The characterization of the Caenorhabditis elegans mitochondrial thioredoxin system uncovers an unexpected protective role of TRXR-2 in -amyloid peptide toxicity

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Given the difficulty of studying the function of the mitochondrial thioredoxin system in the context of a complete animal using the mouse or Drosophila models (due to embryonic or larval lethal phenotypes, respectively), we turned our interest to the nematode Caenorhabditis elegans, an excellent genetically tractable model where the mitochondrial thioredoxin system is highly homologous to that of mammals.

We first demonstrated that C. elegans trx-2 and trxr-2 genes encode proteins that are targeted to mitochondria in vivo, driven by putative MTS at their respective N-terminus. Interestingly, the rather ubiquitous expression pattern of the trxr-2 gene deeply contrasts with the highly restricted one of the trx-2 gene in AIYL/R and ASEL neurons and muscle cells, which suggests that TRXR-2 might have additional substrates other than TRX-2. We identified TRX-2 on the basis of its conserved active site sequence WCGPC and it cannot be ruled out that other thioredoxin family members with a more divergent active site sequence are also present in mitochondria. Furthermore, it has been reported that human mitochondrial GRX-2 is a substrate for mitochondrial thioredoxin reductase (22), a function that could also be conserved in worms (at least 4 glutaredoxin genes have been reported in C. elegans, see below). Likely, other TRXR-2 substrates not functionally or structurally related to thioredoxins and glutaredoxins might also exist in mitochondria. It is worth noting that TRX-2 is not detected in the intestine under unstressed conditions (FIG. 2A-C and 2J-K) but it is expressed in this organ upon UPR^mito induction (FIG. 6), indicating that TRX-2 expression might be turned on in additional tissues depending on stress conditions.

C. elegans is, to date, the only metazoan in which the mitochondrial thioredoxin system is not essential for survival. More surprisingly, trx-2 and trxr-2 single and double mutants do not show enhanced sensitivity to different stressors. Collectively, this lack of phenotype supports the idea that a redundant system exists to counterbalance the absence of the mitochondrial thioredoxin system under both unstressed and stressed conditions. One alternative could be that the C. elegans cytosolic thioredoxin reductase gene trxr-1 might produce an isoform targeted to mitochondria as it happens in other organisms (48,59). However, we have ruled out this possibility as trxr-1; trxr-2 double mutants are also fully viable (51). The viability of the trxr-1; trxr-2 double mutants raises the interesting question of which enzyme(s) maintain the different worm thioredoxins in their reduced active state in the absence of both thioredoxin reductases. An obvious candidate is the glutaredoxin system based on the numerous reports of functional redundancy of both thioredoxin and glutaredoxin systems (either cytoplasmic or mitochondrial) in several organisms (12,58). We have recently uncovered an example of such functional redundancy in C. elegans as trxr-1 animals feeding on glutathione reductase (gsr-1) RNAi bacteria display a highly penetrant larval arrest phenotype due to defective cuticle ecdysis (51). However, RNAi downregulation of gsr-1 or any of the four worm glutaredoxins (glrx-5, glrx-10, glrx-21 and glrx-22) in trx-2 and trxr-2 single and double mutant backgrounds did not result in any obvious synthetic phenotype (Supplemental Table 3). As RNAi feeding penetrance can be highly variable depending on genetic backgrounds and tissue expression, combinations of glutaredoxin system mutants with those of trx-2 and trxr-2 will be needed to unequivocally identify the redundant system for the mitochondrial thioredoxin system. Other plausible explanations for the lack of phenotype of the mitochondrial thioredoxin system mutants could be that the system is dispensable under normal laboratory growth conditions while being essential in the wild or that, alternatively, the mitochondrial thioredoxin system plays a modulatory role in non-essential ROS-mediated signaling mechanisms.

The induction of trx-2 and trxr-2 upon UPR^mito activation suggests that the mitochondrial thioredoxin system is part of the chaperone machinery required to cope with the load of unfolded/misfolded proteins that accumulate and aggregate when mitochondrial proteostasis is compromised. Most likely the function of the mitochondrial thioredoxin system under UPR^mito is aimed at reducing incorrect disulfide bonds in these misfolded proteins to either facilitate native refolding or their export from mitochondria and subsequent degradation by proteasome (56). The maintenance of cellular and subcellular proteostasis is essential for organismal survival and its imbalance dramatically affects the function of all cellular organelles, including mitochondria, eventually leading to enhanced progression of aging and development of neurodegenerative diseases such as Alzheimer´s, Parkinson´s or Huntington´s disease (26).

In this work, we have found a protective role of the trxr-2 gene on the aging-dependent progressive paralysis phenotype caused by Ab aggregation in worm muscle cells, a well established model for Alzheimer´s disease (31). This protective role is not shared by the trx-2 gene despite it is also expressed in muscle cells, reinforcing the notion that additional substrates for TRXR-2 might mediate such a protective function. We have shown here that while trxr-2 downregulation clearly enhances paralysis, increased TRXR-2 expression does not alleviate the paralysis onset. On the other hand, trxr-2 downregulation does not substantially modify the total levels of Ab species nor amyloid deposits which, in turn, are dramatically diminished in TRXR-2 overexpressing animals. The lack of correlation between total amyloid loads and the paralysis rate has been previously reported in this AD worm model (15,32) although no molecular mechanism has been proposed yet to clarify this fact. A plausible explanation relates to the recent finding that muscle mitochondria of Ab worms are highly fragmented (CD Link, unpublished data). Our preliminary unpublished results indicate that decreased TRXR-2 levels further enhance the mitochondrial fragmentation of Ab worms, which might account for the increased paralysis phenotype in trxr-2 mutants or trxr-2 RNAi treated Ab worms without provoking noticeable changes in total Ab load or amyloid deposits. Alternatively, despite the fact that higher levels of TRXR-2 strongly reduce both total Ab load and amyloid deposits, it is possible that the residual amount of Ab species detected in both immunoblots and immunostainings of overproducing TRXR-2 Ab worms might still be sufficient to induce paralysis, thus explaining why overexpressing TRXR-2 does not improve paralysis onset.

Elucidating the mechanism(s) by which TRXR-2 overexpression degrades most Ab load is of enormous interest. For instance, it is conceivable that TRXR-2 might interact with proteins implicated in the specific degradation of the Ab peptide and amyloid deposits in mammals, such as angiotensin-converting enzyme, insulin-degrading enzyme or neprilysin (2), all of which have close orthologues in worms. Alternatively, an interesting possibility relates to the finding that reduced insulin signaling improves paralysis of Ab worms by promoting autophagic degradation of Ab (13) Thus, it could be possible that trxr-2 overexpression triggers a decrease of insulin signaling resulting in Ab autophagic-dependent degradation. As a whole, it will be very important to gain deeper insight into the molecular mechanisms underlying the dual role of TRXR-2 in alleviating paralysis and decreasing amyloid deposits in Ab worms and the potential application of this knowledge to the human scenario in Alzheimer´s disease

In conclusion, the characterization of the C. elegans mitochondrial thioredoxin system reported in this work provides a new tool to further investigate the function of this redox system in metazoan physiology and pathology. The genetic amenability of C. elegans paves the way to use the mitochondrial thioredoxin mutants in genetic screens to identify other systems involved in the maintenance of organismal redox homeostasis, and particularly opens new possibilities to understand the role of mitochondrial redox systems in the pathology of neurodegenerative diseases.