научная статья по теме DIFFERENTIAL EXPRESSION PATTERNS OF PEROXIREDOXINS IN OLFACTORY NEURONS Биология

Текст научной статьи на тему «DIFFERENTIAL EXPRESSION PATTERNS OF PEROXIREDOXINS IN OLFACTORY NEURONS»

БИОЛОГИЧЕСКИЕ МЕМБРАНЫ, 2014, том 31, № 4, с. 288-295

УДК 577.29

DIFFERENTIAL EXPRESSION PATTERNS OF PEROXIREDOXINS

IN OLFACTORY NEURONS

© 2014 A. S. Kolesnikova, A. A. Khokhlov, R. R. Romanov, M. F. Bystrova*

Institute of Cell Biophysics, Russian Academy of Sciences, ul. Institutskaya, 3, Pushchino, Moscow oblast, 142290 Russia;

*e-mail: marinabystrova@rambler.ru Received 31.03.2014

Peroxiredoxins (Prdxs) comprise a large family of cysteine-based peroxidases that are involved in a wide variety of physiological processes. At the single-cell level co-expression of Prdxs has not been studied. We performed gene expression profiling in mouse olfactory neurons and found that individual cells express distinct prdxs in multiple combinations. In the case of conventional interpretation of RT-PCR data, differential expression patterns indicate that each olfactory neuron selects specific set of prdxs to express. However, a new concept of stochastic dynamics of gene expression may provide an alternative interpretation of our data. Here we suggest that the lack of synchronization in transcriptional states of individual genes may give rise to temporal shifts in gene expression pattern, thus constituting a plausible source of variability detected at the single-cell level. If so, the olfactory neuron is likely to transcribe genes for each of the six Prdxs yet with entirely different transcriptional phases. Based on the discontinuous mode of gene expression, the composition of Prdx transcripts within a cell may be variable, and therefore, the outcome of gene expression analysis is likely to depend on the moment of cell harvesting. This assumption easily reconciles the apparent discrepancy between transcriptional profiles derived from single cells and from population of cells.

Keywords: peroxiredoxin, olfactory neuron, single cell, cellular heterogeneity, stochasticity.

DOI: 10.7868/S0233475514040045

INTRODUCTION

The olfactory neuroepithelium of vertebrates is the only neuronal tissue that is continually exposed to the oxidant-rich environment. Reactive oxygen species (ROS) are generated endogenously during aerobic cellular metabolism and exogenously from inhaled airborne toxicants, such as ozone and cigarette smoke. Furthermore, the oxidative metabolism of the airborne volatile odorants, which traverse mucus layer covering olfactory epithelium to reach olfactory receptors on olfactory cilia, may contribute to ROS generation. For instance, the olfactory epithelium contains a high concentration of multiple cytochrome P450 enzymes that have been suggested to transform lipophilic odorous molecules, thus making them less membrane permeable and preventing their direct entry into the brain [1]. Cytochrome P450 enzymes are known to catalyze the oxidation of endogenous and exogenous compounds and represent a significant source of ROS even under basal conditions [2]. Thus, redox control is particularly essential for the normal functioning of olfactory epithelium cells. Three major classes of antioxidant enzymes operate in mammalian cells to catab-olize hydroperoxides: catalase, glutathione peroxi-dase, and peroxiredoxins. The relative contribution of peroxide-degrading enzymes is currently under de-

bate; however, the importance of Prdxs for peroxide metabolism in neural cells and their involvement in protecting neurons from oxidative insults are increasingly recognized [3, 4]. Antioxidant status of rat olfactory epithelium was earlier studied by [5]. Based on immunohistochemical evidence, the authors declared that in the olfactory epithelium, glutathione peroxi-dase and catalase were largely confined to sustentacu-lar cells and Bowman's glands. The possible inference from these data is that the peroxidase capacity of olfactory neurons depends critically on the Prdx enzymes. Prdxs comprise a large family of cysteine-based peroxidases that control a wide variety of cellular and physiological processes. They are known principally for their pivotal role in antioxidant defense but their ability to regulate redox-sensitive signal transduction pathways may be just as important. Prxds are constitu-tively abundant and ubiquitously present in all living organisms from bacteria through higher eukaryotes. Prdxs participate in cellular antioxidant defense by removing hydrogen peroxide, organic hydroperoxides, and peroxynitrite but can also act as molecular chap-erones to prevent protein misfolding during oxidative stress [6, 7]. All Prdxs contain an absolutely conserved N-terminal cysteine residue in the active site that cycles between the reduced and oxidized states and is essential for catalytic activity of these enzymes. The re-

markable feature of the thiol-based redox catalysis is that the enzyme's own substrate may cause over-oxidation of the active-site cysteine to sulfinic acid, resulting in inactivation of the enzyme. The sensitivity to the loss of function due to post-translational modifications such as over-oxidation or phosphorylation provides the basis for the involvement of Prdxs in peroxide sensing and cellular signaling [8]. There are six Prdx proteins in mammals that are encoded by six distinct genes. Wide-spread distribution and high abundance of mammalian Prdxs have been well-documented. They constitute approximately 0.2—1% of soluble proteins in most tissues and cultured mammalian cells [9]. Some cell types demonstrated selective expression of Prdxs. Red blood cells were shown to contain Prdx 1, 2, and 6 [10]. Cell-type specific expression was reported for Prdx 1 and 2 in mouse testis [11] and for Prdx 5 in degenerative human tendon [12]. Differential distribution of individual Prdxs among distinct cell types of brain was shown with the use of immuno-histochemistry [13—16]. Prdxs 2, 3, 4, and 5 were detected in neuron, while Prdx 1 and 6 were localized to glia. The neuronal localization of Prdxs 2—5 and the glial expression of Prdx 1, Prdx 4, and Prdx 6 were also confirmed by [17]. The confinement of individual Prdx to a discrete population of cells may reflect its specific role in cellular physiology. One of us and coauthors earlier showed that Prdx 6 is ubiquitously distributed in epithelial tissues and particularly enriched in the olfactory epithelium [18]. Prdx 6 was identified as the essential component of cellular antioxidant defense system in this tissue. Using immunogold-elec-tron microscopy, we demonstrated the presence of Prdx 6 in rat olfactory neurons. Note that there is no means to assess the presence of each of the six Prdxs in the same cell at the protein level. Immunohistochem-istry and fluorescent in situ hybridization techniques allow for monitoring the co-expression of no more than three genes or gene products at once at tissue sections. Single-cell gene expression analysis provides an experimental solution to the problem of monitoring multiple mRNA transcripts present in individual cells at a given time. It is generally assumed that a single cell contains approximately 10—20 pg of total RNA, 1—3% of which represent mRNA. The problem of limiting RNA can be resolved with the use of RNA amplification techniques that enable generating sufficient targets from sub-picogram quantities of starting sample [19]. This study was aimed to determine the expression profile of Prdx genes in individual olfactory sensory neurons (OSNs). Information about individual cell specific gene expression patterns may help to elucidate the question of whether multiple Prxd subtypes offer redundant or additive protection of neurons against oxidative insults. To date, the co-localization of six Prdxs at the single-cell level has not been studied. We were interested in determining whether individual cells mirror expression profile of the whole cell population. For the first time, we attempted to apply a con-

cept of stochastic gene expression for the interpretation of the data of single-cell analysis.

MATERIALS AND METHODS

Isolation of single cells. Olfactory epithelium was dissected from adult male C57Bl/6J mice. All animal studies were conducted in accordance with the EC Directive 86/609/EEC for animal experiments. Olfactory epithelial turbinates were soaked in an extracellular solution (in mM: 135 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 glucose, and 10 HEPES, pH 7.4 with NaOH) to remove superficial blood and debris. The turbinates were rinsed 3 times at 3 min intervals. Olfactory epithelium was dissociated for 15 min in a solution containing 0.05% trypsin, 140 mM NaCl, 5 mM KCl, 1 mM EGTA,

1 mM EDTA, 10 mM HEPES (pH 7.0) at room temperature. The tissue was then removed and rinsed 3 times with the extracellular solution. To isolate individual cells, a piece of the olfactory epithelium was first shredded by a steel needle and then gently triturated by a fire-polished glass pipette. Dispersed cells were then plated on dishes. Cells that were adhered to the dish were carefully rinsed with the bath solution containing 140 mM NaCl, 2.5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 10 mM HEPES (pH 7.4 with NaOH) to minimize possible contaminations with RNA from ly-sed cells. Dissociated cells were visualized under a Ziess Axioscope-2 microscope and identified by their characteristic morphology. A single olfactory neuron was sucked into a fire-polished glass micropipette with an opening of 4—10 ^m, expelled into a PCR tube filled with a solution for first-strand cDNA synthesis, snap-frozen, and stored at —70°C. Cell lysis was subsequently performed at 70°C.

Reverse transcription and amplification of the whole-cell transcriptome. The cellular material was utilized for the SMART (switching mechanism at the 5' end of RNA templates) cDNA synthesis using Mint cDNA synthesis kit (Evrogen) based on slightly modified protocols described in the user manual. Specifically, a modified oligo-dT primer containing a T7 promoter sequence was used in reverse transcription reaction (5'-AAACGACGGCCAGTGAATTGTAATACGACTCA-

strand cDNA was synthesized directly fr

Для дальнейшего прочтения статьи необходимо приобрести полный текст. Статьи высылаются в формате PDF на указанную при оплате почту. Время доставки составляет менее 10 минут. Стоимость одной статьи — 150 рублей.

Показать целиком