Molecular Brain Research 108 (2002) 143–146www.elsevier.com/ locate/molbrainres

Short communication

1 ,25-Dihydroxyvitamin D increases striatal GDNF mRNA and protein3

expression in adult ratsa b b b˜Begona Sanchez , Elena Lopez-Martin , Carmen Segura , Jose L. Labandeira-Garcia ,

a ,*Roman Perez-FernandezaDepartment of Physiology, School of Medicine, University of Santiago de Compostela, 15782 Santiago de Compostela, Spain

bDepartment of Morphological Sciences, School of Medicine, University of Santiago de Compostela, 15782Santiago de Compostela, Spain

Accepted 9 October 2002

Abstract

Glial cell line-derived neurotrophic factor (GDNF) has been postulated as a possible candidate for therapeutic treatment in Parkinson’sdisease (PD). Recent in vitro data suggest that 1,25-dihydroxyvitamin D3 [1,25(OH) D ] treatment may enhance GDNF mRNA2 3

expression. In the present study, using semiquantitative RT-PCR and Western blot, we have shown that 1,25(OH) D administration2 3

intraperitoneally, significantly increases GDNF mRNA and protein levels in the striatum of adult rats. 2002 Elsevier Science B.V. All rights reserved.

Theme: Disorders of the nervous systems

Topic: Degenerative disorders: Parkinson’s

Keywords: 1,25-Dihydroxyvitamin D3; GDNF; Striatum; Rat; Parkinson’s disease

Parkinson’s disease (PD) is characterized by a progres- 1,25(OH) D is to stimulate calcium transport in bone,2 3

sive degeneration of midbrain nigral dopaminergic (DA) intestinal tract and kidney. However, specific receptors forneurons and subsequent loss of DA input to the striatum. 1,25(OH) D have been detected in other tissues (such as2 3

Glial cell line-derived neurotrophic factor (GDNF) is a pituitary, thyroid, liver, breast, muscle or brain), suggestingmember of the transforming growth factor-b superfamily other functions of the hormone beyond bone metabolismwhich has been reported to have neurotrophic effects and mineral homeostasis [14]. Interestingly, it has beenparticularly on the dopaminergic nigrostriatal system demonstrated that VDR expression is present throughout[2,13]. Recent studies have indicated that endogenous large portions of the central nervous system (CNS) of rats,neurotrophic factors can be regulated by different pharma- including striatum [16,11], suggesting that 1,25(OH) D2 3

cological treatments, and a procedure to increase striatal through interactions with VDR may play a role in theGDNF levels is of great interest as potential therapy for development and function of the CNS.PD. Recently, it has been shown that 1,25-dihydrox- Ten adult Sprague–Dawley rats were used in this study;yvitamin D3 [1,25(OH) D ] increases GDNF expression five of them were used as control and the other five rats2 3

in C6 glioma cells in vitro [10], and increases GDNF were treated with 1,25(OH) D . The 1,25(OH) D -2 3 2 3

levels in the rat cortex [17]. treated animals (Calcitriol, Sigma, MO, USA) were intra-The biological effects of 1,25(OH) D , the most active peritoneally (i.p.) injected at dose of 1mg/kg/day for 72 3

metabolite of vitamin D, are mediated by their binding to consecutive days. The control rats received 1 ml /kg/dayspecific intracellular receptors (vitamin D receptor, VDR) of vehicle (1:1, v /v saline–ethanol). After 7 days ofpresent in target cells [12]. The classical role of injection with 1,25(OH) D or vehicle, animals were2 3

deeply anaesthesized with chloral hydrate (400 mg/kg)and killed, and striatum removed quickly and stored at*Corresponding author. Tel.:134-981-582-658; fax:134-981-574-280 8C. mRNA from striatal tissue was isolated using145.

E-mail address: fsropefe@usc.es(R. Perez-Fernandez). mRNA Isolation Kit (Roche Diagnostics, IN, USA) ac-

0169-328X/02/$ – see front matter 2002 Elsevier Science B.V. All rights reserved.PI I : S0169-328X( 02 )00545-4

144 B. Sanchez et al. / Molecular Brain Research 108 (2002) 143–146

cording to manufacturer’s instructions. RT-PCR was per- vehicle-treated rats (0.560.07) (P,0.01) (Fig. 1E). Weformed as previously described [4]. Briefly, cDNA syn- also evaluate GDNF protein expression in 1,25(OH) D -2 3

thesis was generated under the following conditions: 10ml treated rats vs. saline-treated rats. The anti-GDNF antibodyof total mRNA was incubated for 50 min at 378C, for 15 recognized a strong band of approximately 38 kDa (corre-min at 428C and for 5 min at 958C. Fiveml of the cDNA sponding to GDNF dimer) and a faint band of 34 kDawere amplified by PCR (948C for 1 min; 578C or 628C (probably non-glycosylated GDNF dimer) in homogenatesfor 1 min for GDNF and GAPDH, respectively; and 728C from striatum of vehicle and 1,25(OH) D -treated rats2 3

for 1 min) during 35 cycles for GDNF or 27 cycles for (Fig. 1C and D, respectively). The densitometric analysisGADPH. For western blot analyses, striatum was shown that systemic (i.p) administration of 1,25(OH) D2 3

homogenized at 48C in 1 ml of lysis buffer (50 mM for 7 consecutive days increased GDNF protein levelsHEPES, pH 7.5, 150 mM NaCl, 5 mM EGTA, 1.5 mM (0.5960.05) in the striatum of treated adult rats withMgCl , 1% SDS, 10% glycerol, 1% Triton X-100, 10 mM respect to the vehicle-treated rats (0.4260.04) (P,0.01)2

sodium orthovanadate, 4 mM phenylmethyl sulfonyl (Fig. 1F).fluoramide and 50mg/ml aprotinin). Further lysate prepa- It is well known that GDNF has trophic effects onration, electrotransfer to nitrocellulose membrane, and dopaminergic neurons [2,13]. GDNF specifically promoteddetection of proteins by enhanced chemiluminescence the survival of dopaminergic neurons in dissociated rat(ECL) were done as described previously [4]. The primary embryo midbrain cultures and increased the morphologicalantibody, GDNF (1:100) was obtained from Santa Cruz differentiation of tyrosine hydroxylase neurons [7]. It hasBiotechnologies (Santa Cruz, CA, USA). been shown in ours and other laboratories, that GDNF has

To determine the relative amounts of GDNF mRNA in both trophic and tropic influence on the nigrostriataleach sample, GDNF PCR product was compared with the dopaminergic system during normal development as wellGADPH PCR product and quantified using Gel Doc 1000 as on developing striatal grafts, in vivo [13,8]. We haveDocumentation System (Bio-Rad Laboratories, CA, USA). shown that, during normal development, dopaminergicTo determine the relative amounts of GDNF protein in nigrostriatal terminals concentrate in the striatal areas,western blot, we compared and quantified GDNF in saline- showing the highest levels of GDNF, and that in intras-treated rats versus vitamin D-treated rats. All values of triatal striatal and mesencephalic co-grafts, dopaminergicrelative optical density (OD) obtained from GDNF and neurons and fibers concentrate in areas showing the highestGAPDH PCR products or GDNF (obtained from western levels of GDNF [8]. In addition, several studies haveblot) are expressed as mean6S.D. Differences between shown that GDNF has remarkable neuroprotective effectscontrol and vitamin D treated rats were evaluated by the against neurotoxins such as 6-OHDA (6-hydroxy-Mann–WhitneyU-test. Primer sequences for GDNF PCR dopamine) or MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahy-amplification were as follows: primer A (59- dropyridine) in rodent and primate models of PD [1,15,3].CCAATATGCCCGAAGA TTATCC-39) was a 22-mer It is particularly interesting to remark that recent studiescorresponding to nucleotides 201–222 of GDNF cDNA [1,6] have shown that injection of GDNF into the striatumand primer B (59-TTCGTAGCCCAAACCCAAG-39) was has significant protective effect both on nigrostriatalan antisense 19-mer corresponding to nucleotides 436–454 pathway and its function, while intranigral administrationof GDNF cDNA. The PCR product obtained was 254 bp in had protective effect on the nigral cell bodies but not onlength. The rat glyceraldehyde-phosphate dehydrogenase the striatal innervation or functional benefit, and intraven-(GAPDH) was used as ‘internal reference’. Sequences of tricular injection had no effects. Therefore, the increase ofthe primers were: forward primer, 59-TGATGACAT- striatal GDNF levels appears to be particularly effectiveCAAGAAGGTGGTGAAG-39, located at residues 758– for a possible treatment of PD. Recently, it has been782 of the rat coding sequence, and reverse primer, 59- described that 1,25(OH) D administration to C6 rat2 3

TCCTTGGAGGCCATGTAGGCCAT-39, located at res- glioma cell line for 48 h elicited a significant increase inidues 974–997 of the coding sequence. The PCR product the level of GDNF mRNA, indicating that 1,25(OH) D is2 3

obtained was 239 bp in length. a potent inducer of GDNF expression and may contributeTo investigate the effect of the systemic 1,25(OH) D to the regulation of GDNF in vivo [10]. Accordingly, it has2 3

administration on the GDNF mRNA expression in the also been reported that 1,25(OH) D administration, for 82 3

striatum of adult rats, we evaluate the expression of this days, to rats with a ischemic brain injury, produces agene with respect to GADPH gene using semiquantitative significant increase in cortical GDNF levels and reducesRT-PCR. As shown in Fig. 1A and B, bands corresponding ischemia-induced brain damage [17]. However, there areto the expected size of GDNF cDNA (254 bp) and no data on a possible effect of 1,25(OH) D administra-2 3

GAPDH cDNA (239 bp) were amplified by PCR and tion on GDNF levels in the striatum, which is of particularvisualized in agarose ethidium bromide gel. The systemic interest to consider GDNF induction as a potential treat-(i.p) administration of 1,25(OH) D for 7 consecutive ment for PD (see above). In the present study, we2 3

days significantly increased GDNF mRNA levels determined GDNF mRNA levels in the striatum, using(0.6860.05) in the striatum of adult rats with respect to the RT-PCR, and GDNF protein levels, using Western blot,

B. Sanchez et al. / Molecular Brain Research 108 (2002) 143–146 145

Fig. 1. Effect of 1,25(OH) D treatment on GDNF mRNA and protein levels in rat striatum. Adult rats were treated with 1,25(OH) D or vehicle for 72 3 2 3

consecutive days and then the striatum was removed for RT-PCR analysis or western blot. (A) GDNF and GAPDH PCR products obtained from striatum ofvehicle-treated rats. Lane 1: 1000 bp ladder; lanes 2, 4, 6, 8, 10: GDNF PCR product; lanes 3, 5, 7, 9, 11: GAPDH PCR product. (B) GDNF and GAPDHPCR products obtained from striatum of 1,25(OH) D -treated rats. Lane 2: Negative control of retrotranscription (RT). mRNA obtained from striatum was2 3

retrotranscribed to cDNA without reverse transcriptase enzyme to discard genomic DNA contamination. Lane 3: Negative control of PCR. PCR wasperformed as described in the text, but cDNA was not added. Lanes 4, 6, 8, 10, 12: GDNF PCR product; lanes 5, 7, 9, 11, 13: GAPDH PCR product. (C,D) Western blots analysis of immunoreactive GDNF in striatum of vehicle- and 1,25(OH) D -treated rats, respectively. The major 38 and 34 kDa2 3

immunoreactive bands are indicated by arrows. (E) The relative GDNF mRNA expression in striatum of adult rats was calculated from theGDNF/GAPDH relation obtained from the densitometric values of A and B (*P,0.01). (F) The relative GDNF protein expression in control- and1,25(OH) D -treated rats was calculated from densitometric values of C and D (*P,0.01). Values are expressed as mean6S.D.2 3

and we observed a significant increase in GDNF striatal dose to induce responses in CNS in rats. Furthermore, thislevels (both, mRNA and protein) of 1,25(OH) D -treated regimen of 1,25(OH) D dosing does not change mean2 3 2 3

rats with respect to control vehicle-injected rats. The VDR blood pressure, blood glucose, hemoglobin and blood gasbelongs to the large family of ligand-activated transcription values, although significantly increases total and free-cal-factors defined as the nuclear receptor family. The mem- cium levels in serum in rats [17]. However, the doses ofbers of this family activate or repress gene transcription 1,25(OH) D or vitamin D analogs must be properly2 3 3

through direct binding to discretecis-acting elements, adjusted in future studies for a potential therapeutic use intermed hormone response elements [12]. VDR expression humans.is present throughout large portions of the developing and In the present and the above mentioned studies [10,17],adult CNS [16,11], suggesting that 1,25(OH) D through it has been shown that 1,25(OH) D induces an increase in2 3 2 3

interactions with the VDR may play a role not only in the the expression of GDNF in the CNS. However, otherdevelopment but also in the normal function of the adult mechanisms may be involved in a possible neuroprotectiveCNS, including a neuroprotective role. Data from our function of vitamin D. Thus, it has been suggested thatpreliminary experiments (unpublished data) and from other 1,25(OH) D may also induce nerve growth factor syn-2 3

studies [17,9] indicated that 1mg/kg (i.p.) is a suitable thesis [18], or facilitate cellular functions that reduce

146 B. Sanchez et al. / Molecular Brain Research 108 (2002) 143–146

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protein increase is directly produced by 1,25(OH) D2 3 [8] E. Lopez-Martin, H. Caruncho, J. Rodriguez, M.J. Guerra, J.L.treatment through VDR at transcriptional level or indirectly Labandeira-Garcia, Striatal dopaminergic afferents concentrate inthrough other transcription factors. GDNF-positive patches during development and in developing

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This study was supported by grants from Spanish [10] P. Naveilhan, I. Neveu, D. Wion, P. Brachet, 1,25-DihydroxyvitaminD3, an inducer of glial cell line-derived neurotrophic factor,´Ministerio de Ciencia y Tecnologıa (BFI2000-0334 andNeuroReport 7 (1996) 2171–2175.PM98-0226).

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