D Kasprowicz

T Runka

A Majchrowski

E Michalski

Low-temperature vibrational properties of KGd(WO 4 ) 2 : (Er, Yb) single crystals studied by Raman spectroscopy

Keywords: A. Optical materials, C. Raman spectroscopy, D. Lattice dynamics

Temperature-dependent polarized Raman spectra of KGd(WO 4 ) 2 : (Er, Yb) single crystals have been analyzed over the 77 -292 K temperature range. The A g and B g spectra obtained are discussed in terms of factor group analysis. The spectra have been found to reveal the bands related to internal and external vibrations of 2-4 WO ,WOW and WOOW molecular groups. Strong depolarization of the majority of the Raman bands has been observed in the whole temperature range. Some anomalies in the spectral parameters of selected Raman bands below 175 K have been discussed in terms of the local distortion of 2-4 WO ions in KGd(WO 4 ) 2 : (Er, Yb) crystals.

Introduction

A c c e p t e d m a n u s c r i p t

Potassium gadolinium tungstate crystals KGd(WO 4 ) 2 (KGW) doped with rare earth ions are very attractive solid state laser materials with interesting optical properties [START_REF] Kaminskii | New crystalline lasers on the base of monoclinic KR(WO 4 ) 2 :Ln 3+ tungstates (R=Y and Ln)[END_REF][START_REF] Pujol | NikoloY ; 6RODQV 0 $TXLOȩ ) 'íaz, Crystalline structure and optical spectroscopy of Er 3+ -doped KGd(WO 4 ) 2 single crystals[END_REF][START_REF] Jxloȩ ) 'íaz | Growth and characterization of monoclinic KGd 1-x RE x (WO 4 ) 2 single crystals[END_REF][START_REF]DUDSRQ 9 9RUOLþHN 0 -HOLQHN 0 ýHUĖDQVNǤ 6WUXFWXUDO DQG fluorescence properties of thin films fabricated by pulsed laser deposition technique from Nd:KGW single crystal[END_REF][START_REF] Mateos | Aguilȩ -0DVVRQV ) 'íaz, Crystal growth, optical and spectroscopic characterization of monoclinic KY(WO 4 ) 2 co-doped with Er 3+ and Yb 3+[END_REF]. The up-conversion processes in these materials produce the highly efficient stimulated emission in the visible spectral range with infrared laser diode excitation.

The Er 3+ doped KGW crystals show the efficient green photoluminescence excited by diode lasers in the 795 -805 nm and 970 -990 nm spectral range. They can be also used in construction of the eye-safe diode-pumped lasers emitting at 1.5 Pm. In order to improve the diode-pumping efficiency of the Er 3+ doped KGW crystal, it is co-doped with Yb 3+ ions. The Yb 3+ ions are characterized by a strong and broad absorption band in the 900 -1025 nm range that allows resonant energy transfer between Yb 3+ and Er 3+

ions. The excitation tuning in the 920 -1000 nm wavelength region in KGW: Er 3+ crystals co-doped with Yb 3+ ions and the large overlap between ytterbium emission and erbium absorption produce the optimal up-conversion efficiency and laser emission [START_REF] Rico | MassoQV & =DOGR 0 $JXLOȩ ) 'íaz, Yb sensitizing of Er 3+ up-conversion emission in KGd(WO 4 ) 2 :Er:Yb single crystals[END_REF][START_REF] Mateos | Infrared-to-green up-conversion in Er 3+ , Yb 3+ -doped monoclinic KGd(WO 4 ) 2 single crystals[END_REF]. The energy transfer between Yb 3+ and Er 3+ ions are phonon-assisted processes, influenced by the number of excitations related to the crystal field of the host material.

The high energy stretching modes of the tungsten-oxygen vibrations in KGW crystals can be effective in these processes. The vibrational properties some tungstates doped with trivalent rare earth ions have been already investigated [START_REF] Basiev | Spontaneous Raman spectroscopy of tungstate and molybdate crystals for Raman lasers[END_REF][9][START_REF]/ 0DFDOLN -+DQX]D $$ .DPLĔVNL 3RODUL]HG 5DPDQ VSHFWUD RI WKH RULHQWHG NaY(WO 4 ) 2 and KY(WO 4 ) 2 single crystals[END_REF][START_REF]ND -+DQX]D 6 .RMLPD -+ 9DQ GHU 0DDV 9LEUDWLRQDO VWXG\ RI SKDVH transitions in KIn(WO 4 ) 2[END_REF][START_REF] Macalik | Polarized infrared and Raman spectra of KGd(WO 4 ) 2 and their interpretation based on normal coordinate analysis[END_REF][13]. However, still little is known on their vibrational properties in the low-temperature region.

KGW crystals have the monoclinic structure with elsewhere [START_REF] Majchrowski | Top seeded solution growth of KHo(WO 4 ) 2 single crystals[END_REF]. The doping concentration of Er 3+ and Yb 3+ ions in the starting melt were 1 at.% and 5 at.%, respectively. The concentrations of the dopants in the as-grown single crystals were not measured. However, according to literature data, the distribution coefficient between the crystal and the melt of lanthanide doped KGW crystals is between 0.88 and 1.02 [START_REF] Jxloȩ ) 'íaz | Growth and characterization of monoclinic KGd 1-x RE x (WO 4 ) 2 single crystals[END_REF], so the concentration of Er

Results and discussion

The crystal structure of KGW : (Er, Yb) is isomorphous with the KGW structure. The primitive unit cell of KGW crystals includes two KGd(WO 4 ) 2 units and contains 24 atoms giving 72 fundamental vibrations. From the factor group analysis of ) the activity of the 3 acoustic modes A u + 2B u , 15 translational modes 2A g + 4 B g + 4A u + 5B u , 6 librational modes 3A g + 3B g and 48 internal modes 12A g + 12B g + 12A u + 12B u is predicted.

Among them 17A g + 19B g are Raman-active modes [START_REF] Rousseau | Normal mode determination in crystals[END_REF]. The detail assignment of the modes for KGd(WO 4 ) 2 crystals was proposed by Macalik et al. [START_REF] Macalik | Polarized infrared and Raman spectra of KGd(WO 4 ) 2 and their interpretation based on normal coordinate analysis[END_REF].

From the spectroscopic point of view, the WO tetrahedron has the T d symmetry, giving 15 vibrational degrees of freedom, which can be divided into four internal

Q 1 (A 1 ), Q 2 (E), Q 3 (F 2 ) and Q 4 (F 2 ) and two external: rotational -Q R (F 1 ) and translational -Q T (F 2 ) modes.
It was found that the vibrational modes of

2-4

WO anion in aqueous solutions are located

at 931 cm -1 (Q 1 (A 1 )), 320 cm -1 (Q 2 (E)), 833 cm -1 (Q 3 (F 2 )
) and 380 cm -1 (Q 4 (F 2 )) [START_REF] Hayes | Scattering of light by crystals[END_REF]. For tungstate crystals, in which WO are recorded at 348, 372 cm -1 as B g modes, and the mode observed at 342 cm -1 is recorded as an A g mode.

The peaks at 314 and 401 cm -1 are attributed to the out-of-plane and in-plane bending of WOW, respectively. Moreover, the bands connected with the bending vibrations of the oxygen WOOW bridge bonds appear at 272, 278 and 298 cm -1 . Below 260 cm -1 , the lattice modes (librations of WO ions located at 89 cm -1 . Most of the bands in this spectral range are active with higher intensity in the y(xx)z geometry, except for the modes at 80, 103, 191 and 252 cm -1 , which are fully polarized and observed in the y(xy)z geometry. The detailed assignment all of the modes is presented in Table 1. To determine the influence of temperature on the Ramana spectra, the crystals were investigated on cooling process from 293 K down to 77 K. The spectral analysis of the bands shows minor frequency changes but significant intensity changes in the majority of them. We observed the higher intensity and stronger depolarization of some of them at 77 K than at room temperature. WO ions from tetrahedral symmetry and the imperfect orientation of molecular groups with respect to the crystallographic axes.

Moreover, it can be also caused by the optical activity of KGW : (Er, Yb) crystals leading to rotation of the polarization plane of the incident light, which was earlier observed by us [START_REF] Kasprowicz | Elastic properties of KGd(WO 4 ) 2 :Ho 3+ single crystals studied by Brillouin spectroscopy[END_REF]. On cooling the deviation of their orientations grows and the The strongest temperature changes in the Raman spectra of KGW:(Er, Yb) crystals were observed in the 650 -850 cm -1 spectral range (Fig. 3).

Fig. 3

In this spectral range we detected the peaks assigned to the internal stretching vibrations of

2-4

WO ions and the oxygen bridge bonds WOOW and WOW. As shown in Fig. 3, that 744 and 756 cm -1 modes, shift insignificantly towards higher frequencies, while the band at 765 cm -1 is evidently shifted to lower frequencies at 77 K. Moreover,

A c c e p t e d m a n u s c r i p t

10 the depolarization of 744 and 765 cm -1 is significantly higher at 77 K than at room temperature (see Table 1). The bending modes of 2-4

WO ions located at 343, 348 and 372 cm -1 do not significantly change their position on cooling to 77 K (Fig. 6). However, we observe the small anomaly in frequency at about 175 K, which results from distortion of In the region of the lattice modes we observed a significant increase in the intensity of most of the bands. In Fig. 7 The mode centered at 206 cm -1 shows a slight shift to a higher frequencies with decreasing temperature, while the frequency of the mode at 89 cm -1 insignificantly decreases by about 2 cm -1 on cooling. This strong lattice mode practically does not change its position and FWHM. However, its integral intensity shows a significant increase, with an anomaly at about 175 K. Probably at this temperature the slightly disturbed translational motion of the whole tungstate -oxygen layers appears.

Conclusions

The results obtained have revealed the depolarization of the majority of the Raman bands in the spectra of KGW : (Er, Yb) crystals at room temperature. The depolarization some of stretching and bending modes is due to the distortion of abbreviations: vw -very weak; w -weak; m -medium; s -strong; vs -very strong; sh -shoulder; Qstretching in-plane modes; G -bending in-plane modes; J -out-of-plane bending modes; T' -translatory modes; L -libratory modes.

A c c e p t e d m a n u s c r i p t

A c c e p t e d m a n u s c r i p t 3 distinctA c c e p t e d m a n u s c r i p t 4

 34 room temperature. The lattice cell parameters are: a = 10.652 Å, b = 10.374 Å, c = 7.582 Å, E = 130.80 o[14]. In KGW structure the tungsten and oxygen atoms form octahedral anionic complexes of C 1 symmetry. The potassium K + and gadolinium Gd 3+ ions, occupy the equivalent crystallographic positions of C 2 symmetry. Due to the difference in the size of K + and Gd 3+ ionic radii, of 1.33 and 1.11, respectively, they occupy the sites in an ordered manner. The structure of KGW is formed by chains the c-axis and joined to one another by oxygen atoms with two W-O distances equal to 1.758 Å and 2.359 Å (WOW oxygen bridge bonds). which are connected by four W-O bonds of two different lengths equal to 1.955 Å and 2.109 Å (WOOW double oxygen bridge bonds). The lengths of the remaining two W-O bonds are 1.747 Å and 1.825 Å[START_REF] Pujol | NikoloY ; 6RODQV 0 $TXLOȩ ) 'íaz, Crystalline structure and optical spectroscopy of Er 3+ -doped KGd(WO 4 ) 2 single crystals[END_REF]. Along the b axis the tungstate -oxygen layers are alternate with the cationic layers made of K + and Gd 3+ ions. In the ytterbium and erbium ions doped KGW crystals the Gd 3+ ions can be efficiently substituted by Yb 3+ and Er 3+ ions[START_REF] Pujol | NikoloY ; 6RODQV 0 $TXLOȩ ) 'íaz, Crystalline structure and optical spectroscopy of Er 3+ -doped KGd(WO 4 ) 2 single crystals[END_REF][START_REF] Macalik | Polarized infrared and Raman spectra of KGd(WO 4 ) 2 and their interpretation based on normal coordinate analysis[END_REF]. For the doping concentration used in this work the crystal structure does not change. The slight change in the cell parameters observed can be a consequence of the different ionic radii of Gd, Er and Yb atoms[START_REF]DUDSRQ 9 9RUOLþHN 0 -HOLQHN 0 ýHUĖDQVNǤ 6WUXFWXUDO DQG fluorescence properties of thin films fabricated by pulsed laser deposition technique from Nd:KGW single crystal[END_REF].The study was undertaken to determine the temperature-dependent vibrational properties of the Er 3+ doped KGW crystals co-doped with Yb 3+ ions (KGW : (Er, Yb)) by the Raman scattering method in the 77 -292 K temperature range. The results obtained are discussed in terms of the influence of temperature on the lattice dynamics and crystal structure.2. ExperimentalKGW : (Er, Yb) single crystals used in the Raman study were obtained using the Top Seeded Solution Growth method from 25 mol % solutions of KGW in K 2 W 2 O 7 on[010] oriented seeds. The detailed description of the technique used can be found

  3+ and Yb 3+ ions in asgrown KGW : (Er, Yb) single crystals is close to the concentration in the melt. The crystallographic orientation of the samples was established by the Laue method using the stereographic Wulff net, described in details by Michalski et al.[START_REF] Michalski | Analytical use of the stereographic Wolff net for single-crystal orientation for 1-, 2-and 3-rotated cuts[END_REF]. The oriented samples of the sizes 5u 4u 3 mm 3 with faces perpendicular to the [100], [010] and[001] directions were used to collect Raman spectra. The scattered light in the Raman spectrometer used was analyzed by a double grating monochromator and detected by the photon counting system. The argon laser line O = 488 nm was used for excitation in the Raman experiment. The polarized Raman spectra were recorded in the spectral range 20 -1000 cm -1 with the spectral slit-width of 2 cm -1 . The temperature dependent polarized Raman spectra were measured for the y(xx)z and y(xy)z scattering geometry over the 77 -292 K temperature range. Taking into regard the Bose-Einstein population factor, the fitting procedure was applied to obtain the band parameters of temperature Raman spectra.

A c c e p t e d m a n u s c r i p t 5 the

 5 zone-center modes for the monoclinic structure (

2 - 4 WO

 24 ions are the most important molecular groups in the crystals investigated, as their activity is revealed in the majority of bands in the Raman spectra. The free 2-4

2 - 4 WOA c c e p t e d m a n u s c r i p t 6 otherFig. 1 TheA c c e p t e d m a n u s c r i p t 7 at 4 WO

 246174 Fig.1

  + , Gd 3+ and W 6+ ions, in-plane and out-of-plane bending of WOOW and WOW bridge bonds, wagging vibrations of the whole W 4 O 20 complex) are observed in the Raman spectra. The dominant band in this spectral range is attributed to the strong translational mode of 2-4

  distorted, which is visible as increased depolarization of particular bands. The WOOW double bridge bonds have two orientations in the crystal structure, which are less sensitive to temperature changes. So, the bands at 527 and 683 cm -1 do not change their polarization on cooling from 293 to 77 K.

A c c e p t e d m a n u s c r i p t 9 4 WO 4 WO

 944 Figs 2(a) and (b)

Fig. 4 (A c c e p t e d m a n u s c r i p t 11 temperature

 411 Fig. 4(a) and (b)

  the other hand no anomaly in the temperature dependence of FWHM is observed, which indicates very slight influence of temperature on the bending vibrations

Fig. 6

 6 Fig.6

A c c e p t e d m a n u s c r i p t 12 Fig. 7 (

 127 Fig. 7(a) and (b)

4 WO

 4 the tetrahedral symmetry and their imperfect orientation with respect to the crystallographic axes. On cooling from 293 to 77 K, most of the bands show minor frequency changes.However, the 765 cm -1 mode exhibits an anomalous shift towards lower frequency. The anomalous shift of the frequency of this mode with lowering temperature is due to the elongation of the W-O bonds in the WOOW bridge bonds, which results from stronger intermolecular interactions between the adjacent 2-4 WO ions. Some anomalous changes in the spectral parameters of modes related to the tungstate-oxygen vibrations in the oxygen tetrahedra2- 4 WO and the oxygen bridge bonds WOOW in the temperature range 150 -175 K have been revealed. The rapid increase in the integral intensity of the 899 cm -1 mode below 175K, the nonlinear narrowing and increase in the integral intensity of -1 band with lowering temperature and the small anomaly in frequency of the bending modes at 343, 348 and 372 cm -1 observed at about 175K, result from the stronger distortion of the 2tetrahedra in the low-temperature range. The bands at 529 and 685 cm -1 assigned to the stretching vibrations of the WOOW oxygen bridge bonds seem to be less temperature sensitive. The anomalies of spectral parameters observed in the temperature range studied are weak, but clearly indicate the lowering of the local symmetry of the KGW : (Er, Yb) crystal structure.

Fig. 1 .Fig. 2 .Fig. 3 .

 123 Fig. 1. Raman spectra of KGW : (Er, Yb) crystals for the y(xx)z and y(xy)z scattering

Fig. 4 .Fig. 5 .Fig. 6 .- 4 WOFig. 7 .A c c e p t e d m a n u s c r i p t

 45647 Fig. 4. Temperature dependencies of the stretching vibrational modes of KGW : (Er, Yb) crystals (a) frequency of 744, 756 and 765 cm -1 mode, (b) integral intensity and FWHM of 765 cm -1 mode.

Table 1 A

 1

	c c e p t e d	m a n u s c r i p t
	8

  To describe these phenomena we determined the degree of depolarization D as the intensity ratio I xy /I xx and I xx /I xy of the Raman bands detected in the y(xx)z and y(xy)z scattering geometries for A g and B g modes,

	crystals is due to the distortion of	2 4	-

respectively

[START_REF] Kasprowicz | Characterization of bismuth triborate single crystal using Brillouin and Raman spectroscopy[END_REF]

. The calculated values of D are presented in Table

1

, both at 293 and 77 K.

According to Table

1

, the depolarization of the majority of Raman bands is higher at 77 K than at room temperature, except for the 527 and 683 cm -1 ones, which are strongly depolarized also at room temperature due to the existence of two different crystallographic nonequivalent orientations of the WOOW oxygen bridge bonds in the KGW crystal structure. The observed depolarization of the bands of KGW : (Er, Yb)

Table 1 .

 1 The wavenumber values [cm -1 ], degree of depolarization D and assignment of Ramanactive modes of KGW : (Er ,Yb) crystals at 293 and 77 K.

	Mode	y(xx)z	y(xy)z	D	y(xx)z	y(xy)z	D							Assignment
			293 K			77 K
	B g		928 vw			928 w		Q	WO -2 4	Q	WOW
	A g	899 vs	899 m	0.33	899 vs	900 s	0.70	Q	WO	2 4	-
	B g	805 w	805 s	0.12	806 m	806 s	0.32	Q	WOW Q	WO	2 4	-
	A g	765 vs	766 m	0.41	761 vs 761 vs	0.69	Q	WOOW
	B g		756 sh			757 sh		Q	WOOW Q	WO	2 4	-
	A g	744 m	744 sh	0.21	747 s	747 m	0.49
	B g	685 w	683 w	0.59	687 m	687 m	0.62	Q	WOOW
	B g	529 w	527 w	0.64	529 m	527 m	0.63
	A g	436 m	436 w	0.43	437 s	437 m	0.72	G	WOW
	B g		410 vw			410 w
	A g	401 vw 401vw	0.56	402 w	402 w	0.93	Q	WOW
	B g	371vw 372 m	0.09	373 w	374 s	0.18	G	WO	2 4	-
	B g	348 w	348 m	0.11	348 m	348 s	0.29
	A g	342 m	343 w	0.41	342 m	341 m	0.59
	A g	314 w 314 vw	0.76	313 m	313 m	0.99	J	WOW
	B g	297 vw 298 w	0.13	299 w	299 m	0.25	G	WOOW G	WO	2 4	-
	B g		278 vw		280 vw 281 vw	0.24
	A g	272 vw			273 vw
	B g		252 vw			255 vw		T'	K
	A g	235 m	236 w	0.40	236 m	236 m	0.77
	A g	206 m	206 w	0.55	208 m	208 m	0.95	T'	Gd	3
	B g		192 w			191 w
	A g	175 m	175 w	0.42	178 m	178 m	0.76	J	WOOW	T'	K
	A g	150 w 150 vw	0.12	151 w	151 w	0.96
	B g		142 vw			140 vw
	B g		131 vw
	A g	123 w 123 vw	0.58	122 w 122 vw	0.37
	A g	115 m 115 vw	0.25	113 m	113 w	0.30	L	(	WO	6	)	J	WOW
	B g		103 vw			102 vw		J	WOOW	T'	W	6 Gd ,	3

Acknowledgments

This work was financed by the Research Project: MNiI 1 P03B 058 27.

A c c e p t e d m a n u s c r i p t


F Allagnat

D Cunha

F Moore

J M Vanderwinden

D L Eizirik

Alessandra K Cardozo

Mcl-1 downregulation by pro-inflammatory cytokines and palmitate is an early event contributing to beta cell apoptosis

Myeloid cell leukemia sequence 1 Bcl-2 JNK ER stress eIF2alpha apoptosis pancreatic beta cell diabetes bax translocation intrinsic pathway

Introduction

Diabetes mellitus arises from the inability of the pancreatic beta cells to preserve metabolic homeostasis via the regulated secretion of insulin. The two main forms of diabetes are type 1 (T1D) and type 2 (T2D) [START_REF] Cnop | Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities[END_REF]. T1D is primarily caused by an autoimmune attack leading to beta cell destruction and insulin deficiency. T2D is triggered by the combination of insulino-resistance and impaired beta cell function and survival, mostly secondary to metabolic factors through a process referred to as glucolipotoxicity [START_REF] Cnop | Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities[END_REF]. Both pathologies are characterized by decreased beta-cell mass secondary to apoptosis; beta-cell loss is more marked in T1D and is a relative late event in T2D, probably contributing to secondary failure of oral therapies [START_REF] Cnop | Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities[END_REF]. The mitochondrial intrinsic pathway of apoptosis controlled by the Bcl-2 protein family [START_REF] Youle | The BCL-2 protein family: opposing activities that mediate cell death[END_REF] plays a major role in pancreatic beta cell death both in T1D and T2D [START_REF] Thomas | Beta cell apoptosis in diabetes[END_REF].

The Bcl-2 protein family can be divided into three groups, the anti-apoptotic proteins (Bcl-2, Bcl-XL, Bcl-w, Mcl-1 and A1), the multi-domain pro-apoptotic proteins (Bax, Bak and Bok) and the pro-apoptotic BH3-only proteins (including Bim, Bid, DP5, Puma, Bad and Noxa). Upon death stimulus, Bax translocates from the cytosol to the mitochondria and oligomerizes with Bak, resulting in mitochondrial outer membrane permeabilization (MOMP). This allows proteins located in the mitochondrial intermembrane space, such as cytochrome c, to be released in the cytosol, where they associate with cytosolic proteins to activate downstream executioner caspases resulting in apoptosis [START_REF] Kroemer | Mitochondrial membrane permeabilization in cell death[END_REF]. The Bax/Bak activation is regulated by a delicate equilibrium between antiapoptotic Bcl-2 proteins and pro-apoptotic BH3-only proteins. Among the anti-apoptotic proteins, overexpression of Bcl-2 and Bcl-XL have been shown to prevent beta cell apoptosis in a variety of models [START_REF] Carrington | Islet beta-cells deficient in Bcl-xL develop but are abnormally sensitive to apoptotic stimuli[END_REF][START_REF] Rabinovitch | Transfection of human pancreatic islets with an anti-apoptotic gene (bcl-2) protects beta-cells from cytokine-induced destruction[END_REF][START_REF] Zhou | Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion[END_REF], but they may hamper beta cell function [START_REF] Zhou | Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion[END_REF]. The molecular role of the myeloid cell leukemia sequence 1 (Mcl-1) in beta cells remains to be characterized. Mcl-1 is structurally unique among the Bcl-2 family; it contains two PEST sequences (polypeptide sequences enriched in proline [P], glutamic acid [E], serine [S] and threonine [T]) which favor Mcl-1 degradation, thereby reducing its half-life. Mcl-1 activity is also regulated by phosphorylation via the JNK and ERK pathways in response to a variety of cell stimuli [START_REF] Hf | Mcl-1: a highly regulated cell death and survival controller[END_REF]. This highly regulated state suggests that Mcl-1, rather than other Bcl-2 pro-survival proteins, is an early pivotal protein in the regulation of cell survival in response to extra-cellular signals.

Pro-inflammatory cytokines (IL-1β, TNF-α and IFN-γ) and high concentrations of saturated free fatty acids (FFAs) such as palmitate are candidate mediators of beta cell apoptosis in T1D and T2D, respectively [START_REF] Cnop | Mechanisms of pancreatic beta-cell death in type 1 and type 2 diabetes: many differences, few similarities[END_REF]. In the present study we investigated the effect of cytokines, FFAs and chemical ER stressors on Mcl-1 expression and, using both

Results

Pro-inflammatory cytokines and palmitate decrease Mcl-1 expression in pancreatic beta cells.

To evaluate the effect of pro-inflammatory cytokines on Mcl-1 expression, INS-1E cells were treated for 24 hours with IL-1β, TNF-α and IFN-γ, alone or in combination, as indicated (Figure 1a). Western blot analyses revealed the presence of a doublet of Mcl-1 isoforms around 40/42kDA, as previously described [START_REF] Yang | The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2[END_REF]. IL-1β alone decreased Mcl-1 expression by 30%. IFN-γ and TNF-α had no effect individually, but induced a 40% decrease in Mcl-1 expression when combined. IFN-γ also potentialized the effect of IL-1β, leading to a 60% reduction of Mcl-1 expression (for quantification see supplemental data; Figure S1a). We have previously shown that cytokines induce ER stress in beta cells [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF]. To test whether ER stress contributes to Mcl-1 decrease, INS-1E cells were exposed to the ER stressors thapsigargin, CPA and tunicamycin for 15 hours, resulting in a similar 40 to 60% decrease in Mcl-1 expression (Figures 1b andS1b). Salubrinal, a selective inhibitor of the cellular phosphatase complex that dephosphorylates the eukaryotic translation initiation factor 2 subunit α (eIF2α) (11) also significantly decreased Mcl-1 expression, although by 25% only (Figures 1b andS1b). INS-1E cells were also incubated for 15 hours in presence of the FFA oleate and the saturated FFA palmitate, the latter a known beta cell death effector [START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF][START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF] production is instrumental in cytokine-mediated ER stress and beta cell apoptosis, while palmitate and thapsigargin do not induce NO production in these cells [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF]. The NO synthase blocker LNMMA, which fully prevents cytokine-induced NO production in beta-cells (data not shown; [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF]), had no effect on thapsigargin-mediated Mcl-1 decrease, whereas it completely prevented the cytokine effects on Mcl-1 expression (Figure 2c). On the other hand, forskolin, an adenylate cyclase activator which partially protects beta cells against cytokine-and palmitate-induced apoptosis [START_REF] Cunha | Glucagon-like peptide-1 agonists protect pancreatic beta-cells from lipotoxic endoplasmic reticulum stress through upregulation of BiP and JunB[END_REF][START_REF] Bregenholt | The long-acting glucagon-like peptide-1 analogue, liraglutide, inhibits beta-cell apoptosis in vitro[END_REF], failed to prevent Mcl-1 degradation (Figure 2c). INS-1E cells then were transfected with a siRNA targeting the PKR-like endoplasmic reticulum kinase (PERK), which is activated upon ER stress and phosphorylates the elongation factor eIF2α, resulting in decreased protein synthesis [START_REF] Boyce | A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress[END_REF].

PERK knockdown using two different siRNA (#1 and #2) decreased total PERK expression by 50% and nearly completely abolished thapsigargin-induced PERK phosphorylation (Figure 2d). This resulted in a 50% decrease in basal and stress-induced eIF2α phosphorylation (Figures 2d-e andS1d). PERK knockdown using siPERK#1 prevented thapsigargin-, palmitate-and IL-1 β-induced Mcl-1 downregulation (Figures 2e andS1d). However, it only partially reversed the inhibitory effects of the cytokine combinations (IL-1β+IFN-γ and TNF-α+IFN-γ) on Mcl-1 expression (Figures 2e andS1d). Similar observations were made using siPERK#2 (Figure S1e). JNK has been reported to inactivate Mcl-1 [START_REF] Inoshita | Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress[END_REF] and cytokines induce a strong and rapid (1h) JNK phosphorylation in INS-1E cells [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Gurzov | Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis[END_REF], leading to a peak of c-JUN phosphorylation between 6 and 8 hours. We thus exposed INS-1E cells to IL-1β+IFN-γ for 8 hours in presence of the JNK inhibitor peptide (JNKi) [START_REF] Bonny | Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death[END_REF] or the chemical JNK inhibitor SP600125 [START_REF] Gurzov | Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis[END_REF]. Both inhibitors partially prevented the effect of cytokines on Mcl-1 expression, suggesting that JNK is involved in the cytokine-induced Mcl-1 downregulation (Figure 2f). Western blot analysis of c-Jun phosphorylation confirmed the efficiency of the inhibitors in blocking JNK activity (Figure S1f). ER stress has been shown to induce a late JNK activation in beta cells [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF], however the JNKi peptide did In other cell types Mcl-1 stability has been shown to be modulated through phosphorylation by the extracellular signal-regulated kinase-1,2 (ERK1,2) and the glycogen synthase kinase 3 β (GSK-3β) [START_REF] Hf | Mcl-1: a highly regulated cell death and survival controller[END_REF]. IL-1β+IFN-γ but not TNF-α+IFN-γ stimulate ERK phosphorylation in INS-1E cells [START_REF] Moore | PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis[END_REF][START_REF] Ortis | Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation[END_REF] The peak of IL-1β+IFN-γinduced ERK activation occurs after 4 to 8 hours of treatment, which is concomitant with

Mcl-1 downregulation. However, blocking IL-1β+IFN-γ-induced ERK activity using PD98059 had no effect of Mcl-1 down-regulation after 8 hours exposure to IL-1β+IFN-γ (figure S2a). Time course experiments indicated that INS-1E cells have a high basal GSK-3α,β phosphorylation, suggesting a low GSK-3α,β activity since the phosphorylated form is inactive (Figure S2b). IL-1β+IFN-γ further stimulated GSK-3α,β phosphorylation after 8hours, and the levels of phospho-and total GSK-3β were decreased at later time points (16 and 24 hours). TNF-α+IFN-γ had no significant effect on P-GSK-3α,β and total GSK-3β (Figure S2c). We next analyzed Mcl-1 protein expression in the presence of two specific GSK-3β inhibitors (SB216763 and BIO) [START_REF] Coghlan | Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription[END_REF][START_REF] Sato | Maintenance of pluripotency in human and mouse embryonic stem cells through activation of Wnt signaling by a pharmacological GSK-3-specific inhibitor[END_REF] in INS-1E cells treated or not for 8 hours with IL-1β+IFN-γ. As a control for the effects of the compounds on GSK-3 activity, we evaluated β-catenin protein expression; β-catenin is targeted for proteosomal degradation upon phosphorylation by GSK-3 [START_REF] Coghlan | Selective small molecule inhibitors of glycogen synthase kinase-3 modulate glycogen metabolism and gene transcription[END_REF]. Both SB216763 and BIO increased beta-catenin expression in INS-1E cells, indicating that these compounds efficiently inhibit GSK-3 activity (Figure S2d). On the other hand, the two inhibitors had

no effect on Mcl-1 expression, indicating that GSK-3-mediated Mcl-1 phosphorylation is not involved in Mcl-1 downregulation in INS-1E cells.

Mcl-1 knockdown increases beta cell apoptosis

To elucidate the role of Mcl-1 in beta cells, INS-1E cells were transfected with two siRNAs targeting the rat Mcl-1. Both siRNAs efficiently decreased Mcl-1 expression

(Figure 3a). We then studied the effect of Mcl-1 knockdown on beta cell survival.

Transfection with Mcl-1 siRNA#1 (Figures 3b andS3a) or #2 (Figure S2b) significantly increased basal and induced apoptosis in all conditions and at all time-points studied in INS-1E cells. Similar data were obtained in FACS-purified primary beta cells using Mcl-1 siRNA#1 (Figures 3c andS3c).

Western blot analyses revealed that knocking-down Mcl-1 increased caspase 3 cleavage in control, cytokine-, thapsigargin-or palmitate-treated cells (Figure 4). Of note, Mcl-1 knockdown did not affect Bcl-2 or Bcl-XL expression, and did not modify the effects of cytokines, thapsigargin or palmitate on the expression of the ER stress markers BiP, CHOP (Figure 4) and XBP-1s (data not shown).

Mcl-1 overexpression prevents beta cell apoptosis

We 2), Bak co-localized with the mitochondrial marker CoxIV, whereas Bax was located in the cytosol (Figure 5b). Interestingly, Bcl-2 and Bcl-XL were present both in the cytosolic and mitochondrial fractions, in line with previous reports that Bcl-2 and Bcl-XL have a broader cellular distribution as compared to Mcl-1 [START_REF] Yang | The intracellular distribution and pattern of expression of Mcl-1 overlap with, but are not identical to, those of Bcl-2[END_REF]. We next evaluated the effect of Mcl-1 overexpression on beta cell survival. As compared to non-infected

(NI) or Ad-LUC-infected cells, Ad-Mcl-1-infected INS-1E cells displayed a 50%
reduction in apoptosis following exposure to IL-1β+IFN-γ, TNF-α+IFN-γ, thapsigargin, tunicamycin or palmitate (Figure 5c). Similar results were obtained in FACS-purified primary beta cells after treatment with IL-1β+IFN-γ, thapsigargin or palmitate (Figure 5d). Time-course experiments revealed that the protection was continuous from 8 to 48 hours treatment with IL-1β+IFN-γ (Figure S3a). Mcl-1 overexpression also reduced by 50% the caspase 3 cleavage induced by IL-1β+IFN-γ (Figure 6a), thapsigargin (Figure 6b) or palmitate (Figure 6c), confirming the anti-apoptotic action of Mcl-1. Similar to Mcl-1 knockdown, Mcl-1 overexpression had no impact on Bax, Bak, Bcl-2, Bcl-XL, CHOP and BiP expression (Figure 6).

Mcl-1 overexpression prevents Bax translocation to the mitochondria

Mcl-1 has been shown to modulate Bax/Bak-mediated MOMP, release of cytochrome c and apoptosis [START_REF] Germain | MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction[END_REF]. We studied Bax and cytochrome c localization by immunostaining in INS-1E cells overexpressing Mcl-1 and exposed to IL-1β+IFN-γ. Upon apoptosis (determined by nucleus morphology using DNA labeling by Hoechst), INS-1E cells displayed a diffuse cytosolic cytochrome c staining whereas it was discrete and typically mitochondrial in living cells, co-localizing with the mitochondrial marker apoptosis inducible factor (AIF) (Figure 7a). The mitochondrial morphology, monitored using AIF (Figure 7a) or ATP synthase (Figure 7b), changed drastically between live and apoptotic cells, the latter showing disintegration of the tubular mitochondrial network and formation of punctiform, fragmented mitochondria. In contrast with the cytochrome c staining, the Bax labeling changed from a homogeneous cytosolic staining in live cells to a discrete punctate staining that co-localized with the mitochondrial marker ATPsynthase in apoptotic cells (Figure 7b). Quantitative assessment revealed that Mcl-1 overexpression reduced by 70% Bax translocation and cytochrome c release induced by IL-1β+IFN-γ (Figure 7c), as compared to Ad-LUC-infected cells. Similar data were obtained after a 15 hours exposure to thapsigargin (supplemental data, Figure S4), or palmitate (Figure S5), suggesting that Mcl-1 protects beta cells from different proapoptotic stimuli by preventing Bax translocation and subsequent cytochrome c release (Figure 8).

Acute and short-term changes in Mcl-1 expression don't affect beta cell function or proliferation

Bcl-XL overexpression has been shown to inhibit glucose-induced insulin secretion [START_REF] Zhou | Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion[END_REF].

In contrast, neither Mcl-1 knockdown (Figure S6a) nor overexpression (Figure S6b) Mcl-1 transgenic mice have increased beta cell mass [START_REF] Matsushita | Islet cell hyperplasia in transgenic mice overexpressing EAT/mcl-1, a bcl-2 related gene[END_REF]. Under the present experimental conditions, however, a 48-hour period of Mcl-1 overexpression did not modify INS-1E cells proliferation, as measured by BrDU incorporation (supplemental data, Figure S7).

Discussion

The aim of this study was to elucidate the role of the Bcl-2 protein family member Mcl [START_REF] Hf | Mcl-1: a highly regulated cell death and survival controller[END_REF]. In this study we confirm our previous findings that palmitate [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Cnop | Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis[END_REF] and cytokines [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF] induce the PERK-eIF2α pathway in beta cells, leading to decreased protein translation [START_REF] Boyce | A selective inhibitor of eIF2alpha dephosphorylation protects cells from ER stress[END_REF]. We presently show using salubrinal that phosphorylation of eIF2α is sufficient to significantly reduce Mcl-1 protein level. Knocking down PERK further demonstrate that eIF2α phosphorylation is instrumental in palmitate-and thapsigargin-induced Mcl-1 downregulation. This is in line with previous reports of eIF2α-dependent Mcl-1 downregulation in other cell-types [START_REF] Fritsch | Translational repression of MCL-1 couples stress-induced eIF2 alpha phosphorylation to mitochondrial apoptosis initiation[END_REF][START_REF] Snyder | Nitric oxide induces cell death by regulating anti-apoptotic BCL-2 family members[END_REF]. The effect of cytokines is, however, more complex since blocking eIF2α phosphorylation only partly prevents cytokine-induced Mcl-1 downregulation. Mcl-1 stability in other cell types is influenced through phosphorylation of various residues by multiple kinases, including JNK, ERK and GSK-3β [START_REF] Hf | Mcl-1: a highly regulated cell death and survival controller[END_REF]. Cytokines induce JNK in beta cells [START_REF] Gurzov | Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis[END_REF][START_REF] Bonny | IB1 reduces cytokine-induced apoptosis of insulin-secreting cells[END_REF] Many reports suggest that ER stress plays a major role in palmitate-induced beta cell apoptosis [START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF][START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Cnop | Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis[END_REF][START_REF] Akerfeldt | Cytokine-induced beta-cell death is independent of endoplasmic reticulum stress signaling[END_REF]. The present data support this hypothesis and provide new insights in the mechanism linking palmitate, ER stress and the intrinsic pathway of apoptosis through eIF2α phosphorylation and Mcl-1 degradation. On the other hand, the role of ER stress in cytokine-induced beta cell apoptosis remains controversial [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Akerfeldt | Cytokine-induced beta-cell death is independent of endoplasmic reticulum stress signaling[END_REF][START_REF] Huang | High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes[END_REF][START_REF] Oyadomari | Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[END_REF]. We presently demonstrate that the cytokine effects on Mcl-1 require ER stressdependent and -independent mechanisms (JNK activation), suggesting that cytokines may, depending on the severity and duration of exposure, induce apoptosis via ER stressdependent and independent mechanisms. This may account for the contradictory findings in this field [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Akerfeldt | Cytokine-induced beta-cell death is independent of endoplasmic reticulum stress signaling[END_REF][START_REF] Huang | High expression rates of human islet amyloid polypeptide induce endoplasmic reticulum stress mediated beta-cell apoptosis, a characteristic of humans with type 2 but not type 1 diabetes[END_REF][START_REF] Oyadomari | Nitric oxide-induced apoptosis in pancreatic beta cells is mediated by the endoplasmic reticulum stress pathway[END_REF]. Altogether, our data point out Mcl-1 degradation, whether it is due to ER stress or JNK activation, as a novel and common mechanism for beta cell apoptosis triggered by potential death effectors involved in the onset of T1D and T2D (Figure 8). New strategies to prevent the development of diabetes require improving beta cell survival without impairing function, i.e. glucose-stimulated insulin secretion (GSIS). Bcl-XL overexpression improves beta cell survival but impairs glucose oxidation and GSIS [START_REF] Zhou | Overexpression of Bcl-x(L) in beta-cells prevents cell death but impairs mitochondrial signal for insulin secretion[END_REF] and the BH3-only protein BAD is also an important modulator of GSIS [START_REF] Danial | Dual role of proapoptotic BAD in insulin secretion and beta cell survival[END_REF], suggesting that some members of the Bcl-2 family have "day" and "night" jobs in beta cells, affecting both cell survival and function [START_REF] Hetz | The daily job of night killers: alternative roles of the BCL-2 family in organelle physiology[END_REF]. This is apparently not the case for

Mcl-1 as neither Mcl-1 knockdown nor overexpression altered GSIS. These observations strongly suggest that the role of Mcl-1 in beta cells is restricted to the control of apoptosis

RESEARCH DESIGN AND METHODS

Materials

The following chemicals were purchased from Sigma-Aldrich NV/SA (Bornem, Belgium) and used as indicated: MG-132 (1µM), tunicamycin (10µg/ml), cyclopiazonic acid (CPA 20µM), thapsigargin (100nM), salubrinal (75µM), forskolin (10µM), SP600125 (10µM), SB216763 (5µM), bromoindirubin-3'-oxime (BIO; 1µM), PD98059 (30µM) were dissolved in DMSO. NG-Methyl-L-arginine (L-NMMA, 1mM) was dissolved in distilled water. Oleate and palmitate (sodium salt; Sigma) were dissolved in 90% ethanol, heated to 60 °C, and diluted (final concentration 0.5 mM) in RPMI 1640 with 1% BSA and 1% fetal calf serum (cFFA medium) [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Cnop | Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis[END_REF]. The peptides JNK inhibitor L-TAT-JNKi and control peptide L-TAT (a kind gift of C. Bonny and M. Mathieu; XigenPharma, Lausanne, Switzerland) were dissolved in culture medium and used at 10µM, as previously described [START_REF] Bonny | Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death[END_REF]. The following cytokine concentrations were used: recombinant human IL-1β (gift from C. W.Reinolds, National Cancer Institute, Bethesda, USA) at 10U/ml in INS-1E cells and 50U/ml in primary cells; recombinant rat IFN-γ (R&D systems, Abingdon, U.K.) at 100U/ml (0.0072µg/ml) in INS-1E cells and 500 U/ml (0.036µg/ml) in primary cells [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF]; recombinant human TNF-α (R&D systems, Abingdon, U.K.) at 1000U/ml in INS-1E cells and primary cells. The selected concentrations of ER stressors, FFA and cytokines are based on previous dose-response studies from our group [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF][START_REF] Cnop | Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis[END_REF]

Cell culture

The rat insulinoma cell line INS-1E (kindly provided by Prof. Claes Wollheim, CMU, University of Geneva) was maintained in the complete RPMI 1640 medium as previously described [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF].

Male Wistar rats (Charles River Laboratories, Brussels, Belgium) were housed and manipulated according to the guidelines of the Belgian Regulations for Animal Care; all performed experiments were approved by the local Ethical Committee. Rat islets were isolated by collagenase digestion followed by hand picking under a stereomicroscope.

Islets were then dispersed and primary beta cells were purified by autofluorescenceactivated cell sorting (FACS; FACS Aria, BD Bioscience, San Jose, CA., U.S.A.) [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF] The preparations used in the present study contained 90.1 ± 3.8 % beta cells (n=11).

Dispersed islets or FACS-purified beta cells were pre-cultured in complete beta cell medium supplemented with 5% heat-inactivated fetal bovine serum (FBS) from 20 to 48 hours for recovery [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF]. Experiments were then conducted in medium without serum [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF].

Western Blot Analyses

Cells were washed once with cold PBS and directly lysed with Laemmli buffer. Lysates were then resolved by SDS-PAGE and transferred to a PVDF membrane. Immunoblot analyses were performed as previously described [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Gurzov | Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis[END_REF] using the following antibodies: polyclonal anti-rat Mcl-1 from Biovision, Gentaur, Brussels, Belgium; polyclonal anti-CHOP/GADD153, monoclonal anti-β-catenin (D-10) and polyclonal anti-Bax (P- [START_REF] Bonny | Cell-permeable peptide inhibitors of JNK: novel blockers of beta-cell death[END_REF] from Santa Cruz Biotechnology, Inc., Santa Cruz, CA., U.S.A.; polyclonal anti-P-GSK-3α,β, polyclonal anti-GSK-3β, polyclonal anti-P-ERK1,2, polyclonal anti-ERK1,2, polyclonal anti-P-PERK, polyclonal anti-PERK, polyclonal anti-P-eIF2α, polyclonal anti-CoxIV, polyclonal anti-BiP, polyclonal anti-cleaved caspase 3, polyclonal anti-Bcl-2 and polyclonal anti-Bcl-XL from Cell Signaling, Boston, MA., U.S.A.; polyclonal anti-Bak from BD Biosciences-Europe, Erembodegem, Belgium; monoclonal anti-α-tubulin and horseradish peroxidase-conjugated goat anti-rabbit or anti-mouse IgG from Sigma-Aldrich.

RNA interference

The following siRNAs were used in this study: GUAUCCAUAUGACAACGGU from Ambion. Allstars Negative Control siRNA (Qiagen, Venlo, Netherlands), which has little effect on beta cell gene expression and no effect on viability [START_REF] Moore | PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis[END_REF]. siRNA transfection were conducted according to a protocol developed in our laboratory [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Moore | PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis[END_REF] using DharmaFECT 1 (Thermo Fisher Scientific, Aalst, Belgium) with a final concentration of 30 nM siRNA. The efficiency of transfection was >90% [START_REF] Cunha | Initiation and execution of lipotoxic ER stress in pancreatic beta-cells[END_REF][START_REF] Moore | PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis[END_REF]. Cells were then cultured for a 48-hour recovery period before being collected or treated as indicated.

Mcl-1 siRNA#1: rat Mcl-1 ON-

Generation of recombinant adenoviruses and cell infection.

Control adenovirus encoding luciferase (Ad-LUC) was purchased from Vector Biolabs, Philadelphia, PA, U.S.A. The adenovirus encoding rat Mcl-1 was generated by Vector Biolabs using rat myeloid cell leukemia 1 complete cds (cDNA clone MGC:93459; IMAGE:7121699; Genbank:BC078835) inserted in the pExpress-1 vector backbone (purchased at ImaGenes GmbH, Berlin, Germany). INS-1E cells and FACS-purified beta cells were infected as previously described [START_REF] Heimberg | Inhibition of cytokine-induced NF-kappaB activation by adenovirus-mediated expression of a NF-kappaB super-repressor prevents beta-cell apoptosis[END_REF].

Assessment of cell viability.

The percentage of viable, apoptotic and necrotic cells was determined using the DNAbinding dyes Propidium Iodide (PI, 5 µg/ml) and Hoechst 33342 (HO, 5 µg/ml, Sigma-Aldrich) [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF]

mRNA extraction and quantitative RT-PCR

Poly(A)+ mRNA was isolated from dispersed islet cells using the Dynabeads mRNA DIRECT™ kit (Invitrogen, Paisley, U.K.) and reverse transcribed as previously described [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF]. Quantitative PCR was performed using the IQ™ SYBR Green Supermix (BIO-RAD, Nazareth Eke, Belgium), in an IQ5 instrument (BIO-RAD). Expression values were corrected for the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Cytokines, FFAs or ER-stressors treatments do not modify GAPDH expression in insulin producing cells under the present experimental conditions [START_REF] Cardozo | Cytokines downregulate the sarcoendoplasmic reticulum pump Ca2+ ATPase 2b and deplete endoplasmic reticulum Ca2+, leading to induction of endoplasmic reticulum stress in pancreatic beta-cells[END_REF][START_REF] Kharroubi | Free fatty acids and cytokines induce pancreatic beta-cell apoptosis by different mechanisms: role of nuclear factor-kappaB and endoplasmic reticulum stress[END_REF][START_REF] Cnop | Selective inhibition of eukaryotic translation initiation factor 2 alpha dephosphorylation potentiates fatty acid-induced endoplasmic reticulum stress and causes pancreatic beta-cell dysfunction and apoptosis[END_REF] The primers used in this study were: rat Mcl-1 Fw : 5'-CCTCCAGCCACCAACTACAT-3', rev : 5'-CCACTTTCTTTCTGCCGTGTTA-3' and rat GAPDH: 5'-AGTTCAACGGCACAGTCAAG-3', rev: 5'-TACTCAGCACCAGCATCACC-3'.

Subcellular fractionation

One million INS-1E cells plated in a 6 well plate were infected using Ad-LUC or Ad-

Mcl

  siRNA strategy and adenoviral-mediated Mcl-1 overexpression, explored the role of Mcl-1 in beta cell function and survival. The data obtained indicate that Mcl-1 is a key antiapoptotic protein in beta cells which is rapidly degraded in response to pro-apoptotic stimuli, leading to increased Bax translocation to the mitochondria, cytochrome c release, cleavage of executioner caspase 3 and apoptosis in beta cells. These novel observations place Mcl-1 as key determinant of beta-cell fate during severe stress.

  ). Similar to what we observed in INS-1E cells, Mcl-1 expression was decreased in primary rat islets treated for 48 hours with TNF-α+IFN-γ or IL-1β+IFN-γ or for 24 hours with thapsigargin, CPA or palmitate (Figure 1d). Time-course experiments of thapsigargin or IL-1β+IFN-γ treatment indicated that Mcl-1 protein expression is rapidly decreased (after 2 and 4 hours of thapsigargin or IL-1β+IFN-γ treatment, respectively) as compared to the two other major anti-apoptotic proteins, Bcl-2 and Bcl-XL, whose expressions were decreased only after 15 and 24 hours of treatment with thapsigargin or IL-1β+IFN-γ, respectively (Figure 1ef). On the other hand, Mcl-1 mRNA expression was upregulated by cytokines, thapsigargin and palmitate, indicating that the Mcl-1 protein decrease is a posttranscriptional event (Figure 2a). Mcl-1 is constitutively targeted for degradation by the proteasome (8). INS-1E cells treated with the proteosomal inhibitor MG-132 displayed increased basal Mcl-1 expression and the inhibitor prevented IL-1β+IFN-γ-, TNF-α+IFN-γ-, thapsigargin-and palmitate-induced Mcl-1 degradation (Figures 2b and S1c), indicating that proteosomal degradation is involved in Mcl-1 downregulation. Cytokine-induced nitric oxide (NO)

  not prevent the effects of a 15 hours exposure to thapsigargin or palmitate on Mcl-1 downregulation. At this time point, JNK inhibition fully prevented the Mcl-1 downregulation induced by IL-1β alone or TNF-α+IFN-γ, and partially prevented the effect of IL-1β+IFN-γ (Figure 2g and S1c). These data indicate that palmitate and thapsigargin down-regulate Mcl-1 protein through ER stress-mediated inhibition of translation, while cytokines decrease Mcl-1 through the combined action of translation inhibition and JNK activation.

  next evaluated whether Mcl-1 overexpression increases beta-cell viability following exposure to different stressful agents. For this purpose, an adenovirus overexpressing rat Mcl-1 (Ad-Mcl-1) was generated. As compared to non-infected (NI) cells or cells infected with the control virus encoding Luciferase (Ad-LUC), INS-1E cells infected with the virus encoding Mcl-1 (Ad-Mcl-1) at multiplicity of infection (MOI) 2, 5 and 10, displayed dose-dependently increased levels of Mcl-1 (Figure 5a). Western Blot analyses of mitochondrial and cytosolic fractions extracted from INS-1E cells infected with Ad-LUC or Ad-Mcl-1 revealed that the exogenous Mcl-1 is mostly localized in the mitochondrial fraction. The endogenous Mcl-1 protein was also located in the mitochondrial fraction and absent from the cytosolic fraction. As previously described in other cell-types (

  altered the basal or glucose-stimulated insulin-secretion (GSIS). Mcl-1 overexpression did not prevent the GSIS inhibition induced by a 15 hours treatment with IL-1β+IFN-γ (Fig S6c).

- 1 in

 1 beta cell function and survival. Knocking-down Mcl-1 sensitized beta cells to apoptosis while overexpressing it protected these cells against pathophysiologically relevant agents (pro-inflammatory cytokines or palmitate), as well as chemical ER stressors, demonstrating that Mcl-1 has an important anti-apoptotic role in beta-cells. Interestingly, Mcl-1 was rapidly degraded by cytokines, palmitate and chemical ER stressors, as compared to the other anti-apoptotic proteins Bcl-2 and Bcl-XL, setting Mcl-1 decrease as a precursor event in beta cell apoptosis. Mcl-1 downregulation was not a transcriptional event but was secondary to ER stress-mediated inhibition of translation for palmitate and ER-stressors, and to the combined action of translation inhibition and JNK activation for cytokines. Altogether, these novel observations strongly suggest that Mcl-1 degradation may be a decisive step leading to beta cell apoptosis in T1D and T2D. Mcl-1 is a very labile protein with a 30-90 min half-life and inhibition of de novo Mcl-1 synthesis results in rapid Mcl-1 down regulation

  and phosphorylation of Mcl-1 by JNK facilitates Mcl-1 degradation in HEK293 cells[START_REF] Inoshita | Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress[END_REF]. We presently observed that JNK contributes to cytokine-mediatedMcl-1 downregulation. Cytokine-induced JNK phosphorylation is a rapid and transitory event (peak at 1hour) probably contributing to the early Mcl-1 degradation (4h -8h). The role of JNK, however, is partial and restricted to cytokines (blocking JNK did not prevent palmitate-or thapsigargin-induced Mcl-1 degradation), indicating that translation arrest is the main event leading to Mcl-1 down-regulation. ERK-mediated phosphorylation has been proposed to stabilize Mcl-1 and we previously showed that IL-1β+IFN-γ induce ERK in beta-cells[START_REF] Moore | PTPN2, a candidate gene for type 1 diabetes, modulates interferon-gamma-induced pancreatic beta-cell apoptosis[END_REF][START_REF] Ortis | Cytokine-induced proapoptotic gene expression in insulin-producing cells is related to rapid, sustained, and nonoscillatory nuclear factor-kappaB activation[END_REF]. However, blocking ERK activity had no effect on Mcl-1 expression, suggesting that ERK activation is not a major player in cytokine-induced changes in Mcl-1 expression in beta cells. This is in accordance with previous studies showing that ERK plays a minor role in Mcl-1 regulation in other models[START_REF] Inoshita | Phosphorylation and inactivation of myeloid cell leukemia 1 by JNK in response to oxidative stress[END_REF][START_REF] Rahmani | The kinase inhibitor sorafenib induces cell death through a process involving induction of endoplasmic reticulum stress[END_REF]. GSK-3 has also been shown to phosphorylate Mcl-1, thereby facilitating its degradation (32), however our present data suggest that INS-1E cells have low GSK-3β activity, and that the kinase is not involved in Mcl-1 regulation. Altogether, our data demonstrate that palmitate and thapsigargin decrease Mcl-1 expression via ER stress-dependent inhibition of translation, while cytokines induce Mcl-1 downregulation via a combination of decreased translation and increased JNK activity (Figure 8).Increasing evidence support the involvement of Bcl2 family members in beta cell apoptosis in response to pro-inflammatory cytokines, saturated FFA and ER stress(3, 5- 7, 13, 20, 33). Here, we demonstrate that cytokines, palmitate and thapsigargin induce Bax translocation, cytochrome c release and caspase 3 cleavage, independently of changes in Bcl-2, Bcl-XL, Bax and Bak expression levels, but partly as a consequence of Mcl-1 downregulation (Figure8). Our data are in agreement with previous studies demonstrating that Mcl-1, in spite of its preferential mitochondrial localization (34), is able to prevent Bax activation and translocation[START_REF] Germain | MCL-1 inhibits BAX in the absence of MCL-1/BAX Interaction[END_REF]. The fact that Mcl-1 is unlikely to directly interact with Bax (34) suggest that additional factors are involved in the inhibitory action of Mcl-1 on Bax translocation. Truncated Bid (tBid) is one of the factors contributing to Bax conformational changes allowing oligomerization and translocation to the mitochondria[START_REF] Roucou | Bax oligomerization in mitochondrial membranes requires tBid (caspase-8-cleaved Bid) and a mitochondrial protein[END_REF] and tBid contributes to cytokine-induced beta cell apoptosis[START_REF] Mckenzie | Proapoptotic BH3-only protein Bid is essential for death receptor-induced apoptosis of pancreatic beta-cells[END_REF]. Mcl-1 has been demonstrated to sequester tBid, thereby blocking Bax translocation and cytochrome c release[START_REF] Clohessy | Mcl-1 interacts with truncated Bid and inhibits its induction of cytochrome c release and its role in receptor-mediated apoptosis[END_REF]. Further studies remain to be performed to determine whether Mcl-1 overexpression results in tBid sequestration, and whether this contributes to the blockade of Bax translocation. In vitro studies suggest that Mcl-1 may interact with other BH3-only proteins including Puma, Noxa, Bim and DP5[START_REF] Chen | Differential targeting of prosurvival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function[END_REF], suggesting that additional proteins involved in Bax translocation might be sequestered by Mcl-1. In line with this hypothesis, we recently demonstrated that the pro-apoptotic BH3-only proteins DP5/Hrk and PUMA, which are rapidly overexpressed in response to ER stress and cytokines in beta cells, are involved in beta cell apoptosis[START_REF] Gurzov | Signaling by IL-1beta+IFN-gamma and ER stress converge on DP5/Hrk activation: a novel mechanism for pancreatic beta-cell apoptosis[END_REF][START_REF] Gurzov | Puma activation contributes to pancreatic beta cell apoptosis induced by pro-inflammatory cytokines and endoplasmic reticulum stress[END_REF]. Taken together, these data suggest that, at least in the case of cytokines, proteins of the Bcl-2 family are regulated at several levels in beta cells; while Mcl-1 is degraded, BH3-only proteins (tBid, DP5, PUMA …) are activated, thereby tipping the pro/anti-apoptotic balance toward apoptosis. Alternatively, other pro-apoptotic mechanisms involving additional Bcl-2 family members (Bim, NOXA …) and/or other pathways (XIAP, FLIPs …) may contribute to beta cell apoptosis.

  The cells were examined by inverted fluorescence microscopy (Axiovert 200, Carl Zeiss, Zaventem, Belgium). A minimum of 500 cells was counted in each experimental condition by two independent observers, one of them unaware of sample identity. There was no difference in viability between non-treated cells (ctrl), cells exposed to the ER stressors solvent DMSO (0.005 to 0.1%) or cells incubated in the FFA medium (data not shown), thus the control (ctrl) data showed in viability figures are mean ±SEM of the different controls used in the experiments. The necrosis levels were low throughout the experiments and were not significantly changed between different experimental conditions in INS-1E cells (1.32±0.22%: mean necrosis levels in all conditions) and FACS-purified primary beta-cells (2.03±0.34%: mean necrosis levels in all conditions). Apoptosis was confirmed by caspase 3 cleavage (described above in Western blot) and Bax translocation (see below in immunofluorescence)

Figure 1

 1 Figure 1 Mcl-1 protein expression is rapidly decreased upon beta cell exposure to

Figure 2

 2 Figure 2 Regulation of Mcl-1 expression

Figure 3

 3 Figure 3 Mcl-1 knockdown increases beta cell apoptosis

Figure 4

 4 Figure 4 Mcl-1 knockdown increases caspase 3 cleavage

Figure 5

 5 Figure 5 Mcl-1 overexpression prevents beta cell apoptosis

Figure 6

 6 Figure 6 Mcl-1 overexpression prevents caspase 3 cleavage

Figure 7

 7 Figure 7 Mcl-1 overexpression prevents Bax translocation to the mitochondria and

Figure 8

 8 Figure 8 Scheme depicting the central role of Mcl-1 in apoptosis induced by

  ). Palmitate, but not oleate,

	decreased Mcl-1 expression by 50% (Figures 1c and S1b

  TARGETplus SMARTpool siRNA (Dharmacon RNAi technologies, Thermo Fisher Scientific, Aalst, Belgium). The SMARTpool is a mixture of

	4	different		siRNA:		#1:GUAAGGACGAAGCGGGACU;
	#2:GUAGAACAAAUCCGAGUUA;		#3:GAAUUGUGGCUAACGAGAA;
	#4:GAUCAGUUCUAGUGUAUAU.
	Mcl-1	siRNA#2:	rat	Mcl-1	Silencer®	Select	pre-designed	siRNA:
	CGAGGACGAUGUUAAAUCU from Ambion (Applied Biosystems, Lennik, Belgium).
	PERK siRNA#1: rat Eif2ak3 ON-TARGETplus SMARTpool siRNA (Dharmacon);
	#1:CAGGAUACGUGUCCCGAUA;		#2:GAACAGGAGUCACGCGCGA;
	#3:CCGUCAGGUCUCGGAAAA; #4: AUACAGUAAUGGUGCGCUU.
	PERK	siRNA#2:	rat	Eif2ak3 Silencer® Select pre-designed siRNA:

  -1. 48 hours later, cells were extracted using the PIERCE Mitochondria Isolation Kit for Cultured Cells (Thermo Fisher Scientific, Aalst, Belgium) according to manufacturer's instructions.

	INS-1E cells were plated at a density of 150,000 cells/condition. 48 hours after infection
	or transfection, cells were incubated for 30 min in glucose-free INS-1E medium. Cells
	were washed in KRBH (Krebs-Ringer/bicarbonate-HEPES) buffer and preincubated for
	Immunofluorescence 30 min in glucose-free KRBH (Krebs-Ringer/bicarbonate-HEPES) buffer (20mM NaCl,
	INS-1E cells grown on glass culture slides (BD Biosciences Europe, Erembodegem, 3.5mM KCl,0.5mM NaH2PO4, 1.5mM CaCl2, 5mM NaHCO3, 10mM HEPES and 0.1%
	Belgium) were fixed for 15 min in fresh 4% paraformaldehyde, rinsed in PBS and BSA). Cells were then incubated for 30 min in KRBH supplemented with 1.67 mM
	permeabilized for 5 min in PBS-Triton X-100 0.1%. Slides were then blocked using PBS-glucose, 16.7mM glucose or 16.7mM glucose + 10μM Forskolin. Insulin release was
	goat serum 5% and incubated overnight at 4°C in the presence of polyclonal rabbit anti-measured using the High Range Rat Insulin ELISA according to manufacturer's
	Bax (1/1000; Santa Cruz Biotechnology, Inc.), monoclonal mouse anti-cytochrome c or instruction (Mercodia AB, Uppsalla, Sweden).
	anti-ATP Synthase β (1/2000; BD Biosciences). For the proliferation assay using BrdU,
	INS-1E cells were incubated for 6 hours in a 100µM BrdU solution prior to fixation.
	Statistical Analyses
	After PBS washing, DNA was denaturated by incubating cells in HCl 2N for 30min. Acid
	Data are presented as means ± SEM. Comparisons were performed by two-tailed paired
	solution was neutralized by washing twice in borate buffer 0.1M. Slides were then
	Student's t-test or by ANOVA followed by t-tests with Bonferroni correction for multiple
	blocked using PBS-goat serum 5% and incubated overnight at 4°C in the presence of
	comparison. A p value ≤0.05 was considered statistically significant.
	purified mouse anti-BrDU (1/500; BD Biosciences). Cells were then washed and further
	exposed for 1h to appropriate Alexa fluor 488 or 555-conjugated antibodies (1/1000;
	N.V. Invitrogen SA, Merelbeke, Belgium). After washing, cells were stained with
	Hoechst, mounted and photographed using fluorescence microscopy (Axio Imager, Carl
	Zeiss, Zaventem, Belgium). A minimum of 600 cells was counted in each experimental
	condition by two independent observers, one of them unaware of sample identity.
	Insulin secretion measurements

and underline Mcl-1 as a promising target for the development of new strategies aiming to prevent beta cell apoptosis in diabetes.

Acknowledgements

This work was supported by grants from the Juvenile Diabetes Research Foundation (JDRF) International (1-2008-536), Fonds National de la Recherche Scientifique (FNRS -FRSM) Belgium, the Communauté Française de Belgique -Actions de Recherche Concertées (ARC), the European Union (Integrated Projects EuroDia in the Framework Programme 6 [FP6] and Naimit in the FP7 of the European Community) and the Belgium Program on Interuniversity Poles of Attraction initiated by the Belgium State (IUAP P6/40). A. K. Cardozo is a Research Associate of the FNRS. F. Allagnat is supported by the JDRF. F. Moore is the recipient of a Post-Doctoral Fellowship from FNRS, Belgium. J.M. Vanderwinden is a Research Director of the FNRS. We thank M. A. Neef, G. Vandenbroeck, M. Urbain, R. Leeman, A. M. Musuaya, R. Makhnas, and S. Mertens from the Laboratory of Experimental Medicine, ULB, for excellent technical support.


Johannes Reichsoellner

Julia Larch

Iris Unterberger

Judith Dobesberger

Giorgi Kuchukhidze

Gerhard Luef

Gerhard Bauer

MD, MSc Eugen Trinka
email: eugen.trinka@uki.at

Idiopathic generalised epilepsy of late onset -A separate nosologic entity?

Keywords: idiopathic generalised epilepsy, late onset, seizure onset

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

INTRODUCTION

Idiopathic generalised epilepsies (IGE) account for approximately 15-20% of all epilepsy cases. [START_REF] Jallon | Epidemiology of idiopathic generalized epilepsies[END_REF] IGE classification is based mainly on clinical and EEG features. Aside from a genetic predisposition, no other etiology is known.[2] IGE can be divided into eight age related epilepsy syndromes according to the still valid classification of the International League Against Epilepsy (ILAE).[2] Four of these syndromes occur after the neonatal period:

Childhood absence epilepsy (CAE), juvenile absence epilepsy (JAE), juvenile myoclonic epilepsy (JME) and epilepsy with grand mal seizures on awakening (EGMA). Age at onset and seizure types are taxonomic criteria: According to the ILAE classification CAE-patients usually have their seizure onset between the age of 6 and 7 years, JAE and JME -during puberty and EGMA -during the second decade of life. [2,[START_REF] Janz | The idiopathic generalized epilepsies of adolescence with childhood and juvenile age of onset[END_REF] A considerable number of patients may not fulfil these criteria related to the limits of seizure onset age. IGE cases with a later than usual onset have been described. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF][START_REF] Gilliam | Adult myoclonic epilepsy: a distinct syndrome of idiopathic generalized epilepsy[END_REF][START_REF] Loiseau | Idiopathic generalized epilepsy of late onset[END_REF][START_REF] Luef | Idiopathic Generalized Epilepsy of Late Onset: A New Epileptic Syndrome[END_REF][START_REF] Marini | Idiopathic generalised epilepsy of adult onset: clinical syndromes and genetics[END_REF][START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF][START_REF] Panayiotopoulos | Idiopathic generalised epilepsy in adults manifested by phantom absences, generalised tonic-clonic seizures, and frequent absence status[END_REF][START_REF] Yenjun | EEG in adult-onset idiopathic generalized epilepsy[END_REF] In these studies, late onset groups were defined arbitrarily and some authors set the age limit at 20, others at 16 or 18 years. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF][START_REF] Marini | Idiopathic generalised epilepsy of adult onset: clinical syndromes and genetics[END_REF][START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF][START_REF] Panayiotopoulos | Idiopathic generalised epilepsy in adults manifested by phantom absences, generalised tonic-clonic seizures, and frequent absence status[END_REF][START_REF] Yenjun | EEG in adult-onset idiopathic generalized epilepsy[END_REF] It has been suggested, that IGE with late onset shares common biologic determinants with IGE while etiologic factors and relations between each IGE syndrome are not clearly understood and discussed controversially. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF][START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF][START_REF] Yenjun | EEG in adult-onset idiopathic generalized epilepsy[END_REF] Therefore, we aimed to study a large population with IGE in a single centre for determining the age range at disease onset and comparing clinical features of late onset patients with those who had classical age at onset.

METHODS

We identified 798 patients with IGE in the database of the epilepsy unit at the Department of Neurology, Medical University of Innsbruck, Austria. IGE was defined according to the ILAE classification (Commission on Classification, 1989). Inclusion criteria for the study sample were: Diagnosis of IGE, more than two follow up visits at our department (at least one visit after 1985), duration of follow up period more than one year, normal brain imaging, no late de-novo absence status and no signs of focal epilepsy.[2, [START_REF] Bauer | Absence status in the elderly as a late complication of idiopathic generalized epilepsies[END_REF][START_REF] Thomas | Absence status epilepsy[END_REF]] Three hundred and six patients were excluded due to the following reasons: Less than two follow up visits (n=130), patient files could not be retrieved (n=43), last patient visit before 1985 (n=54), follow up period of less than 1 year (n=15) focal or cryptogenic epilepsy additional to IGE (n=5), brain trauma (n=14), meningitis (n=4). In addition, 41 patients with uncertain diagnosis were excluded.

All patients had EEG and neuroimaging (CT or MRI). Patient charts included a detailed medical history.

We analysed biographic data, especially age at seizure onset and date of the first and the last follow-up. Patients' case histories were screened for seizure types (myoclonic seizures, generalised tonic-clonic seizures and absence seizures at the onset and during the course of epilepsy) and status epilepticus. Syndromes were diagnosed according to the ILAE criteria. Patients with evolving syndromes, for example CAE with late onset who first had absences and later on developed generalised tonic clonic seizures were classified as CAE .

Patients with absences in childhood and later myoclonic jerks with or without grand mal seizures on awakening were classified as JME [START_REF] Trinka | Absences in adult seizure disorders[END_REF]. We analysed family history (first degree relatives with epilepsy), history of febrile seizures, EEG characteristics, neurological and psychiatric findings, compliance, social status, anti-epileptic drug (AED) treatment at the last follow up visit and neuroimaging data. Alcohol, sleep deprivation and stress were regarded as seizure triggers.

As not all original EEGs were retrievable over the long observation period, we extracted the most characteristic records and rated the following EEG phenomena: Generalised spikes and sharp waves, localised spikes and sharp waves, diffuse slowing and local slowing and absence status epilepticus.

Psychiatric comorbidities and cognitive disturbancies were categorised as "behavioural disorder/affective disturbance", "psychosis" and "learning disabled". Social status was grouped into "independent (from assistance)", "partially dependent", "dependent" and "not identified". Compliance ranged from non-compliance to good compliance.

Seizure outcome was qualified as (i) seizure freedom for more than one year at the last follow up (ii) seizure freedom on their initial AED-treatment (immediate responders) and (iii) patients in terminal remission (seizure freedom for more than 5 years).

Data were processed with MS Excel 2003 and SPSS 14.0. χ² test was applied for group comparisons (significance level 0.05). Bonferroni-Holm correction was used as multiple testing was performed (α level given in tables, α 1 -α 20 ). Spearman Rank Order was used for correlation of onset age with the variables.

RESULTS

Four-hundred and ninety-two patients (299 women, 193 men, significantly more women than men -p< 0.001) met the inclusion criteria. Age at seizure onset varied from one to 55 years of age (mean -15 years). Even 80/492 (16.3%) patients had their onset after the age of 20 years. The mean period of ongoing seizure activity was 11 years ranging from less than one year up to 63 years.

One hundred and ninety-two patients had EGMA (39%; age at seizure onset -0.1 -51.7 years, mean 17.6, SD 8.7), 112 patients had JAE (22.8%; age at seizure onset -3 -39 years, mean 15.6, SD 5.6), 75 patients had JME (15.2%; age at seizure onset -4 -39 years, mean 14.3, SD 5.1) and 113 patients had CAE (23%; age at seizure onset -0.3 -55 years, mean 9, SD 7.5).

The ages of seizure onset were normally distributed up to the age of 30 years.

According to the age at seizure onset the population was divided into three groups (fig. 1): In group I, seizure onset was between 1-15 years (n=284), in group II -between 15 -30 years (n=180) and in group III -above 30 years (n=28). The proportion of men and women did not differ significantly throughout the groups (table 1, p=0.203). Two hundred and ninety eight out of all patients (60.6%) were seizure free and 144/492 (29.3%) were in terminal remission at the last follow-up visit (table 3).

DISCUSSION

The age of seizure onset in our large group of patients with IGE showed a broad age range -from the first year of life up to 55 years with a normal distribution up to the age of 30.

From the fourth decade on, the distribution was skewed with only very few patients (n=6) older than 40. Overall only 28/492 (5.7%) of patients had seizure onset after the age of 30 years.

A relatively high proportion of patients with seizure onset after the second decade of life (n=80, 16.3%) in our study sample is in line with the ILAE classification.

[2] It demonstrates, however, that a late onset is not as rare as it has been stated in previous studies. [START_REF] Loiseau | Idiopathic generalized epilepsy of late onset[END_REF][START_REF] Gastaut | Individualsation des epilepsies dites 'benignes' ou 'fonctionnelles' aux différents âges de la vie[END_REF] We found no statistically significant differences related to prognosis, EEGfindings and characteristic clinical features other than seizure types which are in fact the taxonomic criterion for the classification system. Previous studies suggesting that late onset cases share most of characteristic features with IGE based their assumptions on arbitrarily defined cut-off ages which explains most of the differences between the studies. [4-6;8;9;11] Ages of 16, 18 and 20 years were used as cut-off values. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF][START_REF] Marini | Idiopathic generalised epilepsy of adult onset: clinical syndromes and genetics[END_REF][START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF][START_REF] Panayiotopoulos | Idiopathic generalised epilepsy in adults manifested by phantom absences, generalised tonic-clonic seizures, and frequent absence status[END_REF][START_REF] Yenjun | EEG in adult-onset idiopathic generalized epilepsy[END_REF] Therefore, the proportion of patients with so-called late onset IGE varied substantially between 8.5 and 28%. [START_REF] Marini | Idiopathic generalised epilepsy of adult onset: clinical syndromes and genetics[END_REF][START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF] In the present study, cut-off ages were not defined a priori. They were based on the observation of the distribution of the ages at onset. None of the clinical features except for seizure types differed significantly in group comparison, underlining the concept of agerelated occurrence of seizure types in epilepsy syndromes. [START_REF] Janz | The idiopathic generalized epilepsies of adolescence with childhood and juvenile age of onset[END_REF] However, there are several uncertainties: First, "minor seizures" like brief absences may be overlooked and other seizures may be regarded as non-epileptic (myoclonic jerks). Therefore, the true age at seizure onset may not always be easily detected. For example, in the not yet recognized syndrome of denovo absence status in the elderly, a certain number of patients may have had minor seizures in childhood. [START_REF] Bauer | Absence status in the elderly as a late complication of idiopathic generalized epilepsies[END_REF] Secondly, the predominating seizure type may change over time within the syndromes. For example, absences and MS in JME tend to remit, while GTCS may be persistent for years. [START_REF] Janz | The idiopathic generalized epilepsies of adolescence with childhood and juvenile age of onset[END_REF] Similar observations have been made for the evolution of AS which may be less pronounced in later ages (phantom absences). [START_REF] Panayiotopoulos | Idiopathic generalised epilepsy in adults manifested by phantom absences, generalised tonic-clonic seizures, and frequent absence status[END_REF] Hence, the patients' age has to be taken under consideration (mean age at the last follow-up was 34.5 years) when comparing different groups. In our study, the age at the last follow-up did not differ significantly between the groups. Finally, in many patients additional neuroimaging MRI was performed, since the diagnosis of generalised seizures, especially absence in adults is not always straightforward.

Nevertheless, the occurrence of additional generalized seizure types and a characteristic EEG pattetrn are the most reliable clue to the syndrome diagnosis.

The treatment response to AEDs was favourable in the majority of patients. The numbers of seizure free patients and those in terminal remission were comparable to other studies. [START_REF] Nicolson | A comparison of adult onset and classical idiopathic generalised epilepsy[END_REF][START_REF] Mohanraj | Outcomes of newly diagnosed idiopathic generalized epilepsy syndromes in a non-pediatric setting[END_REF] The proportion of patients who became seizure free after the initial drug treatment was only 23.8% which is lower than expected. [START_REF] Mohanraj | Outcomes of newly diagnosed idiopathic generalized epilepsy syndromes in a non-pediatric setting[END_REF] This may reflect a selection bias towards more difficult to treat patients as some of the seizure free patients may have been treated by general neurologists and were lost for follow up at our institution.

In agreement with previous findings, 8.9% of patients had focal abnormalities in the EEG and 14.6% in neurological examination. The presence of focal findings should not preclude from diagnosing idiopathic generalised epilepsies if all other features are typical.

Nevertheless, the concept of "generalised" epilepsies has been challenged. [START_REF] Craiu | Are absences truly generalized seizures or partial seizures originating from or predominantly involving the pre-motor areas? Some clinical and theoretical observations and their implications for seizure classification[END_REF] Psychiatric comorbidity in epilepsies, including IGE, have been repeatedly investigated. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF][START_REF] Akanuma | Psychiatric comorbidity in adult patients with idiopathic generalized epilepsy[END_REF][START_REF] Gelisse | Psychiatric disorders in juvenile myoclonic epilepsy[END_REF][START_REF] Trinka | Psychiatric comorbidity in juvenile myoclonic epilepsy[END_REF] These studies consistently show that about 30% of patients will experience a psychiatric disturbance at least once in their life time. This is also reflected in our study where 28.5% of patients had affective disturbances rated by the treating physician on clinical grounds and 6.1% received anxiolytics or antidepressants. Despite the possible influence of psychiatric comorbidities on seizure outcome, we were not able to address this issue specifically in this retrospective survey. [START_REF] Cutting | Adult-onset idiopathic generalized epilepsy: clinical and behavioral features[END_REF] Alcohol (consumption and withdrawal) has been discussed as an effective seizure trigger in literature. [START_REF] Gordon | Alcohol and marijuana: effects on epilepsy and use by patients with epilepsy[END_REF] Differences within the age groups could either result from a psychosocial phenomenon or a confounding mechanism: Due to the age related habits of drinking, one could hypothesize that alcohol might be more common seizure trigger in later years of life. Since patients are instructed to abstain from alcohol consumption at seizure onset, patients aged younger than 15 at seizure onset are less likely to drink at legal ages (and in the course never experience alcohol as a seizure trigger). Other seizure triggers as sleep deprivation and stress do not play a more important role in late onset cases.

The majority of patients (66.1%) were treated with VPA. Although there are yet no Class I studies comparing the efficacy of VPA with other AED in IGE, two large class III studies (SANAD B and KOMET trial) confirmed its good efficacy and tolerability in these syndromes, supporting VPA as the first choice in IGE. [START_REF] Marson | A randomised controlled trial examining the longer-term outcomes of standard versus new antiepileptic drugs. The SANAD trial[END_REF][START_REF] Trinka | The KOMET study: an open-label, randomized, parallel-group trial comparing efficacy and safety of levetiracetam with sodium valproate and carbamazepine as monotherapy in subjects with newly diagnosed epilepsy[END_REF] This study bears considerable methodological limitations in being a retrospective study. To achieve better comparability the ILAE criteria of 1989 were applied, even though some patients were diagnosed with IGE earlier and reclassified later. Diagnostic techniques have improved substantially in the past 20 years and the rate of patients who received a straight forward diagnosis has increased. Although this epilepsy centre can be regarded as a primary care centre for patients with epilepsy (majority of patients are in adolescence and adulthood) a selection biases towards more severe cases cannot be excluded.

CONCLUSION

In this large cohort of patient with IGE, the age of seizure onset had a loose upper limit with 16.3% after the second decade and 5.7% after the third decade of life. Later onset of seizures was associated with differences in seizure types (emphasizing the age related onset) but not regarding other clinical issues. We cannot confirm the assumption that IGE of late onset might be a separate nosologic entity.

Table 1

 1 Description of population and comparison of age groups concerning seizure types and syndromes GTCS) and myoclonic seizures (MS) in the course of the period with seizure activity differed throughout all groups (see table 1). The age specific distribution of seizure types at disease onset (see fig. 2, table 1) differed in group comparison (p<0.001). GTCS occurred in 93.7% of all patients in the course of the disease, AS in 52.6% and myoclonic seizures in 28.9% (see table 1). At least one episode of status epilepticus occurred in 7.7% of patients (group comparison was n.s., table 1). compared to 43/284 (15%) patients with seizure onset under the age of 15 (group I) (table 2). Seizures were provoked by stress in 51/492 (10.4%, n.s.) patients and by sleep deprivation in 172/492 (35%, n.s.).

	Groups			Total	Group 1	Group 2	Group 3	p-value
	(age of onset, years)			(<15 yrs)	(15 -30yrs.)	(> 30yrs.)
	No. of patients			492 (100) 284 (57.7) 180 (36.6)	28 (5.7)
	Median age of onset (yrs.)	13.9	10.25	17.6	35.4
	Gender m	m		193 (39.2) 102 (35.9) 78 (43.3)	13 (46.4)	0.203*
		F		299 (60.8) 182 (64.1) 102 (56.7)	15 (53.6)
	Family history			138 (28)	73 (25.7)	52 (28.9)	13 (46.2)	0.065*
	Febrile seizures			21 (4.3)	14 (4.9)	6 (3.3)	1 (3.6)	0.697*
	Seizure		GTCS	241 (49.2) 101 (35.6) 117 (65)	24 (85.7)	<0.001
	type(s) at disease	MS	87 (17.7)	40 (14.1)	45 (25)	2 (7.1)	0.004
	onset		AS	193 (39)	154 (54.2) 34 (18.9%)	4 (14.3)	<0.001
	Occurring seizure	GTCS	461 (93.7) 261 (91.9) 175 (97.2)	25 (89.3)	0.044
			MS	142 (28.9) 75 (26.4)	63 (35)	4 (14.3)	0.027
	types
			AS	259 (52.6) 193 (68)	58 (32.2)	8 (28.6)	<0.001
			EGMA	192 (39)	75 (25.7)	96 (53.3)	21 (75)
	Syndrome		JME	112 (22.8) 55 (19.4)	54 (30)	3 (10.7)	<0.001
			JAE	75 (15.2)	51 (18)	23 (12.8)	1 (3.6)
			CAE	113 (23)	103 (36.3) 7 (3.9)	3 (10.7)
	Status epilepticus		38 (7.7)	26 (9.2)	8 (4.4)	4 (14.3)	0.073*
	Numbers are n(%) unless stated, percentages relate to groups, p-value of group comparison according to χ² test,
	*α-level after Bonferroni-Holm correction for multiple testing.
	GTCS, generalized tonic clonic seizures; MS, myoclonic seizure; AS, absence seizures; EGMA, epilepsy with
	grand mal on awakening; JME, juvenile myoclonic epilepsy; JAE, juvenile absence epilepsy; CAE, childhood
	absence epilepsy.

Absences (AS) were the most common seizure type in group I (193/284, 68%, p<0.001, fig.

2

) and correlated with an early onset of IGE (p<0.001). The occurrence of generalised tonic-clonic (JME, 23 (12.8%) -JAE and 7 (3.9%) -CAE. In group III (n=28), three patients (10.7%) had JME, one (3.6%) -JAE and three (10.7%) -CAE (table

1

).

Generalised spike and wave or polyspike wave EEG patterns were detected in 84.8% of all patients (417/492). Their occurrence did not differ between the groups. Twenty three patients (4.7%, n.s.) had also localised EEG abnormalities. In 31/492 (6.3%, n.s.) patients, local slowing was seen in EEG, while EEG of 99/492 (20.1%) patients showed diffuse slowing. Only 27/492 (5.5%) patients had normal EEG. In 7/492 (1.4%) patients, EEG documented a non-convulsive status epilepticus. analysed as co-variables in group comparison) no other variable showed a positive correlation (correlation coefficient 0.146, p=0.001) with the age at seizure onset (table 2). In patients with seizure onset between 15 -30 years (group II) 49/180 (27%) patients experienced alcohol triggered seizures, The proportion of patients with psychosocial problems did not differ significantly in group comparison. Behavioural disorders/affective disturbances were observed in 140/492 (28.5%): Mild affective disturbances were noted in 113/492 (23.1%, n.s.) patients, 32/492 (6.5%, n.s.) had learning disabilities and 12/492 (2.4%) had a history of psychosis. Social status was rated as "independent" in the majority of patients 471/492 (95.7%, group comparison n.s.). At the last follow up 15/492 (3%) patients received antidepressants or anxiolytics, while 30/492 (6.1%) patients had a history of treatment with antidepressants or anxiolytics. Compliance was good in 326/492 (66.3%) patients.

Table 3

 3 Outcome variables in group comparison

	Groups	Total	Group 1	Group 2	Group 3	p-value
	(age of onset, years)		(<15 yrs)	(15 -30 yrs.)	(> 30yrs.)
	Seizure Freedom	298 (60.6)	173 (60.9)	108 (60)	17 (60.7)	0.981*
	Remission	144 (29.3)	78 (27.5)	57 (31.7)	9 (32.1)	0.589 *

ACKNOWLEDGEMENTS

(1) This work was presented at the 8 th European Congress on Epileptology (Berlin, September 21 st -25 th , 2008) as a platform presentation. [23] (2) The Corresponding Author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees) on a worldwide basis to the BMJ Publishing Group Ltd and its Licensees to permit this article (if accepted) to be published in the Journal of Neurology, Neurosurgery & Psychiatry editions and any other BMJPGL products to exploit all subsidiary rights, as set out in our licence ( http://jnnp.bmjjournals.com/misc/ifora/licenceform.shtml ).