinositide 3-kinase substrateAkt within few minutes (Han and Holtzman 2000; Willaime-Morawek et al. 2005).

It is well established that phosphorylation of Akt isassociated with CGCs survival (Gunn-Moore et al. 1997;Favata et al. 1998; Finkbeiner 2000; Zhu et al. 2004). Ithas been reported that the survival of mature CGCs afterserum and K+ withdrawal can be maintained by severaldifferent survival factors, all converging on the activation ofthe Akt (Vaillant et al. 1999; Kumari et al. 2001). Amongthese, exposure to growth factors such as IGF-1-induceactivation of Akt (D’Mello et al. 1993; Dudek et al. 1997).Using an antibody against phospho-Akt we observedin TLQP-21-treated CGCs, within 15 min, a significantincreased level of the active form of this enzyme knownto be involved in promoting the survival of differentcell types.

Protein kinase C, present in different isoforms in CGCs, isanother kinase involved in CGCs apoptosis induced by lowpotassium. In K5 conditions, levels of some PKC isoformswere markedly decreased, some not altered or even increasedduring the occurrence of death, suggesting a pivotal role for

this kinase in the maintenance of CGCs survival (Dekker andParker 1994; Lin et al. 1997).

We observed, by western blot analyses, that treatment withTLQP-21 reduced PKC phosphorylation of one isoform at15 min of incubation, time corresponding to the maximalactivity of both Erk1/2 andAkt, sharing similar qualitative andquantitative properties to the action of IGF-1. These resultsare in agreement with recent evidences showing that inhibitionof PKC promotes survival of CGCs in low potassium condi-tions through an Akt-dependent pathway (Zhu et al. 2004).

To further identify the mechanisms responsible for theneuroprotective effect of TLQP-21, we examined the effectsof modulating the activity of other death signal transductionpathways, previously shown to be involved, both in vivo andin cell culture systems in neuronal apoptosis, such as thestress-activated protein kinase JNK pathway (Miller andJohnson 1996; Watson et al. 1998; Coffey et al. 2002). Thestress-activated kinase JNK is known to regulate c-jun geneexpression and phosphorylation (Karin 1995; Ip and Davis1998; Coffey et al. 2002). As previously demonstrated,different constitutive JNK pools coexist in neurons, repre-senting different JNK isoforms (Coffey et al. 2002). In thepresent work, we demonstrated that TLQP-21 enhanced thephosphorylation of one of the three isoforms revealed by theanti-p-JNK and corresponding to 44 kDa.

The data reported in this work corroborate our findingsthat TLQP-21 acts on neuronal targets by eliciting a number

0 0 1 0 2 0 3 0 4 0 5 0 6

0

1

2

3

Time (min)

Rat

io34

0/38

0

mL / g n 5 2 - F G I mol/L m 6 5 - l C K

0 1 1 2 - r c S µ 0 1 - 1 2 - P Q L T mol/L µ mol/L

(a)

(c)

(b) 1 2 - P Q L T

F G I 6 5 K

0 10 20 30 40 50 600

1

2

3

0

1

2

3

Time (min)

0 10 20 30 40 50 60

Time (min)

0

1

2

3

0 10 20 30 40 50 60

Time (min)

(d)

1 2 - P Q L T F G I 6 5 K

1 2 - P Q L T F G I 6 5 K

0 1 0 1 3 1

Fig. 8 Effect of TLQP-21 on intracellular calcium concentration

([Ca2+]i) increase as measured by fura-2AM. (a) Trace corresponding

to the average of 40 TLQP-21 responsive cerebellar granule cells

(CGCs) from different cultures. Addition of TLQP-21 (10 lmol/L)

induced a fast [Ca2+]i increase in 6 days in vitro (DIV) CGCs, while the

scrambled peptide (Scr-21, 10 lmol/L) was completely inactive.

Insulin-like growth factor (IGF) (25 ng/mL) used as positive control,

provoked a higher, fast, and persistent effect respect to TLQP-21. KCl

56 mmol/L was used to confirm the cell viability. The scale on the left

indicates the values of ratio (340/380). (b) A representative trace

showing the [Ca2+]i of a TLQP-21 responsive CGC and (c) of a not

responsive cell. (d) Representative trace of [Ca2+]i increase induced

by different doses of TLQP-21 (1, 3, and 10 lmol/L), IGF (25 ng/mL)

and KCl 56 mmol/L in one responsive cell.

Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

542 | C. Severini et al.

of signaling cascades that may share similar properties of theaction of growth factors, possibly acting as a physiologicalregulator of cell survival for CGCs.

Besides the similarities between TLQP-21 and IGF-1 inthe neuroprotective action, several differences betweenthem in the effectiveness to CGCs apoptosis can bepointed out.

While Akt seems to be the most important pathwayinvolved in the mechanism of action of IGF-1, Erk1/2 are themain kinases implicated in the signal transduction of TLQP-21, as demonstrated by the sustained phosphorylationof Erk1/2 induced by this peptide after 24 and 48 h.Corroborating the specific involvement of the MAPK–Erk1/2 pathways in the TLQP-21 neuroprotection, UO126treatment reduced TLQP-21-mediated Erk1/2 phosphoryla-tion and completely prevented the neuroprotective effectexerted by TLQP-21, as reported by neuronal viability data,confirming that this way is crucial for the TLQP-21mechanism of action.

Finally, using fura-2AM Ca2+ imaging we demonstratedthat TLQP-21 was able to induce a significant increase in[Ca2+]i in a subpopulation of CGCs.

Sustained levels of cytosolic calcium were suggested toplay a key role in the prevention of apoptosis in differentneuronal models such as rat sympathetic neurons (Koikeet al. 1989), dorsal root ganglion neurons (Eichler et al.1992), and rat CGCs (Galli et al. 1995; Marshall et al.2003). Conducting live cell imaging using fura-2AM Ca2+

imaging allowed us to visualize the [Ca2+]i rise in individualneurons, showing that TLQP-21 was able to induce a slow[Ca2+]i rise in about 60% of the total number of analyzedCGCs, while the entire population of CGCs was responsiveto IGF-1, with a more rapid and sustained action.

Once again, the molecular mechanism for TLQP-21 rescueappears to operate through a modality of action typical toneurotrophic factors such as IGF-1, although with a reducedeffectiveness.

In conclusion, the present paper has provided clues for thepossible signaling pathways that are activated by TLQP-21 toexecute its protective function in CGCs.

As previously reported, in primary cultures of rat CGCsthe processing of VGF occurs in a post-endoplasmicreticulum compartment and the resulting products arepreferentially accumulated in secretory vesicles from whichthey are released upon cell membrane depolarization.Indirect immunofluorescence in CGCs clearly demonstratesthat VGF carbossi-terminal forms are strongly enriched insecretory vesicles present in neurites. Accordingly, only theseshort C-terminal form peptides are released on cell depolar-ization (Trani et al. 1995), suggesting that TLQP-21 may beone of these secreted peptides, acting as an autocrine/paracrine anti-apoptotic factor.

As a final point, because low-molecular-weight VGFforms were previously demonstrated to be present in the rat

cerebellum, it is tempted to speculate that TLQP-21 may actalso in vivo as a physiological regulator of cell survival forCGCs.

Acknowledgements

We would like to thank Dr Lionel Marlier for critically reading this

manuscript. This work was supported by grants from FIRB

RBNE013XSXSJ-004 and FIRB RBNEO1JKLF-006 to R. Possenti

and partially by Regione Lazio funding for ‘Sviluppo della ricerca

sul cervello’.

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Journal Compilation � 2007 International Society for Neurochemistry, J. Neurochem. (2008) 104, 534–544� 2007 The Authors

544 | C. Severini et al.