BS407-Written Report
A Revelation by the Yeast Model System
Introduction
Friedreich Ataxia (FA) is the most prevalent hereditary ataxia, affecting 1 in every 50000 individuals worldwide (Puccio2001, Calabrese2005). Individuals affected with this neurodegenerative disease are enfeebled by loss of coordination (especially gait and limb disturbance), muscle weakness and deterioration of sensory responses (Knight1999, Puccio2001, Calabrese2005). Additional non-neurological consequences include cardiomyopathy, diabetes mellitus and scoliosis (Knight1999, Foury1997, Puccio2001, Calabrese2005). These symptoms worsen with aging, and patients rarely reach forty years of age (Knight1999).
The disease is inherited in an autosomal recessive fashion due to mutations in the human FA gene FRDA1 (also known as X25), which encodes for the protein frataxin (Foury1997, Puccio2001, Calabrese2005). Though frataxin is expressed ubiquitously throughout the body, its expression occurs predominantly in the neurons (especially in the dorsal root ganglia and cerebellum) as well in some non-neuronal sites like the pancreas, the heart and the skeletal muscles (Knight1999, Puccio2001, Calabrese2005). However, the effects of FA mainly arise in cells that are non-dividing and irreplaceable, in cells relying exclusively on aerobic metabolism, and in cells
with high mitochondrial activity and number, like the neurons and cardiomyocytes (Knight1999, Puccio2001).
The causative mutation for most cases of FA is the hyperexpansion of GAA trinucleotide repeats in the first intron of FRDA1, which forms a 'sticky' DNA triple helix structure that prevents transcriptional initiation of the gene (Knight1999, Puccio2001, Calabrese2005). Occasionally, point mutations can also inactivate the frataxin gene (Knight1999). In the case that both gene copies have the GAA hyperexpansion, the severity of disease is correlated to the degree of expansion in the smaller copy (Knight1999, Calabrese2005). Albeit the exact physiopathological mechanism of FA remains elusive, numerous research efforts dealing with FA and its causal gene have extensively increased our understanding of the disease. Thus this review outlines the advances in the knowledge of FA provided by the yeast model, along with supporting evidence from studies in other organisms. Therapeutic advances to combat FA and future prospects are also discussed.
Yeast as a genetic model for Friedreich Ataxia
Yeast (Saccharomyces cerevisiae) as a genetic model has been very useful for the comprehension of the molecular basis of FA. As a simple unicellular eukaryote, not only is it easy to
genetically manipulate but it also shares many of its cellular functions with humans, and frataxin is no exception. Although frataxin's amino acid sequence did not show similarity to any known functional domains at the time of its discovery, its sequence is highly conserved evolutionarily from yeast to humans (Foury1997, Knight1999, Puccio2001). In fact, it has been proven that human frataxin can rescue yeast frataxin mutants from the FA phenotype (Puccio2002, Irazusta2006).
Early studies in yeast involved experimental deletions of the FRDA1 yeast orthologue (YFH1, also known as YDL120). YFH1- knockout mutants showed a reduction in respiratory activity and an inability to grow in non-fermentable carbon sources, suggesting a role for frataxin in oxidative phosphorylation (Foury1997, Knight1999, Puccio2001, Puccio2002). It was also confirmed then that both human and yeast frataxin has a mitochondria localization signal peptide at the N-terminus, which is removed via two sequential cleavages upon arrival in the mitochondria (Foury1997, Knight1999, Puccio2001). Thus, frataxin's role has long been pinpointed to oxidative respiration in the mitochondria.
The Role of Iron
Even though frataxin was already speculated to be involved in mitochondrial respiration, how the loss of frataxin causes FA remained uncertain. But then, Fe2+ chelation studies boosted scientific interest on FA by remarkably demonstrating a 10 to 15-fold accumulation of iron in the mitochondria of YFH1- mutants compared to the wildtype (Foury1997, Knight1999, Calabrese2005). Further research discovered that the high-affinity mitochondrial iron import system in YFH1- mutants is constitutively active, causing an upsurge in mitochondrial iron levels while shrinking the amount of cytosolic iron (Knight1999, Puccio2001, Puccio2002). Additionally, it was found out that iron accumulation in YFH1- mutants was accompanied by an increase in the synthesis of reactive oxygen species (Calabrese2005). Thus, in support with biochemical data, it was suggested that these elevated iron levels in the mitochondria lead to increased oxidative stress in cells due to Fe2+ catalyzed production of highly reactive hydroxyl radicals
via the Fenton reaction (Foury1997, Knight1999, Radisky1999, Puccio2001, Puccio2002). Meanwhile, in vitro experiments mimicking heart tissues of FA patients have demonstrated that only a specific form of iron, Fe2+ (but not Fe3+), mediates oxidative toxicity in the mitochondria (Puccio2001). These connections between oxidative stress and FA was further substantiated with observations that exposure of YFH1- yeast to oxidative agents (H2O2 and diamide) and oxidant-generating agents (Fe2+ and Cu2+) caused diminished growth and loss of mtDNA (i.e. induction of rho- mutants) (Foury1997).
Similar evidence has been obtained in humans. Electron microscopy of heart tissues of FA patients reveals the presence iron deposits associated with mitochondrial damage (Knight1999, Puccio2002, Calabrese2005). Moreover, it has been demonstrated that FA patients have elevated levels of free radicals (Puccio2002). Thus, the connection between iron, oxidative stress and the pathogenesis of FA could not be ignored both in yeast and humans.
Though it was previously accepted that iron overload in the mitochondria brings about the increased production of hydroxyl radicals, recent discoveries have acquired divergent results from this view. New experiments have shown that oxidative stress in YFH1- yeast only occurs under aerobic conditions, regardless of iron levels in mitochondria (Bulteau2007). While this implies that oxygen is essential in the production of free radicals in FA, this also suggests the revolutionary idea that iron accumulation is non-essential in generating oxidative stress in YFH1- mutants. This notion was even supported by findings showing that defects in Fe-S cluster biosynthesis lead to iron accumulation in the mitochondria but not oxidative stress (Muhlenhoff2000, Bulteau2007). Conversely, it was also discovered that most of the iron in the mitochondria is not in its ferrous ion form, and YFH1- mutant and wild-type yeast show equivalent Fe2+ levels (Puccio2001, Bulteau2007). Since only Fe2+ triggers toxicity, a good explanation why oxidative stress might not be a direct consequence of iron accumulation in the mitochondria is that the type of iron accumulated in the mitochondria of frataxin mutants cannot induce cytotoxicity.
The function of frataxin
Several clues about frataxin's function have already been unraveled (especially in iron regulation and oxidative stress), but its exact function was still in question.
Given that iron accumulates in the mitochondria of YFH1- mutants, it was reasonable to suspect that frataxin operates in mitochondrial iron efflux. This hypothesis was supported by studies where reintroduction of frataxin in YFH1- mutant yeast led to a gradual decrease in mitochondrial iron (Radisky1999). Since analysis of its amino acid sequence point out that frataxin contains no transmembrane domains, it was speculated that frataxin mediates its activity through an ABC transporter of iron (Radisky1999). On the other hand, it is equally logical to think that frataxin is involved in iron influx. This suggestion is backed by findings that the expression of YFH1 repressed the expression of 'iron regulon' members, especially FET3, which is important in the high-affinity iron transport system into the mitochondria (Radisky1999, Puccio2002). But either way, it is very likely that frataxin participates in regulation of mitochondrial Fe2+ concentrations, seeing that iron accumulation is the main phenotype observed in FA. In fact, this theory seems to be defended by evolution itself, for the reason that recently, frataxin expression was found to be dependent on environmental iron concentrations, which indicates that evolution has adapted mechanisms to counter the detrimental effects of too much iron through frataxin (Seguin2009).
Studies have also detected a loss of mitochondrial DNA (mtDNA) in certain YFH1- mutant strains, suggesting that frataxin also plays a role in protection of mtDNA integrity (Foury1997). This observation was considered to be in agreement with the iron efflux hypothesis. Supposing that (1) oxidative stress in FA is brought about by iron accumulation in the mitochondria, and (2) oxidative stress can cause the destabiliazation and mutation of mtDNA, frataxin can defend mtDNA by preventing the increase in oxidative stress through regulation of mitochondrial iron concentrations (Knight1999). Though mtDNA mutation may not be a direct outcome of frataxin loss, the mutation or loss of mtDNA could contribute to the pathophysiology of FA, as mtDNA infidelities have been
linked to other neurodegenerative disease such as myoclonic epilepsy and ragged red fiber syndrome (Knight1999).
Another proposed role for frataxin was promoting antioxidant defense against free radical damage. Supporting evidence include: (1) elevated frataxin levels can detoxify reactive oxygen species by activation of glutathione peroxidase and elevation of reduced thiol levels in mouse models (Calabrese2005), (2) overexpression of frataxin in humans and yeast increases resistance to oxidative damage and decreases reactive oxygen species production (Seguin2009), (3) FA patients experience impairment in antioxidant defenses including glutathione and superoxide dismutase (SOD) (Puccio2002, Calabrese2005), (4) Decreased SOD activity has been observed in mice with FA (Irazusta2006), and (5) human fibroblasts from FA patients fail to induce action of the antioxidant enzyme manganese superoxide dismutase (MnSOD) (Puccio2002, Calabrese2005, Irazusta2006).
However, more recent experiments infer yet another function for frataxin. Using recombinant yeast frataxin, it was shown that frataxin monomers self-assemble in the presence of increasing Fe2+ concentrations into progressively larger homomultimers that can accommodate more and more iron. (Adamec2000, Park2003, Gakh2008). Since this displays a striking similarity to the iron-storage protein ferritin, frataxin could act by sequestering and storing iron (Adamec2000, Park2003, Gakh2008). Some biochemical studies also support the theory of frataxin as a mitochondrial iron-storage protein (Puccio2001).
The multimer assembly of frataxin is highly controlled in vivo and responds dynamically to changes in mitochondrial iron levels and stress exposure, indicating how important this process is (Gakh2008). This assembly is even more particularly important during stress conditons as mutations affecting frataxin assembly prevent frataxin-mediated defense from cell damage (Gakh2008). Since the cell gives so much attention in the regulation of this process, it can be considered that frataxin may not only work as an iron-storage protein. In fact, there is evidence that the sequestration of Fe2+ by frataxin complexes can also prevent production of free radicals via the Fenton
pathway, which is probably because frataxin oxidizes its sequestered Fe2+ into the Fe3+ form when mitochondrial iron is in excess (Adamec2000, Park2003). In addition, sequestered Fe2+ can be delivered directly to ferrochelatase and ISU-type proteins to support heme and Fe-S cluster biosynthesis in both yeast and humans, which also indicates that sequestered Fe2+ is actually bioavailable (Park2003, Ramazzotti2004). Therefore, this iron sequestration complex formed by frataxin may not simply serve as an iron store but may have additional functions. With the knowledge that there are already various proteins that were found to have multiple functions, it is possible that seemingly contradictory hypothesises about the functions of frataxin can indeed be reconciled. For example, provided that sequestered Fe2+ in frataxin multimers is bioavailable, we can put forward the idea that frataxin may also be able to pass iron to the mitochondrial iron export system to enhance iron efflux.
Effect on iron-dependent proteins
Another noteworthy observation in the yeast model of FA was that despite the high concentrations of iron in the mitochondria in YFH1- mutants, there is a reduction in the activity of heme-containing proteins and Fe-S proteins such as mitochondrial respiratory complexes and aconitase (Knight1999, Puccio2001). However, Fe-S proteins are particularly susceptible to rapid oxidative degradation by free radicals. Hence, it was suggested that this deficiency is a result of oxidative damage produced by the increased levels of free radicals in the mitochondria (Puccio2002, Bulteau2007). The inactivation of these proteins may then further aggravate the iron overload in the mitochondria due to release of their bound iron atoms (Knight1999). Contrastingly, there is also striking evidence against this hypothesis. FA mouse models have shown that iron-dependent protein deficiency occurs prior to free radical production (Puccio2002). Moreover, frataxin overexpression increases resistance to oxidative damage but results in defects in Fe-S assembly, signifying that oxidative damage is not a consequence of defective Fe-S proteins (Seguin2009).
Meanwhile, several reports have strongly suggested that frataxin can act as an iron donor for the in vivo synthesis of Fe S clusters and heme groups (including the previous statement that frataxin complexes can pass Fe2+ to ferrochelatase and ISU-type proteins to support heme and Fe-S cluster synthesis) (Park2003, Ramazzotti2004). In addition, it has been proven that iron homeostasis strongly depends on Fe-S cluster assembly, and consequently, any defect in Fe-S cluster biogenesis can lead to mitochondrial iron accumulation (Seguin2009, Muhlenhoff2000). Hence, if frataxin really supports Fe-S cluster synthesis, mitochondrial accumulation of iron can be explained by defective Fe-S cluster synthesis in FA. Furthermore, oxidative stress can also arise from this since several components of the respiratory machinery are Fe-S proteins and defects in respiration would result to higher levels of reactive oxygen species (Calabrese2005). These results provided an alternative theory on the effects on iron-dependent proteins in FA.
But then again findings from later research disagree with this notion. In yeast YFH1- mutants, both MnSOD and iron-sulfur enzymes was observed to get inactivated, and recovery from both phenotypes occurred when yeast are supplied with high manganese levels, even if high iron concentration and frataxin absence is maintained (Irazusta2006). This implies that neither frataxin paucity nor high levels of iron directly lead to Fe-S protein deficiency. However, a decrease in iron levels was also shown to rescue mutants from both phenotypes as well, denoting that the apparent manganese shortage occurs downstream of iron accumulation (Irazusta2006). Taking these observations together, it was proposed that the accumulation of iron due to frataxin deficiency impairs MnSOD, possibly by the displacement of manganese atoms in MnSOD by iron (Irazusta2006). Consequently, antioxidant defense activity by MnSOD is lost, allowing accumulation of free radicals and oxidative damage to Fe-S proteins (Irazusta2006).
Additional evidence in support this theory (which puts iron accumulation upstream of Fe-S protein deficiency) is that as long as iron accumulation does not occur, Fe-S proteins (specifically the respiratory complexes) do not show decreased activity (Puccio2001, Puccio2002). In contrast, while iron
chelation experiments in YFH1- mutants have rescued respiratory complex activity as expected, this does not happen for t he Fe-S protein aconitase (Puccio2001, Puccio2002). This observation has two implications: (1) aconitase inactivation is not a consequence of iron accumulation, and as aconitase inactivation is an indicator of increased intracellular oxidative damage, iron accumulation does not cause the increased formation of hydroxyl radicals, and (2) aconitase deficiency must be directly linked to the absence of functional frataxin, which is supported by studies demonstrating that frataxin can interact with aconitase directly to prevent oxidative disassembly of aconitase's Fe-S clusters and donate iron for aconitase's reactivation (Bulteau2004). In addition, this observation that aconitase is directly dependent on frataxin led to the conjecture that aconitase could act as a regulator of the iron import system (which becomes constitutively active in YFH1- mutants), with the basis that other aconitases have been reported to have regulatory functions (Irazusta2006).
Other mutations that produce similar phenotypes
While the consequences of YFH1 mutation has been extensively studied in yeast, certain mutations in other yeast genes were also found to produce phenotypes similar to FA, specifically mitochondrial iron accumulation and mtDNA damage (Knight1999, Puccio2001).
One example is SSQ1, which encodes for the mitochondrial heat shock protein 70 (HSP70) and acts as a chaperone involved in regulation of protein conformation, import of mitochondria-tagged proteins into the mitochondria, and Fe-S cluster assembly (Puccio2001, Puccio2002, Calabrese2005). Experiments with SSQ1- mutants reveal impaired kinetics in the 2nd cleavage of frataxin 'post-mitochondrial import' processing (Knight1999). This suggests that SSQ1 is important in the processing of frataxin to its mature form (Knight1999). Furthermore, the dense bodies observed upon electron microscopy of SSQ1- mutants are suspected to contain not only iron but also toxic aggregates of misfolded proteins, which may contribute to FA's pathophysiology (Knight1999). SSC1, a
homologue of SSQ1 in yeast, is also speculated to contribute to the FA phenotype (Knight1999).
Another example is ATM1, which encodes for an ATP-binding casette transporter involved in the export of Fe-S clusters (Puccio2001). It is considered that frataxin may promote iron efflux through ATM1, though a direct link remains to be established (Radisky1999). Therefore, further investigation on the characteristics of ATM1 is necessary to elucidate its role in FA. In contrast, XQ13, the human counterpart of ATM1, has been associated with X-linked sideroblastic anemia with ataxia (Knight1999). Though ataxia is also observed in this disease, the iron accumulation phenotype mainly occurs in human red blood cells, a feature not witnessed in FA (Knight1999). Thus, ATM1 characterization may also lead to the elucidation of the mechanism of tissue-specificity in FA.
There are also some mutations in other human genes that bring about FA-like phenotypes. For example, mutation of the gene encoding for α-tocopherol lead to the inherited neurodegenerative disease AVED (ataxia with isolated vitamin E deficiency), which produces a phenotype that closely resembles FA (Knight1999). Although the relationship between AVED and FA has not been found yet, the antioxidant property of vitamin E is suspected to counteract the oxidative stress resulting from the loss of frataxin in FA.
Therapeutic advances
Despite years of intensive research on the pathogenesis and disease mechanisms of FA, no effective therapy to fight this disease has been formulated hitherto. However, several approaches to counteract the detrimental effects of FA have been proposed.
First, the development of Fe2+ chelators may provide a good treatment for FA patients since Fe2+ might be mediating the effects of FA (Radisky1999, Puccio2001, Puccio2002). However, chelators like desferrioxamine may cause detrimental side effects, and may sometimes displace iron-mediated toxicity rather than combating it (Puccio2001, Puccio2002). Recently, development of PCIH (2-pyridylcarboxaldehyde isonicotinoyl hydrazone) analogues, which can chelate iron
specifically from the mitochondria, has commenced to hopefully provide a better iron chelation therapy for FA (Calabrese2005).
It has been noticed that the pathology of FA is similar to deficiencies in vitamin E, which is an antioxidant that protects neurons from oxidative damage (Foury1997). Thus, another alternative contended is the use of antioxidants to slow down the progression of the disease (Puccio2001, Puccio2002, Calabrese2005). The antioxidant idebenone may be a promising potential drug against FA as it prevents iron-induced injury, does not interfere with aconitase activity, and diffuses freely through membranes (including the blood-brain barrier) (Puccio2001, Puccio2002). Clinical trials with idebenone and other antioxidants such as Coenzyme Q10 and Vitamin E have provided very encouraging results (Puccio2002, Calabrese2005). In addition, the development of mitochondria-targeting antioxidants has proven to be even much more effective than their general analogues (Calabrese2005).
Heat-shock protein (HSP) induction is also seen as a plausible therapeutic approach as HSPs can provide stress tolerance and cytoprotection against various metabolic disturbances (Calabrese2005). Supporting evidence for this approach includes findings that in several neuronal models, overexpression of HSPs, especially HSP70, protect cells from injury leading to apoptosis or necrosis (Calabrese2005).
Finally, manganese administration, which was considered due to research that demonstrated the recovery from Fe-S protein deficiency by manganese supplication, may also be feasible therapeutic approach (Irazusta2006). However, care must be taken if this should be applied since high doses of manganese can be toxic (Irazusta2006).
Further research
Many questions remain unsolved regarding FA, as neither the physiological action of frataxin nor the pathological mechanism of the disease has been unraveled completely. Though it is clear that mitochondrial iron accumulation, oxidative stress and Fe-S protein deficiency are involved in the pathology of FA, how they interact to give rise to the FA phenotype is still
uncertain. Other observed characteristics that can be put into consideration as contributing factors to FA pathophysiology include mtDNA damage, manganese-dependent protein inactivation and toxic protein aggregation. Nonetheless, it is always possible that frataxin carry out multiple functions in the cell that embraces some, if not all, of the above.
Further research to elucidate the complete picture of FA need not be limited to frataxin. In fact, other genes that cause similar phenotypes to FA (such as ATM1, AVED-causative gene, etc.) may provide crucial information in solving this conundrum.
With all the results presented in this review (which cannot even possibly cover all the current knowledge on FA)
References
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