Photoperiod affects the cerebrospinal fluid proteome: a comparison between short day– and long day–treated ewes

https://doi.org/10.1016/j.domaniend.2015.04.003Get rights and content

Highlights

  • Ovine CSF proteome was determined under short days and under long days.

  • A total of 103 proteins were identified.

  • Forty-one were differentially expressed: 18 were more abundant in long days, 23 in short days.

  • Their functions could be important for seasonality.

Abstract

Photoperiod is the main physical synchronizer of seasonal functions and a key factor in the modulation of molecule access to cerebrospinal fluid (CSF) in animals. Previous work has shown that photoperiod affects the transfer rate of steroids and protein hormones from blood to CSF and modulates choroid plexus tight junction protein content. We hypothesized that the CSF proteome would also be modified by photoperiod. We tested this hypothesis by comparing CSF obtained from the third ventricle of mature, ovariectomized, estradiol-replaced ewes exposed to long day length (LD) or short day length (SD). Variations in CSF protein expression between SD- or LD-treated ewes were studied in pools of CSF collected for 48 h. Proteins were precipitated, concentrated, and included in a polyacrylamide gel without protein fractionation. After in-gel tryptic digestion of total protein samples, we analyzed the resulting peptides by nanoliquid chromatography coupled with high-resolution tandem mass spectrometry (GeLC-MS/MS). Quantitative analysis was performed using 2 methods based on spectral counting and extracted ion chromatograms. Among 103 identified proteins, 41 were differentially expressed between LD and SD ewes (with P < 0.05 and at least a 1.5-fold difference). Of the 41 differentially expressed proteins, 22 were identified by both methods and 19 using extracted ion chromatograms only. Eighteen proteins were more abundant in LD ewes and 23 were more abundant in SD ewes. These proteins are involved in numerous functions including hormone transport, immune system activity, metabolism, and angiogenesis. To confirm proteomic results, 2 proteins, pigment epithelium-derived factor (PEDF) and gelsolin, for each individual sample of CSF collected under SD or LD were analyzed with Western blots. These results suggest an important photoperiod-dependent change in CSF proteome composition. Nevertheless, additional studies are required to assess the role of each protein in seasonal functions.

Introduction

Seasonal functions are synchronized by several parameters, among which duration of night is the most important. This parameter is translated into a chemical signal by release of melatonin from the pineal gland. In sheep, an experimental model widely used for studies of seasonal biological rhythms, melatonin acts on several target sites in the brain including the pars tuberalis, the mediobasal hypothalamus, and the choroid plexus (CP) [1]. These latter, together with the cerebral endothelium, are part of the blood–brain barrier (BBB) system, and both are involved in blood filtration and in the synthesis of cerebrospinal fluid (CSF).

Proteins in the CSF originate from blood filtration by the CP, from local CP synthesis, and from drainage of the brain-borne compounds [2]. We have previously demonstrated that CSF renewal rate is modified by photoperiod and that long days (LD; 16L:8D) promote a smaller CSF production rate than short days (SD; 8L:16D) [3]. The passage of molecules through the BBB and therefore through the CP also differs with photoperiod in sheep [4], [5]. For example, peripheral estradiol (E2) and progesterone passage from blood to brain is greater in LD than SD animals; this phenomenon is dependent on melatonin because pinealectomized animals do not show specific photoperiodic regulation. In rams, a protein hormone, leptin, has a higher transfer rate from blood to CSF under LD conditions [5]. More recently, we attempted to explain the cellular mechanisms involved in the regulation of blood–CSF transfer and found downregulation of specific tight junction proteins (afadin-6, zona occludens 1 and 2, and cadherin) of the CP under LD conditions [6]. This suggests that the permeability of the CP is decreased during SD periods.

We hypothesized that proteins passing through the CP, secreted by the CP, or drained from the brain may vary according to photoperiod. To test our hypothesis, we did a quantitative proteomic analysis on the CSF of ovariectomized, estradiol-replaced ewes kept under SD or LD conditions. We used an approach combining sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and trypsin digestion followed by nanoflow liquid chromatography tandem mass spectrometry (GeLC-MS/MS), associated with 2 label-free quantitative methods based on spectral counting (SC) and extracted ion chromatograms (XICs). Reliability of quantitative proteomic data obtained from pooled CSF was then checked for 2 proteins using Western blots.

Section snippets

Animals, light treatment, and surgery

All animal experiments were conducted in accordance with French Authorization No. 37801 for Animal Experimentation and Surgery and approved by the Val de Loire Local Ethics Committee. Adult Ile-de-France ewes (n = 10) of similar age (2.5-yr old) and weight (52.5 ± 3.0 kg) obtained from UEPAO (Unité expérimentale PAO n°1297 [EU0028], INRA Centre Val de Loire, Nouzilly, France) were maintained indoors in separate pens under artificial lighting conditions and fed a constant maintenance diet of

CSF protein concentration

We found higher protein concentration in LD CSF (211 ± 21 μg/mL) than in SD CSF (121 ± 18 μg/mL; P = 0.0317, Mann–Whitney U test).

CSF proteome

To establish the inventory of proteins in ovine CSF, the LD and SD samples included in polyacrylamide gel (without fractionation) were analyzed by GeLC-MS/MS and a total of 103 nonredundant proteins were identified, considering one peptide and a false discovery rate <1% (Table S1). To establish the relative abundance of the proteins in the ovine CSF, emPAI was

Discussion

To our knowledge, this is the first report to propose a comparison of ovine CSF protein content between SD (8L:16D) and LD (16L:8D) photoperiod conditions using the high throughput analysis method. We identified a total of 103 proteins in ovine CSF; 25 proteins have previously been described [16] and 88 were newly identified in this study. Comparison of these data with previously published human proteome data [17] using the CSF Proteome Resource (CSF-PR, http://129.177.231.63/csf-pr/) revealed

Acknowledgments

The authors thank Dr Massimiliano Beltramo for his critical revision of the article. L.D. and J.S. received a travel grant for scientific exchanges from PHC Polonium from the French Embassy in Poland/Ministry from Foreign Affairs and International Development. The high resolution mass spectrometer was financed (SMHART project) by the European Regional Development Fund (ERDF), the Conseil Régional du Centre, the French National Institute for Agricultural Research (INRA), and the French National

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  • The authors A.-P.T.G. and G.H. contributed equally to this study.

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