Peroxiredoxin expression in the central nervous system and oxidative modifications : potential role in neurodegeneration

With its large oxygen consumption, the brain is exposed to oxidative stress which is also thought to contribute to neuron loss in neurodegenerative diseases (NDDs). A tight regulation of intracellular levels of reactive oxygen and nitrogen species is thus needed to maintain redox homeostasis. The role of peroxiredoxins (Prdxs) in this regulation is currently believed to be central among cellular peroxidases. To better understand the functions of Prdxs in the brain, we used immunohistochemistry to study the distribution of the six mammalian members (Prdx1-6) in cell types and regions of the mouse brain. Prdx2-5 were found in neurons and Prdx1, 4 and 6 in glia. The brain regional analysis also revealed an uneven distribution among neuronal populations. Interestingly, areas affected in NDDs exhibit particularly low levels of Prdxs. This could contribute to the specific vulnerability of these neurons. Moreover, while Prdx impairment in Parkinson's disease is thought to occur through posttranslational modifications, we showed that Prdxs undergo hyperoxidation in neuroblastoma cells upon exposure to peroxynitrite donor SIN-1, but not with the respiratory inhibitor MPP+. This provides the first evidence of Prdx hyperoxidation by peroxynitrite. We also confirmed a divergent sensitivity to hyperoxidation among Prdxs. Prdx5 is the only isoform for which no KO mice and no Prdx5 overexpressing mice exist, thereby limiting the study of Prdx5 in vivo functions. The first stages for the generation of Prdx5 transgenic mouse models are reported here. However, additional work will be necessary to obtain and characterize the transgenic animals. In conclusion, though Prdx1-6 are all peroxide scavengers, our work reinforces the idea that they may be complementary in the brain as a result of their different substrate specificity, cellular/subcellular localization and posttranslational regulation.(BIOL 3) -- UCL, 201

Silencing of peroxiredoxin 3 and peroxiredoxin 5 reveals the role of mitochondrial peroxiredoxins in the protection of human neuroblastoma SH-SY5Y cells toward MPP+.

Peroxiredoxins (PRDXs) are a family of peroxidases well conserved throughout evolution. Human PRDX3 and PRDX5, two mitochondrial PRDXs, have been implicated in several pathologies associated with oxidative stress. However, the individual role of PRDX3 and PRDX5 in cellular antioxidant defense has never been well established due to their overlapping peroxidatic activities. We investigated the expression and function of mitochondrial PRDXs in human neuroblastoma SH-SY5Y cells. Our results show that PRDX3 and PRDX5 are expressed constitutively in these neuronal cells. To examine further the function of mitochondrial PRDXs, we silenced the expression of PRDX3 and/or PRDX5 using small hairpin RNAs. Our results show that mitochondrial PRDX-depleted cells are more prone to oxidative damages and apoptosis induced by MPP(+), a complex I inhibitor which provides an experimental paradigm of Parkinson's disease

Peroxiredoxin 5: structure, mechanism and function of the mammalian atypical 2-Cys peroxiredoxin

Peroxiredoxin 5 (PRDX5) was the last member to be identified among the six mammalian peroxiredoxins. It is also the unique atypical 2-Cys peroxiredoxin in mammals. Like the other five members, PRDX5 is widely expressed in tissues but differs by its surprisingly large subcellular distribution. In human cells, it has been shown that PRDX5 can be addressed to mitochondria, peroxisomes, the cytosol, and the nucleus. PRDX5 is a peroxidase that can use cytosolic or mitochondrial thioredoxins to reduce alkyl hydroperoxides or peroxynitrite with high rate constants in the 10(6) to 10(7) M(-1)s(-1) range, whereas its reaction with hydrogen peroxide is more modest, in the 10(5) M(-1)s(-1) range. PRDX5 crystal structures confirmed the proposed enzymatic mechanisms based on biochemical data but revealed also some specific unexpected structural features. So far, PRDX5 has been viewed mainly as a cytoprotective antioxidant enzyme acting against endogenous or exogenous peroxide attacks rather than as a redox sensor. Accordingly, overexpression of the enzyme in different subcellular compartments protects cells against death caused by nitro-oxidative stresses, whereas gene silencing makes them more vulnerable. Thus, more than 10 years after its molecular cloning, mammalian PRDX5 appears to be a unique peroxiredoxin exhibiting specific functional and structural features

Insights into mammalian peroxiredoxins : expression in the central nervous system with a focus on neuronal populations affected in neurodegenerative disorders.

Peroxiredoxins (PRDXs or Prxs) are peroxidases able to reduce peroxides with a high efficiency, conferring to these enzymes functions in cytoprotection and redox signaling. PRDXs are abundant and widely expressed in most mammalian tissues. Recently, a modified pattern of PRDXs has been reported in neurodegenerative disorders such as Parkinson’s disease (PD), in term of expression level or post-translational modifications, with consequences for their activity. Emerging evidence point to the potential role of PRDXs in the pathogenesis of these neurodegenerative diseases. No systematic analysis of PRDX expression in the central nervous system (CNS) has been conducted so far. In this work, we first confirmed PRDX cell type expression in the mouse CNS by immunohistochemistry (IHC) through co-localization with specific markers such as NeuN, GFAP, Olig2 and Mac1. Several PRDXs are mainly expressed in glial cells (PRDX4 and PRDX6), but most of them are expressed in neurons (PRDX2 to PRDX5). Moreover, focusing on neuronal PRDXs, we report a complete characterization of PRDX expression levels in the most representative neuronal populations of the murine CNS with IHC procedures. Finally, tyrosine hydroxylase-PRDX double immunofluorescence showed that PRDX2 to PRDX5 are moderately to weakly expressed in the dopaminergic neurons from substantia nigra pars compacta, the neuronal population affected in PD. This faint expression could contribute to the vulnerability of these neurons in the disease. At the opposite, PRDXs are strongly expressed in motor neurons of the spinal cord which degenerate in the Amyotrophic Lateral Sclerosis (ALS). Further investigations will be needed to determine whether PRDXs play a major role in neurodegeneration

Abolition of peroxiredoxin-5 mitochondrial targeting during canid evolution.

In human, the subcellular targeting of peroxiredoxin-5 (PRDX5), a thioredoxin peroxidase, is dependent on the use of multiple alternative transcription start sites and two alternative in-frame translation initiation sites, which determine whether or not the region encoding a mitochondrial targeting sequence (MTS) is translated. In the present study, the abolition of PRDX5 mitochondrial targeting in dog is highlighted and the molecular mechanism underlying the loss of mitochondrial PRDX5 during evolution is examined. Here, we show that the absence of mitochondrial PRDX5 is generalized among the extant canids and that the first events leading to PRDX5 MTS abolition in canids involve a mutation in the more 5' translation initiation codon as well as the appearance of a STOP codon. Furthermore, we found that PRDX5 MTS functionality is maintained in giant panda and northern elephant seal, which are phylogenetically closely related to canids. Also, the functional consequences of the restoration of mitochondrial PRDX5 in dog Madin-Darby canine kidney (MDCK) cells were investigated. The restoration of PRDX5 mitochondrial targeting in MDCK cells, instead of protecting, provokes deleterious effects following peroxide exposure independently of its peroxidase activity, indicating that mitochondrial PRDX5 gains cytotoxic properties under acute oxidative stress in MDCK cells. Altogether our results show that, although mitochondrial PRDX5 cytoprotective function against oxidative stress has been clearly demonstrated in human and rodents, PRDX5 targeting to mitochondria has been evolutionary lost in canids. Moreover, restoration of mitochondrial PRDX5 in dog MDCK cells, instead of conferring protection against peroxide exposure, makes them more vulnerable

Cytotoxicity induced by hydrogen peroxide (A–B) and <i>t</i>-BHP (C–D) in MDCK cells overexpressing human PRDX5.

<p>After one hour exposure to high levels of peroxide, cell death was evaluated by LDH release. The total LDH release (100% cell death) was determined after lysis of the cells with 2% Triton X-100. Assays were performed three times for each clone and values are means ± SEM. Significance is designated as *p<0.05, **p<0.01, ***p<0.001. <i>t-</i>BHP, <i>tert</i>-butyl hydroperoxide.</p

Dog PRDX5 subcellular distribution.

<p>(A) Intracellular localization of dog PRDX5 was assessed by liver subcellular fractionation. Post-nuclear (E), cytosolic (S), mitochondrial (Mito) and peroxisomal (Perox) fractions were analyzed for marker enzyme and PRDX5 content by Western blotting, using anti-Hsp90 (cytosolic), anti-COX4 (mitochondrial), anti-catalase (peroxisomal) and anti-PRDX5 antibodies. Fifteen µg of proteins from each fraction were loaded. PRDX5 subcellular localization was also assessed by immunofluorescence detection of endogenous PRDX5 in MDCK cells (B, E) with co-localization with MitoTracker Red (C) or with peroxisomal catalase (F). Nuclei were counterstained with DAPI. <i>Hsp90</i>, heat shock protein 90; <i>COX4</i>, cytochrome c oxidase subunit 4.</p